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BIOTECHNOLOGY

JULIAN Ε.

DAVIES,

Editor

Pasteur Institute
Paris, F r a n c e

Editorial

Board

L. B o g o r a d
J. B r e n c h l e y
P. Broda

H a r v a r d University, C a m b r i d g e , U S A
P e n n s y l v a n i a State University, University P a r k , U S A
University of M a n c h e s t e r Institute of Science and T e c h n o l o g y ,
M a n c h e s t e r , United K i n g d o m

A.L. Demain
D . E . Eveleigh
D . H . Gelfand
D.A. Hopwood
S.-D. Kung
J . - F . Martin
C. Nash
T. Noguchi
W . Reznikoff
R.L. Rodriguez
A . H . Rose
P. Valenzuela

M a s s a c h u s e t t s Institute of T e c h n o l o g y , C a m b r i d g e , U S A
Rutgers University, N e w B r u n s w i c k , U S A
C e t u s C o r p o r a t i o n , E m e r y v i l l e , California, U S A
J o h n Innes Institute, N o r w i c h , United K i n g d o m
University of M a r y l a n d , C o l l e g e P a r k , U S A
University of L e o n , L e o n , Spain

D. Wang

S c h e r i n g - P l o u g h C o r p o r a t i o n , Bloomfield, N e w J e r s e y , U S A
S u n t o r y , L t d . , T o k y o , Japan
University of W i s c o n s i n , M a d i s o n , U S A
University of California, D a v i s , U S A
University of Bath, B a t h , United K i n g d o m
C h i r o n , I n c . , E m e r y v i l l e , California, U S A
M a s s a c h u s e t t s Institute of T e c h n o l o g y , C a m b r i d g e , U S A

BIOTECHNOLOGY SERIES
1. R. S a l i w a n c h i k

Legal Protection for Microbiological
Genetic Engineering
Inventions

2. L . V i n i n g (editor)

Biochemistry
and Genetic
Commercially
Important

3 . K . H e r r m a n n and
R. S o m e r v i l l e
(editors)

Amino Acids:
Regulation

4 . D . W i s e (editor)

Organic

5 . A . L a s k i n (editor)

Enzymes and Immobilized
Biotechnology

6. A . D e m a i n and
N . S o l o m o n (editors)

Biology

7. Z . V a n ë k and
Z . H o s t â l e k (editors)

Overproduction
of Microbial
Metabolites:
Strain Improvement
and Process
Control
Strategies

8. W . Reznikoff and

Maximizing

Gene

Expression

Mammalian

Cell

Technology

Regulation
of
Antibiotics

Biosynthesis

Chemicals

and

from

of Industrial

and

Genetic

Biomass
Cells

in

Microorganisms

L. G o l d (editors)
9. W . Thilly (editor)
10. R. R o d r i g u e z and
D . D e n h a r d t (editors)

Vectors:

1 1 . S . - D . K u n g and
C . A r n t z e n (editors)

Plant

12. D . W i s e (editor)

Applied

13. P . Barr, A . B r a k e , and
P . V a l e n z u e l a (editors)

Yeast Genetic

14. S. N a r a n g (editor)

Protein Engineering:
Approaches
to the
Manipulation
of Protein
Folding

15. L . G i n z b u r g (editor)

Assessing

16. N . First and
F . Haseltine (editors)

Transgenic

Vectors

A Survey

of Molecular

and Their

Cloning

Uses

Biotechnology

Biosensors
Engineering

Ecological
Animals

Risks of

Biotechnology

ÏV

Biotechnology Series

17. C . H o and D . W a n g
(editors)

Animal

Cell

Bioreactors

18. I. G o l d b e r g and
S. R o k e m (editors)

Biology

of

Methylotrophs

19. J. G o l d s t e i n (editor)

Biotechnology

2 0 . R. Ellis (editor)

Vaccines: New Approaches
to
Immunological
Problems

2 1 . D . Finkelstein and

Biotechnology
Technology

C . Ball (editors)

of

Blood

of Filamentous
and
Products

Fungi:

Biotechnology of
Filamentous Fungi
Technology and Products

Edited

by

David B. Finkelstein
Christopher Ball
Panlabs Incorporated
Bothell, Washington

Butterworth-Heinemann
Boston

London

Oxford

Singapore

Sydney

Toronto

Wellington

Copyright © 1992 by Butterworth-Heinemann, a division of Reed Publishing (USA) Inc. All rights
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Library of Congress Cataloging-in-Publication Data
Biotechnology of filamentous fungi : technology and products / edited by David B.
Finkelstein and Christopher Ball,
p.
cm. — (Biotechnology)
Includes bibliographical references and index.
ISBN 0-7506-9115-8 (case bound)
1. Molds (Fungi)—Biotechnology. I. Finkelstein, David Β. II. Ball, Christopher.
III. Series: Biotechnology
TP248.27.F86856 1991
660'.62—dc20
91-16555
CIP

British Library Cataloguing in Publication Data
Finkelstein, David B.
Biotechnology of filamentous fungi : technology and products.
I. Title II. Ball, Christopher
589.2
ISBN 0-7506-9115-8

Butterworth-Heinemann
80 Montvale Avenue
Stoneham, MA 02180
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Printed in the United States of America

CONTRIBUTORS

Herbert L. Holland
D e p a r t m e n t of C h e m i s t r y
Brock University
St. C a t h a r i n e s , O n t a r i o
Canada

James Β. Anderson
C e n t r e for Plant B i o t e c h n o l o g y
University of T o r o n t o
Erindale Campus
Mississauga, Ontario
Canada

Paul A . H o r g e n
C e n t r e for Plant B i o t e c h n o l o g y
University of T o r o n t o
Erindale C a m p u s
Mississauga, Ontario
Canada

C h r i s t o p h e r Ball
Panlabs, Inc.
Bothell, Washington
Paul B a y m a n
D e p a r t m e n t of Biology
T u l a n e University
N e w O r l e a n s , Louisiana

Sirkka Keränen
VTT
Biotechnical L a b o r a t o r y
E s p o o , Finland

Joan W . Bennett
D e p a r t m e n t of B i o l o g y
T u l a n e University
N e w Orleans, Louisiana

Jonathan K . C . Knowles
G l a y o Institute for M o l e c u l a r
Biology
G e n e v a , Switzerland

R a m u n a s Bigelis
A m o c o Research Center
N a p e r v i l l e , Illinois

L o u i s e S. L e e
U S D A A R S Southern Regional
Research C e n t e r
New Orleans, Louisiana

Arnold L. Demain
D e p a r t m e n t of Biology
MIT
Cambridge, Massachusetts

P r a k a s h S. M a s u r e k a r
M e r c k and C o . , I n c .
R a h w a y , N e w Jersey

David B. Finkelstein
Panlabs, Inc.
Bothell, Washington

Clayton W . McCoy
University of Florida
Institute of F o o d and Agricultural
Citrus Research
L a k e Alfred, Florida

Wayne A. Gardner
D e p a r t m e n t of E n t o m o l o g y
University of G e o r g i a
G e o r g i a E x p e r i m e n t Station
Griffin, G e o r g i a

Helena Nevalainen
VTT
Biotechnical Laboratory
E s p o o , Finland

Randolph L. Greasham
M e r c k and C o . , Inc.
R a h w a y , N e w Jersey

vii

viîî

Contributors

Kerry Ο'Donneil
USDA ARS
T h e National C e n t e r for Agricultural
Utilization R e s e a r c h
P e o r i a , Illinois

Robert T. Rowlands
P e n l a b s , Inc.
Bristol, United K i n g d o m

M e r j a Penttilä
VTT
Biotechnical Laboratory
E s p o o , Finland

C l a u d e P . Selitrennikoff
D e p a r t m e n t of Cellular and Structural
Biology
University of C o l o r a d o Health
Sciences C e n t e r
Denver, Colorado

Stephen W . Peterson
USDA ARS

T u u l a T . Teeri
VTT

T h e National C e n t e r for Agricultural
Utilization R e s e a r c h
P e o r i a , Illinois

Biotechnical Laboratory
E s p o o , Finland
Mary Jo Zidwick

John A. Rambosek
P a n l a b s , Inc.
Bothell, W a s h i n g t o n

Cargill, Inc.
Cargill R e s e a r c h
Minneapolis, Minnesota

ACKNOWLEDGMENTS

W e w o u l d like to thank the following P h . D . colleagues at P a n l a b s for h e l p in
r e v i e w i n g the chapters: G r a h a m B y n g , M i c h a e l Cantrell, M a r k C r a w f o r d , L i n d a
L a s u r e , B r u c e Maloff, Phyllis M c A d a , J o h n R a m b o s e k , C h r i s t o p h e r R e e v e s ,
C h a r l e s S o l i d a y , and D e a n T a y l o r .

xv

CHAPTER

1
Editorial Introduction
David B. Finkelstein
Christopher Ball

W e p e r c e i v e a need for this text despite ever-increasing material discussing the
e m e r g i n g b i o t e c h n o l o g y - b a s e d industries. Earlier publications on the b i o t e c h n o l o g y
of filamentous fungi mainly describe ideas d e v e l o p e d in the 1970s (Smith a n d Berry
1975 and 1983). O t h e r texts h a v e dealt with the evolution of b i o t e c h n o l o g y ideas
d u r i n g the 1980s, but usually as specialized s u b t o p i c s , for e x a m p l e , the genetics of
filamentous fungi as part of publications dealing with the genetics of other o r g a n i s m s (Ball 1984; Bennett and L a s u r e 1985; H e r s h b e r g e r et al. 1989).
T h i s text a i m s to b e relatively c o m p r e h e n s i v e , but inevitably it has s o m e bias
t o w a r d m o l e c u l a r b i o l o g y , g e n e t i c s , and biochemistry of filamentous fungi. N e v e r t h e l e s s , a n u m b e r of general principles of b i o c h e m i c a l e n g i n e e r i n g such as p r o c e s s
design and scaleup are dealt w i t h , as is the isolation and preservation of these
o r g a n i s m s . T h e m a i n e m p h a s i s in this b o o k is on n e w e r d e v e l o p m e n t s with fungi of
c o m m e r c i a l r e l e v a n c e . C o n s e q u e n t l y , certain traditional topics are d e - e m p h a s i z e d
or omitted but this should in n o w a y detract from their i m p o r t a n c e . In this respect,
w e believe that for a description of industrial applications the texts of S m i t h and
Berry (referred to previously) are very c o m p r e h e n s i v e as are various v o l u m e s in the
current B i o t e c h n o l o g y Series.
T h e history of c o m m e r c i a l l y relevant strain d e v e l o p m e n t using filamentous
fungi is e x t e n s i v e and w e will limit the discussion to a few highlights. W i t h the
e x c e p t i o n of m u s h r o o m s , the origins of this p r o c e d u r e can be traced to the O r i e n t ,
w h e r e the Koji p r o c e s s used Aspergillus
oryzae to m a k e saki, soy s a u c e , and m i s o
on semisolid cereal p r e p a r a t i o n s . T h i s led to the use of this o r g a n i s m for e n z y m e
production ( T a k a m i n e 1914).
1

2

Editorial Introduction

In the s e c o n d d e c a d e of this c e n t u r y , F l e m i n g discovered antibiotic activity in
surface culture by a Pénicillium
species. This activity w a s later s h o w n to b e
penicillin, an o u t s t a n d i n g contribution to the welfare of m a n k i n d . It w a s followed
about 2 0 years later by the d e v e l o p m e n t of s u b m e r g e d culture t e c h n o l o g y for
industrial production of penicillin. A l s o at the time of W o r l d W a r II, the b i o c h e m i cal genetics of Neurospora
crassa ( T a t u m and B e a d l e 1942) e m e r g e d . T h i s led to
the elucidation of the b i o c h e m i c a l genetics of a w i d e variety of o r g a n i s m s and
evolution of the o n e - g e n e , o n e - e n z y m e h y p o t h e s i s . It also led indirectly to the
d i s c o v e r y of a diploid p h a s e in Aspergillus
nidulans ( R o p e r 1952), and a l l o w e d
identification of a parasexual cycle in the o r g a n i s m . T h e latter discovery w a s
e x t e n d e d to c o m m e r c i a l filamentous fungi, eventually enabling detailed genetic
analysis to be carried out via the parasexual c y c l e . Originally, such analysis had
b e e n an intractable p r o b l e m d u e to the lack of an identifiable sexual cycle in m a n y
c o m m e r c i a l fungi.
T h e genetics of c o m m e r c i a l filamentous fungi w e r e studied in detail. T h i s
included empirical mutation p r o g r a m s to i m p r o v e penicillin production in Pénicillium chrysogenum
( B a c k u s and Stauffer 1955), and parasexual b r e e d i n g p r o g r a m s
in the s a m e o r g a n i s m as well as in the citric acid p r o d u c i n g Aspergillus
niger and
the c e p h a l o s p o r i n C p r o d u c i n g Cephalosporium
acremonium
(Ball 1984).
T h e evolution of c o n t i n u o u s culture technology (Pirt and R i g h e l a t o 1967)
paralleled the w o r k in classical g e n e t i c s . It studied the relationship of fungal
g r o w t h , and product formation to g r o w t h rates and specific growth-limiting s u b strate supply. At the s a m e t i m e , studies on fungal biosynthesis of c o m m e r c i a l l y
important m o l e c u l e s w e r e initiated (Ball 1984).
F o r proprietary r e a s o n s , publications from industry h a v e b e e n relatively s c a r c e ,
but o n e publication of note is the description of a c o m m e r c i a l penicillin strain
i m p r o v e m e n t p r o g r a m (Lein 1986). Here is a very g o o d illustration of h o w fund a m e n t a l k n o w l e d g e can be empirically applied in a c o m m e r c i a l context. T h e s a m e
p h i l o s o p h y has been used by m a n y c o m m e r c i a l organizations together with significant n e w techniques such as r e c o m b i n a n t D N A ( H e r s h b e r g e r et al. 1989).
In this b o o k , u n i q u e aspects of filamentous fungi are highlighted together with
aspects that are c o m m o n to m o s t m i c r o o r g a n i s m s studied in industries using
b i o t e c h n o l o g y . T h e true u n i q u e n e s s of filamentous fungi in b i o t e c h n o l o g y is their
versatility and breadth of utility. F i l a m e n t o u s fungi can generate a w i d e r a n g e of
industrial products including primary metabolites such as o r g a n i c a c i d s , s e c o n d a r y
metabolites such as /3-lactam antibiotics, nonantibiotic d r u g s , and e n z y m e s for use
in food p r o d u c t i o n . W h o l e o r g a n i s m s such as m u s h r o o m s can be used as well as
o r g a n i s m s used as insecticides and herbicides. F i l a m e n t o u s fungi also qualify as
potential hosts for secretion of certain h e t e r o l o g o u s proteins such as m a m m a l i a n
p r o t e i n s . H o w e v e r , not all things about fungi are of benefit. M y c o t o x i n s p r o d u c e d
by fungi can b e lethal to h u m a n s , and there is also a need for d e v e l o p m e n t of
antifungal agents to destroy fungi that c a n , by various m e c h a n i s m s , kill a n i m a l s and
p l a n t s . T h e latter topics are important aspects of the b i o t e c h n o l o g y of filamentous
fungi and are dealt with in this text.
T h e c o m m e r c i a l significance of filamentous fungi can also be appreciated w h e n

or agricultural c h e m i c a l s . L. (1967) Appl. this is particularly true in genetic studies w h e r e Neurospora crassa and Aspergillus nidulans h a v e historically reigned as p r o t o t y p e o r g a n i s m s (Bennett and L a s u r e 1985). CRC Press.. Butterworths. In c o n c l u s i o n . H e r e filamentous fungi are used to study i m p r o v e m e n t of existing p r o c e s s e s . eds. American Society for Microbiology. and Hegeman. Lein. eds. T o o u r k n o w l e d g e . 14-15. S. and Hostalek. 4 2 9 ^ 6 3 . ed. or the d i s c o v e r y of n e w p r o d u c t s . (1955) Mycologia 41. like o r g a n i s m s such as a c t i n o m y c e t e s . M. J. and waste treatment industries. L. S.. A d r a m a t i c illustration of b i o c o n v e r s i o n by filamentous fungi is their critical impact on the steroid industry (Sebek and P e r l m a n 1979).. C . Academic Press. (1984) Genetics and Breeding of Industrial Microorganisms. h o w e v e r . fungi. Roper.References 3 o n e realizes h o w m a n y industries use t h e m . Hershberger. c h e m i c a l . Pirt. R.. food. T h e s e include the p h a r m a c e u t i c a l . J. the e s t a b l i s h m e n t of n e w p r o c e s s e s . REFERENCES Backus.. Z. 1283-1290. eds.. 15. J. A s exemplified a b o v e . P. F. J. W. a g r i c u l t u r e . G. and Righelato. Queener. Stoneham. L. W. (1989) Genetics and Molecular Biology of Industrial Microorganisms. (1986) in Overproduction of Microbial Metabolites—Strain Improvement and Process Control Strategies (Vanek. J. and Stauffer. and Lasure. D u r i n g the last d e c a d e it h a s b e e n suggested that these o r g a n i s m s m i g h t be used c o m m e r c i a l l y as hosts for p r o d u c t i o n of m a m m a l i a n p r o t e i n s . fungal catalytic p r o p e r t i e s — t h o s e of w h o l e o r g a n i s m s or of e n z y m e s — c a n be h a r n e s s e d in a cost-effective w a y and with less toxic e n v i r o n m e n t a l i m p a c t . N e v e r t h e l e s s . fundamental research continues to p r o v i d e v a l u a b l e information for a p p r o a c h i n g the study of c o m m e r c i a l filamentous fungi... Z. Washington. E n v i r o n m e n t a l impact can often be a p r o b l e m w h e n using synthetic c h e m i s t r y o n an industrial scale. can be r e g a r d e d as c h e m i s t s with low salaries! T h e y are able to p r o d u c e a variety of s e c o n d a r y metabolites with low m o l e c u l a r weight c h e m i c a l structures that can serve as antibiotics. A n o u t s t a n d i n g e x a m p l e described in this text in the c h a p t e r on therapeutic metabolites is the p r o d u c t i o n of d r u g s that are h y d r o x y m e t h y l g l u t a r y l ( H M G C o A ) reductase inhibitors w h i c h l o w e r h u m a n cholesterol levels. an introduction to a text on b i o t e c h n o l o g y of filamentous fungi w o u l d not b e c o m p l e t e without a c k n o w l e d g i n g w o r k d o n e on so-called " a c a d e m i c " filam e n t o u s fungi as m o d e l s y s t e m s . F i n a l l y . w e are sure that the readers of this text will b e optimistic about the future of the b i o t e c h n o l o g y of filamentous fungi on m a n y different fronts and that these o r g a n i s m s will a l w a y s be of u n i q u e value to industry and w o r t h y of fundamental scientific investigation. W i t h regard to n e w p r o c e s s e s . C.. this potential has not b e e n realized. W i t h regard to d i s c o v e r y of n e w p r o d u c t s .). DC. e n z y m e . (1952) Experientia 8. Bennett. Ball. 105-139. MA. A. Boca Raton. FL. . New York. C. pp. Microbiol. other therapeutic d r u g s . (1985) Gene Manipulations in Fungi.

eds. Chem. E. London.. J. J. 483-496. London. Academic Press. L. London. . pp. (1983) The Filamentous Fungi.. Eng. Edward Arnold. D. (1975) The Filamentous Fungi. O. New York. Fungal Technology. eds. J. and Berry. Smith. E. E. 824-828. K. 234-243. Vol. R. and Berry. Natl.. and Beadle. G. 6. (1979) in Microbial Technology—Microbial Processes. USA. D. (1942) Proc. Industrial Mycology. R. and Perlman.. Takamine. Edward Arnold. IV. second edition. Smith. D. Sei. Vol.4 Editorial Introduction Sebek.. (1914) Indust. Acad. I. Tatum. San Francisco. W. I.. 28. Vol.

m o s t filamentous fungi g r o w as b r a n c h e d filaments t e r m e d hyphae ( s i n g . Mannarelli. Labeda during the preparation of this manuscript. Sylvester for the photographic illustrations.S. . 7 . and antibiotics and other p h a r m a c e u t i c a l s . 1987. w h i c h are collectively called mycelia ( s i n g . 1989. organic a c i d s . F u n g i are an e x t r e m e l y diverse g r o u p of heterotrophic m i c r o o r g a n i s m s that are exploited b y h u m a n s for various biotechnological applications: they are used in the p r o d u c t i o n of foods. Peterson T h e p u r p o s e of this c h a p t e r is to p r o v i d e biotechnologists with an introduction to s o m e of the basic t e c h n i q u e s used in the isolation and preservation of filamentous fungi and to give a brief introduction to the t a x o n o m y of filamentous fungi with selected references to s o m e of the m o r e important t a x o n o m i c literature.P.M. m y c e l i u m ) .CHAPTER 2 Isolation. F i l a m e n t o u s fungi p r o d u c e a vast array of s e c o n d a r y m e t a b o l i t e s and s o m e species h a v e highly efficient protein secretion m e c h a n i s m s that can be exploited to e x p r e s s h o m o l o g o u s or h e t e r o l o g o u s g e n e p r o d u c t s (Cullen et al. h y p h a ) . and Taxonomy Kerry O'Donnell Stephen W. L e o n g and B e r k a 1990). fungi. K n o w l e s et al. Department of Agriculture over other firms or similar products not mentioned. p o l y s a c c h a r i d e s . e n z y m e s . S a u n d e r s et al. as agents of biological control of pest insects. . A s the n a m e i m p l i e s . B. Preservation. Berry 1988. b e v e r a g e s . and w e e d s . Special thanks are due Charles E. L e o n g and B e r k a 1990). The mention of firm names or trade products does not imply that they are endorsed or recommended by the U. We are pleased to acknowledge the constructive criticisms of Doctors C P . 1 9 8 1 . Needham for preparing the graphic art and to Ray F. F i l a m e n t o u s fungi a c c o u n t for virtually all m e m b e r s of the fungal k i n g d o m . and D. and in b i o m a s s c o n v e r s i o n ( O n i o n s et al. 1987. Kurtzman.

a class of biflagellate. T h e cell walls contain at least s o m e chitin. S o m e yeasts form h y p h a e and/or p s e u d o h y p h a e .8 Isolation. T h e s e o r g a n i s m s h a v e cellulosic cell walls (Bartnicki-Garcia 1970) and r D N A s e q u e n c e s that indicate p h y l o g e n e t i c affinities to the algae ( G u n d e r s o n et al. Preservation. uni. heterotrophic o r g a n i s m s that h a v e b e e n traditionally classified as fungi within the division M a s t i g o m y c o t i n a . t a x o n o m i c treatments of these o r g a n i s m s are located in the m y c o l o g i c a l literature. Correct identification of an u n k n o w n isolate to the division level generally requires the p r e s e n c e of teleomorphic (sexual state. the O o m y c e t e s will be treated here with the fungi.2 ) that are f o r m e d . M o s t notable a m o n g these fungi are such serious h u m a n p a t h o g e n s as Histoplasma capsulatum. Coccidiomyces immitis ( c o c c i d i o m y c o s i s ) . Divisions of the E u m y c o t a are defined according to the a n a m o r p h i c (Figure 2 . together with mycelial features (Table 2 . (E) Few-spored sporangia of Thamnidium (Zygomycotina) borne on a branched sporangiophore. formerly called imperfect state) spore stages ( H e n n e b e r t and W e r e s u b 1977). (C) Multispored sporangia of Absidia (Zygomycotina) borne apically on unbranched aerial sporangiophores. and Taxonomy H o w e v e r .v a n Rij ( 1 9 8 4 ) . w h i c h forms b r a n c h e d filaments that exhibit polarized apical g r o w t h . m a y b e septate (divided by transverse cross-walls) or coenocytic ( n o n s e p t a t e ) . R e p r o d u c t i o n is either asexual or sexual. T h i s definition of fungi w o u l d e x c l u d e the O o m y c e t e s . 1987). yeasts represent a g r o u p of essentially unicellular fungi that g r o w vegetatively and divide either by b u d d i n g (Saccharomyces cerevisiae) or b y fission (Schizosaccharomyces pombe). see K r e g e r . (D) Multispored sporangium of Mucor (Zygomycotina). (B) Conidial variation in the Deuteromycotina. M a n i p u l a t i o n of cultural c o n d i t i o n s F I G U R E 2-1 Morphological diversity of anamorph spores and spore-bearing structures. 2. All of these thermotolerant fungi g r o w as the yeast p h a s e in the host at 37°C and as a m y c e l i u m at 24°C.1 ) and t e l e o m o r p h i c spore stages (Figure 2 . A few species of fungi are d i m o r p h i c . as they are frequently e n c o u n t e r e d w h e n isolating fungi from terrestrial and aquatic e n v i r o n m e n t s . Fusarium (F) and Alternaria (A) are multicelled.1 TAXONOMY F u n g i are classified in the s u b k i n g d o m E u m y c o t a of the k i n g d o m F u n g i ( W h i t t a k e r and M a r g u l i s 1978). T h e m y c e l i u m . Yeasts are phylogenetically diverse and h a v e r e p r e sentatives a m o n g the A s c o m y c o t i n a and B a s i d i o m y c o t i n a ( K r e g e r . (A) Uniflagellate zoospore of Blastocladiella (Mastigomycotina). A s a t a x o n o m i c c o n v e n i e n c e . and Wangiella (Blastomyces) dermatiditis (blastomycosis) ( M c G i n n i s 1980). formerly called perfect state) or a n a m o r p h i c (asexual state. m e a n i n g that they can g r o w either as d i v i d i n g yeast-like cells. E u m y c o t a are e u k a r y o t i c . etiologic agent of h i s t o p l a s m o s i s .to multicellular heterotrophic o r g a n i s m s with absorptive nutrition. Pénicillium (Ρ) is unicellular. F o r m o r e information on yeasts that will not b e considered further in this chapter.v a n Rij 1984). V e g e t a t i v e g r o w t h in s o m e yeasts is not exclusively unicellular. . F u r t h e r m o r e . (F) Conidiophore of Aspergillus (Deuteromycotina). U n i or biflagellated spores ( z o o s p o r e s ) / g a m e t e s are p r o d u c e d in o n e division ( M a s t i g o m y c o t i n a ) . d e p e n d i n g on the g r o w t h c o n d i t i o n s . or they m a y p r o d u c e a m y c e l i u m .1 ) .

1 Taxonomy 9 .2.

10 Isolation. teleomorphic stages h a v e not been o b s e r v e d in pure culture for m a n y homothallic taxa. (B) Four asci of Sordaria (Ascomycotina) in which the eight ascospores within each ascus are uniseriate. (D) Basidium of the mushroom Coprinus (Basidiomycotina) upon which four basidiospores are borne. a pattern of sexuality in which strains are selfsterile. E v e n t h o u g h h o m o t h a l l i c strains are selffertile. a b s e n c e of t e l e o m o r p h i c spore stages m a y be d u e to suboptimal cultural conditions or h e t e r o thallism. Preservation. (C) Asci of the fission yeast Schizosaccharomyces (Ascomycotina) in which the ascospores are unordered. h o w e v e r . and Taxonomy F I G U R E 2-2 Morphological diversity of teleomorph spore stages. T h e conidial state is the p r e d o m i n a n t . (A) Zygospore of Absidia (Zygomycotina) borne between opposed suspensors in which one suspensor has a whorl of appendages. m a y stimulate production of such stages in s o m e strains. Ascospore color variations are due to mutations.

conidial anamorph often present (Figures 2-1 b. motile uni. hyphomycetes) Mycelium unicellular to multicellular. regularly septate. A u t h o r citations are a p p e n d e d to every binomial and these represent the n a m e of the author(s) of the b i n o m i a l . B e c a u s e m o s t m e m b e r s of the D e u t e r o m y c o t i n a are p r o b a b l y linked to the A s c o m y c o t i n a . 2-2c). For e x a m p l e . motile cells absent Ascomycotina (ascomycetes) Mycelium unicellular to multicellular. regularly septate. is d e t e r m i n e d by the International C o d e of Botanical N o m e n c l a t u r e ( I C B N ) ( V o s s et al.2.l a ) produced during life cycle Zygomycotina (zygomycetes) Mycelium typically coenocytic. anamorph (asexual state) predominant stage consisting of uni. with only a few species containing a b a s i d i o m y c e t o u s or m a s t i g o m y cotinous mycobiont. motile cells absent Deuteromycotina (fungi imperfecti. k n o w n as lichens o r lichenized fungi. w h e r e k n o w n . teleomorph spores are ascospores formed inside ascus (Figures 2-2b. 2-If). motile cells absent r e p r o d u c t i v e stage in laboratory culture a n d . 2. S y m b i o t i c associations of fungi ( m y c o b i o n t ) and algae ( p h y c o b i o n t ) . motile cells absent Basidiomycotina (basidiomycetes) Mycelium unicellular to multicellular. 1983). o v e r t w o thirds of all fungal taxa are A s c o m y c e t e s . are not included in the synoptic key but are m e n t i o n e d here b e c a u s e m a n y obligately lichenicolous fungi p r o d u c e novel s e c o n d a r y m e t a b o lites (Elix et al. ( 1 9 8 0 ) . teleomorph spores are basidiospores formed outside basidium (Figure 2-2d). called nomenclature.1.1 TABLE 2-1 Eumycota Taxonomy 11 Common Names and Characteristics of the Five Divisions of the Mastigomycotina (aquatic or zoosporic fungi) Thallus unicellular or mycelial. see K e n d r i c k ( 1 9 7 9 ) . W h e r e k n o w n .or biflagellate zoospores (Figure 2 . regularly septate.1 Introduction to Taxonomy T a x o n o m y is the systematic classification of o r g a n i s m s . teleomorph (sexual state) absent. T h e b i n o m i a l . conidial anamorphs common (Figure 2 . F o r information on a n a m o r p h i c t e l e o m o r p h i c relationships.l e ) . m o s t conidial anam o r p h s represent asexual stages of a s c o m y c e t o u s t e l e o m o r p h s . or scientific n a m e consists of t w o Latin or latinized n a m e s : the generic n a m e and the specific epithet.l b . and Sugiyama (1987). teleomorph (sexual state) spores are zygospores (Figure 2-2a) although frequently absent. T h e set of rules that g o v e r n h o w fungi and plants are n a m e d .l c through 2 . the fungal c o m p o n e n t of m o s t lichens is a s c o m y c e t o u s . D o m s c h et al. M y c o l o gists and botanists h a v e a d o p t e d a binomial system of n o m e n c l a t u r e for n a m i n g taxa a c c o r d i n g to rules a d o p t e d and published in the I C B N . mycelium coenocytic. the t e l e o m o r p h i c state of the . 2-If). 1988).and/or multispored sporangia (Figures 2 .

12 Isolation.500 n e w species are described a n n u a l l y .8) 17.2 Numbers of Fungal Taxa T h e r e are a p p r o x i m a t e l y 6 .1. R e c o g n i z i n g s y n o n y m y eventually leads to an easier. E n g l a n d . F o r e x a m p l e . K e w . et T h i r u m . and these n a m e s are c o m p i l e d in the Index of Fungi published b y the C o m m o n w e a l t h M y c o l o g i c a l Institute. k n o w l e d g e of s y n o n y m y is n e c e s s a r y to access t a x o n o m i c literature for the p u r p o s e of identifying strains. the a n a m o r p h i c filamentous fungus Acremonium chrysogenum ( T h i r u m . therefore. 2. increase o u r capacity to m a k e accurate predictions about the b e h a v i o r of the taxa u n d e r study. B o o t h has an asexual state of Acremonium butyri (van B e y m a ) W .2 Approximate Number of Genera and Species in Each Division of the Eumycota Division No. it w o u l d take TABLE 2 . and Percent Genera No.680 (28.104 (18. and Tilachidium butyri van B e y m a . (1983) h a v e e s t i m a t e d that there m a y be as m a n y as 2 5 0 .5) Ascomycotina 2. 0 0 0 fungal g e n e r a that contain 6 0 . G a m s . such as E. are k n o w n as p l e o m o r p h i c fungi. several n a m e s or s y n o n y m s of a single species of biotechnological interest m a y b e used in the current literature. For n o w . resulting in n a m e c h a n g e s of g e n e r a and species as discussed in C a n n o n ( 1 9 8 6 ) . Cephalosporium khandalense S u k a p . et T h i r u m .6) 28.2) Deuteromycotina 1. the term synanamorph is applied. (1983). F u n g i with t w o or m o r e distinct reproductive states.000 (25. Preservation.2 ) . n o m e n c l a t u r a l p r o b l e m s are recognized and resolved. tonophilum. At this r a t e . viridescens. T h e t e l e o m o r p h i c fungus Nectria viridescens C . 1. has a s y n o n y m of Cephalosporium chrysogenum T h i r u m . of w h i c h o v e r 7 0 % are yet to be described.2) Zygomycotina 145 (2. n a m e c h a n g e s ideally reflect greater u n d e r s t a n d i n g of the genetic relationships of the taxa a n d . and Percent Species Mastigomycotina 190 (3.170 (1. G a m s .9) 16. from w h i c h penicillin and c e p h a l o s p o r i n s C and Ρ are d e r i v e d .720 (46.2) . This a n a m o r p h has t w o s y n o n y m s . A s a result of continuing study of fungal taxa and m y c o l o g i c a l t a x o n o m i c literature.8) 765 (1. apud S u k a p . both s y n o n y m s w o u l d be i m p r o p e r to use for the a n a m o r p h of N. et S u k a p . A l t h o u g h n o v i c e s and specialists alike find s y n o n y m s confusing. If the life cycle of these fungi includes m o r e than o n e asexual form of reproduction or a n a m o r p h . ) W . O v e r 1. 0 0 0 to 7 0 .000 (26. 0 0 0 species of fungi (Table 2 . m o r e streamlined identification s y s t e m . 0 0 0 currently accepted species ( H a w k s w o r t h et al.0) Basidiomycotina 1.8) Data from Hawksworth et al.650 (45. 1983). T h e r e f o r e . H a w k s w o r t h et al. et S u k a p . and Taxonomy conidial a n a m o r p h i c species Aspergillus tonophilus O h t s u k i is Eurotium tonophilum O h t s u k i .

3 ) . In excess of 2 0 0 collections h a v e b e e n established w o r l d w i d e . catalogue available Identification service for a fee Institute for Fermentation (IFO) 7.: (09) 368-3377.: 28-5614. T o this e n d . Baron-Hay Court South Perth. and (3) literature references to m o n o g r a p h i c treatments of the m o r e c o m m o n genera of biotechnological imp o r t a n c e in w h i c h k e y s to species can be found.000 strains 17-85 Juso-Honmachi 2-chome Yodogawa-ku.2. see Guide to World Data Center on Microorganisms—A List of Culture Collections in the World. first edition. Osaka 532.3 Fungal Culture Collections A w e a l t h of fungal g e r m p l a s m is readily accessible to biotechnologists from e s tablished fungal culture collections (Table 2 .3 Resource Centers with More Than 4.750 strains Institute of Microbiology Academia Sinica. W F C C W o r l d D a t a C e n t e r on M i - TABLE 2 . (2) literature references listed by t a x o n o m i c g r o u p or b y specialized subject matter. and these h o u s e a p p r o x i m a t e l y 2 0 0 . 1989.1. a literature reference section is included ( A p p e n d i x ) that contains three subsections: (1) a list of general reference b o o k s . biotechnologists interested in identifying u n k n o w n isolates need ready access to "user-friendly" k e y s and illustrated descriptions of the m o r e c o m m o n species. Zhongguancun Beijing. Western Australia 6151 Tel. 2. T a x o n o m i c k e y s to species at present rely primarily on m o r p h o l o g i c al features.: 06-302-7281.000 Strains Culture Collection Department (CCCM) 4. 100080 China Tel.1 Taxonomy 13 o v e r a century for all taxa of the fungi to be described! B y w a y of contrast. catalogue available Plant Research Division Culture Collection (WA) 5. Japan Tel. Telegrams: AGDEP Perth Telex: AA 93304 . 0 0 0 species. G i v e n the large n u m b e r of species descriptions scattered t h r o u g h o u t the m y c o logical literature. m o l e c u l a r tools are available for d e v e l o p i n g identification kits for taxa of biotechnological significance ( C h e h a b and K a n 1989). including several introductory m y c o l o g y t e x t b o o k s for researchers with limited formal training or practical e x p e r i e n c e in systematic m y c o l o g y . 0 0 0 p u r e .000 strains Plant Pathology Branch Western Australian Department of Agriculture. H o w e v e r . authenticated strains representing o v e r 7 . For a c o m p l e t e listing of the c o l l e c t i o n s . virtually all flowering plant species h a v e been described. A n asterisk m a r k s highly r e c o m m e n d e d t e x t s .

: (613) 996-1665 Fungal Genetics Stock Center (FGSC) 5.: (301) 881-2600. catalogue available Identification service for a fee 29.14 Isolation. Illinois 61604. Germany Tel. Canada Tel.: 2154-11841. Maryland 20852.3 (Continued) Centraalbureau voor Schimmelcultures (CBS) Oosterstraat 1 PO Box 273 3740 AG Baarn.000 strains 5. UK Tel. and Taxonomy TABLE 2 . Preservation. Canada Tel. USA Tel. B-1348 Belgium Tel. Kew. USA Telex 898055.200 strains Data from Hawksworth and Kirsop (1988) and Kirsop and Kurtzman (1988). catalogue available Identification service for a fee Mycothèque de l'Université Catholique de Louvain (MUCL) 15. Freiherr-vom-Stein Allee 2 Postfach 16/329. Weimar 5300.400 strains Sektion Biologie Pilzkulturensammlung. USA Tel.: (403) 987-3054. catalogue available 44. Ontario. The Netherlands Tel.: 32-10-43 37 42. Telcom Gold/Dialcom: 84: CAU009 Telex: 265871 (MONREF G). . Peoria. University Street.000 strains Biosystematics Research Centre. University of Alberta Edmonton. catalogue available Agricultural Research Service Culture Collection (NRRL) Northern Regional Research Center 1815 N. Alberta T6G 2E1. USA Tel. Surrey TW9 3AF.500 strains CAB International Mycological Institute (IMI) 12. Saunders Building Agriculture Canada.: Weimar 3498 American Type Culture Collection (ATCC) 12301 Parklawn Drive Rockville.: (913) 588-7044. DIALCOM 142: CDT0109 Identification service for a fee 22. Kansas 66103.: (309) 685-4011 University of Alberta Microfungus Collection (UAMH) Devonian Botanic Gardens.000 strains Canadian Collection of Fungus Cultures (CCFC) 12. Ottawa K1A OC6.500 strains Ferry Lane.500 strains Department of Microbiology University of Kansas Medical Center Kansas City.: 01-940 4086.000 strains Place Croix du Sud Louvain-la-Neuve. catalogue available Identification service for a fee Friedrich-Schiller-Universitat Jena (MW) 5. Telex: UCL Β 59037.

S a i t a m a .1 Taxonomy 15 c r o o r g a n i s m s . Obrancu mini 10. S w e d i s h U n i versity of Agricultural S c i e n c e s . Waterloo Road London SEI 8XY.3 3 0 0 B r a u n s c h w e i g . D S M . . Fax. J a p a n (available from U N E P / U n e s c o / I C R O . England Tel. 01-940 4086 Dialcom 84:CAU009 Data from Hawksworth and Kirsop (1988) and Bower (1989). for yeasts). 6023/4 Telex 2966662818. D . S w e d e n ) .4 ) . F o r a m o r e c o m p l e t e listing of fungal culture c o l l e c t i o n s . DTI Cornwall House. Saitama 351-01. England Tel. Culture collection data b a n k s are b e i n g integrated internationally u n d e r the Microbial Strain D a t a N e t w o r k ( M S D N ) to facilitate location and ordering of specific strains (Table 2 . Japan Tel.: 23407 Dialcom/Memocom 75:DBrl0154 World Data Center for Microorganisms (WDC) Life Science Research Information Section RIKEN 2-1 Hirosawa Wako. England Tel.: +81484621111. U p p s a l a . M a s c h e r o d e r W e g 1 b . In addition to these culture c o l l e c t i o n s . TABLE IrA Culture Collection Databases European Culture Collections Organization (ECCO) Czechoslovak Collection of Microorganisms (CCM) 662 43 Brno tr. G e r m a n y ) offer an identification service for a p r e a r r a n g e d fee.: + 4 4 1 211 0267 TELECOM GOLD 75:DB10015 Microbial Information Network Europe (MINE) CAB International Mycological Institute Ferry Lane Kew. for filamentous fungi) and K i r s o p a n d K u r t z m a n ( 1 9 8 8 . Fax + 4 4 223 277605 Dialcom/Memocom 75 :DB 10001 or DB10005 Microbial Culture Information Service (MiCIS) Laboratory of the Government Chemist. see H a w k s w o r t h and K i r s o p ( 1 9 8 8 . +81 484 62 1554 Dialcom 42:CDT0007 Microbial Strain Data Network (MSDN) Institute of Biotechnology Cambridge University 307 Huntingdon Road Cambridge CB3 OJX.D e u t s c h e S a m m l u n g v o n M i k r o o r g a n i s m e n u n d Zellkulturen G m b H .: +44223276622 Telex 81240CAMPSL G.2. other collections (for e x a m p l e . Czechoslovakia Tel. Surrey TW9 3AF. S-750 0 7 . Ext.

References in T a b l e 2 . In addition to sale of cultures and c a t a l o g u e s of strains. F o r course information. a habitat rich in fungi currently being exploited by biotechnologists. with the n u m b e r of participating collections e x p e c t e d to increase in the future ( B o w e r 1989).16 Isolation. In addition. or C M I ) or a culture collection federation (see T a b l e 2 .2 ISOLATION Pure cultures are a prerequisite prior to any attempt to characterize the biological or b i o c h e m i c a l activities of an isolate. a n u m b e r of fungal culture collections offer identification services for a fee (see T a b l e 2 . H y a t t s v i l l e . T h e primary focus of the isolation section of this chapter deals with the soil mycoflora. Preservation. S o m e of the most important items include l a m i n a r flow h o o d or transfer r o o m with U V germicidal l a m p s . T e c h n i q u e s for isolation of fungi vary according to the ecological habitat and/or substratum from which they are s a m p l e d . B C C M . A T C C . G e o r g i a . permits to transport plant or animal p a t h o g e n s can be o b t a i n e d from the United States D e p a r t m e n t of A g r i c u l t u r e .3 ) are recognized as an International Depository Authority u n d e r the B u d a p e s t Treaty ( 1 9 8 1 ) . Should strains of either plant or animal p a t h o g e n s be n e e d e d . informed advice on strain selection. S h o u l d t a x o n o m i c assistance be required in obtaining a definitive species identification. Permits for h u m a n p a t h o g e n s can be obtained from the C e n t e r s for D i s e a s e C o n t r o l . A P H I S .2. fungal culture collections m a y offer identification services.5 ) . authentic strains can be obtained directly from established c o l l e c t i o n s . A x e n i c cultures are also required to obtain an accurate species identification of m a n y fungal taxa and for virtually all of the microfungi. contact a culture collection (for e x a m p l e . This treaty w a s formulated in 1977 to obviate the p r o b l e m of h a v i n g to deposit a culture in each country in which a patent w a s applied. p e r m i t s are usually required and these can be obtained from the appropriate g o v e r n m e n t a l a g e n c y . Atlanta. Collections also offer further support to b i o t e c h n o l o g y as a patent depository. autoclaves or pressure c o o k e r s . 2. and Taxonomy Several major culture collections h a v e on-line electronic mail service for ordering of cultures t h r o u g h M S D N .3 ) . A T C C .6 should be consulted for details of isolation m e t h o d s for other specific habitats or t a x o n o m i c groups. In the United States. M a r y l a n d . refrigerators and freezers (preferably walk-in if . C B S . D S M . and strain m a i n t e n a n c e and preservation. incubators. culture collections and other institutes periodically offer short c o u r s e s on the identification of industrially important microfungi. see T a b l e 2 . research and d e v e l o p m e n t contract services in diverse areas of systematic and applied m y c o l o g y . N R R L . If biotechnologists h a v e k n o w l e d g e of which taxa m a y possess the desired physiological p r o p e r t y . Several of the major collections ( C B S . 2.1 Laboratory Equipment Required T h e e q u i p m e n t required for the m e t h o d s outlined in this chapter is c o m m o n to m o s t m o l e c u l a r or microbiological laboratories.

Germany United States Federation for Culture Collections (USFCC) Dr. 1980 Basidiomycotina Watling 1971. Schenck and Perez 1990 Entomogenous and nematophagous fungi Barron 1977. Eastern Point Road Groton. Hale and Savory 1976. Dagmar Fritze (editor) DSM-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH Mascheroder Weg lb D-3300 Braunschweig. Bacon 1990. Frisvad 1986 Aquatic fungi Fuller and Jaworski 1987. L. Phaff 1990 Mastigomycotina Fuller and Jaworski 1987 Zygomycotina Benjamin 1959-1965. Kreger-van Rij 1984. 25 rue de Docteur Roux 75724 Paris Cedex 15.2. Seifert 1990. Stevens 1981 Soil fungi Barron 1972. France TABLE 2 . USA United Kingdom Federation for Culture Collections (UKFCC) Division of Microbiological Reagents & Quality Control Central Public Health Laboratory 61 Colindale Avenue London NW9 5HT. Johnson and Curl 1972 Foodborne fungi Pitt and Hocking 1985. H. Huang (editor) Central Research Pfizer Inc. Fair et al. Couch and Bland 1985. Kohlmeyer and Kohlmeyer 1979 Plant pathogens Tuite 1969. Samson et al.6 Literature Guide to Isolation Methods General methods Booth 1971. CT 06340. 1983. 1989 Mycorrhizal fungi and Endophytes Schenck 1982. 1981. 1988 Wood decay fungi Maloy 1974.2 TABLE 2-5 Isolation 17 Culture Collection Federations World Federation for Culture Collections (WFCC) Dr. Kemp 1985 . Stalpers 1978 Yeasts Barnett et al. Lichtwardt 1986 Ascomycotina and Deuteromycotina Domsch et al. MacLean 1982. England European Culture Collections Organisations (ECCO) Collections Nationale de Cultures de Microorganismes Institut Pasteur.

T h e s e t e c h n i q u e s are generally applicable for the isolation and pure culture of filamentous fungi from other substrates. h o m o g e n i z e r s . dissecting and c o m p o u n d m i c r o s c o p e s . Preservation.3 A method for isolating pure fungal cultures from single spores.2. .2 General Notes on Isolation T h e basic techniques for isolating filamentous fungi are presented with the focus directed primarily on soil fungi (Figure 2 . 2. p H m e t e r s . and b a l a n c e s . distilled w a t e r and v a c u u m are also n e e d e d . a source of d e i o n i z e d . F u n g a l isolation techniques can be broadly defined as direct or indirect. In addition.18 Isolation. plasticware and g l a s s w a r e . and other c o m m o n laboratory e q u i p m e n t such as vortex m i x e r s . A d e q u a t e space is required so that field s a m p l e s can be physically separated from the pure culture w o r k area. and Taxonomy v o l u m i n o u s s a m p l e s are to be stored). FIGURE 2 .3 ) .

2. it is advisable to k e e p detailed collection notes and s p e c i m e n s if species identifications are required.4 ) .2 Isolation 19 Sporulating Colony Microscope Slide Λ— Agar Square ' (cell-side up) Concavity Mountant • Ci) Cover-glass Cover-glass FIGURE 2 . F o r m a n y a s c o m y c e t e s and b a s i d i o m y c e t e s . or streaked directly o n t o agar m e d i a to obtain p u r e c o l o n i e s derived from single s p o r e s . A cover-glass is then added. B e c a u s e m a c r o f u n g i such as m u s h r o o m s frequently d o not p r o d u c e diagnostic sporulating stages in p u r e c u l t u r e . W i t h direct isolation. h o w e v e r .3 Collecting Soil Samples L a t e s u m m e r to fall m a y b e the best time to collect soil s a m p l e s in t e m p e r a t e r e g i o n s .3 a ) An agar square ( 5 x 5 mm x 1 mm thick) containing sparse growth with desired reproductive stages is excised from an agar plate and is placed in a single-well depression slide cell-side up. ( l a . as the soil mycoflora is m o s t active at that t i m e .4 Two methods of rapid preparation of fungal material for observation with a compound microscope. such as a fresh m u s h r o o m . T o assist researchers interested in identifying microfungi from p u r e c u l t u r e s . if the spore density is h i g h . using aseptic techn i q u e .b a s e d bleach in w a t e r . T h e s e spores can then be serially diluted in sterile w a t e r .2. spores or s p o r o p h o r e tissue (for e x a m p l e .2 0 % solution of s o d i u m h y p o c h l o r i t e . Liquid such as nutrient broth is added to the well to exclude air bubbles which obscure observation. can be used to establish a pure culture (Watling 1981). Tissue excised from within a surfacesterilized s p o r o p h o r e ( w a s h e d with 7 0 % ethanol or a 1 0 . t w o simple m i c r o s c o p e slide preparative t e c h n i q u e s are illustrated (Figure 2 .2. followed by a sterile water w a s h ) . discharged spores can be collected o n a sterile substrate. (lb—3b) The adhesive side of transparent tape is gently pressed against a sparse area of growth containing desired reproductive stages. collections . fleshy fungi such as m u s h r o o m s ) are transferred directly to the isolating m e d i u m . The tape with attached fungal material is mounted cell-side down on a microscope slide.

500 mg/1 Gram ( + ) bacilli. the fungal isolation area should b e physically separated from TABLE 2-7 List of Commonly Used Antibiotics and Specific Inhibitors InhibitorΊΑ ntibiotic Working Oxgall 5 g/1 Pythium.4 Contamination (Bacteria. F a s t .6-Dichloro-4-nitroaniline 25 mg/1 Pénicillium. Preservation. If appropriate sporulating stages are a b s e n t . and m i t e s . Mites) W h e n isolating fungi from natural substrates. Hyattsville. A l t e r n a tively. h y p h a l tips can be isolated aseptically from a culture g r o w i n g on a nutrient dilute agar with the aid of a dissecting m i c r o s c o p e . 2 . and yeast g r o w t h . Antibacterial antibiotics can be incorporated into the m e d i u m if bacterial c o n t a m i n a t i o n is a p r o b l e m (Table 2 . w h i c h can be obtained from the U .2.7 ) . S a m p l e s can be taken at different d e p t h s according to the soil c o m p o s i t i o n . T h e r e f o r e . Dried s a m p l e s can be stored at r o o m t e m p e r a t u r e if they are not i m m e d i a t e l y transported to the laboratory for p r o c e s s i n g . fungi. Importation of soil s a m p l e s into the U n i t e d States requires a p e r m i t . S . s a m p l e s can be dried to prevent anaerobiosis and microbial c o n t a m i n a t i o n . O n c e collected. Fungi. D e p a r t m e n t of A g r i c u l t u r e . 5 ) into the m e d i u m . three types of c o n t a m i n a n t s are likely to b e e n c o u n t e r e d : bacteria. It is r e a s o n a b l e to a s s u m e that all organic s a m p l e s collected from nature m a y h ar b o r adult mites or their e g g s . 2. bacterial. M i x e d fungal cultures can be separated b y streaking spores to single colonies on fresh m e d i a . soil s a m p l e s should be kept cool (preferably at 4°C) in transit to the laboratory to m i n i m i z e c o n t a m i n a t i n g m o l d . 2. Gram (-) cocci Data from Seifert (1990).20 Isolation. Concentration Effective Against Phytophthora Aspergillus Trichoderma Selected filamentous fungi . and Taxonomy should not b e restricted to these m o n t h s . A P H I S .g r o w i n g fungal c o n t a m i n a n t s can be m i n i m i z e d and/or eliminated by plating less material on the p r i m a r y isolation plates together with incorporating inhibitors (see Section 2 . M a r y l a n d . Sodium deoxycholate L-Sorbose ι g/i 40 g/1 Selected filamentous fungi Validamycin 3 g/1 Selected filamentous fungi Benomyl 1-2 mg/1 Ascomycetes Triton X-100 and X-171 0. o-Phenyl phenol 6 mg/1 Trichoderma Rose bengal 30-700 mg/1 Bacteria.4 g/1 Fusarium Tetracycline 25-100 mg/1 Bacteria Streptomycin or kanamycin 100-200 mg/1 Gram (-) bacilli Chloramphenicol 200 mg/1 Bacteria Penicillin G 12.5-1. S a m p l e s should be r e m o v e d from the g r o u n d with a sterile spatula or cylindrical corer and placed in tagged sterile plastic b a g s .

2. or k e r o s e n e ) .2 Isolation 21 p u r e fungal cultures as well as from any materials that m a y c o m e in contact with the p u r e culture w o r k area to avoid mite infestations. A given weight of soil. Mites can c r o s s . J o h n s o n and Curl 1972) e m p l o y baiting t e c h n i q u e s or a highly selective m e d i u m to isolate g r o u p s of fungi with similar physiological or ecological properties. S o m e mite-infested cultures can b e saved by storing t h e m at . 2-6-dichloro-4-nitroaniline. see P a r k i n s o n et al. M e t h o d s d e v e l o p e d to isolate fungi from soil in situ (Barron 1 9 7 1 . they frequently introduce bacterial c o n t a m i n a t i o n . synthetic selective m e d i a can be modified to suit the n e e d s of e a c h isolation strategy. Y C B A . or the soil can b e sprinkled o n t o the surface of the solidified agar m e d i u m .2 0 ml of nutrient w e a k sterile m o l t e n agar (50°C) is a d d e d and swirled to disperse the soil. w h i c h kills mites and their e g g s . Variations of the dilution plate m e t h o d h a v e included incorporating inhibitors [for e x a m p l e . Serial dilutions are m a d e and aliquots are a d d e d to p o u r plates of a nutrient dilute m e d i u m [such as water agar (see following p a r a g r a p h s ) to w h i c h antibiotics are frequently added] or spread o n the surface of a solid agar m e d i u m . Y N B A . a few crystals of dichlorocide can be added directly to a mite-infested c u l t u r e . A n aliquot of soil ( 5 . or b e n o m y l (Pitt and H o c k i n g 1985)] into the m e d i u m to restrict the g r o w t h of fast-growing fungi (see T a b l e 2 . then all cultures in the vicinity m a y be t o o . available from g a r d e n supply c e n t e r s .c o n t a m i n a t e fungal cultures by c r a w l i n g from o n e culture tube to another a n d .7 ) . T h e soil plate method ( W a r c u p 1960) is the simplest and quickest m e t h o d for isolation of fungi from soil.g r o w i n g fungi will not be o v e r g r o w n by fast-growing fungi such as species of Mucor and Mortierella. O r t h o . Soil s a m p l e s should b e plated in duplicate or triplicate on a w i d e s p e c t r u m of . Difco) c a n b e incorporated into the synthetic selective m e d i u m . If a m o r e quantitative estimate of the soil mycoflora is required.2 0 ° C for u p to 3 d a y s . A l t e r n a t i v e l y . usually o v e n dried. M i t e s are killed by this p r o c e d u r e so that a mite-free subculture can b e o b t a i n e d . T h e m o s t w i d e l y used qualitative t e c h n i q u e for the isolation of a large n u m b e r of different taxa from soil is the soil dilution plate method.2. a u n i q u e nitrogen (yeast-carbon b a s e . it is advisable to destroy all cultures by a u t o c l a v i n g and to clean the w o r k area with a miticide (for e x a m p l e .2. is b l e n d e d in sterile distilled w a t e r c o n t a i n i n g a nonionic detergent (such as 0 .m m Petri dish to w h i c h 1 0 .5 0 m g dry weight) is a d d e d to a sterile 1 0 0 . ( 1 9 7 1 ) .5 Isolation from Soil T h e t w o m o s t frequently used m e t h o d s to isolate fungi from soil are the soil dilution plate method and the soil plate method ( W a r c u p 1960). in the p r o c e s s . 2 % T w e e n 80) to obtain a h o m o g e n e o u s s u s p e n s i o n . s l o w e r . D i f c o . Detroit) or c a r b o n source (yeast-nitrogen b a s e . c h l o r a m p h e n i c o l . O n c e cultures b e c o m e c o n t a m i n a t e d with m i t e s . or the use of heat and/or c h e m i c a l t r e a t m e n t s of the soil prior to plating. w h i c h is then tightly sealed o v e r n i g h t . r o s e b e n g a l . At the correct dilution. A final w o r d of caution: it is r e a s o n a b l e to a s s u m e that if o n e culture is mite infested. Isotox Insect S p r a y . F o r e x a m p l e . W i t h the dilution plate and soil plate m e t h o d s .

T h e m e d i a listed are given in alphabetical order. F o r m u l a t i o n s of the most c o m m o n agar m e d i a (for e x a m p l e . p s y c h r o p h i l e s ( 0 . such as tetracycline or a u r e o m y c i n . at 5 0 mg/1. C M A . C Z A . but d o i n g . 2. O A . most fungi are m e s o p h i l i c ( 1 5 .p o s i t i v e bacteria. can be added to sterilized m e d i a cooled to 50°C prior to p o u r i n g . and Pitt and H o c k i n g 1985). A l t h o u g h g r o w t h conditions m a y vary from species to s p e c i e s .22 Isolation. H A . C M I ) .2 0 ° C ) . A T C C . B C C M .7 .2. A l s o . F o r most m e s o p h i l e s and t h e r m o p h i l e s . R e c i p e s for all m e d i a listed are for 1 1. h u m i d i t y . Stevens 1 9 8 1 . Light.8 ) . care should be taken to ensure that the agar m e d i u m d o e s not dry out. V 8 A ) m a y be required to obtain sporulating stages. D e s i c c a t i o n of agar plates can be avoided by pouring thicker p l a t e s . Β r o a d . and t h e r m o p h i l e s ( 3 5 ^ 5 ° C ) . or c h l o r a m p h e n i c o l at 100 mg/1 can be autoclaved with the m e d i u m . M e d i a listed here m a y be generally characterized as either rich or dilute. plates should be incubated for at least 2 w e e k s . T h e r m o p h i l i c and psychrophilic fungi should be given special considerations.6 Growth Conditions F o r routine isolation and m a i n t e n a n c e of filamentous fungi. M A ) are available c o m m e r c i a l l y as dried preparations a n d . M o s t saprobic fungi g r o w well on rich m e d i a (see T a b l e 2 . recipes for s o m e of the c o m m o n l y used m e d i a can be found in culture collection catalogues (for e x a m p l e . Preservation.s p e c t r u m antibiotics. Y M A .3 0 ° C ) and g r o w well on a solid culture m e d i u m rich in c a r b o h y drates with a slightly acidic p H ( 5 . it m a y be necessary to routinely incorporate antibacterial antibiotics into the m e d i u m to m i n i m i z e bacterial c o n t a m i n a t i o n . these items m a y be cost-effective. and aeration are other factors that should be taken into consideration b e c a u s e n o r m a l laboratory conditions for these factors m a y not be optimal for g r o w t h and/or sporulation for all strains. 0 ) . H o w e v e r . P C A .3 5 ° C ) . It is not a l w a y s necessary to dissolve the agar prior to sterilization. and incubating s a m p l e s in a h u m i d e n v i r o n m e n t . N a C l should be a d d e d to the m e d i u m to maintain a high o s m o t i c p r e s s u r e . It is advisable to test a n u m b e r of m e d i a prior to c h o o s i n g o n e s for routine use. using 2 % Difco Bacto agar and distilled w a t e r . T h e s e antibiotics should be a d d e d to sterilized m e d i a after cooling to 60°C and prior to p o u r i n g . Detailed c o m p i l a t i o n s of agar m e d i a d e v e l o p e d for the isolation. P D A . S m i t h and O n i o n s 1983. and Taxonomy m e d i a . a list of these formulations is given (see T a b l e 2 . and incubation duration and t e m p e r a t u r e should be varied to obtain the b r o a d e s t s p e c t r u m of isolates: m e s o p h i l e s ( 1 5 . b e c a u s e m y c o l o g i c a l culture collections routinely use fresh materials to prepare m e d i a . 0 .8 ) . P D A . W h e n used together. Booth 1 9 7 1 . For halophilic fungi. a relatively small r a n g e of m e d i a can be e m p l o y e d (Table 2 . w h e r e a s slower g r o w i n g p s y c h r o p h i l e s generally require a longer incubation period. Y M A + ) but nutrient-weak "starvation m e d i a " ( W A . streptomycin and penicillin at 100 mg/1 are effective against g r a m negative and g r a m . and identification of broad g r o u p s or specific fungal taxa are available (Tuite 1969. For p r i m a r y isolation from natural substrates. for m a n y laboratories. m a i n t e n a n c e .8 for definitions and formulations of m e d i a . C M A . Should plastic d i s p o s a b l e Petri dishes be u s e d . by increasing the p e r c e n t a g e of agar from 2 to 4 % .

Adjust volume to 1 1. distilled water. Add agar and melt it prior to sterilization.5 g.5-7. Czapek Agar (CZA) Components N a N 0 3 .5 g.0 g of glucose per liter. 0. 20 g. 20 g. bottle. agar. 1 g.000 ml. 2 g. adjust pH to 6. Use Pénicillium and Aspergillus. Use Fusarium. Preparation Heat malt extract in 500 ml of water until dissolved. 20 g. Bottle and sterilize. glucose.0 with NaOH. Optional addition of 5.0 g of yeast extract per liter. agar. and melt prior to sterilizing. 3 g. 50 g. distilled water. Preparation Dissolve ingredients and sterilize. . 1 1. 1 1. Use Excellent rich general purpose medium. KCl. 0. F e S 0 4 . M g S 0 4 .2. agar. 1 g. 1 1. glucose (optional). and sterilize. 1. agar. Use Pénicillium and Aspergillus. 1 1. Preparation Dissolve all chemicals.7 H 2 0 . 20 g. 30 g. and add filtrate to dissolved agar ( ± glucose) in 500 ml of water.7 H 2 0 . 0. 3 g.2 TABLE 2-8 Isolation 23 Formulations of Commonly Used Culture Media Corn meal agar (CMA) Components corn meal. then heat to dissolve. Use General purpose medium. Preparation Cook corn meal in 500 ml of water at 60°C for 30 min. Hay extract agar (HA) Components hay. Cornmeal Plus agar (CMA + ) Components corn meal filtrate (see above). 20 g. filter through cheesecloth. "starvation" medium for induction of sporulation. 20 g. Preparation Steam hay in 500 ml of water for 30 min. filter through cheesecloth. add agar.5 with NaOH and bring filtrate up to 1 1. yeast extract. Malt extract agar 2% (MA) Components malt extract.01 g. 20 g. agar. K 2 H P 0 4 . especially wood inhabiting basidiomycetes. sucrose. Use General purpose. Czapek yeast extract (CZYA) Components Czapek agar with 5. distilled water. add sucrose and agar. When cool adjust pH to 6. 30 g. sucrose. distilled water.

agar. and sliced. Sterilize. Potato-dextrose agar (PDA) Components potatoes (washed. 1 1. 30 g. distilled water. filter through cheesecloth. 20 g. add glucose and dissolve. 20 g. 20 g. 1 1. 20 g.0 g. Use Fusarium. Preparation Cook potatoes in 500 ml of water for 60 min. plant pathogens. 1 1. Use Starvation medium. filter through cheesecloth. Preparation Combine all materials and melt agar by heating prior to sterilization. V-8 juice agar (V8A) Components V-8 juice. carrot (washed. sporulation induction. Preservation. distilled water. Preparation Steam or autoclave potatoes and carrots in 1 1 of water for 30 min. C a C 0 3 . peeled. . agar. agar. 1 1. cellulolytic fungi. Add potato filtrate to 500 ml of water containing melted agar. Use Rich general purpose medium.24 Isolation. sporulation induction medium. bottle. avoid new potatoes). agar. Phytophthora. distilled water. filter through cheesecloth. 20 g. 20 g. and other Oomycetes. and sliced. bring volume up to 1 1. distilled water. Melt agar in 500 ml of water and add to oatmeal filtrate and bring volume up to 1 1. 20 g. bring filtrate up to 1 1. Potato-sucrose agar (PSA) Components potato filtrate (see PDA above). Add agar and melt. 20 g. Preparation Dissolve all ingredients prior to sterilizing. Preparation Steam oatmeal in 500 ml of water for 60 min. 175 ml. 5. peeled. 20 g. 1 1. sucrose. 20 g. Use Fusarium. Potato-carrot agar (PCA) Components potatoes (see PDA). and sterilize. agar. Use General purpose. and Taxonomy TABLE 2-8 (Continued) Oatmeal agar (OA) Components oatmeal flakes. glucose. Pythium.

agar. M e d i a should b e sterilized for 15 m i n at 15 psi ( 1 2 1 ° C ) . yeast. distilled water. m a y be kept metabolically active by regular transfers. 1 1. d e s i r a b l e . 15 g.3 Preservation 25 Water agar (WA) Components agar. 1 g. o n c e o b t a i n e d . basidiomycetes. 2. m a i n t e n a n c e of the culture is essential for the investigatory p r o c e s s . M g S 0 4 · 7 H 2 0 . Use Starvation medium. 1 1. glucose 10 g. 1 1. T h e a d d e d a d v a n tage of the culture collection is the separation in time and space of the act of o b t a i n i n g the culture and the investigation of its properties. K 2 H P 0 4 . s o m e t i m e s c h a n g i n g . so that the culture is available to the microbiologist at the t i m e and place that is m o s t c o n d u c i v e to productive research. Data from Stevens (1981). 4 g. melt agar in 500 ml of water and combine with dissolved chemicals. or in a state of m e t a b o l i s m on agar slants that are c o v e r e d with w a t e r or mineral oil. Preparation Dissolve agar in 500 ml of water by heating. Sterilize. so obviates the p r o b l e m of obtaining u n d i s s o l v e d c l u m p s of agar in sterilized m e d i a . unfortunately. 3 g. 20 g. . 20 g. distilled water. 20 g. 0. F o r fungi that m a y o c c u r only in exotic or t e m p o r a l habitats. T h e s e are a l w a y s active to s o m e extent a n d . Preparation Combine all chemicals in 500 ml of water and melt by heating. either in an autoclave or pressure c o o k e r . 3 g. The Cultures lowered cultures l o n g . tap or distilled water. Preparation Melt agar prior to sterilization. soluble starch. Use Thermophilic fungi. agar.3 PRESERVATION T h e large a m o u n t of t i m e and labor involved in isolating pure cultures from nature m a k e s the perpetuation of the isolates. w h i c h m a y i m p e d e solidification of the agar. malt extract.2. 5 g. dissolve other ingredients in 500 ml and combine with melted agar. Sterilize. Yeast phosphate soluble starch agar (YPSS) Components yeast extract. Oomycetes.5 g.t e r m storage of fungi is achieved in o n e of three general w a y s . Use Rich general purpose medium. Yeast malt agar (YMA) Components yeast extract. peptone.

Cultures are revived by aseptically b r e a k i n g o p e n the a m p u l e . frozen rapidly in a dry-ice b a t h .2 Storage Under Mineral Oil or Water Intermediate-length storage of cultures can sometimes be achieved by growing the fungus out on an agar slant and then covering the agar with sterile water or mineral oil.3. T h e r e is n o risk of m u t a t i o n . Preparations of this sort h a v e g o o d viability after 4 0 or m o r e y e a r s . is o n e of the m o s t widely established m e t h o d s of these t e c h n i q u e s (Figures 2 . or arthropods (mites). selection. Also. transfer of strains to fresh media. incubator. or c o n t a m i n a t i o n in these metabolically inactive preparations. 2. N o t all fungi can be stored in this m a n n e r .3. T h e small pellet is sealed inside. if properly dried.3 Storage by Lyophilization D e h y d r a t i o n of cultures is a c c o m p l i s h e d by several m e a n s . Covering the cultures slows the rate of metabolism markedly by reducing available oxygen and also prevents the dehydration of the agar and fungal material. The strains do eventually lose viability. other fungi. It requires the regular preparation of agar slant tubes. the fungus m u s t sporulate and form small spores . It is. < 2 0 0 strains) with limited resources. and the possibility that perpetual transfer selects for genotypes that grow well on the agar slant under the conditions of culture. and Taxonomy S p o r e s or other fungal e l e m e n t s m a y be d e h y d r a t e d and stored without overt m e t a b o l i c activity. variations of these three techniques h a v e b e e n reported that h a v e a d v a n t a g e s for different taxa. placing the pellet in sterile broth or distilled water.m m glass tubing with o n e end sealed. and contamination with other fungi and bacteria is an occasional problem.1 Storage on Agar Slants Perpetual transfer is a suitable method for maintenance of small culture collections (for example.5 and 2 . labor intensive and thus suitable only for limited numbers of species. This method requires little equipment other than a refrigerator. however. and paraphernalia for preparing media. spores of the fungus are s u s p e n d e d in a nonfat p o w d e r e d milk solution or in sterile b o v i n e s e r u m .3. T h e strain is recovered by scraping the agar slant surface with a wire loop and streaking the fungal material onto suitable agar media. Preservation. and then dried u n d e r v a c u u m . strains that are metabolically active are susceptible to mutation and loss of particular characteristics.26 Isolation. and for different b u d g e t s . 2. The drawbacks of perpetual transfer are the possibility of contamination of cultures by bacteria. or freeze-drying. 2. u n d e r v a c u u m . L y o p h i l i z a t i o n . O v e r the past 5 0 y e a r s . T h e w h o l e fungus m a y b e frozen and m a i n t a i n e d at low t e m p e r a t u r e . by heating the a m p u l e neck and is stored at refrigerator t e m p e r a t u r e . followed (usually) by storage at 4°C for several months between transfers. inspection of the tubes to assure growth. and then streaking the r e h y d r a t e d s u s p e n s i o n o n t o suitable agar m e d i a . 6 . In this m e t h o d . for different laboratory e n v i r o n m e n t s . placed in small sterile tubes (for e x a m p l e .6 ) . In g e n e r a l . or other a m p u l e s ) .

V a c u u m is applied and the culture is dried. and an o x y g e n . In this m e t h o d .>1 h after ampules raised) Use bidirectional oxygen-natural gas torch to cut off ampules FIGURE 2-5 Lyophilization method for preserving fungi. Cultures of yeasts prepared in this manner have very good viability after 5 .1 0 years and are probably equivalent to lyophilized preparations for longevity.g a s torch for sealing a m p u l e s u n d e r v a c u u m . B o t h of these drying p r o c e s s e s require a g o o d quality v a c u u m p u m p .c a p glass vials.3. < 1 0 μνη d i a m e t e r ) for successful lyophilization. but instead of freezing prior to drying. v a c u u m m a n i f o l d s .2. lyophilization offers a low-cost m e a n s of storing large n u m b e r s of strains with little c h a n g e in characteristics and very low m a i n t e n a n c e . absorbs m o i s t u r e within a few h o u r s to a few d a y s . T h e d r y i n g m a t e r i a l . T h e a m p u l e s are stored at 4°C for increased longevity. sterile ampule Attach ampules to manifold and freeze in 50% ethylene glycol-13% ethanol-dry ice bath precooled to -40 to -50°C Apply vacuum Raise manifold when bath reaches 0°C (2-3 h) to remove ampules from ice bath Monitor drying with vacuum gauge until 5-15 millitorr (. F o r species that can be p r e s e r v e d in this m a n n e r .3 Preservation 27 Harvest spores I Suspend spores in bovine serum I Aliquot 50-100/iL per prelabeled. 2. the ampule neck is heated.5 Storage in Silica Gel or Soil A simpler m e t h o d of d e h y d r a t i n g spores and m y c e l i u m is through the use of a n h y d r o u s silica gel (Figure 2 . T h e cotton plug and drawn-out neck reduce the evaporation rate. drawn out. (for e x a m p l e . L a r g e r spores or h y p h a e usually d o not maintain viability through the lyophilization p r o c e s s and m u s t b e stored in s o m e other m a n n e r .4 Storage by L-Drying A similar technique is L-drying. fungal material is s u s p e n d e d in a nonfat p o w d e r e d milk solution. and a tight fitting cotton plug inserted. 2.3. w h i c h is inert. preventing the suspension from boiling under reduced pressure. In this process. and put o n t o a n h y d r o u s silica gel particles p r e sterilized for 3 h in a 160°C o v e n in s c r e w .7 ) . the spore or mycelium preparation is placed in an ampule as described in the preceding section. T h e caps of the .

High capacity vacuum pump (not shown) located on floor beneath lyophilization apparatus. An in-line vacuum gauge (not shown) is normally attached to the left-most outlet (not shown). Preservation. .28 Isolation. and Taxonomy FIGURE 2-6 (A) Lyophilization apparatus used in the ARS Culture Collection at the Northern Regional Research Center for preservation of microorganisms. A plastic safety shield (SS) covers the pyrex glass manifold to which 30 ampules (A) are attached. using a cross-fired torch. Cold trap (CT) filled with dry ice is covered with plastic safety shield. Oxygen cylinder ( 0 2 ) and natural gas outlet (G) for sealing ampules with cross-fired torch. Ethanol/ethylene glycol/dry ice bath (IB) for freezing ampules. (B) Sealing ampoules under vacuum.

A n o t h e r m e t h o d . Parafilm®. a few crystals of the silica gel are shaken out of the tube o n t o a suitable agar plate and a l l o w e d to g r o w out. Neurospora crassa can b e m a i n t a i n e d with n o loss of viability for at least 6 years and p r o b a b l y for m u c h longer periods of t i m e . A s u s p e n s i o n of fungal material is pipetted o n t o the sterile soil.5% Carnation® instant nonfat dry milk Prepare Silica Gel • Sterilize loosely-capped 1 -dram vials. A m e r i c a n C a n C o m p a n y . tubes are closed and often sealed with laboratory film (for e x a m p l e . half-full of 6-12 mesh silica gel. 1 -2 days Viability Test Store • Tightly-capped @ 4°C To revive strain sprinkle a few crystals onto agar or broth medium FIGURE 2-7 Silica gel method for preserving fungi.3 Preservation 29 Prepare Spore Suspension • Use wire loop to harvest spores • Mix spores with 0. G r e e n w i c h . similar to the silica gel t e c h n i q u e . is the preservation of fungi in sterile soil. T h e cultures are r e c o v e r e d by shaking soil particles o n t o a suitable agar plate and a l l o w i n g the fungus to g r o w . T h e silica gel tube can b e resealed and used m a n y t i m e s w i t h o u t loss of viability. C T ) .2. 2 h in 160°C oven • Chill @ 0°C Chill @ 0°C / Agar Slant of Sporulating Strain m Add spore suspension to labeled chilled vials Ice @ 0°C 30 min Desiccate • Loosely-capped @ room temp. and either a l l o w e d to g r o w for 1-2 w e e k s or is i m m e d i a t e l y sealed and stored. . W h e n an active culture of the strain is n e e d e d .5 mL sterile ice-cold 7.

remove cryovial from LN 2 freezer and rewarm with constant agitation in 37°C water bath until contents completely thawed Transfer part or all of cryovial contents to suitable agar or broth medium If a cryovial is returned to LN 2 freezer. or even agar plugs c o n t a i n i n g fungal Prepare homogenous suspension in 10% final cone.3.1 0 to . cost-effective m e c h a n i s m for preservation. T h e other c o m m o n m e c h a n i s m of freezing cultures is with liquid nitrogen (Figure 2 . labeled.3 0 ° C after g r o w t h on an agar slant.1 7 6 to . and Taxonomy Cryopreservation T h e m o s t robust and generally applicable m e t h o d of preserving fungal strains is b y freezing either in a m e c h a n i c a l freezer at . 6 . m y c e l i u m and s p o r e s . screw-cap cryovials Cell Suspension Β Allow cells to equilibrate in cryoprotectant for 15-30 min @ room temp. .6 Isolation. Preservation. T h i s m e t h o d has the o b v i o u s a d v a n t a g e of r e d u c e d h a n d l i n g and is effective o v e r periods of several y e a r s .1 2 m o n t h s ) m a y be an attractive.1 9 6 ° C . Place vials in -55 to -80°C freezer Passively cool 1V6-2 h Store in mechanical freezer @ -55 to -80°C —- Transfer vials to liquid-nitrogen (LN 2) freezer t Viability test 1 Viability test To revive strain. but in the short term (that is.8 ) . T y p i c a l l y . passively cool as above and record this data since viability may be affected FIGURE 2-8 Cryopreservation of fungi.30 2.1 0 to . T h e strain is r e c o v e r e d by t h a w i n g the slant and transferring the culture to fresh agar.3 0 ° C or in a liquid nitrogen freezer at . It m a y not b e applicable to all fungi and has a limited effective time for the r e c o v e r y of viable c u l t u r e s . glycerol m t V ι ι Immediately dispense aliquot into presterilized. S o m e species are readily stored in m e c h a n i c a l freezers at .

G. ed. . 81-103. C . (1989) Trends Biotechnol. Orlando. Strains stored in the v a p o r p h a s e of the freezer retain viability as well as those stored in the liquid p h a s e . P. 3 2 1 ^ 3 3 . 285-287. and then placed in the liquid nitrogen freezer. Bower. Acad. . and Kan. ed. McGraw-Hill. 5. E. F. Couch. J. Booth.). T u b e s that will b e i m m e r s e d in liquid nitrogen m u s t seal c o m p l e t e l y (that is.). USA 86. L. Cultures are revived by rapid t h a w i n g in a 37°C w a t e r bath and plating of the fungal material on suitable agar m e d i a . C. L.. REFERENCES Bacon. R. F. (1971) in Methods in Microbiology (Booth. Sei. Benjamin.. Bartnicki-Garcia. Barron. T h e costs for the freezers and supply of liquid nitrogen m a k e this s o m e w h a t m o r e e x p e n s i v e than other m e t h o d s .1 9 6 ° C in the liquid p h a s e . 111-116. N . P. T u b e s with any leaks will partially fill with liquid nitrogen and can boil explosively w h e n rapidly t h a w e d . (1989) Proc. Chehab. . Guelph. 273-322. D. Sei. 259-282. New York. M a n y liquid nitrogen freezers h a v e a platform o v e r the liquid nitrogen w h e r e the tubes are stored. and Bland. D. Academic Press.p h a s e freezers h a v e important safety a d v a n t a g e s o v e r liquid-phase freezers as m e n t i o n e d p r e v i o u s l y . New York. 1-94.3 0 ° C (1°C per m i n u t e ) . W. Cambridge University Press.References 31 m a t e r i a l . heat-sealed glass a m p u l e s ) and m u s t b e tested for l e a k a g e prior to i m m e r s i o n in liquid nitrogen. C. C . but kept in the v a p o r p h a s e .). Payne.. Academic Press. B . Topics in Mycobiology 1. J. D. K. Academic Press.1 7 6 ° C versus . (1971) in Methods in Microbiology (Booth. (1959-1965) Aliso 4. 6. Liquid nitrogen storage is generally accepted as the m e t h o d of c h o i c e for the preservation of p h y s i o l o g i c a l and m o r p h o l o g i c a l features. N e a r l y all fungal species can be stored in a liquid nitrogen freezer.). T h e t e m p e r a t u r e in the v a p o r p h a s e of the freezer is about . World Intellectual Property Organization. W. (1977) The Nematode-Destroying Fungi.. Α. pp. and Yarrow. Palo Alto. G. New York.c a p plastic tubes rather than heat-sealed glass a m p u l e s . (1983) Yeasts: Characteristics and Identification. (1986) Microbiol. J. C. (1985) The Genus Coelomyces. Cambridge. Barnett. Blackwell Scientific Publications. J. 3. Academic Press. pp. T u b e s of fungal material stored in this type of freezer are n e v e r i m m e r s e d in liquid n i t r o g e n . 11-19. R. pp. pp.. New York. but for s o m e fungi this is the only m e a n s for successful strain preservation. 1-10. Geneva. ed. 4 0 5 ^ 2 7 . D. Canadian Biological Publications. are s u s p e n d e d in 1 0 % glycerol or d i m e t h y l sulfoxide. T h i s allows the use of very c o n v e n i e n t s c r e w . (1988) Physiology of Industrial Fungi. Barron. F. S. 168-171. 399 pp. 7. Berry. T h e longevity of fungal material stored this w a y is indefinite. Cannon. Natl. W. (1990) in Isolation of Biotechnological Organisms from Nature (Labeda. 9178-9182. Y. R. Budapest Treaty (1981) Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure 1977 and Regulations 1981. and v a p o r . cooled gradually to . (1970) in PhytochemicalPhytogeny (Harborne. ed.

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Y. New York. . I l l . (1989) Trends Biotechnol. Sugiyama. N. M . Evans. H. St. ed. (1978) BioSystems 10. pp.. P. J. Utrecht. Whittaker. Seattle. New York. Stevens. Paul. Bürdet. (1988) Atlas of Entomopathogenie Fungi. Liverpool University Press. and Ward. Tuite. MN. Stalpers. Saunders. Liverpool. R. McGraw-Hill. Berlin. ed. J. (1983) International Code of Botanical Nomenclature. New York. ed. University of Washington Press. Regnum Vegetable Series No. CA. and Margulis. and Holkema. pp. C . .. H. Watling. Mycol. (1987) Pleomorphic Fungi: The Diversity and Its Taxonomic Implications. 16. N. Picknett. (1981) How to Identify Mushrooms to Genus. . and Ward. pp. 3-18. C . M.). D. ed. Chaloner. Fungi and Bacteria. Schenck. (1969) Plant Pathological Methods. Tuite. (1978) Stud. R. Tokyo.. 2 1 . P. W. pp. 237-258... J. J. 219-236. et al. J. H. Schenck. (1990) in Isolation of Biotechnological Organisms from Nature (Labeda. 1-248.. Bohn.5 1 . T. Α. S. B . J. Springer-Verlag. (1982) Methods and Principles of Mycorrhizal Research. Warcup... V: Cultural and Developmental Features. Minneapolis. ML. (1990) in Isolation of Biotechnological Organisms from Nature (Labeda. C . and Perez... Ε. (1960) in The Ecology of Soil Fungi (Parkinson. M. R. Α. Kodansha. eds. H. (1981) Mycological Guidebook. D. 283-287. P. G. G. A. and Latege. (1971) in Methods in Microbiology (Booth.2 1 . 187 pp. American Phytopathological Society Press. Scheltema. ed. Eureka. D. Watling. McGraw-Hill. ed. Κ. Burgess.). L.References 33 Samson. .). 7. Voss.). R. C . F . G. 3 . Mad River Press.. Academic Press. R. Seifert.

J. 3 vols. W. New York. (1979) Introductory Mycology. New York. . W. ed. (1987) CBS Course in Mycology. England.) *Ainsworth. 3rd ed. C. (1981) Moulds—Their Isolation. References to Specialized Subject Matter and Taxonomic Groups Medical Mycology Emmons. T.. (1976) Fundamentals of Mycology. and Stalpers.. M. B. An Advanced Treatise. (Keys to genera. 2.. P. Kew. R. (1983-1985) Fungi Pathogenic for Humans and Animals. Vol. Burnett. Canadian Biology Publications. K. (Keys to genera. *Domsch. (1980) Laboratory Handbook of Medical Mycology. Subsection 2. 2nd ed. K.) Alexopoulos. L. D. Sparrow.. (1977) The Nematode-Destroying Guelph. Philadelphia. Kew. Note: An asterisk (*) indicates the most essential references. Vol. von (1981) The Genera of Fungi Sporulating in Pure Culture. I. J. J. Academic Press. A.. W. 7th ed. Α.. Marcel Dekker. L. Academic Press. D. eds. New York. General References *Ainsworth. New York. 3rd ed. and Ainsworth. F. J. Academic Press. Samson. Academic Press. 621 pp. Sparrow. C. Toronto. Gams. Baarn. G. G. An Advanced Treatise. H.. Α. J. J. S. (1983) Ainsworth & Bisby s Dictionary of the Fungi. and Anderson. A. McGinnis. C . Preservation. Howard. Vol. (1973) The Fungi. M... 2nd ed.. Edward Arnold. A Taxonomic Review with Keys: Basidiomycetes and Lower Fungi. K. Entomogenous and Nematophagous Fungi Barron. Fungi. 3rd ed. A. Vol. New York. *Arx. (1973) The Fungi. Webster. and Mims. Binford.. J. C ... Cultivation and Identification. (1979) Biology of the Fungi. J. W. (1980) Compendium of Soil Fungi. New York. M. A. C. D. J Cramer. and Taxonomy APPENDIX: LITERATURE REFERENCES Subsection 1. van der Plaats-Niterink. Cambridge. University of Toronto Press.34 Isolation.. van der Aa. Utz. C . C. H... *Ellis. A. G. Commonwealth Mycological Institute. Vaduz.. Cambridge University Press. (1977) Medical Mycology. A. and Sussman.. Kew.. K. B. *Ellis. Lea & Febiger. eds. New York.. K. Commonwealth Mycological Institute. 1. IVA. A Taxonomic Review with Keys: Ascomycetes and Fungi Imperfecti. Centraalbureau voor Schimmelcultures. McGraw-Hill.-H.. Commonwealth Mycological Institute. IVB. (1971) Dematiaceous Hyphomycetes. Gams. Sutton. 3rd ed.. and Sussman. London. England. and Kwon-Chung. Η. *Hawksworth. F. R.. (1976) More Dematiaceous Hyphomycetes.. H. England. G. John Wiley. H. Malloch. Ross. (1980) Introduction to Fungi. B. S.

H. St. MN. Beihefte zur Nova Hedwigia no.. (1961) Fungi in Oceans and Estuaries. E. Marine Mycology Johnson. American Phytopathological Society Press. L. A. Edward Arnold. G. Freeman. . Blackwell Scientific Publications. I. and Latge. A. Y. NY. G. Y. (1985) Fungi and Food Spoilage. A.. Fair. E. Samson. Allsopp. (1990) Molecular Industrial Mycology.. A. Huntington. eds.. and Eggins. Samson. and Rossman. (1986) The Trichomycetes: Fungal Associates of Arthropods. Chamuris.. L. L. Baarn.. K. I. 1-41. Food Microbiology King. J. Pitt.Appendix: Literature References 35 Hoog.. Cramer. (1972) The Genera of Hyphomycetes from Soil. J. (1979) Marine Mycology. and Corey.. 2 volumes. 7th ed.. P. Lichtwardt. R. and Berka. D. W. F. New York.. (1981) Smith's Introduction to Industrial Mycology. von (1987) Plant Pathogenic Fungi. C . Academic Press. Domsch. Bills. R. Κ. E.. Α. M. and Hocking. and Kohlmeyer. (1988) An Atlas of Entomopathogenic Fungi. Berlin. Evans. F. T. M. SpringerVerlag.. A. 3rd ed. The Higher Fungi. and Sparrow. O. D .. and Anderson. H. (1989) Fungi on Plants and Plant Products in the United States. Gams. W. London. W. Beuchat.. F. Marcel Dekker. E. Paul. W. Palo Alto. New York. Academic Press.. Α. Onions. and Emerson. Plant Pathogenic Fungi Arx. New York. S. St.. S. Soil F u n g i Barron. 1.. Kohlmeyer. D. T. J. Springer-Verlag. J. G. H. San Francisco. S. 87. H. (1980) Compendium of Soil Fungi. G. and Spielman. New York. W. Weinheim. R.. American Phytopathological Society Press. R. Mycol. D . New York. Cramer. . G. eds. Leong. J. Pitt. . J. (1987) A Literature Guide for the Identification of Plant Pathogenic Fungi.. R. J. R. (1964) Thermophilic Fungi. J. Thermophilic Fungi Cooney. Centraalbureau voor Schimmelcultures. and van Reenen-Hoekstra. Sydney. R. ed. Rossman.. D. S. (1988) Introduction to Food-borne Fungi. E. Krieger Publishing. P.. Α. H. Industrial Mycology Berry. (1986) Methods for the Mycological Examination of Food. de (1972) Stud. 2nd ed. (1988) Physiology of Industrial Fungi. L. Academic Press. Berlin. Paul.. Plenum Press. R. J. Palm. A. Jr. MN...

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(1980) Genera of Hyphomycetes. Stuttgart. Moser. F. Eureka. (1984) Mycol. (1978) Identification of wood-inhabiting fungi in pure culture.. Br. I. 1-43. A. and Marasas. W. W. Stud. Watling. P. M. Fr. J. 1-406. 32. J. Gustav Fischer Verlag. A. Ellis.. Mycol. C .. Reptr. O. D. A. University Park. J. Morquer.. J. M. Amsterdam. L... and Stalpers. W. Stuttgart. J. 1-96. Mycol. W. Kew. 16. Academic Press. (1976) More Dematiaceous Hyphomycetes. Izvo Naukova Dumka.. England. 364 pp. . Α. Mycol. (1963) Bull. Carmichael. Heterobasidiomycetes. R.. BerlinDahlem 209. G. Jülich. London. Stalpers. (1975) Trans.. University of Alberta Press. G. Lehre. Dutton and Co. Gams. 296-298. Cramer. ed. Mycol. North Ryde. M. Rouch. and Nirenberg. G. 15. Ellis. Kiev. R. Fisher. E. (1971) The Genus Fusarium. an Illustrated Manual for Identification. Pitt. MacLeod. Vol. (1972) Penitsillii. Kew. E. W. Fischer-Verlag. W. Soc. W. B. Watling. New York. 1 (Jülich. (1987) Illustrated Genera of Smut Fungi. (1982) The genus Fusarium—A Pictorial Atlas. Booth. Gallertpilze und Bauchpilze. (1983) Keys to Agarics and Boleti. CA. (1984) Die Nichtblaetterpilze. Bot.. R. Ν. Fayret. Aphyllophorales. (1972) Mushrooms of North America. (1980) The Resupinate Νon-poroid Aphyllophorales of the Temperate Northern Hemisphere. Eureka. M. (1980) A Literature Guide for Identifying Mushrooms. (1985) A Laboratory Guide to Common Pénicillium Species. Kew. J. (1971) Cephalosporium-artige Schimmelpilze (Hyphomycetes).. CAB International Mycological Institute. C. and Watling. Nelson. von (1970) A Revision of the Fungi Classified as Gloeosporium. J. (1954) Can. A. J. Pennsylvania State University Press. 1-248. 159 pp. I. K. (1979) The Genus Pénicillium and its Telemorphic States Eupenicillium and Talaromyces. M. J. R. Kendrick. Gams. H. Τ.Appendix: Literature References 37 Jülich. Joly. B. (1971) Dematiaceous Hyphomycetes. B. and Ainsworth. NSW. Cooke. 818-890. London. L. Viala. Mad River Press. Roger Phillips.). Gerlach. G.. Miller. Deuteromycotina Arx. 389-404. I. 64. J. P. W. V: Cultural and Developmental Features.. New York. J. Pidoplichko. (1977) Stud. Cryptogamic Studies. Mordur. J. 137-241. Papers 154. C. Edmonton. E. Huntington. Hermanides-Nijhof. M. and Berge. G.. CAB International Mycological Institute. 5 1 . CA. (1983) Fusarium Species. England. Russia. Conners. K. NY. Soc. Ο. North-Holland Publishing. W. 79. E. E. and Sigler. Pitt. 2nd ed. Toussoun. 141-177. Barron.. Gasteromycetes. England. Mad River Press. Vanky. Kreiger.. (1967) Plant Dis. CAB International Mycological Institute. Mitteilungen aus der Biologischen Bundesanstadt für Land-und Fortwirtschaft.. (1962) Mycopathology 17. P. Ε. (1981) How to Identify Mushrooms to Genus. B. W. CSIRO Division of Food Research. (1972) The Genera of Hyphomycetes from Soil.

T.J. M. Amsterdam. Smith. (1965) The Genus Aspergillus. D.. D. (1977) Stud. B. where known.38 Isolation. von. and Taxonomy Raper.. W. (1980) von Arx et al. (1980) Ellis ( 1 9 7 1 . Α. References to Monographic Treatments of Genera of Biotechnological Importance (Note all hyphomycete genera listed below. Barnett. Payne. Preservation.. M. Elsevier Science. J. A. New Delhi. have ascomycete teleomorphs) Absidia (zygomycete) M i l ' k o (1970) Z y c h a et al. R. and Yarrow. Williams & Wilkins. de. R. Cambridge. (1969) Acremonium (hyphomycete) G a m s (1971) G a m s (1975) ( T e l e o m o r p h s : Epichloe. V. (1986) .W. C. Williams & Wilkins. Chalaropsis. CAB International Mycological Institute. Kreger-van Rij. R. A. and Thorn. (1979) Stud. and Fennell. Neosartorya) Aureobasidium (hyphomycete) C o o k e (1962) Hermanides-Nijhof (1977) Beauveria (hyphomycete) M a c L e o d (1954) H o o g (1972) Ceratocystis (ascomycete) D o m s c h et al. Subsection 3. 1-38. Nectria. Mycol.. R. Baltimore. Yeasts Arx. Eurotium. eds. England. Mycol. Mycol. Papers 116. ed. A. Cambridge University Press. Indian Council of Agricultural Research. I. (1986) Advances in Pénicillium and Aspergillus Systematic s. Subramanian.. J. C. Miranda. Raper. (1980) The Coelomycetes. 1976) Aspergillus (hyphomycete) R a p e r and Fennell (1965) S a m s o n and Pitt (1986) ( T e l e o m o r p h s : Enter ice lia. 1^2. (1983) Yeasts: Characteristics and Identification. R. Acervuli and Stromata. Thielaviopsis) Chaetomium (ascomycete) D o m s c h et al. N. Α. (1949) A Manual of the Penicillia. B. I. A. B. and Pitt. Rifai. 6.. (1980) O l c h o w e c k i and Reid (1974) ( A n a m o r p h s : Chalara. D. 1-119. Neocosmospora) Alternaria (hyphomycete) Joly (1967) D o m s c h et al. (1974) Stud. (1984) The Yeasts—A Taxonomic Study. Baltimore. and Yarrow. 14. Κ. Fungi Imperfecti with Pycnidia. Samson. C. (1972) Hyphomycetes. Plenum Press. Kew. New York. 18... J. Samson. Samson. 1-56. (1969) Mycolog. Κ. Sutton.

(1980) W a t e r h o u s e (1968) Plaats-Niterink (1981) Rhizopus (zygomycete) D o m s c h et al. ( 1 9 8 3 ) B o o t h (1971) G e r l a c h and N i r e n b e r g (1982) D o m s c h et al. Venturia) Colletotrichum (ascomycete) von A r x (1970) Sutton (1980) (Teleomorph: Glomerella) Fusarium (hyphomycete) N e l s o n et al. 1985) (Teleomorphs: Eupenicillium. Hamigera) Phanerochaete (basidiomycete) Stalpers (1978) (Anamorph: Sporotrichum) Phytophthora (oomycete) N e w h o o k et al. (1980) (Teleomorph: Hypocrea) Verticillium (hyphomycete) D o m s c h et al.Appendix: Literature References Cladosporium (hyphomycete) D o m s c h et al. (1980) (Teleomorphs: Nectria. (1980) ( T e l e o m o r p h s : Nectria. Trichoma. Cordyceps. (1978) W a t e r h o u s e (1970) Pythium (oomycete) D o m s c h et al. 1976) (Teleomorphs: Mycosphaerella. (1980) Ellis ( 1 9 7 1 . (1963) D o m s c h et al. (1980) (Teleomorphs: Nectria. Gibberella) Gliocladium (hyphomycete) M o r q u e r et al. Torrubiella) . Talaromyces) 39 Pénicillium (hyphomycete) R a p e r and Thorn (1949) P i d o p l i c h k o (1972) S a m s o n (1979) D o m s c h et al. (1980) Schipper and Stalpers (1984) Trichoderma (hyphomycete) Rifai (1969) D o m s c h et al. (1980) Pitt ( 1 9 7 9 . Talaromyces. Sphaerostibellä) Mortierella (zygomycete) B e n j a m i n (1978) G a m s (1977) Mucor ( z y g o m y c e t e ) Schipper (1978) Paecilomyces (hyphomycete) Samson (1974) (Teleomorphs: Byssochlamys.

M o r p h o l o g y ( w h e t h e r the strain is pelletted or filamentous in 41 .1 . G e n e t i c stability is an important o n e b e c a u s e any gains in productivity d u e to increased biosynthetic potential will b e lost again if the strain d e g e n e r a t e s d u r i n g the s o m e t i m e s e x t e n s i v e g r o w t h stage required to d e v e l o p b i o m a s s for the production scale fermentation. A l t h o u g h increased productivity is of c o u r s e p a r a m o u n t . in d r a w i n g u p this short list it is important to c o n s i d e r a n u m b e r of other. or in s o m e other w a y r e d u c e p r o d u c t i o n c o s t s .CHAPTER 3 Strain Improvement and Strain Stability Robert T. Clearly this is not possible b e c a u s e of the e n o r m o u s e x p e n s e it w o u l d involve in t e r m s of lost p r o d u c t i o n each time a culture w a s w o r s e than the control. qualitative strain characteristics. S o it is n e c e s s a r y to resort to smaller.1 REQUIREMENTS FOR INDUSTRIAL STRAIN IMPROVEMENT T h e objective of an industrial strain i m p r o v e m e n t p r o g r a m is to p r o d u c e and detect genetically altered cultures that give increased productivity. laboratory scale screening to eliminate all but those cultures m o s t likely to s u c c e e d in the production scale test. Rowlands 3. using fermentation vessels intermediate in size b e t w e e n the laboratory and p r o d u c t i o n scales. In an ideal w o r l d . T h i s is usually d o n e by p r o d u c i n g a short list of potentially successful cultures from the laboratory screens w h i c h are scaled u p (their fermentation tested and re-optimized as appropriate) via a pilot plant. T h e m o s t important or frequently e n c o u n t e r e d of these are listed in T a b l e 3 . the best w a y of d o i n g this w o u l d b e to test all n e w cultures on the production scale until such an i m p r o v e d o n e is found.

w h i c h . but also by the fermentation e n g i n e e r responsible for getting the i m p r o v e d strains to express their m a x i m u m productivity on the p r o d u c t i o n scale. A l t h o u g h it is important for the fermentation geneticist to r e c o g nize that it will be too e x p e n s i v e for the fermentation e n g i n e e r to redesign c o m pletely the process for each n e w strain. W A ) penicillin strain line b e c a u s e this allows less p o w e r to b e u s e d . A similar i m p r o v e m e n t w a s introduced in 1990 with strain P 1 4 . d e p e n d i n g on the particular fermentation p r o c e s s . Both of these i m p r o v e m e n t s resulted in significant substrate cost savings and other process e c o n o m i c s for penicillin manufacturers. O t h e r characteristics m a y also be important. sugar or oil to the fermentation) or other substrate converted into product as o p p o s e d to being. but it is also possible to select for strains that give increased productivity u n d e r low o x y g e n c o n d i t i o n s . giving increased substrate c o s t s . Dissolved o x y g e n levels in fermentation broth are related to viscosity. n o longer wastefully oxidizes phenylacetic acid (the penicillin G side-chain precursor) but incorporates it almost 1 0 0 % into penicillin G (Lein 1986). a reduced c a r b o n efficiency.B 1 2 which s h o w s m a r k e d l y reduced oxidation of p h e n o x y a c e t i c acid (the penicillin V side-chain precursor). taken from the P a n l a b s Penicillin C l u b . m a i n t e n a n c e e n e r g y . and resistance to shear stress or certain m e d i u m c o m p o n e n t s that m a y inhibit either g r o w t h or production. and a highly viscous strain m a y be too e x p e n s i v e in t e r m s of the p o w e r required to maintain a d e q u a t e dissolved o x y g e n levels in the fermentation broth. or allows m o r e p r o d u c t i v e b i o m a s s to be built up for the s a m e p o w e r input.B 4 . P i g m e n t or other undesirable metabolite production can be a p r o b l e m in the extraction and purification p r o c e s s . is the mutation introduced into the strain line in 1981 with culture P 1 4 . could nullify any benefits d u e to increased productivity.42 Strain Improvement and Strain Stability TABLE 3-1 Some Characteristics to Consider When Choosing an Improved Production Strain Productivity Oxygen requirement Genetic stability Pigment production Morphology Shear sensitivity Substrate efficiency s u b m e r g e d culture) d e t e r m i n e s viscosity. or simply wastefully m e t a b o l i z e d . A n o t h e r e x a m p l e of qualitative c h a n g e s in a strain. it will also be apparent that. the e n g i n e e r must r e c o g n i z e that a n e w . for e x a m p l e . Genetically altering the strain's t e n d e n c y to p r o d u c e a filamentous s u b m e r g e d form into a tendency to p r o d u c e a pelletted form has been an important feature of the success of the Panlabs ( B o t h e l l . m a k i n g this m o r e e x p e n s i v e . T h e s e include ability to p r o d u c e s p o r e s . O n the other h a n d . H a v i n g g o n e through this list it is important to m a k e the point that these factors need to be considered not only by the fermentation geneticist responsible for p r o d u c i n g the i m p r o v e d strains.used for g r o w t h . unlike earlier cultures. as d i s c u s s e d . By substrate efficiency is m e a n t the proportion of carbon (for e x a m p l e .

If they are not. 3.2 Genetic Instability in Industrial Microorganisms 43 strain is a different entity from the previous o n e . T h e geneticist can then terminate p r o b l e m strain lines or selection p r o c e d u r e s . m o s t yield i m p r o v e m e n t and cost reduction c o m e s about by a s e q u e n c e of i m p r o v e d s t r a i n — i m p r o v e d operating c o n d i t i o n s — n e w i m p r o v e d strain—further i m p r o v e d o p e r a t i n g c o n d i t i o n s . W h e t h e r it b e c o m e s a big or a small p r o b l e m relates to the frequency of such undesirable m u t a t i o n s and the intensity of positive selection for the undesirable t y p e s . w h i c h m a y not a l w a y s be e c o n o m i c a l l y feasible. can advise the geneticist on the r e s p o n s e of the n e w strain to the production scale. T h e p u r p o s e of this c h a p t e r is to describe in m o r e detail the principles such as those d e s c r i b e d in the earlier p a r a g r a p h s that g u i d e the fermentation geneticist in generating and isolating i m p r o v e d industrial strains. h o w e v e r . and w h i c h e l e m e n t s of the p r o d u c t i o n scale p r o c e s s are too e x p e n s i v e to modify to a c c o m m o date the n e w strain. in turn.3. B a s e d on his laboratory scale e x p e r i m e n t s . then they h a v e lost the capacity to be m u t a t e d . e t c . the ideal strain m i g h t be o n e with a low s p o n t a n e o u s mutability for stability but a high inducible mutability for yield i m p r o v e m e n t ! Its effects can be m i n i m i z e d or eliminated by c h a n g i n g the selection forces present in the e n v i r o n m e n t . W h a t w e see as genetic instability is simply the result of the natural p r o c e s s of s p o n t a n e o u s m u t a t i o n . the effects of genetic instability can be mitigated by a p r o g r a m of regular re-isolation. This m e a n s c h a n g e s to the fermentation m e d i u m and other aspects of the fermentation p r o c e s s . as well as possible c h a n g e s in the seed d e v e l o p m e n t stages. and describing a strain as " s t a b l e " or " u n s t a b l e " is a c o m p a r a t i v e statement. T h e e n g i n e e r . etc. . and will require at least s o m e r e . feeding r e g i m e n s . S a u n d e r s et al. T h e term instability as used in this c h a p t e r refers to true genetic instability.o p t i m i z a t i o n of b a t c h e d m e d i u m ingredients and feeding r e g i m e n s . already referred to briefly. M u c h can b e d o n e to deal with the p r o b l e m of genetic instability. I n d e e d . C a t c h e s i d e 1974. the fermentation geneticist can r e c o m m e n d certain m e d i u m r e q u i r e m e n t s . seed d e v e l o p m e n t r e g i m e n s . and redesign the screens being used to p r o d u c e n e w strains that scale u p m o r e successfully. and so o n . Of c o u r s e . to the engineer. It can be altered by m u t a t i o n (Jansen 1972. It should b e clear b y n o w that an important e l e m e n t in the successful relationship b e t w e e n the fermentation geneticist and the fermentation e n g i n e e r is frequent feedback of information. s o m e strains are m o r e stable than o t h e r s . c o n t a m i n a t i o n . not instability of fermentation results d u e to such factors as variation in seed d e v e l o p m e n t p r o c e d u r e s . All strains are inherently u n s t a b l e . and n o further genetic yield i m p r o v e m e n t will be p o s s i b l e . 1982). as d e s c r i b e d in a s u b s e q u e n t section. and by reducing the n u m b e r of subcultures . p r o c e s s e r r o r s . T a l m u d 1977. In all c a s e s .2 GENETIC INSTABILITY IN INDUSTRIAL MICROORGANISMS A n integral part of the process of industrial strain i m p r o v e m e n t is c o p i n g with the p r o b l e m of instability. to give its best p e r f o r m a n c e .

In such c a s e s . F o r each fermentation p r o c e s s .i m p r o v e m e n t or cost-reducing mutations with the m i n i m u m of scaleup p r o b l e m s . In s o m e c a s e s . 3. and i n d e e d . E x c e p t i o n a l l y . but u n fortunately this c a n n o t b e relied on as a p a n a c e a . on agar m e d i u m . as will be discussed in a later section. m u s t b e m a d e the subject of an individual study. W h a t e v e r the source of the n e w strain. about 5 0 times better than F l e m i n g ' s original strain of P. h o w e v e r . In s o m e c a s e s . and occasionally it m a y be possible to d r a w on culture collection strains. m o s t i m p r o v e m e n t s in industrial strains o c c u r through m u t a t i o n of the current production strain. and will also c h a n g e with time at different stages in the p r o c e s s . especially if a sporulation step is involved. T h e b a l a n c e of the forces of mutation and selection will b e different for e a c h p r o c e s s .3 SOURCES OF GENETIC MATERIAL T h e basic genetic resource for the fermentation geneticist is usually the current production strain. T h e ultimate source of all genetic variation is by m u t a t i o n . the geneticist m a y be able to use closely related sister strains from a mutation tree if these are not too divergent (see F i g u r e 3 . before and after m u t a g e n e sis as well as in r e c o m b i n a t i o n and genetic engineering p r o g r a m s . instability m a y be exacerbated by surface subculture. vegetative s u b m e r g e d culture in liquid m e d i u m m a y partly or entirely eliminate the p r o b l e m . notatum ( R â p e r et al. A c o m m o n . and the best w a y s to c o p e with it. pure cultures are required. the genetic b a c k g r o u n d of this strain is best suited for the expression of p r o d u c t i v i t y . T h e p r o b l e m of instability p e r m e a t e s all aspects of the yield i m p r o v e m e n t p r o c e s s . and specific instances will be discussed at the appropriate places t h r o u g h o u t this chapter. the extent of the genetic instability p r o b l e m . w h e t h e r " s p o n t a n e o u s " or i n d u c e d . at the very b e g i n n i n g of a mutation p r o g r a m . in s o m e cases o c c u r r i n g o v e r m a n y y e a r s . s o m e preparation w o r k is required. for e x a m p l e . the fermentation geneticist m a y wish to screen soil s a m p l e s to see if there is a naturally higher p r o d u c i n g variant available. usually for qualitative characteristics to be used in r e c o m b i n a t i o n p r o g r a m s . T h i s w a s in fact d o n e at the b e g i n n i n g of the d e v e l o p m e n t of the penicillin fermentation w h e n a natural isolate of Penicillin chrysogenum w a s found to p r o d u c e about 100 O U / m l of penicillin. 3.1 ) . it m a y be desirable to bring together c o m p o n e n t s of different g e n o t y p e s by r e c o m b i n a t i o n or duplicate specific g e n e s by genetic e n g i n e e r i n g . This strain has been adapted to the existing plant and p r o c e s s b y a s e q u e n c e of selection and p r o c e s s optimization steps. from w h i c h it has to b e " s e l e c t e d " or " s c r e e n e d " out. O c c a s i o n a l l y . F o r this selection process to operate with m a x i m u m efficiency. it usually o c c u r s as a m e m b e r of a p o p u l a t i o n of assorted g e n o t y p e s . T h e r e f o r e . 1944).44 Strain Improvement and Strain Stability b e t w e e n re-isolation steps to an absolute necessary m i n i m u m .4 RE-ISOLATION AND PURIFICATION T o detect an i m p r o v e d strain.

to use m i x e d c u l t u r e s . Note that a so-called mutation "line" is never a single line. that is.1 A typical mutation tree. a l t h o u g h o n e m u s t be alert to the possibility that unstable m o r p h o l o g i c a l types m a y c o n t i n u e to s h o w a low p e r c e n t a g e of variants on s u b c u l t u r e . It is n e c e s s a r y to use m o r e than o n e t y p e of m e d i u m in this exercise b e c a u s e it is often found that w h e r e a s o n e m e d i u m m a y s h o w n o m o r p h o l o g i c a l differences. discarded strain to be re-evaluated. so it is important to always preserve significant strains. a change in operating conditions requires an earlier. S u c h m i x e d cultures can generally b e identified and purified by plating t h e m out on a variety of agar plate m e d i a and testing representative s a m p l e s of the different m o r p h o l o g i c a l t y p e s . without realizing it. as indicated on the left-hand side. Genetically different m o r p h o l o g i c a l types b r e e d t r u e . if not i m p o s s i b l e . In d o i n g this it is important to distinguish b e t w e e n g e n u i n e l y different m o r p h o l o g i c a l t y p e s . Sometimes. Strains on the same horizontal line have been tested concurrently. and the best ones proceeded to the next round of mutation to form new branches. failing in m u t a t i o n p r o g r a m s is. . U s e of such cultures will m a k e the detection of yield i m p r o v e m e n t m u t a t i o n s very difficult.4 Re-Isolation and Purification 45 STARTING CULTURE SISTER CULTURES UNRELATED CULTURES FIGURE 3 . whereas discarded strains are marked with an X. In practice it is more like a tree. m a k i n g it h a r d e r to detect.3. and apparent differences d u e to a s y n c h r o n o u s g e r m i n a t i o n . cultures that look p u r e but that as a result of either p o o r microbiological t e c h n i q u e or instability actually consist of p o p u l a t i o n s of t w o or m o r e g e n o t y p e s . w h e r e a s differe n c e s d u e to a s y n c h r o n o u s g e r m i n a t i o n s h o w a r a n d o m pattern on repeated s u b c u l t u r e s . b e c a u s e the effect of an i m p r o v e m e n t in a quantitative character will be diluted out by the l o w e r p r o d u c i n g g e n o t y p e s present. with several branches being mutated at any one time.

b e c a u s e variations in productivity d u e to different g e n o t y p e s d o not a l w a y s show m o r p h o l o g i c a l correlation. H o w e v e r . such high doses m a y b e undesirable for other r e a s o n s .1 C h o i c e of M u t a g e n . 3. A l t h o u g h mutation is the ultimate source of all n e w genetic variation. 1976. E v e n w h e n n o m o r p h o l o g i c a l variations can be seen with a variety of m e d i a . T h i s exercise will also give a m e a s u r e of the d e g r e e and types of instability that will h a v e to b e c o p e d with as the p r o g r a m p r o c e e d s . T h i s often gives the first " i m p r o v e d " strain in a strain i m p r o v e m e n t p r o g r a m . M u t a t i o n s that give increased productivity are frequently recessive or s e m i d o m i n a n t ( M a c d o n a l d et al. G e n e r a l l y . the very high p r o d u c i n g strains of Pénicillium chrysogenium currently used for c o m m e r c i a l penicillin p r o d u c t i o n tend to p r o d u c e conidia with a multiple and variable n u m b e r of nuclei. it is still w o r t h w h i l e testing a s a m p l e of single-colony re-isolates.46 Strain Improvement and Strain Stability a n o t h e r m a y s h o w t w o or three types of variant. Ditchburn et al. Alternatively multinucleate material of w h a t e v e r n a t u r e — s p o r e s . E v e n if o n e is fortunate to h a v e purely uninucleate material a v a i l a b l e .1.5. as discussed in the mutation section. and thus m a y not be e x p r e s s e d in a h e t e r o k a r y o n . p r o b l e m s m a y o c c u r with c l u m p i n g . or m y c e lial f r a g m e n t s — m a y be subjected to such high doses of m u t a g e n that only o n e n u c l e u s s u r v i v e s . H o w e v e r . 1963. m o r e dilute m e d i a or o t h e r w i s e m o r e stressful conditions are better at s h o w i n g u p m o r p h o l o g i c a l variation. caution is n e c e s sary b e c a u s e Yuill (1950) has s h o w n that not all strains of s u p p o s e d l y uninucleate species of fungi p r o d u c e uninucleate conidia. Mutation of o n e n u c l e u s in a m u l t i n u c l e a t e system can give rise to a heterokaryotic c o n d i t i o n . n o o n e m u t a g e n can be relied on to give all possible types of .1. p r o b l e m s can still o c c u r if m u l t i n u c l e a t e material is used for m u t a g e n e s i s .5. spores and protoplasts. or will h a v e identified the highest p r o d u c i n g g e n o t y p e from the p o p u l a t i o n . C a l a m et al. GENERATION OF NOVEL GENOTYPES Mutagenesis 3. A l s o . After carrying out this re-isolation s t e p . U n i n u c l e a t e protoplasts m a y p e r h a p s be separated from multinucleate protoplasts (enucleate o n e s not being a p r o b l e m genetically) by filtration or density centrifugation. This is w h y it is essential to re-isolate and purify the survivors of a mutation treatment before s c r e e n i n g . giving rise to a mutated and an u n m u t a t e d d a u g h t e r nucleus on division ( A u e r b a c h 1976). 1976. p r o t o p l a s t s . E v e n w h e n a p u r e culture has been o b t a i n e d . o n e either will h a v e c o n f i r m e d that the culture is p u r e . S i m p s o n and C a t e n 1980).5 3. T h e p r o g e n y of a somatic cross and transformants from a genetic e n g i n e e r i n g p r o g r a m also h a v e to be s c r e e n e d . or with m o s a i c i s m w h e r e b y a mutation b e c o m e s fixed in only o n e strand of the D N A helix. and the points relating to re-isolation and purification are equally valid for t h e m . A subculture step to allow segregation of mutated and u n m u t a t e d nuclei before re-isolation might also be desirable ( R o w l a n d s 1984a). In filamentous fungi there are t w o potentially uninucleate states.

Before e m p l o y i n g any m u t a g e n i c treatment in a strain i m p r o v e m e n t p r o g r a m it is essential to d e t e r m i n e (a) w h e t h e r it is m u t a g e n i c to the strain in q u e s t i o n . A l t h o u g h all m u t a g e n s are inherently d a n g e r o u s b e c a u s e of their effect on the D N A . U n f o r t u n a t e l y . a l o w e r i n g of the m e a n v a l u e . e v e n w h e n repaired via the s a m e " e r r o r . H o w e v e r . S u c h m u t a t i o n specificity has b e e n k n o w n for m a n y years (see A u e r b a c h 1976 and R o w l a n d s 1984a for r e v i e w s and d i s c u s s i o n ) . T h e fermentation geneticist c a n n o t rely on s p o n t a n e o u s m u t a t i o n e v e n t s to p r o d u c e e n o u g h variation to use as input into his s c r e e n s . T h e fact that a m u t a g e n i c t r e a t m e n t kills the cells is not. and so o n e or m o r e m u t a g e n i c t r e a t m e n t s h a v e to be used to " i n d u c e " m u t a t i o n s . they should a l w a y s b e neutralized. t o o . e v i d e n c e that it is inducing m u t a t i o n s in the s u r v i v o r s . they can b e a p p l i e d . such as ultraviolet light and nitrous acid. ipso facto. either singly or in c o m b i n a t i o n . In c o n s i d e r i n g w h i c h m u t a g e n i c t r e a t m e n t s to u s e . A useful list of mutation t r e a t m e n t s and the respective safety p r e c a u t i o n s to b e used with t h e m is given b y Baltz ( 1 9 8 6 ) . 1 . and (b) w h a t is the m o s t effective d o s e to u s e . T h i s will e n s u r e that the b r o a d e s t r a n g e of m u t a n t types is o b t a i n e d in successive stages of i m p r o v e m e n t of an industrial strain. 5 . as exemplified by the S O S repair studied in E. to filamentous fungi. on c o m p l e t i o n of the m u t a g e n i c t r e a t m e n t . are easier to protect against.1 M u t a g e n D o s e . A n o t h e r factor that has to be c o n s i d e r e d is the safety of the operators w h e n c a r r y i n g out m u t a g e n i c t r e a t m e n t s . Instead. with a little intelligent modification. a p r o n o u n c e d s k e w t o w a r d the l o w e r v a l u e s . this m a y result in a noticeable and characteristic c h a n g e in the frequency distribution of the productivities of the surviving isolates as c o m p a r e d with the u n m u t a t e d control i s o l a t e s — a flattening of the original bell-shaped c u r v e . s o m e . a m o r e direct m e a s u r e m e n t m u s t b e used. W h e n long-lived c h e m i c a l m u t a g e n s such as ethylm e t h a n e sulfate and n i t r o s o g u a n i d i n e are u s e d . T o the extent that the n u m b e r of these p a t h w a y s in any o n e o r g a n i s m is limited. a strain is found to be refractory to o n e or m o r e m u t a g e n i c t r e a t m e n t s . 2 . O c c a s i o n a l l y . a n u m b e r of theoretical and practical c o n s i d e r a t i o n s h a v e to b e taken into a c c o u n t . for e x a m p l e . and (hopefully) a small increase in the m a x i m u m values ( R o w l a n d s 1984a). c h e c k their effectiveness as described in Section 3 . W h e r e the frequency of productivity . N e a r l y all i n d u c e d m u t a t i o n occurs via p a t h w a y s that repair d a m a g e d D N A .5 Generation of Novel Genotypes 47 m u t a t i o n .3. to protect the o p e r a t o r s and the e n v i r o n m e n t . and use t h e m in rotation.5. coli (Eisenstadt 1988). with s o d i u m thiosulfate.p r o n e " p a t h w a y . it m i g h t b e t h o u g h t that the type of D N A d a m a g e i n d u c e d by different m u t a g e n i c agents is irrelevant. can b e s h o w n to generate a distinct and specific s p e c t r u m of m u t a t i o n s in a given strain (Eisenstadt 1988). 3. C o n s i d e r i n g these various factors. the fermentation geneticist is rarely able to predict w h a t type of m o l e c u l a r alteration to the D N A is required to i m p r o v e a given strain. each type of m u t a g e n i c t r e a t m e n t . A l t h o u g h the protocols used are primarily d e s i g n e d for use with Streptomyces. e v e n a r m e d with such k n o w l e d g e .2. the best advice that can be given to the fermentation geneticist is to find three or four mutation treatments that can be safely u s e d . W h e r e the frequency of m u t a t i o n s affecting productivity is sufficiently h i g h .

the fermentation geneticist has to deal with a m i x t u r e of single and multinucleate material. smaller than a single g e n e in length) into the strain of interest. C o n s e q u e n t l y if a m u t a t i o n d o s e is high e n o u g h that multiple mutations o c c u r within a single n u c l e u s . is to h a v e only o n e m u t a t i o n per n u c l e u s . using an a g a r . such as a m o r p h o l o g y or drug-resistance m a r k e r . the best advice that can be given is to try a r a n g e of d o s e s . T h e transforments can then be screened. such as spores with a v a r y i n g n u m b e r of n u c l e i . T h e s e fragments insert into the host g e n e s and disrupt t h e m by interrupting the transcription units (Figure 3 . T h e r e is little value in using a u x o t r o p h s for this p u r p o s e . low kill rates are better than high (see R o w l a n d s 1983 for a review of the available literature). O n c e a strain has a significant level of productivity. 2 on p r e s c r e e n s ) .7 5 % kill w a s o p t i m a l for obtaining productivity i m p r o v e m e n t mutations for the antibiotic c e p h a losporin C (called A D . for m u t a t i n g uninucleate m a t e r i a l .4 6 4 in their article). O n e e x a m p l e of in vitro m u t a g e n e s i s with a fairly w i d e application is in generating strains b l o c k e d in various g e n e s of their biosynthetic p a t h w a y . as selective m a r k e r s for a r e c o m b i n a t i o n p r o g r a m ) . but is unfortunately not universally applicable. and d e t e r m i n e the o p t i m u m d o s e by using the various m e t h o d s for m e a s u r i n g m u t a g e n e s i s d i s c u s s e d earlier. it is m o s t effective w h e n used in conjunction with classical m e t h o d s of strain i m p r o v e m e n t . If the drug-resistance m a r k e r s are c h o s e n w i s e l y . for e x a m p l e . T h e principle involved is to " s h o t g u n t r a n s f o r m " small fragments of g e n o m i c D N A (that is. w h e r e b y the genes affecting productivity or important qualitative characteristics could be directly targeted (see R o w l a n d s 1983 and 1984a). any positive m u t a t i o n s that o c c u r are likely to b e cancelled out by o n e or m o r e n e g a t i v e m u t a t i o n s in the s a m e n u c l e u s . possibly also c o n t a m i n a t e d with mycelial fragments. A s i d e from traditional r a n d o m m u t a g e n e s i s . A typical result is that of B r o w n and E l a n d e r ( 1 9 6 6 ) . 2 ) or by in vivo or in vitro r e c o m b i n a t i o n t e c h n i q u e s . In V i t r o M u t a g e n e s i s .2 ) . 3 . S u c h m u t a n t s can then b e used to increase productivity by selecting for suppression of the block (see Section 3 . with multinucleate m a t e r i a l . such kill rates m a y give n o detectable productivity-increasing mutations at all. it is possible to get a m e a s u r e of the effectiveness of the m u t a g e n i c treatment b y m e a s u r i n g the increase in frequency of s o m e easily scorable genetic m a r k e r . the drug-resistant m u t a n t s t h e m s e l v e s could b e used as v a l u a b l e input to the screening p r o g r a m (see Section 3 . T h e situation to a i m at.48 Strain Improvement and Strain Stability m u t a t i o n s is too low to noticeably affect the productivity frequency distribution. 7 . it will be a p p a r e n t that there are m a n y m o r e potential mutations that w o u l d result in a d e c r e a s e in p r o ductivity than there are that will result in an increase. fermentation geneticists h a v e sought for m a n y years for an effective m e t h o d of directed m u t a g e n e s i s . 3 . for r e a s o n s discussed earlier. A m a t h e m a t i c a l description of this a r g u m e n t is given by Baltz ( 1 9 8 6 ) . 1 . This d r e a m is n o w possible with the d e v e l o p m e n t of sophisticated t e c h n i q u e s for m a n i p u l a t i n g D N A in vitro. w h o found that 7 0 . Of c o u r s e . S o o n c e a g a i n . A s with m a n y r e c o m b i n a n t D N A t e c h n i q u e s . A n d quite often. 7 . unless they are n e e d e d for other p u r p o s e s (for e x a m p l e . 5 . therefore. M o s t authors agree that.

c D N A c l o n i n g . TRANSFORMING DNA* A B C D V W X Y Z E F G H IJ ΚL M INACTIVATED BIOSYNTHETIC GENE plate b i o a s s a y to identify the b l o c k e d m u t a n t s . w h e r e .2 Protoplasts Protoplasts are of use to the fermentation geneticist in several w a y s . In fungi such as Cephalosporium acremonium. Protoplasts can also b e used for m u t a t i o n . or by c h e m i c a l t r e a t m e n t ( T i m m i s 1 9 8 1 . protoplast fusion is the m o s t efficient w a y of carrying out genetic crosses in the laboratory ( H a m l y n and Ball 1979.3. H a v i n g isolated a g e n e of interest.5. w h e r e n o n e of the natural forms of . and r e c o m b i n i n g fungal protoplasts h a v e b e e n d e s c r i b e d in P e b e r d y ( 1 9 7 9 ) . it integrates correctly and p r o d u c e s the desired p h e n o t y p e . Shortle et al. it b e c o m e s possible to specifically m u t a t e it b y replacing parts of its s e q u e n c e using restriction e n z y m e s . T h e y can be used both as a source of novel genetic material and also as an aid to r e c o m b i n i n g genetic material. or o t h e r r e c o m b i n a n t D N A t e c h n i q u e (see C h a p t e r 6 ) . Birkett and H a m l y n 1985). Protoplast fusion c a n of c o u r s e also be used as a reliable w a y to initiate h e t e r o k a r y o n formation in filamentous fungi e v e n w h e n natural m e c h a n i s m s are available. The transforming DNA could also be a linear fragment. w h i c h h a v e n o c o n v e n i e n t natural m e c h a n i s m for sexual or p a r a s e x u a l genetic e x c h a n g e . 3. 1981). This t e c h n i q u e has b e e n called "insertional i n a c t i v a t i o n " or " m u t a t i o n a l c l o n i n g " ( H o p w o o d and C h a t e r 1984).5 Generation of Novel Genotypes BIOSYNTHETIC GENE ABCDEFGHIJKLM 49 FIGURE 3 . T h e mutated g e n e is then re-introduced into the p r o d u c t i o n o r g a n i s m .2 Production of blocked mutants by insertional inactivation. either by insertional inactivation. fusing. T h e general t e c h n i q u e s for p r e p a r i n g . hopefully. the principle is essentially the same.

o n e should be a w a r e that the stress involved in the production and r e g e n e r a t i o n of protoplasts can by itself c a u s e loss of productivity. Of c o u r s e . but at least s o m e will be u n i n u c l e a t e and h a v e a better c h a n c e of expressing p r o d u c t i v i t y . increase the g e n e d o s a g e of g e n e s c o d i n g for rate-limiting steps (for e x a m p l e . S o if o n e takes a perfectly g o o d production strain and crosses it with a strain that d o e s not fit into the current production p r o c e s s . T h e a d v a n t a g e of genetic e n g i n e e r i n g o v e r somatic crossing is that it can be used to isolate and modify a single g e n e while leaving the parental genetic b a c k g r o u n d intact. W h e n using protoplasts for any of these p u r p o s e s .3 Recombination R e c o m b i n a t i o n in industrial fungi is generally achieved in t w o w a y s : by s o m a t i c crosses initiated with or without protoplast fusion (Ball 1984). either successful crosses in the industrial laboratory tend to be carried out b e t w e e n closely related (sister) cultures (Figure 3 . the additional copies of the g e n e had b e e n inserted at a . It is not clear w h e t h e r this h a p p e n s in fungi. from Aspergillus niger.i m p r o v e m e n t g e n e s . for e x a m p l e . In this w o r k . ( 1 9 8 9 ) . A n interesting recent c o m b i n e d use of both genetic e n g i n e e r i n g and somatic crossing to achieve productivity i m p r o v e m e n t w a s described by Finkelstein et al. T h i s is easiest achieved by m u t a t i o n . the production of the e n z y m e g l u c o a m y l a s e . or modify the structural g e n e itself b y in vitro m u t a g e n e s i s as described earlier. Skatrud et al. it is very unlikely that any of the p r o g e n y will be able to be scaled u p . Finally. or by genetic e n g i n e e r i n g ( C h a p t e r 6 ) . W h a t o n e is really seeking to d o in i m p r o v i n g a production strain is to modify o n e specific g e n e while k e e p i n g the rest of the genetic b a c k g r o u n d the s a m e . In this w a y o n e can transfer individual g e n e s b e t w e e n o r g a n i s m s . mutation of protoplasts could be used as an alternative to using very high d o s e s of m u t a g e n on mycelial fragments. 1 9 8 3 . as described earlier. 1983).50 Strain Improvement and Strain Stability the o r g a n i s m s are suitable. if the o r g a n i s m p r o d u c e s n o or very few s p o r e s . protoplasts are of c o u r s e useful as a t e c h n i q u e for re-introducing D N A into the host strain after in vitro genetic m a n i p u l a t i o n ( C h a p t e r 6 ) . S o .5. It is possible to enrich for uninucleate protoplasts by using various fractionation p r o c e d u r e s . t w o different m u l t i c o p y transformants w e r e crossed together.1 ) . In this instance. or by cloning short s e q u e n c e s of D N A by genetic e n g i n e e r i n g . and there is e v i d e n c e in Streptomyces that the p r o c e s s m a y be m u t a g e n i c ( S u g i y a m a et al. and in s o m e of the p r o g e n y the yield w a s d o u b l e d a g a i n . F o r e x a m p l e . S u b s e q u e n t l y . 1989). 3. I k e d a et al. or it is necessary to repeatedly back-cross the r e c o m b i n a n t with the desirable gene(s) to the original production strain. C o m m e r c i a l l y important strains of fungi almost n e v e r p o s s e s s a d e m o n s t r a b l e sexual c y c l e . w a s initially increased approximately eightfold by introducing multiple copies of the cloned g l u c o a m y l a s e g e n e . S o m a t i c crosses h a v e the d i s a d v a n t a g e in the industrial context that they tend to efficiently r a n d o m i z e the g e n o m e s of the t w o p a r t n e r s . giving an eventual fifteenfold increase o v e r the original starting strain. increase their expression by modifying the p r o m o t e r and terminator s e q u e n c e s . filtration. the protoplasts p r o d u c e d m a y contain n o nuclei t h r o u g h o n e and t w o to several n u c l e i .

A s this expression p a t h w a y requires a finite t i m e . H o w e v e r . U n d o u b t e d l y m u c h of the e x p r e s s i o n p a t h w a y can actually o c c u r during the screening itself. the n u m b e r of m u t a n t s found d e c r e a s e s . w e m a y r e a s o n a b l y a s s u m e that the longer the delay b e t w e e n m u t a g e n e s i s and s c r e e n i n g . can increase p r o d u c t i v i t y . but this is not necessarily the m o s t efficient p r o c e d u r e . In a d d i t i o n .2 Plating Density Effect T h e plating d e n s i t y . if the n e w protein is a modified e n z y m e . 3. .1 Delay of Expression Before any physical or c h e m i c a l c h a n g e to the D N A can result in an altered p h e n o t y p e . o n e has to screen t h e m to find the i m p r o v e d cultures ready for scaling up to p r o d u c t i o n . a n u m b e r of e v e n t s m u s t occur: the initial D N A d a m a g e m u s t b e fixed into a stable m u t a t i o n by the appropriate repair p a t h w a y . T h i s is k n o w n as the plating density effect or G r i g g effect (Grigg 1952. or increasing g e n e d o s a g e and expression by genetic e n g i n e e r i n g . can affect the detection of m u t a n t s . 1972) and is clearly a potential c a u s e of failure to detect i m p r o v e d cultures. the better c h a n c e there is for it to be c o m p l e t e d . A better a p p r o a c h w o u l d be to allow t i m e for fixation and e x p r e s s i o n of n e w m u t a t i o n s before screening c o m m e n c e s . and a simple r a n d o m a s s o r t m e n t and r e s e g r e g a tion of the c h r o m o s o m e s of the t w o parents w a s all that w a s required to get the m a x i m u m p o s s i b l e n u m b e r of g e n e copies into a single strain. 3. A s the plating density is increased. then the n e w D N A m u s t be transcribed and translated into protein. and h o w selective the s c r e e n s . it is necessary to realize that n o matter h o w g o o d a n e w g e n o t y p e m a y b e . and m u t a t i o n can easily p r o d u c e qualitative c h a n g e s as well as quantitative c h a n g e s .3.6 EXPRESSION H a v i n g g e n e r a t e d a selection of novel g e n o t y p e s . this then requires t i m e to exert its effect (a process of diluting out the preexisting metabolites) before a detectable c h a n g e in the p h e n o t y p e of the o r g a n i s m is o b s e r v e d . 4 . n u m b e r of c o l o n y . that is. this is untrue b e c a u s e r e c o m b i n a t i o n b e t w e e n fairly closely related strains with different p r o d u c t i v i t y . It has s o m e t i m e s b e e n argued that r e c o m b i n a t i o n is better for introducing qualitative characteristics into a production o r g a n i s m . Scott et al.i m p r o v e m e n t m u t a t i o n s .6. w h e r e a s m u t a g e n e s i s is better for quantitative characteristics.f o r m i n g units inoculated o n t o a p l a t e . H o w e v e r . T h e effect of m i x e d cultures on e x p r e s s i o n of desirable traits has already b e e n discussed in Section 3 . it will n e v e r b e detected unless it is properly e x p r e s s e d .6.6 Expression 51 different single site in e a c h p a r e n t . 3. there are a n u m b e r of physiological factors that m a y prevent or h i n d e r expression.

even w h e n process modifications are m a d e to a c c o m m o d a t e it. that is.1 SCREENING General Considerations A s e x p l a i n e d earlier. or e v e n inferior t o . .7. and peptides is inhibited b y inorganic p h o s p h a t e (Martin 1977). Penicillin biosynthesis is also regulated b y lysine ( D e m a i n 1957) and valine ( G o u l d e n and C h a t t a w a y 1969). e t c .3 Strain Improvement and Strain Stability Metabolic Regulation Practically all metabolic activities are regulated in s o m e w a y . T h e mixing conditions in the screening e n v i r o n m e n t (dissolved o x y g e n availability. to identify the limiting factors in the production e n v i r o n m e n t — n u t r i e n t availability. 1979). 3. a m i n o g l y c o s i d e s . o t h e r w i s e the screen m a y p r o d u c e a culture that requires m o r e o x y g e n than is available o n the p r o d u c t i o n scale. to give a d e q u a t e levels of expression. T h e m o s t important screen for the industrial geneticist is still the shake flask. . and m a n y e x a m p l e s are k n o w n w h e r e the c o m p o s i t i o n of the g r o w t h m e d i u m can repress the formation of c o m m e r c i a l l y important metabolites. high shear rates. T h e shake flask m e d i u m (including seed stages) must therefore be b a s e d on the production m e d i u m . r e a s o n a b l e levels of penicillin are unlikely to be p r o d u c e d unless a side-chain p r e c u r s o r (phenylacetic acid or p h e n o x y a c e t i c acid) is supplied. such as p H and t e m p e r a t u r e . must also be faithfully replicated. It is vitally important to use the s a m e source of raw materials as is used in the p r o d u c t i o n and pilot plant scales. O t h e r p a r a m e t e r s . but not on both ( S h o m u r a et al. as o p p o s e d to using laboratory g r a d e r e a g e n t s . otherwise o n e risks selecting cultures that d o well u n d e r the conditions of the screen. w h e t h e r this is the o n l y . T h i s is b e c a u s e the productivity of m a n y cultures b e c o m e s m e d i u m d e p e n d e n t as the cultures adapt to the particular set of and balance of m e d i u m c o m p o n e n t s d u r i n g the o n g o i n g selection p r o c e s s . In practice this requires close collaboration b e t w e e n the fermentation geneticist and the fermentation engineer. 1980). but this is a task better a v o i d e d at the outset. or is too sensitive to shear. the object of a screen is to identify a short list of i m p r o v e d cultures for r e c o m m e n d a t i o n to the fermentation e n g i n e e r for s c a l e u p .6. shear rates) m u s t also reflect those of the p r o d u c t i o n scale.7 3. it is important to control such factors as m e d i u m c o m p o s i t i o n . It is often possible to re-adapt an i m p r o v e d culture to a n e w source of m e d i u m ingredients or even to a n e w m e d i u m . and to r e p r o d u c e t h e m in the screen. M e h t a et al. o x y g e n limitation. the current p r o d u c t i o n strain on the large scale. S o m e antibiotics m a y be p r o d u c e d only o n surface or s u b m e r g e d c u l t u r e . S o w h e n d e s i g n i n g a screening m e d i u m . t h o u g h this m a y reflect o u r lack of understanding of the necessary conditions for b i o s y n t h e s i s . F o r e x a m p l e . but are n o better t h a n . as well as p H and t e m p e r a t u r e . the screen m u s t reflect the s a m e e n v i r o n m e n t a l conditions that the i m p r o v e d culture will m e e t o n the production scale. both penicillin and c e p h a l o sporin C biosynthesis are subject to carbon catabolite repression (Soltero and J o h n s o n 1 9 5 3 . or the final level of screening. A l s o . T h e biosynthesis of a w i d e r a n g e of antibiotics including p o l y k e t i d e s . A s s u c h .52 3.

2. Rational or indirect screens involve assaying or m e a s u r i n g s o m e b i o c h e m i c a l feature associated with p r o d u c t i v i t y . as is d o n e with the substitution of lactose for g l u c o s e or sucrose as c a r b o n source for screening for i m p r o v e d penicillin-producing m u t a n t s of P. A l t h o u g h this can be m i m icked in s h a k e flasks it is very i n c o n v e n i e n t . c o l o n i e s m a y be cultured on individual agar plugs (Ditchburn et al. it m a y also b e desirable to select m u t a n t s with increased s t a m i n a . Of c o u r s e . R a n d o m screens m a y b e m o r e properly described as " d i r e c t " screens ( R o w l a n d s and N o r m a n s e i l 1983) b e c a u s e they usually involve direct assay of the p r o d u c t (antibiotic.7. and unfortunately such details . Of c o u r s e .3 ) to select a m u t a n t with an increased rate of biosynthesis. and i n d e e d . or m o r e usually on o n e day t o w a r d the end of the fermentation. but the closer o n e can c o m e to d o i n g s o . ) of interest. increasing the risk of c o n t a m i n a t i o n and also r e d u c i n g the rate of t h r o u g h p u t of cultures in the screen. A n o t h e r w a y to c o p e with this p r o b l e m is to replace a repressing nutrient with a n o n r e p r e s s i n g o n e . Increased rate m u t a n t s are generally m o r e likely to scale u p . b e c a u s e the b i o c h e m i c a l correlate c a n n o t be g u a r a n t e e d to give increased p r o d u c t i v i t y . are less than in p r o d u c t i o n . the m o r e successful the screen will b e . e t c . b e c a u s e of its indirect n a t u r e . T h i s usually m e a n s that the fermentation p e r i o d . H o w e v e r . rather than productivity itself. it has to be accepted that production scale conditions can n e v e r be exactly duplicated in shake flasks. 1 . " such screens can only be treated as p r e s c r e e n s and all cultures selected m u s t be subsequently tested in direct shake-flask s c r e e n s . S u c h agar p r e s c r e e n s are usually followed by o n e or m o r e levels of shake-flask screen d e s i g n e d a c c o r d i n g to the principles discussed in Section 3 . Alternatively. if resources a l l o w . chrysogenum. Types of Screens S c r e e n s are usually classified as r a n d o m (or empirical) and " r a t i o n a l " (or b i o c h e m i cal s c r e e n s ) . and c o n s e q u e n t l y the final titers r e a c h e d . 7 . 1974).3. rather than increased accretion rate. T h e m o s t efficient types are those that kill all cultures with low productivity. T h i s day of harvest should be c h o s e n b y carrying out a time course of the fermentation and identifying the point j u s t before the rate of product accretion declines (Figure 3 . thus a l l o w i n g an increase in t h r o u g h p u t of several orders of m a g n i t u d e o v e r that of the r a n d o m s c r e e n s . although the error i n v o l v e d in detecting c h a n g e s of slope is greater than that involved in detecting c h a n g e s in final level. it is m o r e usual to use b a t c h e d levels of the fed ingredients w h i c h are sufficiently low so as not to inhibit or repress g r o w t h or productivity. S u c h screens m a y consist of an agar plate prescreen or p r i m a r y screen. w h e t h e r by bioassay or c h e m i c a l m e a n s . Instead. is likely to p r o d u c e a large proportion of "false p o s i t i v e s . Rational screens are clearly m o r e effective the m o r e is k n o w n about the nature and regulation of the biosynthetic p a t h w a y .7 Screening 53 Practically all p r o d u c t i o n scale fermentation processes involve feeding of certain m e d i u m ingredients to the fermenting culture. with c o l o n i e s g r o w n in a r a n d o m or o r d e r e d array and overlaid with a bioassay o r g a n i s m or s o m e c h e m i c a l reagent to m e a s u r e productivity (Ball and M c G o n a g l e 1978). Shake-flask cultures m a y b e assayed on several different d a y s . 3.

54 Strain Improvement and Strain Stability F I G U R E 3 . A very useful type of rational screen is to use a toxic a n a l o g u e of a biosynthetic intermediate to select for resistant m u t a n t s . W h e r e it is not possible to find a condition in w h i c h the a n a l o g u e s . Godfrey 1 9 7 3 . b y using a less efficient carbon s o u r c e .3 Optimum day of harvest in shake-flask screening (theoretical time course). S u c h m u t a n t s m a y be resistant d u e to o v e r p r o d u c t i o n of the natural intermediate and thus either lead directly to increased productivity. or p r o v i d e a genetic b a c k g r o u n d in which further productivity imp r o v e m e n t s are m o r e likely to be expressed (for e x a m p l e . so only the m o r e generally applicable types of screen will be discussed here (Table 3 . it w o u l d be e x t r e m e hubris to think that o n e could predict every possible type of productivity i m p r o v e m e n t mutation and design a b i o c h e m i c a l screen to identify it. or nonoptimal t e m p e r a t u r e and/or p H ( M e h t a and N a s h 1979). are very poorly u n d e r s t o o d for the majority of c o m m e r c i a l fermentation p r o d u c t s . C h a n g and E l a n d e r 1979). E v e n w h e r e a biosynthetic p a t h w a y is understood in considerable detail. for e x a m p l e .2 ) . Select at five days (arrow A) for increased rate mutants. H o w e v e r . R o w l a n d s 1984b). it is possible to increase the toxicity of m a n y such substances by increasing the stress on the m i c r o o r g a n i s m . A c o m m o n p r o b l e m in utilizing this t e c h n i q u e is finding a n a l o g u e s that are sufficiently toxic. Select after seven days (after arrow B) for increased stamina mutants. T h e possible variety of rational or b i o c h e m i c a l screens is almost infinite and has been covered in great detail e l s e w h e r e (for e x a m p l e . S o it is a l w a y s essential to use both rational and r a n d o m screens in conjunction to achieve the m o s t efficient rate of progress in yield i m p r o v e m e n t .

to o v e r c o m e feedback repression of the biosynthetic p a t h w a y . n i t r o g e n . T h i s involves finding a c h e m i c a l that is toxic to the p r o d u c i n g o r g a n ism but that is detoxified by the p r o d u c t . it m a y be n e c e s s a r y to carry out such a selection on a regular basis d u r i n g a strain i m p r o v e m e n t p r o g r a m . respectively (Martin et al. H o d g s o n 1982. L u e n g o et al. Specific forms of m e t a b o l i c d e r e g u l a t i o n . n o r m a l for p r o t o t r o p h y . ) they can h a v e a variety of u s e s . R e s i s t a n c e to m a n y toxic c h e m i c a l s can also o c c u r by c h a n g e s in the cell w a l l / m e m b r a n e structure. such as relief of c a r b o n .2 Screening 55 Types of Rational Screens Mentioned in the Text* Resistance to toxic analogues of biosynthetic intermediates Resistance to end product (or toxic analogue) Selective detoxification Resistance to chemicals affecting cell wall/membrane Metabolic derepression Reversion of blocked mutants *For a more exhaustive list see Rowlands 1984b. and h e a v y metal ions b y reaction with /3-lactams. b y progressively increasing the level of toxin.p r o d u c i n g c o m m e r c i a l strains it is very unusual to obtain c o m p l e t e l y b l o c k e d m u t a n t s .p r o d u c t toxicity or inhibition b e i n g a prerequisite for further i m p r o v e m e n t s in productivity ( W o o d r u f f 1966). can also be selected for using 2 . d u e to the multiplicity of m e t a b o l i c p a t h w a y s .7 TABLE 3 . A variant of this t e c h n i q u e is to use a n a l o g u e s of the e n d p r o d u c t . R a n d o m screening p r o g r a m s will naturally tend to p r o d u c e b l o c k e d m u t a n t s . F o r the t e r m s of this discussion any m u t a n t with 1 0 % or less of the original productivity can be c o n s i d e r e d to be a b l o c k e d m u t a n t . i m i d a z o l e . " that is. W h e n these m u t a t i o n s o c c u r in o t h e r w i s e n o r m a l strains (for e x a m p l e . T h e s e c h a n g e s can in turn lead to increased ability to export products from the cell. A rather intellectually satisfying form of rational screening is selective d e toxification. B o w m a n et al. it can b e used c o n t i n u o u s l y t h r o u g h o u t a screening p r o g r a m . and arsenate (or v a n a d a t e ) . for e x a m p l e . 1983). rather than . phenylacetic acid. In h i g h . a variant of this t e c h n i q u e is to use such a n a l o g u e s to inhibit p r o d u c t i o n of z o n e s m e a s u r e d b y . as described earlier. will inhibit g r o w t h sufficiently. W h e n cultures exhibit low productivity as a pleiotropic effect of d e v e l o p m e n t a l m u t a t i o n s c a u s i n g p o o r g r o w t h and sporulation.3. increased resistance to e n d . p i g m e n t a t i o n . 1979). or " n o n p r o d u c e r s . S u c h m u t a n t s can also b e p r o d u c e d to o r d e r by insertional inactivation t e c h n i q u e s . If the concentration of the toxic c h e m i c a l is properly adjusted. bioassay o v e r l a y . the p o l y e n e antibiotics h a v e been cited as b e i n g particularly effective for this ( T a k e d a C h e m i c a l Industries 1977. then only h i g h e r . 1979. e t c . but this is clearly less efficient in t e r m s of t h r o u g h p u t . U n l i k e other b i o c h e m i c a l t e c h n i q u e s . In filamentous fungi. or p h o s p h a t e r e p r e s s i o n . or the e n d product itself w h e r e this is sufficiently t o x i c . m u t a n t s with m a r k e d l y r e d u c e d productivity. E x a m p l e s include the detoxification of h y d r o x y l a m i n e . C h a n g and E l a n d e r (1979) successfully used this t e c h n i q u e to i m p r o v e c e p h a l o s p o r i n C productivity.d e o x y g l u c o s e . m o r p h o l o g y .p r o d u c i n g m u t a n t s will be able to g r o w . I n d e e d . m e t h y l a m m o n i u m salts.

H o w e v er. a screen d o e s not p r o d u c e any useful m u t a n t s .m i n d e d policy of trying all types of s c r e e n s . it is rarely useful (except in the a c a d e m i c sense) to study e a c h n e w strain to identify the nature of the b i o c h e m i c a l c h a n g e that g a v e the i m p r o v e m e n t b e c a u s e that rate-limiting step has n o w g o n e . a useful philosophy to follow that is used at P a n l a b s is that of "rational e m p i r i c i s m " ( R o w l a n d s 1986). a l t h o u g h l a b o r i o u s . in the w o r s e case these m i g h t be all that o n e o b t a i n s . modification of qualitative characters can also result in significant cost r e d u c - . U n o w s k y and H o p p e 1978). Direct. B i o c h e m i c a l characterization of n o r m a l m o r p h o l o g y b l o c k e d m u t a n t s can provide a valuable insight into the biosynthetic p a t h w a y and its regulation ( N o r m a n s e l l et al. r a n d o m screens h a v e the a d v a n t a g e that. Such strains are p e r h a p s m o r e likely to p o s s e s s g e n u i n e increases in biosynthetic activity than those p r o d u c e d by any other techn i q u e . they are best discarded. If a particular form of rational screen does not w o r k . T h e r e are several published e x a m p l e s of successful application of this t e c h n i q u e ( D u l a n e y and D u l a n e y 1967.56 Strain Improvement and Strain Stability as a p r i m a r y m u t a t i o n . h a v i n g b e e n m u t a t e d twice in genes directly affecting p r o d u c t formation. w h e n it stops w o r k i n g it is replaced by another o n e . in rational e m p i r i c i s m . This p h i l o s o p h y takes the view that n o o n e type of screen can ever be predicted a priori to b e successful. and tried again. In d e c i d i n g w h e t h e r to use r a n d o m or rational screens for productivity i m p r o v e m e n t . w h a t is the next rate-limiting factor ("bottleneck") that n e e d s to b e o v e r c o m e . w h i c h m u s t be m a d e to w o r k in the sense that they should reflect the production scale c o n d i t i o n s . T h e objective here is to obtain s u p p r e s s i o n . or stops w o r k i n g after a t i m e . If. it should be stopped and another o n e tried. it should be returned to later. T h u s to a c h i e v e the fastest rate of strain i m p r o v e m e n t the m o s t logical practice is to a d o p t an o p e n . they will detect all and any types of beneficial m u t a t i o n s . only that it d o e s work. after a reasonable p e r i o d . and the next required b i o c h e m i c a l c h a n g e will b e different. Of course they also can be used directly for productivity i m p r o v e m e n t s by r e m u t a g e n e s i s and selecting for r e c o v e r y of productivity. D h a r and B o s e 1968.c o n s u m i n g study of w h y a rational screen did not w o r k (unless an o b v i o u s error w a s m a d e ) . Screens for identifying strains with i m p r o v e d productivity are naturally u p p e r m o s t in the m i n d s of those involved with industrial strain i m p r o v e m e n t . and so m a y other physiological characteristics of the strain. F o r the s a m e r e a s o n s . for e x a m p l e . T h e y are also valuable recipients for cloned D N A in the application of genetic e n g i n e e r i n g to identify clones carrying biosynthetic g e n e s . the rate-limiting step will c h a n g e . T h u s . If a screen is found to be p r o d u c t i v e . in such a w a y as to o v e r c o m p e n s a t e for the original lesion and thus result in increased productivity. it is rarely possible to identify what type of mutation w o u l d b e m o s t beneficial. 1979). it should continue to be used until it n o longer generates useful m u t a n t s . both rational and r a n d o m . This is not true of the direct shake-flask s c r e e n s . so that a screening t e c h n i q u e that w a s previously unsuccessful m a y later generate useful m u t a n t s . This is b e c a u s e after several r o u n d s of m u t a t i o n and selection. It has been explained a b o v e that so-called rational screens tend to p r o d u c e a high percentage of false p o s i t i v e s — i n d e e d . T h e r e is rarely any a d v a n t a g e in the industrial situation in u n d e r t a k i n g a t i m e . o n e d o e s not care w h y a particular screen w o r k s . and in w h i c h c o m b i n a t i o n . A l s o . rather than reversion of the original m u t a t i o n .

or by actually increasing the concentration of these substances in the m e d i u m (taking care that this d o e s not disturb the p H ) . Increased resistance to toxic substances c a u s i n g a particular p r o b l e m to the fermentation can b e selected by incorporating t h e m at the subtoxic level into the screen. Increased shear resistance can b e selected for in shake flasks by increasing the agitation or introducing baffles into the flasks. nitrogen s o u r c e . S o m e t i m e s a laboratory m a y p o s s e s s a n u m b e r of miniature stirred fermentors in w h i c h to c h e c k these strains for any gross physiological c h a n g e s before they g o to the pilot plant. followed by fermentation in shake flasks with r e d u c e d agitation. B e c a u s e the a c c u r a c y of testing increases only by the square root of the d e g r e e of replication ( C a l a m 1964).7. S o m e of these h a v e been t o u c h e d on earlier in this c h a p t e r . usually with little or n o replication. after cultures p o s s e s s i n g the desired qualitative c h a n g e h a v e been selected. Of c o u r s e . a p e r c e n t a g e of t o p isolates from this level ( n o w represented b y a relatively small n u m b e r of strains) can be retested in 2 5 0 . filled with m e d i u m to a higher level than n o r m a l . let it suffice to say that any type of qualitative alteration can be selected for by appropriately modifying the screen. then c h e c k i n g these in shake flasks to o b s e r v e the true s u b m e r g e d m o r p h o l o g y .4 . is given in Figure 3 . C a l a m ( 1 9 6 4 ) and D a v i e s ( 1 9 6 4 ) h a v e justified the use of multilevel screening as d e s c r i b e d h e r e . and the d e g r e e of replication is increased. or with a closure that restricts g a s e o u s e x c h a n g e . increasing . incorporating the various points discussed so far. A typical flow d i a g r a m for mutational strain i m p r o v e m e n t . before r e c o m m e n d i n g the short-listed strains for the smallest scale of pilot-plant testing.3. can b e selected by u s i n g an agar plate prescreen to identify m u t a n t s with altered colonial m o r p h o l o g y .3 Screening Strategies T h e w a y in w h i c h the various screens are applied. particularly w h e n i m p r o v e d m u t a n t s are rare and the testing error of the screen is h i g h . then increasing their concentration gradually as the strain b e c o m e s m o r e tolerant. they m u s t be c h e c k e d for retained (or i m p r o v e d ) productivity.l i m i t e d c o n ditions can b e screened for b y selecting for ability to p r o d u c e u n d e r h e a v y agar o v e r l a y on p l a t e s .7 Screening 57 tions o n the p r o d u c t i o n scale. in w h i c h the cultures first g o through c o a r s e (low-resolution) screens in w h i c h large n u m b e r s of isolates can be p r o c e s s e d rapidly. Finally. but miniature s h a k e flasks or tube ferm e n t a t i o n s c o u l d also be u s e d . R e s i s t a n c e to p h o s p h a t e . A g a r plate or p l u g screens are an o b v i o u s e x a m p l e . M o r p h o l o g y c h a n g e s . and carbon source repression can b e selected in agar plates or shake flasks using the a n a l o g u e s described earlier.m l s h a k e flasks. 3. leading to reduced-viscosity pelletted strains. can b e referred to as the screening strategy. p e r h a p s n o w in 2 5 0 . or o p e r a t e d . it is m o r e efficient to use single replicates in the p r i m a r y stages to allow larger n u m b e r s to b e p r o c e s s e d . and p e r h a p s eliminate s o m e or r e c o m m e n d certain process modifications to b e m a d e d u r i n g their pilot-plant e v a l u a t i o n . Clearly the list is endless.m l s h a k e flasks using a high d e g r e e of replication. A p r e d e t e r m i n e d p e r c e n t a g e of top isolates then p a s s e s to the next level of s c r e e n i n g . Cultures that give i m p r o v e d p e r f o r m a n c e u n d e r o x y g e n . M o s t screens are operated as multilevel s c r e e n s .

W h e n the m a g n i t u d e of the productivity i m p r o v e m e n t s is small in relation to the testing error of the screen. It is essential w h e n using multilevel screens that the s a m e selection pressures are reflected at e a c h level. T h u s . especially if the first level is a b i o c h e m i c a l screen. Soller 1980). S i m p s o n and C a t e n 1979. D a v i e s 1964. as well as those .4 Flow diagram for mutational screening. pooling and recycling the i m p r o v e d strains at a single level of the screen can i m p r o v e the efficiency of selection ( C a l a m 1964. this is a natural w e e d i n g out of false-positives (Ditchburn et al. and the recycling occurs at an early level in the screen. as the best strain from o n e cycle is used as the mutation parent for the next. Ball and M c G o n a g l e 1978). not all strains s h o w i n g an i m p r o v e m e n t on o n e type of screen will s h o w it o n a n o t h e r . o t h e r w i s e the w o r k d o n e at o n e level will be u n d o n e at the next. this t o p 1 0 % of isolates will contain the genuinely i m p r o v e d m u t a n t s . 1974. A n y c o m b i n a t i o n of levels and cutoff points m a y be used. the d e g r e e of replication as the n u m b e r s fall through progression through the v a r i o u s levels. Of c o u r s e . T h e difference here is that a n u m b e r of strains are r e c y c l e d .58 Strain Improvement and Strain Stability Reisolation and Purification Mutagenesis Kill count Plating out Mutation count Reisolation and Purification Screening Reisolation and Purification Further testing F I G U R E 3 . All strain i m p r o v e m e n t p r o g r a m s use a form of r e c y c l i n g . typically the t o p 1 0 % . C a l a m (1964) used three levels with a cutoff of 1 0 % at each level.

and a u t o m a t i o n . h o w e v e r . warts and all. A l s o . rapid recycling is p r o b a b l y m o s t effective w h e n used with multilevel screens ( C a l a m 1964. a m o d u l a r a p p r o a c h to a u t o m a t i o n is safest. E v e n t u a l l y the productivity of the recycling population b e c o m e s significantly h i g h e r than that of the c o n t r o l . they will c o m e to d o m i n a t e the p o p u l a t i o n . although requiring a larger capital o u t l a y . In s u b s e q u e n t c y c l e s . w h i c h will slow d o w n the rate of increase in m e a n productivity of the p o p u l a t i o n ) . 3.8 Yield Improvement by Process and Medium Development 59 cultures that are there by r a n d o m variation or testing error. and so can m e d i u m and p r o c e s s d e v e l o p m e n t . labor costs are very h i g h . and the m a g n i t u d e of the testing error (a larger testing error requiring that a larger rakeoff be u s e d . G e n e r a l l y .3. and also b e c a u s e different lines will a c c u m u l a t e different sets of m u t a t i o n s . and i n d e e d . several p o o l i n g and recycling lines should be run in parallel to allow for t h i s . it is safer to a u t o m a t e an existing screen. T h i s m o d u l a r a p p r o a c h also increases the flexibility of the s c r e e n . o c c u r . A u t o m a t e d e q u i p m e n t and robots take u p a lot of dedicated space and so s o m e d e g r e e of miniaturization is desirable. the m a g n i t u d e of the productivity i m p r o v e m e n t s c a u s e d by the m u t a t i o n s . D a v i e s 1964). care m u s t be taken not to introduce artificial selection pressures for the sake of c o n v e n i e n c e b e c a u s e this will result in a high p e r c e n t a g e of false-positives b e i n g g e n e r a t e d . the faster the rate of strain i m p r o v e m e n t will b e . the latter cultures will b e progressively lost and the g e n u i n e l y i m p r o v e d m u t a n t s e n r i c h e d for. B y this is m e a n t leaving alone those parts of the screen that are difficult to a u t o m a t e or very sensitive in t e r m s of selection p r e s s u r e . and c o n c e n t r a t i n g instead on a u t o m a t i n g such aspects as plating and assay. S o m e t i m e s . T h e chief d a n g e r of using this p o o l i n g and recycling t e c h n i q u e is that if strains with u n d e s i r a b l e s e c o n d a r y characteristics. and w h e r e necessary discard such lines. and a s u b s e q u e n t i m p r o v e m e n t m u t a t i o n will o c c u r in the n e w p o p u l a t i o n . Strain i m p r o v e m e n t is essentially a n u m b e r s g a m e . the size of the rakeoffs. S o the recycling population m u s t be s a m p l e d at regular intervals to c h e c k for this. or false-positives. T h e a u t o m a t e d parts of the screen are then c o n n e c t e d together by m a n u a l o p e r a t i o n s . m a k i n g it c h e a p e r to r e s p o n d to c h a n g e s in screening r e q u i r e m e n t s . If p o s s i b l e . In miniaturizing a screen. that is k n o w n to be effective. s o m e d e g r e e of a u t o m a t i o n of the screening p r o c e s s has b e c o m e an attractive option ( N o l a n 1986). is a w a y of reducing recurrent labor c o s t s . W i t h the advent of m i c r o p r o c e s s o r control and r o b o t i c s . and the m o r e m u t a n t s screened p e r unit t i m e . T h e rate of i m p r o v e m e n t in the m e a n productivity of the recycling p o p u l a tion will d e p e n d on the mutation frequency. and strains can be isolated from it for further testing.8 YIELD IMPROVEMENT BY PROCESS AND MEDIUM DEVELOPMENT G e n e t i c c h a n g e s can lead to i m p r o v e d productivity and reduction in operating c o s t s . to allow for crossing the end p r o d u c t s of sister lines to m a x i m i z e productivity i m p r o v e m e n t . H o w e v e r . w h i c h is enriched for as before. w h e n both a p p r o a c h e s are . in m a n y d e v e l o p e d c o u n t r i e s . All these screening strategies can of c o u r s e be c o m b i n e d in various w a y s to suit the r e q u i r e m e n t s of a particular screening p r o g r a m . T h e s e will c o m e to d o m i n a t e the p o p u l a t i o n .

or production of interfering substances o c c u r . either for a shorter or a longer p e r i o d . addition of a n e w m e d i u m ingredient. or culture storage. and . P r o v i d e d that the principles described in this chapter are adhered t o .o p t i m i z e d . N e w strains can also require modifications in the harvesting and extraction stages. P e r h a p s the m o s t o b v i o u s of these is the m e d i u m . This w o r k is d o n e initially in shake flasks. T h e policy at P a n l a b s is to r e c h e c k the relative concentrations of each m e d i u m ingredient required for each significantly i m p r o v e d strain (so-called " m e d i u m r e b a l a n c i n g " ) . . and all the skill exercised by the fermentation e n g i n e e r to a c c o m m o d a t e the n e w strain.5 . Q u i t e apart from a potentially different physiological m a k e u p . it will be necessary to take the n e w strain b a c k to the laboratory to introduce secondary adaptive mutations to benefit from its imp r o v e d productivity potential. In the case of a strain with an alteration in m o r p h o l o g y . any i m p r o v e d strain will require an increased supply of precursors (such as sulfate and phenylacetic acid in the case of penicillin production. S o m e t i m e s n o c h a n g e s are necessary but it is preferable to b e safe than sorry. p i g m e n t a t i o n . m y c e l i u m . l o w . A n overall s c h e m e for strain i m p r o v e m e n t is given in F i g u r e 3 . T h e general principles and m e t h o d s for preservation of fungi h a v e b e e n adequately described in K i r s o p and Snell ( 1 9 8 4 ) . In s o m e i n s t a n c e s . or s a n d . the effect is synergistic. the n e w strain should not be too different in its r e q u i r e m e n t s from the existing production strain and should give a r e a s o n a b l e p e r f o r m a n c e in its early production trials. is required for o p t i m u m expression of the n e w s t r a i n ' s i m p r o v e d productivity. Agitation conditions ( p o w e r . 3. either at r o o m t e m p e r a t u r e or 4 ° C . C a l a m 1970) to express its increased productivity. . silica g e l . H o w e v e r .2 0 ° C .t e m p e r a t u r e storage at . impeller size.9 PRESERVATION T h e p r o b l e m of genetic instability is also serious in the area of p r e s e r v a t i o n .60 Strain Improvement and Strain Stability c o m b i n e d . each n e w strain is a n e w entity. with different r e q u i r e m e n t s for expressing its m a x i m u m potential. All cultures h a v e to be stored at s o m e t i m e . a r t h r o s p o r e s . and then the results can be used to appropriately modify the pilot plant and production scale p r o c e s s e s by c h a n g i n g the batched levels or feeds as appropriate. A s has been stated before. In addition to the m e d i u m . despite all the care taken by the fermentation geneticist to a c c o m m o d a t e the existing p r o c e s s . and g e o m e t r y ) m a y also h a v e to be modified to a c c o m m o d a t e the n e w m o r p h o l o g y . and storage on agar slants or plates u n d e r oil. storage on agar slants.1 9 6 ° C (liquid nitrogen) in cryoprotectants such as glycerol or sugars.7 0 ° C . Suitable m e t h o d s include lyophilization (freeze-drying). or r e p l a c e m e n t of an existing o n e with a n o t h e r . T h e stage of the life c y c l e used for storage can also be v a r i e d — c o n i d i o s p o r e s . a further increase in p e r f o r m a n c e will c o m e if the various flexible fermentation p a r a m e t e r s are re-optimized for the n e w strain. re-optimization of other process p a r a m e t e r s m a y give e c o n o m i c benefits. the i n o c u l u m d e v e l o p m e n t stages will h a v e to be carefully r e . S o m e t i m e s . usually at r o o m t e m p e r a t u r e . if c h a n g e s in m o r p h o l o g y .

or material is required for a m u t a t i o n p r o g r a m .p r o d u c i n g variants can usually be r e c o v e r e d from d e g e n e r a t e d populations by e x t e n s i v e reisolation p r o g r a m s . for l o n g . before it is used in p r o d u c t i o n . it is possible to d e v e l o p short-term preservation m e t h o d s .t e r m storage very low survival rates m a y b e acceptable as long as productivity is retained. and this is w h y production of s e c o n d a r y m e t a b o lites by c o n t i n u o u s fermentation is impractical. After any long-term storage p r o c e d u r e . and w h y s u b s e q u e n t p r o d u c t i o n fermentations c a n n o t usually b e inoculated with material from the end of p r e v i o u s ones. S u c h d e g e n e r a t i o n of productivity also o c c u r s t o w a r d the e n d of a fermentation for similar r e a s o n s . retention of productivity is j u s t as important as retention of viability. " H i g h .9 Preserved Culture Preservation 61 F I G U R E 3 . W i t h luck and skill it is . or transformation. and several subcultures are likely to be n e e d e d to p r o d u c e a u s a b l e b i o m a s s .g r o w i n g than their parents b e c a u s e they e x p e n d less e n e r g y in (to t h e m ) u n n e c e s s a r y o v e r p r o d u c t i o n of m e t a b o l i t e s . c r o s s i n g . the culture should a l w a y s be re-isolated and purified in the s a m e w a y as for the isolates from m u t a t i o n .5 Flow diagram for strain improvement. \ Reisolation and Purification ι Generation of Novel Genotypes * Reisolation and Purification ι ι Selection Reisolation and Purification * Medium Optimization Scale-Up In c o n s i d e r i n g w h i c h is the best storage m e t h o d for c o m m e r c i a l p r o d u c t i o n strains. M u t a n t s with low productivity m a y h a v e a p p e a r e d during storage. but it is clearly better not to let the population get into this state in the first p l a c e . R a t h e r than g o i n g back to long-term p r e s e r v e d cultures each t i m e a n e w fermentation is r e q u i r e d . Viability is likely to b e l o w .3. C o n s e q u e n t l y they will take o v e r the p o p u l a t i o n and it will a p p e a r to h a v e " d e g e n e r a t e d . and these will frequently b e s t r o n g e r . I n d e e d .

Ball. Marcel Dekker. American Society for Microbiology. (1976) Mutation Research: Problems. either from within o n e ' s o w n c o m p a n y or at industrially orientated scientific m e e t i n g s . it is not possible to p u r s u e a degree c o u r s e in strain i m p r o v e m e n t . In Cephalosporium. it can often be useful to be able to carry out a large n u m b e r of clone fermentations o v e r an e x t e n d e d period of t i m e . 159-188. Birkett.. p h y s i o l o g y ..10 CONCLUSIONS T h e industrial fermentation geneticist is beset by m a n y u n i q u e p r o b l e m s in striving for the goal of i m p r o v i n g production strains. Ν. C .. Boca Raton.). A s yet. London. eds. Bacteriol. (1986) in Manual of Industrial Microbiology and Biotechnology (Demain. F.). In lieu of a c a d e m i c training. C . 45. several w e e k s or p e r h a p s m o n t h s ) . C. (1984) in Genetics and Breeding of Industrial Microorganisms (Ball.and p r o c e s s . T h e general principles of g e n e t i c s . C. Baltz. w h i c h is a prerequisite for continuing success in strain i m p r o v e m e n t . C o n i d i o s p o r e s are generally the hardiest stage of the life cycle for both longand short-term p r e s e r v a t i o n .and short-term preservation m e t h o d s for industrial use will b e strain. P. P. New York. pp. but the interpretation of these principles in the industrial context is a continuing c h a l l e n g e . Washington. T h e best long. arthrospores are a useful alternative. (1978) J. and McGonagle.. b i o c h e m i s t r y . FL. and from literature articles to build up his or her o w n e x p e r i e n c e b a s e . F.d e p e n d e n t . Α.2 0 ° C or . J. DC.). J.. eds. 184-190. 3. pp. (1985) in Fungal Protoplasts: Applications in Biochemistry and Genetics (Peberdy. ed. Chapman and Hall. and Hamlyn. M. O n c e again. pp. bearing in m i n d the considerations discussed previously. the industrial geneticist must d r a w on the e x p e r i e n c e of senior c o l l e a g u e s . Results and Perspectives. . and the practical techniques can only be learned in the industrial e n v i r o n m e n t . S u c h techniques include storage on agar or in glycerol/lactose solutions at low t e m p e r a t u r e s . and Solomon. A. but it is also feasible to store the m y c e l i u m of m a n y strains of fungi in suitable glycerol/lactose solutions at . and Ferenczy. 67-74. A l t h o u g h this introduces o n e m o r e subculture step into the seed d e v e l o p m e n t p r o c e s s . Ball.7 0 ° C . but are not always available. A. L. the best advice that can be given is for the fermentation geneticist to evaluate a variety of these m e t h o d s for suitability. L. REFERENCES Auerbach. and engineering are the s a m e as those taught and researched in a c a d e m i c institutions. Appl. H. T h i s can s o m e t i m e s be used to a d v a n t a g e in preparing aliquots of vegetative m y c e l i u m ready for inoculation into seed stages. 207-223. m i c r o b i o l o g y . R. CRC Press.62 Strain Improvement and Strain Stability usually possible to find a storage technique in which both viability and productivity can b e retained at near 1 0 0 % o v e r the short term (that is.

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and p r o d u c t i v e fungal fermentation p r o c e s s e s . c h e m i c a l s . dissolved o x y g e n . T h u s . scalable. B u c k l a n d 1984). and e n z y m e s . the fungi usually present special c h a l l e n g e s d u r i n g scaleup b e c a u s e of their various morphological forms. A discussion of the m o r p h o l o g i c a l forms that m a y exist u n d e r different g r o w t h conditions is also p r e s e n t e d . the first part of this chapter discusses the g r o w t h kinetics of filamentous fungi on solid m e d i u m and in s u b m e r g e d culture. 1979. B a n k s 1979. and m i x i n g time ( W a n g et al. Greasham F i l a m e n t o u s fungi are well k n o w n as p r o d u c e r s of c o m m e r c i a l l y important p r o d u c t s such as antibiotics. H o w e v e r . p h y s i c a l .CHAPTER 4 Growth Kinetics and Fermentation Scaleup Randolph L. T h e r e are several a p p r o a c h e s to scaling u p fermentation processes including the use of constant v o l u m e t r i c o x y g e n transfer coefficient. T h e r e f o r e . n o single scaleup p r o c e d u r e has been found to be universally effective b e c a u s e m a n y fermentation p r o c e s s e s h a v e their o w n u n i q u e p a r a m e t e r s that m u s t b e a d d r e s s e d d u r i n g s c a l e u p . U n l i k e m a n y unicellular m i c r o b e s . M a n y of these products are p r o d u c e d at large scale as a result of the c o n c e r t e d scaleup efforts of both fermentation microbiologists and b i o c h e m i c a l e n g i n e e r s . A n u n d e r s t a n d i n g of the g r o w t h and m o r p h o l o g i c a l c o m p l e x i t y of the filamentous fungi is i m p o r t a n t for d e v e l o p i n g consistent. i m p e l l e r tip s p e e d . o x y g e n u p t a k e rate. and biological process p a r a m e t e r s that can affect s c a l e u p and p e r f o r m a n c e of filamentous fungal fermentations. 65 . the second part of this c h a p t e r will focus o n the c h e m i c a l .

Microbiol. After a particular incubation period.R. C. A s the h y p h a c o n t i n u e s to g r o w . e x p o n e n t i a l . 57. Reading. U n d e r appropriate c o n d i t i o n s . 4. p r o d u c i n g hyphal filam e n t s referred to as g e r m t u b e s . .G. T h e type of sporulation as well as the m o r p h o l o g y of the spores and s p o r e b e a r i n g structures are key characteristics in fungus identification ( H a w k s w o r t h 1977). A.1. the spores g e r m i n a t e . T h e lag phase is characterized as the p r e g e r m i n a t i o n period during w h i c h spores dispersed on solid m e d i a begin swelling at a linear rate (Trinci 197 l b ) . 11-24. Gen. Reprinted by permission of the publisher from Trinci.66 4. (1969) J. T h e basic vegetative structure of g r o w t h consists of a tubular filament k n o w n as a h y p h a that originates from the g e r m i n a t i o n of a single reproductive spore. Society for General Microbiology.1 (Trinci 1969). Exponential g r o w t h m a y b e defined by the following expression: 0 5 10 15 20 Cultivation Time (hours) F I G U R E 4-1 Colony growth of Aspergillus nidulans.1 Growth Kinetics and Fermentation Scaleup FUNGAL GROWTH F i l a m e n t o u s fungi are m o r p h o l o g i c a l l y c o m p l e x m i c r o o r g a n i s m s . the vegetative m y c e l i u m gives rise to a r e p r o d u c t i v e m y c e l i u m that supports the production of r e p r o d u c t i v e s p o r e s . exhibiting different structural forms t h r o u g h o u t their life c y c l e s . as illustrated in F i g u r e 4 . G r o w t h of the g e r m tubes in length is e x p o n e n t i a l and at a rate that can be several-fold greater than that of the s a m e fungal strain g r o w n in s u b m e r g e d culture (Trinci 1971b). constant growth rate. P.. and constant g r o w t h rate. J.1 Surface Culture G r o w t h of filamentous fungi on solid m e d i u m m a y be dividied into four p h a s e s : l a g . deceleration. England. it frequently b r a n c h e s repeatedly to form a m a s s of hyphal filaments referred to as a m y c e l i u m .

respectively. they begin to b r a n c h r e p e a t e d l y . the rate of increase for h y p h a l tips and h y p h a l _1 1 length w a s 0 . T h e radius of a " m a t u r e " c o l o n y increases linearly with time (Pirt a c c o r d i n g to the equation r. 8 0 9 h for Mucor racemosus (nonseptate hypha).4. 2 7 h " . A s the g e r m tubes c o n t i n u e to g r o w in length. T h e p r o t o p l a s m p r o d u c e d in this region supports apical extension at the hyphal tips. forming m y c e l i a with n u m e r o u s h y p h a l tips. . T h e m y c e l i u m then e x p e r i e n c e s a period of r e d u c e d g r o w t h . and μ is the specific g r o w t h rate ( h ) . F o r P. chrysogenum. the h y p h a e in this zone consist of an apical cell and several adjacent cells with o p e n p o r e septa (Trinci 1971a). t is time ( h ) . Integration of this e x p r e s s i o n gives \n{x2lxx) = μ{ί2 - tx) w h e r e xx and x2 are h y p h a l m a s s e s at tx and t2. T h e length of the peripheral g r o w t h z o n e in septate fungi is c o n s i d e r a b l y shorter than that found in n o n s e p t a t e fungi.1 dxldt Fungal Growth 67 — μχ _ 1 w h e r e χ is h y p h a l m a s s . T h e h y p h a l d o u b l i n g t i m e 0 d ) derived from this equation m a y be expressed as td = In 21 μ. m a r k i n g the formation of a " m a t u r e " c o l o n y c o m p o s e d of differentiated m y c e l i a . Trinci ( 1 9 7 1 b ) found that the specific g r o w t h rate of g e r m tubes can r a n g e from -1 -1 0 . Trinci ( 1 9 7 1 a ) reported the length of this z o n e for P. G r o w t h occurs primarily at the c o l o n y e d g e in the region k n o w n as the peripheral g r o w t h z o n e . E x p o n e n t i a l g r o w t h usually c o n t i n u e s until the mycelial density per unit surface area e x c e e d s the m i n i m u m supply of nutrients. chrysogenum to be 4 9 6 / i m and that of M ucor racemosus Peripheral Growth Zone • ι—ι—ι—Γ Q Septum Li_J Central Hyphal Port Compartment 3 L Apical Cell F I G U R E 4-2 Growth region of a hyphal filament. 2 8 h and 0 . W i t h septate fungi (such as Pénicillium and Aspergillus). Trinci (1974) reported that the n u m b e r of h y p h a l tips and the total h y p h a l length of a m y c e l i u m increase exponentially at similar r a t e s . d i a g r a m m a t i c a l l y illustrated in F i g u r e 4—2. respectively. and Kr is the radial g r o w t h rate constant (/xm/h). respectively. = r0 + Kr{tx - 1967) t0) w h e r e rx and r0 are the c o l o n y radii at times tx and t0. 2 8 2 h for Aspergillus niger (septate h y p h a ) to 0 .

3 w h e r e the g r o w t h of Geotrichum lactis is plotted against time of cultivation during a 28-h fermentation cycle (Trinci 1971a).68 Growth Kinetics and Fermentation Scaleup (nonseptate) to be 3 . (1971) J.1 B a t c h C u l t u r e .2. T h e first three p h a s e s are quite similar to those discussed previously for fungi g r o w i n g on solid m e d i a . G r o w t h kinetics of filamentous fungi in s u b m e r g e d culture are quite similar to those of unicellular m i c r o o r g a n i s m s that r e p r o d u c e by binary fission.ιη. e x p o n e n t i a l .1. Reprinted by permission of the publisher from Trinci. 4. T h e lag p h a s e represents a period d u r i n g w h i c h the fungal cells or spores adapt to a n e w e n v i r o n m e n t . r e d u c e d g r o w t h . Gen. 67.1. Reading. 4 0 6 μ. P. and stationary. T h e s e p h a s e s are illustrated in Figure 4 . T h e s e results suggest that h y p h a l g r o w t h in the peripheral g r o w t h z o n e is e x p o n e n t i a l . Microbiol. MA. H e also s h o w e d that the theoretical specific g r o w t h rate of the h y p h a e in this z o n e ( μ ^ ) w a s a function of both the radial g r o w t h rate and the length of the peripheral g r o w t h z o n e (w) and expressed as /Ah = Krlw U s i n g this e x p r e s s i o n . A d a p t a t i o n includes formation of e n z y m e s and intermediates to s u p - F I G U R E 4-3 Growth kinetics of Geotrichum lactis in batch culture at 25°C. At least four p h a s e s of g r o w t h m a y be o b s e r v e d in batch culture: l a g . . Society for General Microbiology. 325-344. J.2 Submerged Culture 4. A. Trinci ( 1 9 7 1 a ) calculated the specific g r o w t h rate of several different filamentous fungi g r o w i n g on solid m e d i a and found the rates to be quite similar to those o b s e r v e d in s u b m e r g e d culture.

spore inocula require a g e r m i n a t i o n period ( S m i t h a n d C a l a m 1980) and pelleted inocula m a y require a certain d e g r e e of m e c h a n i c a l disruption. 67. Society for General Microbiology.4.0 mg/1. 5 /Xmax. nonseptate hypha. and Ks is the saturation constant equal to the substrate concentration at μ = 0 .1 .1 0 % i n o c u l u m of actively g r o w i n g . 325-344. m i n i m i z a t i o n of the lag p h a s e m a y be a c h i e v e d by u s i n g a 5 . for g r o w t h of Aspergillus on g l u c o s e to b e 5. Usually the cultivation m e d i u m during this p h a s e of g r o w t h contains an e x c e s s of required nutrients a n d is d e v o i d of inhibitors.119 0.1 Fungal Growth 69 port r e s u m p t i o n of g r o w t h . Specific g r o w t h rates of several filamentous fungi in s u b m e r g e d culture are presented in T a b l e 4 . Ks. s is the substrate c o n c e n t r a t i o n .032) a. h o m o g e n i z a t i o n of pelleted g r o w t h prior to inoculation has an unfavorable risk of c o n t a m i n a t i o n associated with it ( S a v a g e and van d e r B r o o k 1946). Gen. T h e r e f o r e . T h e rate of h y p h a l g r o w t h d e p e n d s not only on the strain of f u n g u s .017) (±0. A. P. Reading.181 (±0.1 Mean Specific Growth Rates of Filamentous Fungi in Batch Culture at 25°C Mean Specific Growth l Rate (hr ) Fungi a Pénicillium chrysogenum ä Aspergillus niger 2 Geotrichum lactis b Rhizopus stolonifer b Mucor racemosus b Actinomucor repens 0. Adapted by permission of the publisher from Trinci. T h u s . b.019) (±0.018) (±0. (1971) J. Microbiol. or the a c c u m u l a t i o n of e n d p r o d u c t s of m e t a b o l i s m that are inhibitory. T h e description of exponential g r o w t h disc u s s e d p r e v i o u s l y for surface g r o w t h also holds true for s u b m e r g e d g r o w t h . H o w e v e r . .032) (±0.135 0. P r o v i d e d that the latter t w o c o n ditions are not r e d u c i n g g r o w t h . the effect of limiting substrate concentration o n the specific g r o w t h rate (μ) m a y b e e x p r e s s e d by the M o n o d m o d e l for microbial growth: w h e r e / x m ax is the m a x i m u m specific g r o w t h rate. the d e v e l o p m e n t of an a d v e r s e p H v a l u e . J.026) (±0. T h e length of this p h a s e is d e p e n d e n t not only on the p h y s i o l o g i c a l state of the m i c r o o r g a n i s m . but also on the m o r p h o l o g y and level of the i n o c u l u m . w h e n the g l u c o s e concentration d r o p s b e l o w 10. A reduction in the specific g r o w t h rate occurs w h e n the m i c r o f u n g u s b e g i n s to e x p e r i e n c e an unfavorable g r o w t h e n v i r o n m e n t such as the limitation of a required nutrient. septate hypha. T h e e x p o n e n t i a l p h a s e of fungal g r o w t h is characterized by a significant increase in cell m a s s .0 TABLE 4 .353 0.Pirt (1975) reported the saturation constant.102 0. A s stated p r e v i o u s l y . England.164 0. but also on the p h y s i c o c h e m i c a l e n v i r o n m e n t a l c o n d i t i o n s . dispersed m y c e l i a .

d e t e r m i n a tion of Ks is difficult in batch culture b e c a u s e o n c e this low level of substrate is r e a c h e d . a slight increase in h y p h a l m a s s m a y be o b s e r v e d during e n d o g e n o u s m e t a b o l i s m of these storage m a t e r i a l s . x. H o w e v e r . Like that of unicellular m i c r o o r g a n i s m s . the dilution rate equals fungus. By plotting the v a l u e s of s. a reduction in the specific g r o w t h rate is o b s e r v e d . A l s o . 4 . w h i c h uses nutrient feeding to control the b i o m a s s at a p r e d e t e r m i n e d level. A s e x p e c t e d . H o w e v e r . cultivation of filamentous fungi in a c o n t i n u o u s flow bioreactor is a c h i e v a b l e p r o v i d e d that the right apparatus is used. T h e feeding solution is usually a " b a l a n c e d " m e d i u m with only o n e nutrient. T h e turbidostat. if the hyphal m a s s a c c u m u l a t e s intracellular storage material during the reduced g r o w t h p h a s e . T h e basic operation of the c h e m o s t a t consists of feeding a nutrient solution at a flow rate that equals the rate of w h o l e broth (including cell mass) r e m o v a l . c o n t i n u o u s cultivation of fungi is achieved in a c h e m o s t a t (Pirt and R i g h e l a t o 1967. if the h y p h a e begin to lyse and release a limited nutrient. 1 .70 Growth Kinetics and Fermentation Scaleup mg/1.s) rL-K. B y controlling the c o n c e n t r a tion of the limiting substrate and the addition rate. 2 .L-2-j w h e r e Y is the cellular yield coefficient (cell m a s s formed/weight of substrate used) and sR is the substrate concentration entering the bioreactor. T h e stationary p h a s e m a y be defined simplistically as the b a l a n c e b e t w e e n h y p h a l m a s s increase and d e c r e a s e . Carter and Bull 1969) w h e r e a p r e d e t e r m i n e d nutrient feeding strategy dictates the b i o m a s s level. thus maintaining the culture v o l u m e in the c o m p l e t e l y m i x e d bioreactor at a constant level. has not been successful for microfungi b e c a u s e on-line m e a s u r e m e n t of b i o m a s s is difficult. T h e steady-state (ds/dt = 0) value for the (s) is related to the dilution rate by the expression \Mmax - DJ T h e value for cell m a s s (x) at steady state is defined by the e x p r e s s i o n s χ - = Y(sR . the growth-limiting substrate. at a steady state (dx/dt = 0 ) . at a slightly r e d u c e d level. 2 C o n t i n u o u s C u l t u r e . it is rapidly m e t a b o l i z e d by the large cell m a s s that has a c c u m u l a t e d . and b i o m a s s productivity ( g r a m s per liter per hour) as a function of dilution . it is possible to maintain the fungus in exponential g r o w t h for an e x t e n d e d period and theoretically at a specific g r o w t h rate b e t w e e n 0 and μ ™ χ · T h e dilution rate (D) is defined by the expression D w h e r e / is the nutrient addition W h e n the c h e m o s t a t is operated the specific g r o w t h rate of the limiting substrate concentration =f/v rate and ν is the culture v o l u m e of the bioreactor. n e w g r o w t h could be e x p e c t e d .

1 Fungal Growth 71 Biomass Concentration < CO 1 Dilution Rate. r a n g i n g from dispersed mycelial filaments (including fragm e n t s ) to intricately i n t e r w o v e n . Fil- . S o d e c k et al. shear.1.4. r a t e . d r o p p i n g rapidly to 0 at the critical dilution rate (Dc). W h e n g r o w n in s u b m e r g e d culture. 1954.) culturing conditions ( A t k i n s o n and D a o u d 1976. t e m p e r a t u r e .4 ) . A l s o . Dc is a p p r o x i m a t e l y equal to / i m ax - Fungal Morphology A s d i s c u s s e d p r e v i o u s l y . T h e s e m o r p h o l o g i c a l g r o w t h forms can h a v e a significant effect o n the r h e o l o gy of the fermentation broth and thus the p e r f o r m a n c e of the bioreactor. A s the dilution rate i n c r e a s e s . these fungi exhibit different m o r p h o l o g i c a l f o r m s . the fungal b i o m a s s d e c r e a s e s . At this dilution rate. filamentous fungi g r o w by apical extension of the h y p h a e and b r a n c h i n g . T h e critical dilution rate as a function of sR is given by the expression Because s 4. W h i t a k e r a n d L o n g 1973). but also b y the nature of the i n o c u l u m as well as the c h e m i c a l ( m e d i u m constituents) and physical ( p H . D ( h r ) Dc F I G U R E 4-4 Steady-state relationships in a chemostar (theoretical). F i l a m e n t o u s g r o w t h of Aspergillus niger is preferred for pectic e n z y m e production ( C a l a m 1976). K o n i g and Schugerl (1982) s h o w e d that the pelleted form of P. w h e r e a s the pelleted form is preferred for citric acid production (Steel et al. 1981). T h e particular form exhibited is d e t e r m i n e d not only b y the fungal s p e c i e s . W i t h s o m e fungal fermentations a particular m o r p h o l o g ical form m a y be preferred to a c h i e v e m a x i m a l p e r f o r m a n c e . chrysogenum w a s desired for p r o d u c t i o n of penicillin in a t o w e r bioreactor. the operation of the c h e m o s t a t m a y be graphically presented (Figure 4 . b i o m a s s w a s h o u t o c c u r s and the c o n c e n t r a t i o n of the limiting substrate w h i c h has r e m a i n e d quite low o v e r m o s t of the r a n g e of dilution rates b e c o m e s equal to sR. mycelial m a s s e s referred to as pellets. etc.3 R » Ks m o s t of the t i m e .

7 . Biochem. and polymer additives (Whitaker and Long 1973. mechanical agitation. Proc.72 Growth Kinetics and Fermentation Scaieup TABLE 4 . England. g r o w t h is e x p o n e n t i a l . medium composition.000 5. only the remaining three factors m a y be used to control pellet formation. (1976) Starting investigational and production cultures. A s e x p e c t e d .000 Reprinted by permission of the publisher from Calam. T h u s . p s e u d o p l a s tic (shear-thinning) flow b e h a v i o r ( S o d e c k et al. p H . Pelleted g r o w t h a n d short mycelial fragments exhibit l o w viscosities and a p p r o a c h N e w t o n i a n (independent of shear rate) flow b e h a v i o r (Chain et al. especially the g a s liquid m a s s transfer rate. 1 1 . 1981). Metz and Kossen 1977). Because the fungal strain. h i g h e r p o w e r inputs are required for filamentous versus pelleted g r o w t h in achieving a d e q u a t e agitation a n d o x y g e n transfer.2 ) . size of spore inoculum. and p H are usually fixed for a particular process.800 4. Several contributory factors that have been associated with pellet formation include fungal strain. The addition of cationic polymers (Reten-205 and -210) to the growth medium of A. Initially. only t h e h y p h a e in the peripheral g r o w t h z o n e c o n t i n u e to exhibit active g r o w t h (Pirt 1966).N e w t o n i a n .2 Effect of Spore Concentration on Morphology and Penicillin Biosynthesis by Pénicillium chrysogenum Inoculum (spores/ml) 2 10 3 10 3 2 x 10 4 10 Morphology Penicillin Yield (U/ml) Dense pellets Dense pellets Open pellets Filamentous 500 1. a m e n t o u s g r o w t h results in highly viscous broths with n o n . For example. substantial savings a n d e n h a n c e d p e r f o r m a n c e m a y b e realized at production scale by lowering the viscosity of the fermentation broth. niger changed the morphology of this microfungus from dispersed filaments to pellets (Elmayergi 1975). T. w h e n t h e pellet is quite small and n o nutrient limitations exist. Pellet g r o w t h u n d e r these conditions m a y b e e x p r e s s e d b y c u b e root kinetics: . Process Biochemistry. medium composition. Rickmansworth. G r o w t h of pellets in s u b m e r g e d culture is s o m e w h a t similar to that of c o l o n y g r o w t h o n solid surface. O n e approach that has received considerable interest is the formation of fungal pellets. A s the pellet increases in size and b e c o m e s diffusion limited for nutrients. C. chrysogenum (Table 4 . Although high viscosities caused by substrates or products in the broth m a y be reduced by dilution (addition of water. 1966). T h e high viscosity h a s a negative impact on the m a s s transfer properties of the b r o t h . Taguchi 1971). the level of spores in the inoculum was shown by Calam (1976) to control pellet formation by P. reduction of high viscosities caused by the morphology of the fungi is more difficult.

T h u s .3 (Ryu and H u m p h r e y 1973). chrysogenum w a s d e v e l o p e d b y G b e w o n y o and W a n g ( 1 9 8 3 ) . T h i s t e c h n i q u e consists of g r o w i n g filamentous h y p h a e on p o r o u s celite b e a d s and p r o d u c i n g spherical g r o w t h with r e d u c e d broth viscosity. A list of these variables is presented in T a b l e 4 . A novel t e c h n i q u e for e x t e n d i n g the period of low viscosity during cultivation of P. Identification of the important variables for a particular p r o c e s s is usually achieved through scaleup studies. Unlike pelleted g r o w t h . This s c a l e . A l t h o u g h the nature of fragmentation is not well u n d e r s t o o d . m e c h a n i c a l agitation ( T a g u c h i 1971) and m e d i u m constituents ( D r e w et al. 1976) a p p e a r to be contributing factors. short h y p h a l fragments should not b e c o m e g r o w t h limited d u e to nutrient diffusion. U n d e r these c o n d i t i o n s .2 SCALEUP OF FERMENTATION Successful transfer of a laboratory fermentation process to p r o d u c t i o n scale requires that p r o c e s s scaleup data be generated by well-designed e x p e r i m e n t s p e r f o r m e d in laboratory and pilot-plant scale b i o r e a c t o r s .2 Scaleup of Fermentation 73 where = = t — μ = w = d = X pellet m a s s after t hours pellet m a s s at t = 0 time specific g r o w t h rate depth of the peripheral g r o w t h z o n e pellet density F r e q u e n t l y . H y p h a l fragmentation is c o m m o n l y o b s e r v e d in s u b m e r g e d culture at the e n d of active g r o w t h (Smith et al. small pellets as o p p o s e d to large o n e s w o u l d generally be considered desirable in d e v e l o p ing filamentous fungal fermentations. the fermentation p r o c e s s d e v e l o p e d initially at o n e scale m a y e x p e r i e n c e difficulties during i m p l e m e n t a t i o n at the next scale. 1983).4. A n o t h e r a p p r o a c h at achieving r e d u c e d viscosities is to use short h y p h a l f r a g m e n t s . rapid scaleup is frequently n e e d e d to m e e t material r e q u i r e m e n t s for product e v a l u a t i o n . T h i s autolysis can h a v e a significant effect on both cellular m e t a b o l i s m and p r o d u c t synthesis (Schugerl et al. R e v i s i o n of the p r o c e s s is then required and frequently achieved by scaling d o w n the process (Jem 1989). a p p r o x i m a t e l y twofold higher than that r e a c h e d in free-cell culturing. B e c a u s e of the highly c o m p e t i t i v e nature of the fermentation industry. the h y p h a e in the center of these larger pellets u n d e r g o autolysis as a result of nutrient limitation. A l t h o u g h both solid-substrate and s u b m e r g e d fermentation p r o c e s s e s can be .d o w n t e c h n i q u e m a y also be useful if the e c o n o m i c s of the p r o c e s s w e r e to require i m p l e m e n t a t i o n of the fermentation in an existing facility. S o m e of these variables are m o r e important than o t h e r s . M a n y of these e x p e r i m e n t s are a i m e d at d e t e r m i n i n g the effects fermentation scale has on p r o c e s s variables. 4. A b i o m a s s of 6 0 g/1 w a s r e a c h e d with this confined cell t e c h n i q u e . 1967).

Society of Chemical Industry. ( 1 9 8 3 ) . T h e s e fermentors are c o m m o n l y constructed of glass or stainless steel and . It is in these types of vessels that o n e usually e x p e r i e n c e s difficulties with m a s s transfer and m i x i n g w h e n d e v e l o p i n g a e r o b i c . Biotechnol. Chem.74 Growth Kinetics and Fermentation Scaleup TABLE 4-3 Processes Process Variables in Fermentation Temperature Pressure Agitation speed Power input Air flow rate Feed rate of: nutrients precursors inducers Weight of broth Volume of liquid Viscosity (apparent) Cumulative amount of: acid base antifoam PH Oxidation reduction Dissolved oxygen Dissolved C 0 2 Effluent gas oxygen Effluent C 0 2 Concentrations of: carbohydrate nitrogen mineral ions precursor inducer product metabolites Flow characteristics Power characteristics Energy balance Respiratory quotient Cell concentration Cellular components: protein (enzymes) DNA RNA Specific activity of enzyme Specific rates of: product formation growth oxygen uptake nutrient uptake Oxygen transfer rate ATP NADH Reprinted by permission of the publisher from Ryu. D. fungal fermentation p r o c e s s e s . the following discussion on scaleup will focus only on the cultivation of fungi in well-aerated and agitated bioreactors.Y o u n g et al.. and Humphrey.1 Laboratory-Scale Fermentations 4. A n excellent discussion of solid-substrate fermentations m a y be found in the review by M o o . T h e first step in scaling u p a fermentation p r o c e s s usually involves transferring a shake-flask fermentation process to highly i n s t r u m e n t e d laboratory-scale fermentors with a tank d i a m e t e r (Dij-to-height (H) ratio of 1:1 to 1:2. 4. (1973) J. s u b m e r g e d fermentation is the m o r e c o m m o n l y used process in the industry t o d a y . A. London.1 B i o r e a c t o r s . T h e r e f o r e .2.2. used for the production of c o m m e r c i a l l y important products by filamentous fungi.1. 2 8 3 295. Appl. 23. similar in design to that s h o w n in F i g u r e 4—5.

C o m p l e t e m i x i n g of the culture fluid is a c h i e v e d by m e c h a n i c a l agitation [one to three impellers with an impeller (D/j-to-tank d i a m e t e r (Dt) ratio of 0 . o x y g e n uptake rate.) are strategically placed in the fermentor so that the discharge jet from the impeller can h e l p r e d u c e mycelial g r o w t h on their surfaces. 5 m o u n t e d on a central stirring shaft] and the use of at least four baffles that are constructed for easy r e m o v a l during vessel cleaning. 1985).4. and o x y g e n transfer coefficient ( B u c k l a n d et al. liquid height. O v e r the last several y e a r s . Di. tank height.2 Scaleup of Fermentation 75 Motor I Shatt Baffle QJacket D- -• •— —• 1 t t Dt F I G U R E 4-5 Diagram of a stirred-tank bioreactor. the c o m p u t e r has b e c o m e a key c o m p o n e n t of a u t o m a t i c fermentor s a m p l i n g and analysis ( O m s t e a d and G r e a s h a m 1989.) and s a m p l i n g . M o r e recently. nutrients. H. T h e laboratory vessels are e q u i p p e d with an air sparger designed as an o p e n t u b e . antifoam. T h e s e vessels are also e q u i p p e d with several inlet/outlet ports for additions ( i n o c u l u m . etc. A l t h o u g h a ring sparger supports higher o x y g e n transfer rates (Clark and L e n t z 1963). carbon dioxide evolution rate. tank diameter. T h e inserted p r o b e s used to m o n i t o r s o m e of the fermentation p a r a m e t e r s ( p H . R e d a et al. etc. Typical on-line calculations p e r f o r m e d b y the c o m p u t e r include carbon dioxide and o x y g e n c o n c e n t r a t i o n s . it frequently b e c o m e s fouled with m y c e l i u m w h e n used to cultivate filamentous fungi. impeller diameter. and controlling fermentation p r o c e s s e s . i m p r o v i n g . T h e glass is usually treated with silicone ( S o l o m o n s 1980) and the stainless steel highly polished to m i n i m i z e fungal g r o w t h on the surfaces of these vessels. dissolved o x y g e n . h. the use of c o m p u t e r s y s t e m s to both m o n i t o r and control various fermentation p a r a m e t e r s has p r o v e n quite valuable in d e v e l o p i n g . respiratory q u o t i e n t . . r a n g e in v o l u m e from 1 to 30 1 (Kristiansen and Sinclair 1980). 3 .0 . Dt. 1991).

1976). P a n et al.f o l d ) . and cost (Jem 1989). E x c e s s glucose m a y r e d u c e penicillin biosynthesis by supporting u n d e s i r a b l e . H e r e again. Swartz (1979) s h o w e d that g l u c o s e w a s the m o s t e x p e n s i v e c o m p o n e n t of the penicillin fermentation p r o c e s s . Nutrient feeding. p r o d u c t i v e . d e m o n strating the i m p o r t a n c e of feed rate optimization studies. Squires (1972) used c h a n g e s in dissolved o x y g e n as the b a s e s for g l u c o s e additions. special treatment. O t h e r g l u c o s e feed rates of 0 . O n c e the shake-flask p r o c e s s is transferred to the laboratory-scale fermentors. 5 and 1. e c o n o m i c a l . high specific g r o w t h rates or inhibiting the activity or synthesis of e n z y m e s involved in penicillin biosynthesis ( D e m a i n 1968). and p r e c u r s o r s . Cultivation Media. T h e benefit of nutrient feeding in the penicillin fermentation p r o c e s s is well k n o w n . A n integral part of m e d i u m optimization in stirred vessels is the investigation of nutrient feeding as a t e c h n i q u e for further m a x i m i z i n g the productivity of the fermentation p r o c e s s . 2 Effect of P r o c e s s V a r i a b l e s . A typical fed-batch fermentation for penicillin production is presented in F i g u r e 4—6 ( Q u e e n e r and Swartz 1979). nitrogen. foaming characteristics. the rate of g l u c o s e addition d u r i n g the " p r o d u c t i o n p h a s e " is important for controlling the rate of fungal g r o w t h and c a r b o h y d r a t e m e t a b o l i s m .1 g/l-h yielded smaller i m p r o v e m e n t s (approximately 2 . M e d i u m cost d e s e r v e s special attention during d e v e l o p m e n t b e c a u s e it frequently contributes significantly to the total cost of the fermentation p r o c e s s . further optimization is usually required in stirred vessels to a c h i e v e m a x i m u m p e r f o r m a n c e . a detailed analysis of the fermentation p a r a m e t e r s begins in support of d e v e l o p i n g a consistent. and scalable p r o c e s s . 1 . T h i s a p p r o a c h is especially attractive at this stage b e c a u s e several (10 to 20) laboratory-scale fermentors are usually available. the m o s t c o m m o n c o m p o n e n t is a g r o w t h limiting e n e r g y s o u r c e . 4 . T h e nutrient(s) is a d d e d directly into the fermentation broth to achieve m a x i m u m m i x i n g efficiency. A l t h o u g h cultivation m e d i a for both i n o c u l u m d e v e l o p m e n t and production m a y h a v e been o p t i m i z e d in shake-flask studies prior to transfer. Properties of the g r o w t h m e d i a that are c o n s i d e r e d important during scaleup include viscosity. nutrients m a y also be a d d e d in r e s p o n s e to c h a n g e s in fermentation process variables such as p H and dissolved o x y g e n . . 2 . A l t h o u g h feeding solutions m a y include such c o m p o n e n t s as p h o s p h o r u s . In addition to feeding nutrients at pre-established rates. c o m m o n l y referred to as fed-batch culturing. solubility. usually consists of the c o n t i n u o u s or intermittent addition of o n e or m o r e m e d i u m c o m p o n e n t s during the fermentation c y c l e . t e m p e r a t u r e sensitivity. optimization of critical p a r a m e t e r s m a y be e n h a n c e d by e m p l o y i n g multifactorial design e x p e r i m e n t a t i o n ( G r e a s h a m and I n a m i n e 1986).76 Growth Kinetics and Fermentation Scaleup 4 . K u e n z i and A u d e n (1983) reported that a 3. B e c a u s e of the c o m p l e x interaction a m o n g fermentation p a r a m e t e r s .2-fold increase in c e p h a l o s p o r i n synthesis b y Cephalosporium acremonium w a s achieved by feeding glucose at a constant rate of 0 . (1972) added glucose to the penicillin fermentation p r o c e s s based on p H c h a n g e s . 7 3 g/l-h after 24 h into the fermentation c y c l e . A n o t h e r important nutrient c o m m o n l y fed during the " p r o d u c t i o n p h a s e " to c o n t i n u e penicillin biosynthesis is nitrogen ( L u r ' e et al.

Academic Press. A s discussed p r e v i o u s l y .2 Scaleup of Fermentation 77 Cultivation Time (hours) F I G U R E 4-6 Typical fed-batch fermentation for penicillin production. T i g h t control of nutrient feeding is beneficial not only for achieving higher p r o d u c t levels. and Swartz. Reprinted by permission of the publisher from Queener. s p o r e s . A l s o . vegetative m y c e l i a .. chrysogenum ( M o u and C o o n e y 1983). R. Microbiol. but also to m i n i m i z e substrate levels at harvest. or pellets m a y b e used as inocula for filamentous fungal fermentations. the o p t i m a l size of the i n o c u l u m m u s t be d e t e r m i n e d . W h i c h e v e r m o r p h o l o g i c a l form is d e s i r a b l e . T h e e n v i s i o n e d " s e e d train" at production scale ( n u m b e r of culture transfers) is initially d e v e l o p e d in laboratory-scale fermentors and o p timized further at the pilot-plant scale. New York. transfer criteria in stirred vessels m a y be b a s e d on carbon dioxide evolution rates. B e c a u s e dry mycelial w e i g h t s and turbidity m e a s u r e m e n t s are difficult to m a k e with filamentous fungi. S. Inoculum. D e v e l o p m e n t of a spore i n o c u l u m is conveniently achieved in s u b m e r g e d culture p r o v i d e d the appropriate conditions for sporulation can be identified for the fungus of interest ( H o c k e n h u l l 1980.4. 3. (1979) Econ. D e v e l o p m e n t of a vegetative mycelial i n o c u l u m usually requires several stages of g r o w t h to a c h i e v e desired levels at p r o d u c t i o n scale. T h e latter could represent substantial savings to d o w n s t r e a m processing and waste disposal c o s t s . a consistent process for preparing the i n o c u l u m is required. Broderick and G r e e n s h i e l d s 1981). O n c e . 35-122. T h i s m e t h o d w a s found to be a g o o d g r o w t h indicator for P.

(1981) found that s o m e strains of Trichoderma reesei required an initial 2-day cultivation period at 31°C followed by 28°C for the r e m a i n d e r of the process to p r o d u c e o p t i m u m levels of cellulase. A l s o . rapid destruction of penicillin o c c u r s . (1972) reported that the optimal p H plateau for high levels of penicillin production by industrial strains of P. A n essential r e q u i r e m e n t of an aerobic fermentation p r o c e s s is that an a d e q u a t e supply of o x y g e n be available for g r o w t h and product formation. If the p H e x c e e d e d 7 . the o p t i m u m p H can be e x p r e s s e d as a plateau. the t e m p e r a t u r e optimal for g r o w t h is also o p t i m a l for product formation. m a n y of the m a s s transfer studies in fermentation focus on the m a s s transfer b e t w e e n gas and liquid p h a s e s . mycelial fermentations can be difficult b e c a u s e the solubility of o x y g e n in fermentation broths is quite l o w . with g r o w t h and p r o d u c t formation o c c u r r i n g in the t e m p e r a t u r e range of 2 0 . Pan et al. for m a n y fermentation p r o c e s s e s . Instead of b e i n g a specific v a l u e . kLa. h o w e v e r . 3 . O p t i m i z a t i o n of p H can be difficult b e c a u s e it m a y c h a n g e during the time c o u r s e of the fermentation. reesei a c h i e v e d o p t i m u m p e r f o r m a n c e at a constant t e m p e r a t u r e of 25°C. 0 to < 7 . (1977) stated that for the z e a r a l e n o n e fermentation. This transfer is m e a s u r e d by the v o l u m e t r i c m a s s transfer coefficient. F i l a m e n t o u s fungi are usually m e s o p h i l i c . T h e t e m p e r a t u r e o p t i m u m is initially d e t e r m i n e d at the shake-flask scale. M e e t i n g this r e q u i r e m e n t in v i s c o u s . and Τ is the absolute t e m p e r a t u r e . T h e r e f o r e . Temperature.4 0 ° C .78 Growth Kinetics and Fermentation Scaleup the p r o c e s s is d e v e l o p e d for spore p r o d u c t i o n . Aeration and Agitation. they also found that other strains of T. h o w e v e r . a p p r o x i m a t e l y 9 p p m . B e c a u s e of the low solubility of o x y g e n and its i m p o r t a n c e to microbial m e t a b o l i s m .> w h e r e A is the A r r h e n i u s constant. especially in the p r e s e n c e of a m m o n i u m ion ( H o c k e n h u l l 1963). a t e m p e r a t u r e of 21°C w a s preferred in shake flasks and a p p r o x i m a t e l y 24°C for the fermentors.l i q u i d m a s s 2 3 transfer ( c m / c m ) . B e c a u s e the spores are easily c o u n t e d . w h e r e kL is the m a s s transfer coefficient in the liquid film (cm/h) and a is the specific interfacial area for a i r . H o w e v e r . there are instances w h e r e the t e m p e r a t u r e d o e s not r e m a i n constant t h r o u g h o u t the fermentation period. T h e specific g r o w t h rate ( μ ) of these m i c r o o r g a n i s m s is related to t e m p e r a t u r e by the familiar A r r h e n i u s relationship in w h i c h μ = E/RT Ae<. 5 . the i n o c u l u m size is very r e p r o d u c i b l e . pH. it m a y not be the preferred t e m p e r a t u r e in stirred v e s s e l s . T a n g n u et al. H i d y et al. Ε is the activation e n e r g y ( k c a l / m o l ) . chrysogenum ranged from p H values > 6 . the o p t i m a l t e m p e r a t u r e for each strain requires careful e x a m i n a t i o n during the fermentation cycle as well as during s c a l e u p . T h e m a s s transfer coefficient is related to the volumetric p o w e r input and superficial gas velocity by the equation . R is the universal gas constant. it m a y be scaled up to m e e t i n o c u l u m size r e q u i r e m e n t s at the production scale.

o x y g e n e n r i c h m e n t of the inlet-air stream is not required at this scale but m a y be required at pilot plant and p r o d u c t i o n scales. .C). T h i s relation w a s s h o w n by T a g u c h i et al. Agitation also increases air h o l d u p t i m e and d e c r e a s e s the film thickness at the gas/liquid interface by turbulent shear (Finn 1954). and the o p e r a t i n g b a c k p r e s s u r e . laboratory ferm e n t o r s is usually not a p r o b l e m e v e n if g r o w t h is in the filamentous form. Different levels of dissolved o x y g e n concentrations are achieved by v a r y i n g the s p e e d of agitation.) (μα)k.0 . V is the v o l u m e ( 1 . increasing the gas/liquid interfacial area. Vs is the superficial gas velocity ( c m / m i n ) . Nyiri and L e n g y e l (1965) reported that c a r b o n dioxide levels higher than 4 % inhibited penicillin formation by P. and p r e s s u r e . (1968) to be a r e a s o n a b l e representation of m y c e l i a l . the b y . kLa = κΙ^Α (Υ. can h a v e a detrimental effect on product formation w h e n present at high levels. and a and β are e x p o n e n t s . mean w h e r e C * is the e q u i l i b r i u m o x y g e n concentration (mM/1) and C is the dissolved o x y g e n in the culture broth (mM/1). A l s o important to scaleup is a profile of the o x y g e n uptake rates ( m o l e s of o x y g e n c o n s u m e d b y the b i o m a s s contained in 1 1/h) t h r o u g h o u t the fermentation c y c l e . n o n . T h e r e f o r e . 8 6 β T h e r e are several w a y s that agitation i m p r o v e s the volumetric o x y g e n transfer coefficient. the rate of aeration. T h e o x y g e n u p t a k e rate ( O U R ) m a y b e calculated by the equation O U R = kLa ( C * .N e w t o n i a n broth b e h a v i o r . U s u a l l y . Pg is the gassed p o w e r e d input ( h p ) . T h e level of c a r b o n d i o x i d e in the culture broth is greatly affected by the p H of the b r o t h . the dissolved c a r b o n d i o x i d e c o n c e n t r a t i o n at the b o t t o m of the tank is greater than that at the top d u e to the large hydrostatic p r e s s u r e s . D e t e r m i n i n g the o p t i m u m dissolved o x y g e n concentration for g r o w t h and p r o d u c t formation is important for both p r o c e s s d e v e l o p m e n t and scaleup (Lilly 1983). Pirt and M a n c i n i (1975) confirmed the inhibitory effect of carbon d i o x i d e on penicillin production and r e c o m m e n d e d that the carbon d i o x i d e level b e m i n i m i z e d during the fermentation p r o c e s s . the rotating impeller disperses the air s t r e a m into fine b u b b l e s . chysogenum. the v o l u m e t r i c flow rate of air. the broth t e m p e r a t u r e . M e e t i n g the o x y g e n r e q u i r e m e n t s of fungi in turbine-agitated. Carbon Dioxide. A s air enters the fermentor. in large v e s s e l s .4.2 k a = κί^Α L Scaleup of Fermentation 79 (Vs)e w h e r e AT is a proportionality c o n s t a n t . 0 0 0 1).p r o d u c t of cell m e t a b o l i s m . C a r b o n d i o x i d e . R y u and H u m p h r e y ( 1 9 7 2 ) a d d e d an apparent viscosity t e r m (μα) to this expression to represent the effect of mycelial g r o w t h on kLa.

the m e a n pellet d i a m e t e r of P. especially in the design of the agitation s y s t e m . S o l o m o n s (1980) reported that the m e a n length of actively g r o w i n g h y p h a e of Fusarium graminearum w a s virtually unaffected by the stirrer speeds tested. s o m e strains of fungi appear to be m u c h less sensitive to shear.t o .5 ) . 4. O c c a s i o n a l l y . A l s o . T h e pilot-plant studies continue to e x a m i n e the effects of fermentation scale o n k e y . d e t e r m i n i n g the shear sensitivity of the fungal strain early in process d e v e l o p m e n t can greatly aid scaleup strategies.d i a m e t e r ratio of 2 : 1 to 3 : 1 and are e q u i p p e d with impellers having DilDt ratios of 0 . 3 . T h e vessels usually h a v e a tank h e i g h t . H o w e v e r . T h e y r a n g e in v o l u m e from 7 0 to 19. T h e R u s h t o n turbine impellers that are c o m m o n l y used in the fermentation industry generate high shear forces which can be undesirable in fungal fermentations. T a n a k a (1976) reported that high shear can adversely affect cell g r o w t h and p r o d u c t formation.7 ) w h i c h d o e s not generate high shear forces. A m o r e attractive impeller design for these fermentations is the hydrofoil impeller (Figure 4 .2.000 1.0 . 2 . This impeller will be discussed further in Section 4 .80 Growth Kinetics and Fermentation Scaleup Rushton Turbine Prochem Hydrofoil F I G U R E 4-7 Types of impellers: flat-blade Rushton turbine and Prochem Maxflo hydrofoil. direct transfer of the fermentation p r o c e s s d e v e l o p e d in laboratory-scale fermentors to production scale can be successful. T h e y are usually constructed of stainless steel and geometrically similar to the laboratory-scale vessels (Figure 4 . T h e sensitivity of filamentous fungi to m e c h n i c a l and h y d r o d y n a m i c shear forces is a l w a y s a consideration in establishing a stirrer speed that achieves desired bulk m i x i n g and supplies an a d e q u a t e o x y g e n supply for the culture.2 Pilot Plant Scale Pilot-plant studies are routinely required for efficient transfer of the fermentation p r o c e s s to p r o d u c t i o n . 2 . the pilot-plant tanks are also highly instrumented and c o m p u t e r controlled. 2 . Effect of Shear. 5 . chrysogenwn w a s found by van Suijdam and M e t z (1981) to decrease with an increase in p o w e r input p e r unit v o l u m e . Like the laboratory-scale fermentors. T h u s .

the type and n u m b e r of i m p e l l e r s . T h e technical issues of aeration that w e r e discussed previously with laboratory-scale fermentors also apply at the pilot-plant scale. c o n t i n u o u s (hight e m p e r a t u r e . fungal culture broths presents challenging o p p o r t u n i t i e s . B e c a u s e both t e m p e r a t u r e and t i m e of sterilization r e m a i n constant at increasing scale. 2 .4. three process variables c o m m o n l y affected b y scale are m e d i u m sterilization. agitation and aeration. T h e sensitivity of the g r o w t h m e d i u m to sterilization m a y b e d e t e r m i n e d in s h a k e flasks b y k e e p i n g the t e m p e r a t u r e of sterilization constant and varying the e x p o s u r e p e r i o d s . resulting in areas of varying nutrient c o n c e n t r a t i o n s t h r o u g h o u t the culture b r o t h .2. i n c o m p l e t e m i x i n g c o m m o n l y o c c u r s in large v e s s e l s . scaleup of this m e t h o d is usually straightforward. 2 . If the m e d i u m p r o v e s to contain heat-labile nutrients. G l u c o s e . T h e desirable sterilization t e m p e r a t u r e is achieved with a heat e x c h a n g e r or live steam injections. A s d i s c u s s e d p r e v i o u s l y . .2 Scaleup of Fermentation 81 p r o c e s s variables. W h e n the microfungi are e x p o s e d to these a r e a s . T h i s t e c h n i q u e consists of rapidly heating and cooling the g r o w t h m e d i u m as it flows t h r o u g h a p i p e . 4 . p r e v e n t i n g o p t i m u m fermentation p e r f o r m a n c e . H o w e v e r . H o w e v e r .1 M e d i u m S t e r i l i z a t i o n . and heat transfer. B a t c h sterilization is a basic t e c h n i q u e for sterilizing g r o w t h m e d i a for small-scale fermentations.2. O n e a p p r o a c h that is c o m m o n l y used to m i n i m i z e undesirable reactions is to sterilize o n e o r m o r e of the nutrients separately. reduction of these high viscosities can be a c h i e v e d by broth dilution and maintaining fungal g r o w t h as small c o m p a c t pellets or small filamentous fragments. 4. is routinely sterilized separately to r e d u c e the undesirable Maillard reactions that can occur. especially in large vessels. T h e e x p o s u r e time to high t e m p e r a t u r e is controlled b y the fluid velocity and p i p e length. T h e m i x i n g b e h a v i o r of a fermentor m a y b e e s t i m a t e d by the m i x i n g t i m e . Highly v i s c o u s . both b i o m a s s concentration and fungal m o r p h o l o g y are major contributors to the r h e o l o g y of the culture broth. mycelial culture broths tend to increase m i x i n g t i m e . Factors affecting the m i x i n g t i m e for fungal culture broths include the r h e o l o g y of the b r o t h . defined as the time required to a c h i e v e c o m p l e t e dispersion of an a d d e d tracer. It can also b e used at larger scales p r o v i d e d the critical m e d i u m c o m p o n e n t s are not adversely affected d u r i n g the increased heating u p and c o o l i n g d o w n periods that are associated with scale. In addition to i n o c u l u m d e v e l o p m e n t . a c o m m o n ingredient of fermentation m e d i u m . Special p r e c a u t i o n s are taken w h e n sterilizing g r o w t h m e d i a containing nutrients that react unfavorably with each other during sterilization. they m a y switch their m e t a b o l i s m from o n e metabolic p a t h w a y to a n o t h e r . and the size of the v e s s e l s . unlike laboratory fermentors w h i c h are usually well m i x e d .s h o r t . A c h i e v i n g a d e q u a t e aeration and agitation in highly v i s c o u s .t i m e ) sterilization m a y b e used (Jain and B u c k l a n d 1988). 2 A e r a t i o n a n d A g i t a t i o n .

A l t h o u g h the R u s h t o n impeller provides excellent m a s s and heat transfer capabilities. N o negative impact on product formation w a s o b s e r v e d with the h y d r o foil i m p e l l e r s . Heat r e m o v a l from the fermentor is usually achieved b y transferring the heat through the vessel wall to the surrounding air or to the coolant circulating through the external j a c k e t . the m i x i n g t i m e for a 100. they h a v e p r o v e n to b e quite . Einsele (1978) reported that for a q u e o u s s y s t e m s . especially if the fermentation is a batch p r o c e s s . Scaleup u n d e r these conditions c a n be greatly facilitated by the use of shake-flask controls ( B a n k s 1979). A similar reduction in p o w e r d r a w w a s reported by B u c k l a n d et al.2. m i x i n g p r o b l e m s m a y be e x p e r i e n c e d with viscous filamentous culture broths if cooling coils are placed inside the tank. (1986) reported that the hydrofoil axial flow impellers c o n s u m e d 4 0 % less p o w e r during the fermentation cycle than the standard R u s h t o n impellers w h e n tested with a v i s c o u s . Heat is generated during an aerobic fermentation p r o cess by the metabolic activity of the cells. 0 0 0 1 w a s not a d e q u a t e for heat r e m o v a l . 4.82 Growth Kinetics and Fermentation Scaleup I m p e l l e r designs r a n g i n g from axial flow m a r i n e impellers to radial flow. Sinclair and M a v i t u n a (1983) simplistically illustrated this p r o b l e m by s h o w i n g that the cooling surface of a fermentor greater than 5 0 . 2 .7 ) . T h e s e results clearly support the use of hydrofoil impellers for viscous filamentous fermentations.000-1 bioreactor w a s a p p r o x i m a t e l y fivefold greater than that o b s e r v e d for a 200-1 bioreactor ( 1 0 0 versus 2 0 sec). 4 . A l t h o u g h shake-flask fermentations are relatively c r u d e and physically unrelated to stirred v e s s e l s . filamentous fermentation ( A v e r m e c t i n ) at the 800-1 scale. A n o t h e r factor that affects m i x i n g time is the fluid v o l u m e of the bioreactor.3 Heat Transfer. T h e p e a k p o w e r d e m a n d of the hydrofoil impellers w a s a p p r o x i m a t e l y 6 6 % of that r e a c h e d with the R u s h t o n i m p e l l e r s . As indicated p r e v i o u s l y . R e c e n t l y . 4 S h a k e . and to a lesser extent by gas s p a r g i n g . G b e w o n y o et al. it has unfavorable high p o w e r d e m a n d s and limited bulk m i x i n g capabilities. H o w e v e r . a n e w l y d e s i g n e d impeller with high efficiency hydrofoil blades w a s introduced to the fermentation industry (Figure 4 . (1989) w h e n both types of impellers w e r e tested with a fungal fermentation (lovastatin) at the s a m e scale. m e c h a n i c a l agitation. flat-blade disc turbine impellers (Rushton impeller) h a v e been extensively tested. with the R u s h t o n radial flow impeller being the design m o s t c o m m o n l y used t o d a y .2. 2 . T h e cooling capacity of fermentors m a y be increased by addition of external heat e x c h a n g e r s and cooling coils to the inside of the tank. W i t h v i s c o u s f e r m e n t a t i o n s . the use of R u s h t o n impellers in a large tank can result in alternating z o n e s of high and low turbulence t h r o u g h o u t the bioreactor. This m e t h o d of heat r e m o v a l is usually a d e q u a t e for laboratory and pilot-plant scale fermentors but m a y e x p e r i e n c e difficulties at production scale.F l a s k C o n t r o l s . the urgent need for significant quantities of a product candidate m a y require that the fermentation p r o c e s s be scaled up rapidly to large bioreactors before the important p r o c e s s variables h a v e b e e n appropriately studied.

the impeller speed will h a v e to be d e c r e a s e d to maintain constant tip s p e e d . or a m i n i m u m dissolved o x y g e n c o n c e n tration. Steel and M a x o n (1962) s h o w e d that the o x y g e n availability rate ( O A R . m a i n t a i n i n g g e o m e t r i c a l similarity is usually quite helpful.d i a m e t e r ratio of a p p r o x i m a t e l y 3 : 1 . A third set is equal to the s e c o n d e x c e p t the m e d i u m is also sterilized at large scale. T h e a p p r o a c h ( s ) selected is usually b a s e d o n the important process variables identified at pilot-plant scale. consisting of laboratory p r e p a r e d m e d i u m and i n o c u l u m . defined as "the o x y g e n u p t a k e rate m e a s u r e d u n d e r c o n d i t i o n s in w h i c h the culture is limited in its respiration rate by o x y g e n s u p p l y " ) m a y also b e used to scale u p highly v i s c o u s . W i t h r e d u c e d agitation and significant broth viscosity ( c o m m o n with filamentous g r o w t h ) . O n e set is the laboratory c o n t r o l . requiring scaleup to favor constant volumetric o x y g e n transfer coefficient. A s e c o n d set consists of m e d i u m formulized at large scale. T h e fifth set consists of laboratory p r e p a r e d m e d i u m and large-scale prepared i n o c u l u m .2 Scaleup of Fermentation 83 v a l u a b l e in e v a l u a t i n g i n o c u l u m d e v e l o p m e n t and m e d i u m sterilization during s c a l e u p . p r o d u c t yield. 0 0 0 . calculated by the equation w h e r e Ν is the impeller speed ( 1/sec) and Di is impeller d i a m e t e r ( c m ) . 0 0 0 to > 2 0 0 .N e w t o n i a n fermentation p r o c e s s . T a g u c h i et al. T h e influence of O A R on n o v o b i o c i n yield at fermentation scales r a n g i n g from 2 0 1 to 2 4 .3 Production Scale Efficient transfer of the pilot-plant process to production scale requires that the important p r o c e s s variables for a particular fermentation h a v e b e e n identified. If the microfungus w a s found to b e highly shear sensitive. A s stated p r e v i o u s l y .N e w t o n i a n filamentous fermentation p r o c e s s e s . (1968) studied the effects of aeration and agitation on g l u c a m y l a s e synthesis by Endomyces at various fermentor sizes ( 3 0 . 0 0 0 gallons is presented in F i g u r e 4—8. c o m m o n l y referred to as the " Α Γ (after inoculation of the fermentor) s a m p l e . as the impeller d i a m e t e r increases with scale. m a n y filamentous fungal fermentations are o x y g e n transfer limited. A l s o . Similar controls m a y be used for fermentation processes that require nutrient feeding b y using laboratory-scale fermentors instead of s h a k e flasks. p o o r m i x i n g of the fermentation broth m a y be e x p e r i e n c e d . and inoculated with laboratory g r o w n culture. 0 0 0 1 in v o l u m e with a tank h e i g h t .t o .5 0 .2. several a p p r o a c h e s to p r o c e s s scaleup m a y be e m p l o y e d (Einsele 1978). A s alluded to p r e v i o u s l y . 0 0 0 1) and found that the v o l u m e t r i c o x y g e n transfer coefficient could be used for scalingu p this m y c e l i a l . sterilized in the laboratory. and efficiency of the p r o c e s s . A l s o . T h e scale is usually dictated by the cost. air b u b b l e s tend to increase in size. n o n . r e d u c i n g the v o l u m e t r i c o x y g e n transfer coefficient. T h e bioreactors at p r o d u c t i o n scale can r a n g e from 2 0 . . scaleup activities m a y focus on constant tip speed ( V t i p) . n o n . constant o x y g e n u p t a k e rate.4. H o w e v e r . T h i s evaluation is usually achieved with five sets (at least t w o flasks p e r set) of c o n t r o l s . 4. A fourth set consists of large-scale p r e p a r e d m e d i u m and i n o c u l u m .

4. W.) F I G U R E 4-8 Effect of OAR (viscosity-independent region) on novobiocin yield in various sizes of fermentor. and water. Electricity is c o n s u m e d by air c o m p r e s s o r s and circulating p u m p s . T h e cost of this c o m p o n e n t m a y be lowered by using c o m p l e x sources such as corn starch.84 Growth Kinetics and Fermentation Scaleup 1.000 Gal. and waste disposal costs. A l t h o u g h the . p r o d u c t r e c o v e r y cost. in filamentous fermentations resistance to transport within the mycelial m a s s e s m a y be rate limiting.0 0 10 20 20-L. m e d i u m ingredients deserve special attention b e c a u s e o n e or m o r e m a y contribute significantly to the manufacturing cost of a fermentation p r o d u c t . H o w e v e r . the m o s t e x p e n s i v e c o m p o n e n t of production m e d i u m is the carbon s o u r c e . fermentation variability. Reprinted by permission of John Wiley & Sons. including raw materials and c h e m i c a l s . including increased r a w material storage c o s t s . D. > C _Q Ο > 03 ω er Fermentor Size • Ο Δ • 0. they can h a v e undesirable p r o c e s s effects. 30 40 50 O A R (mM/L.3 ECONOMICS D u r i n g d e v e l o p m e n t of a consistent and scalable fermentation p r o c e s s . Electrical p o w e r can also be a significant contributor to the overall m a n u f a c t u r ing cost of a fermentation p r o c e s s . (1962) Biotechnol. the major c o n s u m e r is the agitator m o t o r . R. electricity.000 Gal. m o l a s s e s . A l t h o u g h gas-to-liquid is usually rate limiting for m a s s transfer. 24. Copyright © John Wiley & Sons. T h u s . 231-240. T h e costs resulting from these process effects could easily offset initial savings. h o w e v e r . Hr. Reprinted from Steel. major efforts are focused on m a x i m i z i n g product formation and m i n i m i z i n g manufacturing c o s t s . U n d e r this c o n d i t i o n . 250-L 10. 4. New York. and Maxon.0 M. and vegetable oils. b e c a u s e the c o m p o s i t i o n of c o m p l e x nutrients is usually poorly defined and variable.2 0. A s stated p r e v i o u s l y . s t e a m .. Bioeng. Inc. U s u a l ly. careful consideration should be given to these potential process effects w h e n introducing a c h e a p e r m e d i u m c o m p o n e n t . scaleup m e t h o d s based on gas-to-liquid m a s s transfer m a y not b e appropriate.

(1986) m Manual of Industrial Microbiology and Biotechnology (Demain. Fastert. 595-619. K. (1976) Appl. Brix..4 8 .. 8. A. 3 9 5 ^ 1 8 . . Buckland. B . 254-274.000-1 tank with a c o m m o n p o w e r input of 2 k W / m w o u l d 4 still c o n s u m e a substantial a m o u n t of electricity ( 2 . REFERENCES Athinson.). pp. and Rossmoore. (1976) in Advances in Biochemical Engineering Vol. DC. Providing fermentation broths with high p r o d u c t c o n c e n t r a t i o n s and low nutrient and b y . Α. 193-199. 126. 193-202... (1979) in Topics in Enzyme and Fermentation Biotechnology Vol. and Lentz. Ferment. (1975) J.). . Bioeng. (1954) Bact. Kaplan...p r o d u c t c o n c e n t r a t i o n s can substantially r e d u c e the cost of product isolation and waste treatment. 11. G. 11. Clark. L. E. 4 1 . 7-12. T w o other m a n u f a c t u r i n g costs that can be greatly affected by the fermentation p r o c e s s are p r o d u c t r e c o v e r y and w a s t e treatment. Gbewonyo. Hallada.. Carter. H. Bioeng. D. England. pp. I. Elmayergi. 281-299. England. Washington. H.. 875-883. and Humphrey. B .References 85 3 p o w e r input per unit v o l u m e ( k W / m ) d e c r e a s e s with increasing bioreactor v o l u m e 3 (Einsele 1978). Microb. R. K. Banks. Gualandi. S.. 134-139. Calam. 41-124. (1985) Bio/Technology 3. (1978) Process Biochem. The Fluid Engineering Centre. 13-14. R. Chain. . Washington. T. Rev..) 3. 5. (1968) Lloydia 3 1 . Einsele. DiMasi. 9 x 1 0 k W h ) during a 6-day fermentation.. K. F.. B . Ellis Horward Limited. M a n y d o w n s t r e a m isolation and purification p r o b l e m s can be solved b y appropriate adjustment of the fermentation c o n d i t i o n s . Biochem. 161-169. D. HunterCevera. R. 13 (July). pp. Ε. 3 1 . and Solomon. C... N. B. pp. A close d i a l o g u e b e t w e e n the fermentation and isolation staff can help e n s u r e that the process d e v e l o p e d achieves high yields. (1977) Mycologist's Handbook. (1976) Proc. and Inamine. (1981) J. (1966) Biotech.. C... Buckland. eds. Hawksworth. England. Environ. (1983) Biotechnol. Α. B. 4 (Ghose. and Daoud. Microbiol. D.. New York. eds. A. DC. D. . Deindoerfer. (1989) in Novel Microbial Products for Medicine and Agriculture (Demain.. (1963) Biotechnol. 785-804. Gen. 177-266. and Bull. Buckland. . Β . Commonwealth Mycological Institute. (1984) Bio/Technology 2. 982-988. 18. G. Broderick. G. et al. o p t i m i z i n g agitation and aeration conditions can yield substantial s a v i n g s .). Springer-Verlag. H. Technol. Gbewonyo. T. pp. Cranfield. Surrey. Microbiol. D. 143-145. Demain. N . and Masurekar. and Demain. (1986) International Conference on Bioreactor Fluid Dynamics. 967-983. Greasham. D. American Society for Microbiology. and Morisi. 53. and Blakebrough. J. Α. Fiechter. P. Bioeng. Finn. . Gbewonyo. G. (1969) Biotechnol.. eds.. Society for Industrial Microbiology. a 100. Α. Somkuti. and Wang. T. T h e r e f o r e . Α. A.. 9. and Buckland... Chichester. Winstanley. (1961) Appl. A. 3 (Wiseman. 722-729. Bioeng. ed. A. 25. and Greenshields.. Drew. C.

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A l t h o u g h they are not essential for the p r o d u c i n g o r g a n i s m ' s g r o w t h in pure c u l t u r e . Demain F i l a m e n t o u s fungi are a m o n g the m o s t prolific p r o d u c e r s of s e c o n d a r y m e t a b o l i t e s . cyclic p e p t i d e s m a d e of n o r m a l and modified a m i n o a c i d s . also k n o w n as idiolites or special m e t a b o l i t e s . S e c o n d a r y metabolites are p r o d u c e d only by 89 . O n e of the r e a s o n s for such an unappreciative attitude t o w a r d fungi w a s the e x t e n s i v e p r o d u c t i o n of growth-inhibitory organic acids w h i c h led m a n y a d i s c o v e r y screening p r o g r a m " d o w n the garden p a t h " to failure. c o m e s from a m o l d .CHAPTER 5 Regulation of Secondary Metabolism Arnold L. they h a v e survival functions in nature ( D e m a i n 1989). B e c a u s e s o m e s e c o n d a r y metabolites are effectors of differentiation ( D e m a i n 1989). usually p o s s e s s bizarre c h e m i c a l structures and unusual c h e m i c a l linkages such as /3-lactam r i n g s . unsaturated b o n d s of p o l y a c e t y l e n e s and p o l y e n e s . Cephalosporium acremonium (= Acremonium chrysogenum). the c e p h a l o s p o r i n s . After these early d i s c o v e r i e s . Pénicillium chrysogenum. S e c o n d a r y m e t a b o l i t e s . it is not surprising that o r g a n i s m s that e n g a g e extensively in m o r p h o l o g i c a l differentiation w o u l d also b e heavily involved in c h e m i c a l differentiation. a r e n e w e d interest has b e e n s h o w n in fungi and m a n y n e w activities h a v e been found in m o l d b r o t h s . and large m a c r o l i d e rings. T h e first e c o n o m i c a l l y important antibiotic. w e r e d i s c o v e r e d in another filamentous fungus. penicillin. W i t h the n e w e m p h a s i s on b r o a d e n i n g the search for useful secondary metabolites a w a y from strictly antibacterial and a n t i t u m o r activities ( D e m a i n 1983) and with the establishm e n t of small c o m p a n i e s that are supplying rare fungi. and the m o s t important g r o u p of antibiotics t o d a y . m o l d s w e r e neglected for m a n y years w h i l e a c t i n o m y c e t e s entered center stage in the quest for n e w antibiotics.

but s o m e t i m e s they o v e r l a p . the higher the nutrient c o n c e n t r a t i o n . chrysogenum. In addition to g r o w t h rate c o n t r o l . 1976) and c h a n o c l a v i n e . " In m a n y idiolite fermentations. and m e d i a supporting rapid filamentous g r o w t h . T r o p h o p h a s e . T h e c o n c e p t of the t r o p h o p h a s e . Claviceps fusiformis.90 Regulation of Secondary Metabolism s o m e species of a g e n u s and as a mixture of m e m b e r s of a c h e m i c a l family b e c a u s e of the low specificity of s o m e e n z y m e s involved in secondary m e t a b o l i s m . see Vining 1973). silicone-treated flasks. R e p r e sentative c o m p o u n d s include m y c o t o x i n s . and p h e r o m o n e s . . T r o p h o p h a s e and idiophase often o c c u r at separate times in batch c u l t u r e .i d i o p h a s e kinetics also o c c u r in the aflatoxin fermentation ( A p p l e b a u m and B u c h a n a n 1979) and in the ergot alkaloid p r o c e s s as carried out by certain strains. p i g m e n t s . T h e g r o w t h p h a s e is called the " t r o p h o p h a s e " and the production p h a s e the " i d i o p h a s e .p h a s e kinetics w e r e clearly seen in Pénicillium urticae by G r o o t W a s s i n k and G a u c h e r (1980) u n d e r carefully designed c o n d i t i o n s . but the t w o p h a s e s o v e r l a p in defined m e d i a supporting slow g r o w t h . the later the a p p e a r a n c e . and Sphaecelia sorghi (for a discussion. the first e n z y m e of the p a t h w a y (6methylsalicylic acid synthetase) a p p e a r e d . that is. and e n d . E x p r e s s i o n of the g e n e s c o d i n g for idiolite biosynthesis d o e s not usually o c c u r at high g r o w t h rates d u e to repression of synthetases during g r o w t h .i d i o p h a s e d y n a m i c s o c c u r in c o m p l e x m e d i a capable of supporting rapid g r o w t h . nutrient r e p r e s s i o n . T h e t w o .1 DELAYED FORMATION OF IDIOLITES In batch cultures containing nutritionally rich m e d i a . antibiotics. use of spores as i n o c u l u m . W h e n g r o w t h stopped (3 h later).i d i o p h a s e transition w a s first put forth d u r i n g studies on the production of the m y c o t o x i n patulin by Β u ' L o c k et al. synthetase d e c a y . Claviceps purpurea. E x a m p l e s of e n z y m e s w h o s e activity appears only in idiophase are penicillin acyltransferase and the side-chain-activating e n z y m e of penicillin biosynthesis in P. A n important characteristic of secondary m e t a b o l i s m is suppression by high specific g r o w t h rates of the p r o d u c i n g cultures. individual biosynthetic p a t h w a y s are affected by regulatory m e c h a n i s m s such as i n d u c t i o n . In a defined m e d i u m supporting only slow g r o w t h . that is.1 . A secondary metabolite is secondary only b e c a u s e it is not essential for vegetative g r o w t h of the p r o d u c i n g culture. E n z y m e s of ergot alkaloid synthesis that a p p e a r only after g r o w t h are dimethylallyltryptophan synthetase (Krupinski et al. a later e n z y m e (ra-hydroxybenzyl alcohol d e h y d r o g e n a s e ) a p p e a r e d . for e x a m p l e . ( 1 9 6 5 ) . T h e timing of product formation should not be used to define a secondary m e t a b o l i t e . 1973). thus favoring production while slow g r o w t h is still o c c u r r i n g .p r o d u c t regulation. A s soon as g r o w t h rate b e g a n to d e c l i n e . s o m e nutritional factor m a y be g r o w t h limiting from the very start of cultivation. 5.c y c l a s e (Erge et al. T h e time of appeara n c e of this latter e n z y m e d e p e n d e d on the a m o u n t of nitrogen source a d d e d to the m e d i u m in an inverse m a n n e r . typical t r o p h o p h a s e . high levels of idiolites are usually p r o d u c e d only after most of the cellular g r o w t h has b e e n c o m p l e t e d .

v a l i n e ] . In addition. lin Ν s y n t h e t a s e . P o l y s a c c h a r i d e s . lactose s h o w s the o p p o s i t e b e h a v i o r (Soltero and J o h n s o n 1953).c o n t r o l l e d fed-batch fermentations of P. G l u c o s e is excellent for g r o w t h of P. S u c h data suggested that g l u c o s e represses e n z y m e s of penicillin b i o s y n t h e sis. G l u c o s e w a s also found to stimulate the c o n v e r s i o n of lysine into proteins and to d e c r e a s e the intracellular concentration of L .D . g a l a c t o s e . c o n v e r s i o n to A C V . In a m e d i u m c o n t a i n i n g a rapidly used c a r b o n source plus a m o r e slowly utilized c a r b o n s o u r c e . H ö n l i n g e r and K u b i c e k ( 1 9 8 9 ) found n o α .a m i n o a d i p y l ) . Exogenous α-aminoadipate could not reverse glucose repression of penicillin biosynthesis although it is taken u p by the m y c e l i a . a- a m i n o a d i p a t e h a s four fates: c o n v e r s i o n to lysine.2. chrysogenum g r o w n on g l u c o s e . In c o m p u t e r . the second c a r b o n source is used for idiolite b i o s y n t h e s i s . the former usually is used first.k n o w n cases are the regulation of penicillin and c e p h a l o s p o r i n p r o d u c t i o n by g l u c o s e . o l i g o s a c c h a r i d e s . usually an excellent c a r b o n source for g r o w t h . idiolite p r o d u c t i o n d o e s not o c c u r in this p h a s e . C a r b o n source regulation of s e c o n d a r y m e t a b o l i s m exists in m a n y fermentat i o n s . In confirmation of the results of Revilla et al.1 Carbon Source Regulation G l u c o s e . the first int e r m e d i a t e of the p a t h w a y . After the favored c a r b o n source is d e p l e t e d . N o effect w a s seen on the formation of penicillin acyltransferase. the second e n z y m e of the t h r e e . T h e m o s t w e l l . sogenum.a m i n o a d i p a t e . T h e classic c h e m i c a l l y defined m e d i u m for penicillin p r o d u c t i o n contains both g l u c o s e and lactose in w h i c h early g r o w t h o c c u r s at the e x p e n s e of g l u c o s e . chrysogenum but d o e s not support e x t e n s i v e penicillin p r o d u c t i o n . or isopenicillin Ν in the intracellular pool of P. the third e n z y m e of the p a t h w a y .2 Effectors of Idiolite Biosynthesis 91 EFFECTORS OF IDIOLITE BIOSYNTHESIS 5.( L . c a t a b o l i s m . and cyclization to extracellular 6-oxopiperidine-2-carboxylic acid. ( 1 9 8 6 ) . interferes with the b i o s y n thesis of m a n y s e c o n d a r y m e t a b o l i t e s .2 5. ( 1 9 8 6 ) found that g l u c o s e represses c y c l a s e (isopenicil- chrysogenum. and sucrose but not lactose (Revilla et al.a . A l s o re pre ssiv e are fructose. chry- w h e r e the g r o w t h rate in t r o p h o p h a s e is controlled b y the g l u c o s e feed r a t e . A C V . and oils are often better c a r b o n sources for p r o d u c t i o n than is g l u c o s e . a h i g h g r o w t h rate results in a low specific production rate of penicillin ( M o u 1983). T h e a m i n o a d i p a t e pool concentration is the limit- ing factor in p r o d u c t i o n of A C V and isopenicillin Ν and s h o w s a strong positive correlation with penicillin G production.e n z y m e p a t h w a y ) and l o w e r s the intracellular level of A C V [ 5 .a . G r o w t h on gluc o s e e l i m i n a t e s the flux to A C V but lactose d o e s not. the first e n z y m e of the penicillin biosynthetic pathway). G l u c o s e d o e s not inhibit the action of penicillin-forming e n z y m e s in P. 1984). and after its e x h a u s t i o n the extensive mycelial m a s s that h a s d e v e l o p e d on g l u c o s e b e g i n s to p r o d u c e antibiotic on the m o r e slowly used lactose.a m i n o a d i p a t e (which is both an intermediate in lysine b i o s y n t h e s i s and a substrate of A C V s y n t h e t a s e .c y s t e i n y l . Revilla et al. g r o w t h o n lactose d e c r e a s e s the flux to lysine ( H ö n l i n g e r and K u b i c e k 1989).5. In jS-lactam-producing fungi.L . Penicillin p r o d u c t i o n can also be supported very well by intermittent or c o n t i n u o u s feeding of g l u c o s e (or sucrose) w h e r e its level n e v e r is high e n o u g h to interfere with antibiotic formation. .

A g a i n the disaccharide w a s not utilized until the glucose w a s e x h a u s t e d . T h e carbon sources that supported the m o s t rapid g r o w t h ( g l u c o s e . T h e 1 g/1 of g l u c o s e s u p p l e m e n t given every 12 h supported the best cephalosporin C p r o d u c t i o n . T h e connection b e t w e e n g l u c o k i n a s e and carbon source repression appears to be indirect. and c y c l a s e ( B a r r e d o et al. g l u c o s e u p t a k e w a s s l o w e r and g r o w t h of the m u t a n t w a s less than that of the parent c u l t u r e . 1989). the highest c e p h a l o s p o r i n _ 1 C titers w e r e o b s e r v e d at the lowest dilution rate tested ( 0 . w h o added different levels of glucose intermittently to C . ( 1 9 7 8 ) . maltose) exerted a strong negative effect on /3-lactam p r o d u c t i o n . penicillin p r o d u c t i o n . In c o n t i n u o u s culture e x p e r i m e n t s u n d e r glucose limitation. T h e higher the concentration of a suppressive carbon source such as g l u c o s e . 7 % glucose for g r o w t h and 3 . 6 % sucrose for antibiotic formation. T h e defined m e d i u m d e v e l o p e d for this p u r p o s e contains 2 . F e d . T h e m u t a n t p r o d u c e d the s a m e level of penicillin as the parent on lactose. s u c r o s e . . the derepression p r o b a b l y o c c u r r i n g as a result of l o w e r concentrations of glucose or catabolites inside the cell. acremonium ( D e m a i n 1963). into the cells. chrysogenum resistant to 2 . Transport capacity appears in the idiophase at the t i m e of penicillin production and starts to disappear w h e n the penicillin p r o d u c t i o n rate r e a c h e s its m a x i m u m ( F e r n a n d e z .d e o x y g l u c o s e ( D O G ) w a s found to be insensitive to g l u c o s e repression of /3-galactosidase. H o w e v e r . chrysogenum is induced by phenylacetate and is strongly repressed by g l u c o s e . penicillin Ν and c e p h a l o s p o r i n C . w h i c h is a d d e d in all penicillin fermentations. g l y c e r o l . T h e Cephalosporium fermentation yields t w o major p r o d u c t s . and certain a m i n o acids but only m o d e r a t e l y by lactose. acremonium after g r o w t h had stopped at 4 0 h d u e to exhaustion of the initial 15 g/1 of g l u c o s e . T h e m u t a n t contains only 1 0 % of the g l u c o k i n a s e activity of the parent strain and also p h o s p h o r y l a t e s D O G at about 1 0 % the rate of the parent. Different carbon sources exerted m a r k e d effects on the production of these antibiotics ( D e m a i n et al. T h e u p t a k e of p h e n y l a c e t a t e by P. Penicillin Ν appeared d u r i n g g r o w t h but cephalosporin C a c c u m u l a t e d only after g r o w t h had c e a s e d .b a t c h cultures with exponential feed of glucose also s h o w e d a linear inverse relationship b e t w e e n g r o w t h or glucose utilization and antibiotic formation. increasing the g l u c o s e concentration up to 8 g/1 in the intermittent feed progressively interfered with antibiotic p r o d u c t i o n . 1988). indeed an inverse relationship w a s found b e t w e e n dilution rate and specific c e p h a l o s p o r i n C p r o d u c t i o n rate. Penicillin Ν is an intermediate in the formation of cephalosporin C but a c c u m u l a t e s extracellularly b e c a u s e the e n z y m e converting it to d e a c e t o x y c e p h a l o s p o r i n C ( e x p a n d a s e = d e a c e t o x y c e p h a l o s p o r i n C synthetase) is a labile e n z y m e requiring c o n t i n u o u s resynthesis t h r o u g h o u t the fermentation. x y l o s e .C a n o n et al.92 Regulation of Secondary Metabolism A n o t h e r site of c a r b o n source repression is the transport of the side-chain p r e c u r s o r . almost equalling the antibiotic production obtained with a single addition of 2 0 g/1 of the n o n s u p p r e s s i v e sucrose. T h e c o n c e p t of carbon source control of c e p h a l o s p o r i n production w a s supported by data of M a t s u m u r a et al. A l t h o u g h the authors claim that g r o w t h of the m u t a n t w a s n o r m a l . 1979). g a l a c t o s e . g l y c e r o l . C a r b o n source repression also occurs in the case of c e p h a l o s p o r i n C formation by C . the p o o r e r w a s antibiotic formation. A m u t a n t of P. it w a s superior only w h e n glucose w a s present. 0 1 h ) .

1989). It is not k n o w n w h e t h e r the purified e n z y m e s are inhibitable. 1984). g l y c e r o l . E x p a n d a s e is a very labile e n z y m e and its constant resynthesis is required for c e p h a l o s p o r i n C p r o d u c t i o n . 1984).l a c t a m formation. O n the other h a n d . that is. F e r m e n t a t i o n s w e r e c o n d u c t e d with high ( 6 . acremonium is m a r k e d l y repressed but not inhibited b y g l u c o s e . T h e s e data further supported the c o n c e p t that the lability of e x p a n d a s e might be responsible for these effects. A p p a r e n t l y the interference is d u e to inhibition of A C V synthetase action in the cell b e c a u s e strong inhibition of this e n z y m e in c r u d e extracts w a s noted with g l u c o s e . T h u s it b e c a m e u n d e r s t a n d a b l e w h y c y c l o h e x i m i d e and carbon sources favored for g r o w t h s h o w e d selective negative effects on cephalosporin p r o d u c t i o n as c o m p a r e d to that of penicillin N .3 . b e c a u s e cyclase is repressed by high c o n c e n t r a t i o n s of g l u c o s e . 1986). G l y c e r o l is a n o t h e r very suppressive carbon source for C. B e c a u s e cyclase is less repressed b y g l u c o s e and is m o r e stable. 1984) led to a study on repression and inhibition of A C V synthetase by glucose and glycerol using the high c e p h a l o s p o r i n .p h o s p h a t e . Later fermentation (that is.p h o s p h a t e . acremonium revealed that c y c l o h e x i m i d e d o e s not inhibit penicillin Ν formation but d o e s interfere with c e p h a l o s p o r i n C p r o d u c t i o n ( K e n n e l et al.5. after trophophase. indeed low g l u c o s e or glycerol stimulated penicillin Ν production but inhibited c e p h a l o s p o r i n formation. T h i s resynthesis is inhibited by c y c l o h e x i m i d e and repressed by g l u c o s e at low or high levels. In this regard it w a s of interest to d e t e r m i n e w h e t h e r suppressive carbon sources differentially affected penicillin Ν versus c e p h a l o s p o r i n C p r o d u c t i o n . 3 % g l u c o s e .p r o d u c i n g strain C . and m a l t o s e ( B e h m e r and D e m a i n 1983. T h u s e x p a n d a s e in C . T h e observation that high levels of g l u c o s e did l o w e r penicillin Ν production ( H e i m et al. 1981). 6 % sucrose). g l u c o s e . Surprisingly.p h o s p h o g l y c e r a t e ( Z h a n g et al. . penicillin Ν synthesis is not inhibited by c y c l o h e x i m i d e or low g l u c o s e . Expandase does not appear until glucose is utilization begins. its c o n t i n u o u s p r e s e n c e might require c o n t i n u e d protein synthesis and it thus could be very sensitive to carbon source repression. acremonium. H e i m et al.2 Effectors of Idiolite Biosynthesis 93 Studies u s i n g resting m y c e l i a of C.6 . e x p a n d a s e w a s inhibited by 3 .1 0 w h o s e cyclase and e x p a n d a s e is s o m e w h a t d e r e g u l a t e d to c a r b o n source repression (Shen et al. 7 % ) c o n c e n t r a t i o n s of g l u c o s e or glycerol and c o m p a r e d to results using the control m e d i u m ( 2 . It w a s s h o w n next that e x p a n d a s e is m a r k e d l y repressed by g r o w t h in 6 . c y c l a s e is m u c h less affected. It w a s found ( B e h m e r and D e m a i n 1983) that c a r b o n source exerted a m u c h greater negative effect on c e p h a l o s p o r i n synthesis than on penicillin Ν formation. and g l y c e r a l d e h y d e . This suggested that e x p a n d a s e activity m i g h t b e unstable in v i v o . O n the other h a n d . 7 % g l u c o s e plus 3 . cyclase w a s repressed to a m u c h lesser d e g r e e by g l u c o s e ( B e h m e r and D e m a i n 1983). Z a n c a and M a r t i n 1 9 8 3 . 3 % ) and low ( 2 . H i g h g l u c o s e or glycerol b r o u g h t penicillin Ν formation d o w n to control levels and almost totally eliminated c e p h a l o s p o r i n C p r o d u c t i o n . high levels of the sugar r e d u c e penicillin Ν production. during growth) cyclase than expandase exhausted and sucrose experiments showed that the earlier appearance of penicillin Ν as compared to cephalosporin is due to the earlier production of (Heim et al. n o repression of A C V synthetase by carbon source w a s detected even t h o u g h g l u c o s e and glycerol did interfere with ß .

e x p a n d a s e w a s repressed and ß . If alkaloid-producing cells are subjected to g l u c o s e addition. chrysogenum ( G . Revilla and J. and sucrose but not by glycerol and s u c c i n a t e . C a r b o n source control also o c c u r s in m y c o t o x i n p r o d u c e r s .1 0 . and that e x c e s s p h o s p h a t e or glucose alone has n o direct negative effect on antibiotic s y n t h e s i s . 1982. p r o d u c e d late in the fermentation.3 . an inverse correlation exists b e t w e e n carotenoid s y n t h e - . Z h a n g et al. G l u c o s e decreases alkaloid p r o d u c t i o n by Claviceps. 5 ' . In Neurospora er as sa. c A M P acts to activate and inactivate e n z y m e s via phosphorylation of e n z y m e s by a c A M P . This kinase is the intracellular receptor of c A M P in fungi (Pall 1984). using a low c o n c e n t r a t i o n of p h o s p h a t e (1 m M ) to limit g r o w t h . T h e e n z y m e . F u r t h e r m o r e . Yet in the high g l u c o s e c a s e . 1973). polyols and organic acids are the preferred carbon and e n e r g y s o u r c e s . production a c c o m p a n i e s g r o w t h ( A u d h y a and Russell 1975). Z h a n g et al. If the slowly m e t a b o l i z e d lactose is the carbon s o u r c e . (1988) found that high concentrations of p h o s p h a t e did not increase the g l u c o s e c o n s u m p t i o n rate in strain C . It is thus wise to terminate the fermentation in industry before sugar is e x h a u s t e d . acremonium. 1977). H o w e v e r .94 Regulation of Secondary Metabolism K u e n z i (1980) c o n c l u d e d that in C. F . If the situation in filamentous fungi is at all similar to that in y e a s t s .d e p e n d e n t protein k i n a s e . 1982) could be d u e to less c a r b o n source repression than with c a r b o h y d r a t e s . T h u s a high concentration of g l u c o s e alone interferes with antibiotic synthesis.p h o s p h a t e ( c A M P ) d o e s not m e d i a t e carbon catabolite repression of secondary m e t a b o l i s m in filamentous fungi. M a r t i n . cyclopium ( L u c k n e r et al. C a r b o n source repression is also of importance in C . f It appears that cyclic a d e n o s i n e . ramanniana is favored by carbon sources that are p o o r for g r o w t h ( A t t w o o d 1971). T h i s suggests that both carbon source repression and inhibition are operative in this organism. personal c o m m u n i c a t i o n ) . It is doubtful that c A M P plays any role in carbon catabolite repression in m o l d s (Arst and Bailey 1977). 1984). production occurs only after g r o w t h . c A M P d o e s not reverse g l u c o s e repression of penicillin biosynthesis in P. A m u t a n t that p r o d u c e s increased levels of cephalosporin C acetylhydrolase c o n v e r t s virtually all of its a c c u m u l a t e d cephalosporin C to the deacetyl form (Fijisawa et al. P r o d u c t i o n of /3-carotene by Mortierella ramanniana var. T h e superiority of methyloleate as a source of c a r b o n for c e p h a l o s p o r i n C p r o d u c t i o n (Pan et al. p h o s p h a t e exerts its negative effect indirectly by regulating the rate of g l u c o s e c o n s u m p t i o n . or of b e n z o d i a z a p e n e alkaloid biosynthesis in P. (1989) found the g l u c o s e c o n s u m p t i o n rate to be the s a m e at both high and low c o n c e n t r a t i o n s of g l u c o s e . Pencillium cyclopium d o e s not p r o d u c e b e n z o d i a z a p e n e alkaloids if g r o w n in a m e d i u m that contains glucose but d o e s with sorbitol and mannitol ( S c h r ö d e r 1978). m a l t o s e . there is an i m m e d i a t e inhibition of alkaloid synthesis by > 5 0 % . A c A M P .d e p e n d e n t protein kinase appears to m e d i a t e the effect of c A M P on d e v e l o p m e n t in Dictyostelium discoideum (Leichtling et al. is repressible by g l u c o s e .l a c t a m p r o d u c t i o n w a s suppressed. acremonium fermentations with respect to the undesirable degradation of cephalosporin C b y an a c e t y l h y d r o lase ( H i n n e n and N ü e s c h 1976). W h e n the enniatin fermentation (Fusarium sambucinum) is carried out with g l u c o s e (which is rapidly utilized).

A m m o n i u m ion is also r e p r e s s i v e . Addition of NH4" re pre s s e s d e a c e t o x y c e p h a l o s p o r i n C synthetase ( " e x p a n d a s e " ) but not c y c l a s e . P r o d u c t i o n of acetate-derived p h e n o l i c idiolites such as t r i h y d r o x y t o l u e n e by Aspergillus fumigatus is u n d e r nitrogen source control ( W a r d and P a c k t e r 1974).b e n z y l . found that g l u t a m i n e at h i g h e r than 1 m M concentration inhibitied resting cell p r o d u c t i o n of penicillin. acremonium. p h e n y l a l a n i n e . 1 9 8 9 a . In 1984. acremonium (Shen et al. L a r a et al. Resting cells fed 0 . derepressed e x p a n d a s e . b ) .B i a n c o et al. and m e t h i o n i n e . 1984) and P. later w o r k of this g r o u p suggests the o p p o s i t e to b e true. T h e authors considered g l u t a m a t e to be an inducer of the p a t h w a y .5. A d d i t i o n of an a m m o n i u m .a m i n o a d i p a t e . H i g h NH4" also d e c r e a s e d g l u t a m i n e synthetase formation by 8 0 % and the g l u t a m i n e p o o l by 3 3 % but increased the g l u t a m a t e pool by twofold.5 mM N H 4 CI. broth a m m o n i a nitrogen levels r e a c h a m i n i m u m at the t i m e of g l u c o s e e x h a u s t i o n . 1987b) h a v e s h o w n A C V synthetase to b e r e p r e s s e d . T h e extent of d e c r e a s e s h o w s a direct relationship to the a m o u n t of carotenoid synthesized.2 Effectors of Idiolite Biosynthesis 95 sis a n d c A M P level in various m u t a n t s (Kritsky et al. chrysogenum twofold as c o m p a r e d to a control with 8. T h e a b o v e results indicated that g l u t a m i n e increases penicillin synthesis and g l u t a m a t e interferes. along with e x p a n d a s e . a m m o n i u m ions s h o w the m o s t potent negative effect ( S a n c h e z et al. ( 1 9 8 2 ) found g l u t a m a t e to stimulate penicillin p r o d u c t i o n in fermentations to a m u c h greater d e g r e e than did g l u t a m i n e or NH4". 5.g l u t a m a t e also stimulated. the side-chain p r e c u r s o r ( M a r t i n e z . H o w e v e r .t r a p p i n g a g e n t . L . chrysogenum ( S a n c h e z et al. T h e repression of antibiotic p r o d u c t i o n and e x p a n d a s e is a c c o m p a n i e d by high a m m o n i a nitrogen levels in the broth t h r o u g h o u t the fermentation. T h e m o s t r e p r e s sive are t y r o s i n e .2 Nitrogen Source Regulation /3-Lactam formation by C. in contrast. Recent studies ( Z h a n g et al. T h e effect is d u e to the a m i n o acids b e c a u s e the c a r b o n c o m p o u n d s resulting from r e m o v a l of the a m i n o g r o u p are inactive. 1981) is strongly regulated by the nitrogen source used. Inhibition of A C V synthetase by N H ^ is negligible.2. S a n c h e z et al.L . b y a m m o n i u m .A s p a r a g i n e and L-arginine are better nitrogen sources than a m m o n i u m for antibiotic formation (Shen et al. Inhibition w a s not reversed b y the three a m i n o acid p r e c u r s o r s of penicillin.m o n o h y d r o x a m a t e and γ . 1982). and effected a rem a r k a b l e increase in jS-lactam s y n t h e s i s . α . t r y p t o p h a n . G l u t a m a t e an al o g s such as L . total inhibition w a s obtained with 10 m M . especially of c e p h a l o s p o r i n s . In P. tribasic m a g n e s i u m p h o s p h a t e . M a t e o s et al. 1984). . Ammonium c o n c e n t r a t i o n s h i g h e r than 110 m M strongly interfere with c e p h a l o s p o r i n p r o d u c tion by C. ( 1 9 8 1 ) found a high (85 m M ) NH4" level to d e c r e a s e penicillin p r o d u c t i o n by P. A l s o induction of synthesis with light leads to a d e c r e a s e in c A M P d u r i n g the lag p h a s e of p h o t o i n d u c tion. 5 m M g l u t a m i n e in the p r e s e n c e of c y c l o h e x i m i d e increased penicillin p r o d u c tion to a greater d e g r e e than g l u t a m a t e or NH4". chrysogenum. in fermentations c o n d u c t e d with low a m m o n i u m c o n c e n t r a t i o n s .g l u t a m i c acid γ . 1988). certain a m i n o acids repress the u p t a k e s y s t e m of p h e n y l a c - e t a t e . to the high a m m o n i u m fermentation l o w e r e d broth n i t r o g e n .

parasiticus can (Eylar and S c h m i d t 1959. s h o w e d that nitrate represses averufin production w h e r e a s a m m o n i u m favors it. Bikaverin production begins only o n e x h a u s t i o n of the nitrogen s o u r c e . 1977) but these claims h a v e to be carefully evaluated for effects on g r o w t h . T h e y s h o w e d that nitrate represses aflatoxin synthesis also in the parent aflatoxin-producing culture. earlier e n z y m e s w e r e not e x a m i n e d (Orrehed et al. D o x t a d e r and A l e x a n d e r 1966. Shih and M a r t h (1974a) reported that aflatoxin formation by Aspergillus parasiticus is suppressed by a concentration of NH4" that is best for g r o w t h . H o w e v e r .96 Regulation of Secondary Metabolism T h e s e polyketides a p p e a r in batch culture only w h e n nitrogen is e x h a u s t e d from the m e d i u m . Shih et al. w o r k i n g with a n o t h e r b l o c k e d m u t a n t a c c u m u l a t i n g the intermediate averufin. 1974). A d d i t i o n of a m m o n i a c o m p l e t e l y interferes with their production without affecting g r o w t h or p H . alanine. nitrate utilization yields only g r o w t h . It w o u l d be of interest to d e t e r m i n e w h e t h e r h e a v y application of nitrate m i g h t prevent aflatoxin formation in the field and in storage. alternariol and alternariol m o n o m e t h y l ether. (1979) on versicolorin production by a blocked A. bikaverin production is d e c r e a s e d . Bikaverin formation by Gibberella fujikuroi is also u n d e r nitrogen regulation ( B u ' L o c k et al. W i t h regard to m y c o t o x i n p r o d u c e r s . m e t h i o n i n e . T h e addition of natural zeolites (which trap free NH4") to Cephalosporium caerulens relieved cerulenin p r o d u c t i o n of nitrogen source control and thus increased production of this antibiotic ( M a s u m a et al. p r o l i n e . and histidine (for r e v i e w . 1974). nitrate production from NH4" or a m i n o a c i d s ) . urticae can be delayed for hours by provision of too great a concentration of nitrogen source ( G r o o t W a s s i n k and G a u c h e r 1980). T h e s e facts suggest that nitrification might h a v e s o m e role in turning off aflatoxin formation in the p r o d u c i n g o r g a n i s m . see M a g g o n et al. A m i n o acids reported to be stimulatory to aflatoxin formation include a s p a r a g i n e . T h e s e w o r k e r s found that w h e r e a s a m m o n i u m salts support both g r o w t h and p r o d u c t i o n . Versicolorin is an intermediate in the aflatoxin p a t h w a y . T h e i r data indicate that nitrogen source regulation is the m a i n nutritional control of aflatoxin b i o s y n t h e s i s . 1982). by Alternaria alternata. repression w a s not a function of p H c h a n g e s or sugar depletion in the m e d i u m . A clue to the identity of a nitrogen source repressor that is m o r e effective than NH4" w a s u n c o v e r e d by the w o r k of Bennett et al. nitrogen source regulation has been studied mainly in the case of aflatoxin. parasiticus m u t a n t . aspartic acid. Basic studies on fungi h a v e s h o w n that nitrogen source regulation is c o m m o n in these o r g a n i s m s . It is interesting that although most fungi cannot carry out nitrification (that is. careful e x a m i n a t i o n of their data s h o w s only a m i n o r differential effect by NH4" on g r o w t h and p r o d u c t i o n . Aspergillus flavus and A. G l u t a m a t e and urea w e r e also active. If glycine is added to a resting mycelial s u s p e n sion. K a c h h o l z and D e m a i n ( 1 9 8 3 ) . Alternariol-O-methyltransferase w a s repressed by nitrate. flavus strains. Nitrate also interferes with production of the polyketide m y c o t o x i n s . A m m o n i u m (or s o m e other readily used nitrogen source) represses e n z y m e s involved in the use of alternate nitrogen sources such as nitrite . T h e a p p e a r a n c e of patulinforming e n z y m e s by P. W h i t e and J o h n s o n (1982) established a correlation b e t w e e n the p r e s e n c e of nitrification and that of aflatoxin production in A. 1988).

5. N i t r o g e n source repression of uricase in N. N R j " . or g l u t a m a t e ( D e B u s k and Ogilvie 1984a and b ) .a m i n o acid d e a m i n a s e . S o m e areA t y p e m u t a n t s h y p e r p r o d u c e the e n z y m e s . Instead. l o w . nitrate r e d u c t a s e . In a g l u t a m i n e m u t a n t . and the p r e s e n c e of the nit-2 g e n e p r o d u c t ( D e B u s k and O g i l v i e 1984a and b ) . In N. M u t a t i o n s affecting nitrogen source regulation are of t w o m a i n t y p e s . T h e regulatory protein appears to b e active u n d e r c o n d i t i o n s of derepression (for e x a m p l e . and the regulatory protein w o u l d a s s u m e an active c o n f o r m a t i o n and bind at the recognition sites of the structural g e n e s . they p r o b a b l y are m o r e easily c o n v e r t e d to g l u t a m i n e than is a m m o n i a . Its expression requires an inducer (one of m a n y a m i n o a c i d s ) . there exists an o th er type of nitrogen control in w h i c h specific a m i n o acids repress or inhibit production of s e c o n d a r y metabolites b e c a u s e . at p r e s e n t . g l u t a m i n e (but not NH4" or glutamate) repressed e n z y m e sy n t h esi s. that is. high a m m o n i u m s u p p l y ) . an o th er g e n e . A g e n e controlling nitrogen source repression (areA) Aspergillus (Marzluf has b e e n identified in 1981). crassa.2 Effectors of Idiolite Biosynthesis 97 r e d u c t a s e . H o w e v e r . w h e n c o m b i n e d with g l u t a m i n e . In o n e t y p e (for e x a m p l e . and g l u t a m a t e . represses nit-2. In the cases of other nitrogen sources that are m o r e repressive than a m m o n i a . these e n z y m e s c a n n o t be repressed by a m m o n i a . crassa is clearly m e d i a t e d by g l u t a m i n e ( W a n g and M a r z l u f synthetase 1979). T h e s e c o m p o u n d s repress the formation of the nit-2 g e n e p r o d u c t w h i c h acts as a positive control agent for use of p o o r e r nitrogen s o u r c e s . lifting of nitrogen source repression. G l u t a m i n e d o e s not a p p e a r to act directly to repress nit-2. T h e g e n e c o d e s for a regulatory protein exerting positive control on transcription. T areA ). nmr-1 f is t h o u g h t to p r o d u c e a protein that. that is. T h e intracellular effector a p p e a r s to be g l u t a m i n e rather than a m m o n i a itself. T h e r e has b e e n c o n s i d e r a b l e c o n t r o v e r s y as to w h e t h e r proteins such as N A D P g l u t a m a t e d e h y d r o g e n a s e or g l u t a m i n e synthetase play a direct role in nitrogen source repression b u t . thus a l l o w i n g the nit-2 g e n e p r o d u c t to d e r e p r e s s synthesis of nitrogen repressible e n z y m e s . G l u t a m i n e p r o d u c e d in the p r e s e n c e of high NH4" is t h o u g h t to bind to the regulatory protein and d e c r e a s e its affinity to the nitrogen recognition sites on each structural gene controlled by nitrogen source r e p r e s s i o n . they m a y not h a v e to be c o n v e r t e d to a m m o n i a before exerting repression. g l u t a m i n e . a r g i n a s e . a large variety of nitrogen sources can n o longer be utilized for g r o w t h and the e n z y m e s catalyzing their usage c a n n o t b e d e r e p r e s s e d . T h u s m u t a t i o n s of nmr-1 allow p r o d u c t i o n of these e n z y m e s in the p r e s e n c e of g l u t a m i n e . T h e p r e c e d i n g p a r a g r a p h s h a v e dealt with a general nitrogen source repression p h e n o m e n o n . In the s e c o n d type (for d e x a m p l e . areA ). g l u t a m i n e c o n c e n t r a tion w o u l d d r o p . all still require d inducer. NH4" and g l u t a m a t e m a y act via g l u t a m i n e formation. extracellular p r o t e a s e . low a m m o n i u m supply) and inactive u n d e r re pre s s ive c o n d i t i o n s (for e x a m p l e . the data favor g l u t a m i n e itself as b e i n g the m a s t e r effector in fungi. nitrogen source repression is exerted by N R j " . O n e such repressible e n z y m e is an extracellular L . the nmr-1 W h e n g l u t a m i n e is g e n e p r o d u c t is inactive as a repressor. O n N H ^ limitation. and a c e t a m i d a s e . the nit-2 g e n e p r o d u c t is a positive effector for e x p r e s s i o n of structural g e n e s c o d i n g for e n z y m e s involved in the utilization of s e c o n d a r y nitrogen s o u r c e s . g l u t a m a t e d e h y d r o g e n a s e .

A direct relationship w a s o b s e r v e d b e t w e e n penicillin titer and intracellular L . D e m a i n (1957) found that lysine is a suppressor of penicillin synthesis by P.3 m M e x o g e n o u s a m i n o a d i p a t e g a v e the m a x imal r e s p o n s e . ( 1 9 8 7 ) did not. T h i s leaves m o r e available for penicillin formation. L-a- a m i n o a d i p a t e . A concentration of 2 . Inhibition in vitro w a s later o b s e r v e d using a firm lysine a u x o t r o p h derived from the leaky m u t a n t ( L u e n g o et al. on this addition. acremonium and Paecilomyces persicinus ( M e h t a et al. a m i n o a d i p a t e addition stimulated penicillin production but cysteine or valine did not. D e m a i n and M a s u r e k a r (1974) later s h o w e d that the first e n z y m e of lysine b i o s y n t h e s i s .a m i n o a d i p a t e s e e m s to b e important. even with the s u p p l e m e n tation. thus interfering with penicillin b i o s y n t h e s i s . chrysogenum. chrysogenum. and to a s e c o n d a r y m e t a b o l i t e on the other. 1986). B e c a u s e . L u e n g o et al.a m i n o adipate and less c o n v e r s i o n to lysine. an attempt w a s m a d e to reverse lysine inhibition with the latter. T h e m e c h a n i s m s by w h i c h the strains attain different intracellular levels of a m i n o a d i p a t e in u n s u p p l e m e n t e d m e d i u m supporting penicillin synthesis is of great interest. In s o m e c a s e s . 1980). the p r i m a r y e n d . High levels of lysine also interfere with c e p h a l o s p o r i n biosynthesis in C. F o r e x a m p l e . (1980) o b s e r v e d repression but Jaklitsch et al. lysine interferes with penicillin and cephalosporin production by fungi. an e x c e s s of lysine limits production of the c o m m o n i n t e r m e d i a t e . Friedrich and D e m a i n (1977) p r o v e d h o m o c i t r a t e s ynthas e to be the crucial site of the negative effect of lysine on penicillin synthesis by s h o w i n g that inhibition w a s reversed by h o m o c i t r a t e . . B e c a u s e the fungal biosynthetic p a t h w a y to L-lysine involves L-a- a m i n o a d i p a t e as an i n t e r m e d i a t e . A m i n o a d i p a t e w a s found not only to reverse the inhibitory effect of l y s i n e . 1961). is susceptible to lysine inhibition in P. intracellular a m i n o a d i p a t e reached the s a m e intracellular concentration ( 0 . In vivo a c c u m u l a t i o n of h o m o c i t r a t e w a s m a r k e d l y d e - p r e s s e d by lysine addition to an early b l o c k e d lysine b r a d y t r o p h i c m u t a n t .a .p r o d u c t feedback regulates the c o m m o n part of the p a t h w a y and thus inhibits idiolite b i o s y n t h e s i s .a m i n o a d i p a t e and (2) the level of the penicillin s y n t h a s e s . In both g r o w i n g and resting c e l l s . B e c a u s e penicillin G and lysine are products of a b r a n c h e d biosynthetic p a t h w a y .a .a m i n o adipate in a m e d i u m supporting penicillin production (Jaklitsch et al. H ö n l i n g e r and K u b i c e k (1989) found that i m p r o v e d strains s h o w r ed u ced c a t a b o l i s m of a . 1979) ( D ' A m a t o and P i s a n o 1976). the level of penicillin synthases m u s t also be a factor in penicillin p r o d u c t i o n . but also to stimulate penicillin synthesis in the a b s e n c e of a d d e d lysine ( S o m e r s o n et al. N o o t h e r a m i n o acid in the pool s h o w e d such a correlation.98 Regulation of Secondary Metabolism they are derived from the s a m e b r a n c h e d p a t h w a y and exert feedback control of that b r a n c h e d p a t h w a y . It did not a p p e a r to be a function of the level of h o m o c i t r a t e sy n t h ase but modification of the flux of α . Penicillin p r o d u c i n g ability in a series of four P. 2 5 m M ) in all four cultures yet the cultures s h o w e d different absolute rates of penicillin p r o d u c t i o n . S u c h p a t h w a y s h a v e an early c o m m o n part that then b r a n c h e s t o the synthesis of a p r i m a r y metabolite on the o n e h a n d . W h e t h e r or not lysine also represses the e n z y m e is c o n troversial. the p r o d u c t of the h o m o c i trate synthase reaction. h o m o c i t r a t e s y n t h a s e . chrysogenum strains differing in penicillin p r o d u c t i o n appeared to be a function of (1) the intracellular c o n c e n tration of L .

5.3 0 0 m M generally supports e x t e n s i v e cell g r o w t h .2. 1988). and e x p a n d a s e . 1982) is subject to p h o s p h a t e c o n t r o l . chrysogenum i m m o b i l i z e d on C e l i t e . w a s repressed b y p h o s p h a t e . but c o n c e n t r a t i o n s of 10 m M and a b o v e suppress the biosynthesis of many secondary metabolites. 1982). c A M P d e p e n d e n t and c A M P i n d e p e n d e n t . acremonium W 53253.l i m i t i n g nutrient in m a n y s e c o n d a r y m e t a b o l i t e f e r m e n t a t i o n s .c y c l a s e in Claviceps S D 5 8 (Erge et al. S t u d y i n g C . e x p a n d a s e b y 6 0 % . F u n g i contain t w o types of protein k i n a s e s . P h o s p h a t e in the r a n g e of 0 . acremonium C W . 5.1 9 . acremonium C-10. H i g h p h o s p h a t e concentrations did not increase the g l u c o s e c o n s u m p t i o n r a t e .p r o d u c i n g strain c o n t a i n s less intracellular A T P during production and less p o l y p h o s p h a t e t h r o u g h o u t the fermentation than the low p r o d u c e r ( N a v a s h i n et al.2 5.3 Effectors of Idiolite Biosynthesis 99 Phosphorus Source Regulation P h o s p h a t e is the crucial g r o w t h .1 . B i k a v e r i n p r o d u c t i o n is d e p r e s s e d by levels of inorganic p h o s p h a t e that h a v e n o inhibitory effect on g r o w t h ( B r e w e r et al. 1973). P a z o u t o v a et al. A C V s y n t h e t a s e . (1982) found that p h o s p h a t e interferes with resting cell p r o d u c t i o n of c e p h a l o s p o r i n C and penicillin Ν in the a b s e n c e of g l u c o s e in C. Martin et al. A T P .2. Kuenzi ( 1 9 8 0 ) c o n c l u d e d that e x c e s s p h o s p h a t e exerts its negative effect indirectly by increasing the rate of g l u c o s e c o n s u m p t i o n and thus e n h a n c i n g carbon s o u r c e r e p r e s s i o n . 1978. A C V synthetase by 5 0 % . 1973). T h e action of these e n z y m e s w a s also inhibited by p h o s p h a t e . T h e a m i n o . h a s b e e n s h o w n to repress the first e n z y m e u n i q u e to alkaloid b i o s y n t h e s i s ( d i m e t h y l a l l y l t r y p t o p h a n synthetase) (Krupinski et al. c y c l a s e . 1983). It h a s b e e n reported that the biosynthesis of /3-lactams by fungi (Martin et al.4 Enzyme Induction M e t h i o n i n e exerts a m a r k e d stimulation of cephalosporin C formation as well as that of penicillin Ν in C. A n important question is w h e t h e r intracellular o r t h o p h o s p h a t e is the ultimate effector or w h e t h e r it m e r e l y regulates the level of s o m e other intracellular effector that controls expression of antibiotic b i o s y n t h e s i s . Z h a n g et al. that is. A m u t a n t of Fusidium coccineum p r o d u c i n g 10 t i m e s m o r e fusidic acid than a l o w . It is also possible that p h o s p h a t e control is exercised on e n z y m e activity by p h o s p h o r y l a t i o n a n d d e p h o s p h o r y l a t i o n via protein kinases a n d p h o s p h o p r o t e i n p h o s p h a t a s e s ( K r e b s and B e a v o 1979). w h i c h at high levels totally inhibits alkaloid formation ( R o b b e r s et al. E x p a n d a s e is also the m o s t repressed e n z y m e of the t h r e e . H o w e v e r . 1976) as well as c h a n o c l a v i n e . but strongly suppressed /3-lactam p r o d u c t i o n . acremonium ( D r e w and D e m a i n 1973 and 1977). (1988) recently studied the p h o s p h a t e effect on /3-lactam biosynthesis by C. and c y c l a s e by 4 5 % in the c a s e of 5 0 m M p h o s p h a t e . formation of all three synthetases exa m i n e d . P h o s p h a t e limitation w a s an important p a r a m e t e r in design of a fluidized b e d p r o c e s s e m p l o y i n g cells of P. R e p e a t e d fed-batch operation a l l o w e d stable and p r o l o n g e d production of penicillin (over 1 m o n t h ) ( O h et al. Inorganic p h o s p h a t e . 3 . for e x a m p l e . and suggested that p h o s p h a t e itself d e c r e a s e s the overall flux t h r o u g h the c e p h a l o s p o r i n biosynthetic p a t h w a y .

septated. M a t s u m u r a et a l . Q u e e n e r and Ellis 1975.100 Regulation of Secondary Metabolism acid is not required for g r o w t h although it can be used as a sole source of nitrogen or sulfur. c y s t a t h i o n i n e .^ h o m o c y s t e i n e —^cystathionine —^cysteine —»Cephalosporin C Î serine ι a-ketobutyrate+NH3 H o w e v e r . both isomers of norleucine are active and the D-form is the more potent of the two. in a defined sulfate-containing med i u m. and fragmentation of the m y c e l i a . T h e greater activity of the D-form for antibiotic synthesis is evidently d u e to its slower u p t a k e and lesser availability for intracellular d e g r a d a t i o n . H i g h levels of m e t h i o n i n e (—0. T h i s difference is particularly significant in v i e w of the finding that initiation of antibiotic synthesis in m e t h i o n i n e . and m o r e highly fragmented. that is. 1976. These studies strongly suggested that the mechanisms of action of methionine and norleucine are identical. O n e of the earliest reasons for questioning m e t h i o n i n e ' s role as solely that of a sulfur d o n o r w a s the inability of other sulfur c o m p o u n d s fully to replace m e t h i o n i n e for c e p h a l o s p o r i n C b i o s y n t h e s i s . N ü e s c h et al.i s o m e r is m o r e effective than the L-form for antibiotic formation. h o m o c y s t e i n e . m e t h i o n i n e . that is. M e t h i o n i n e sulfur 35 is an efficient p r e c u r s o r of the sulfur a t o m of cephalosporin C.5%) m u s t be added to achieve o p t i m u m s y n t h e s i s . irregular. and fragmented h y p h a e (arthrospores) are obtained with m e t h i o n i n e . Of particular i m p o r t a n c e w a s the low activity of the postulated intermediates b e t w e e n m e t h i o n i n e and c e p h a l o s p o r i n C . septation. and cystathionine yield filamentous m y c e l i a w h e r e a s s w o l l e n .g r o w n m y c e l i a are s w o l l e n . T h e high r e q u i r e m e n t is p r o b a b l y the result of m e t h i o n i n e d e g r a d a t i o n by the intracellular a m i n o acid oxidase(s) ( N ü e s c h et al. As with methionine. W h e r e a s m y c e l i a g r o w n in sulfate are filamentous. . before antibiotic synthesis c o m m e n c e s . acremonium can use the nonsulfur analog norleucine as a methionine replacement. D r e w et al. (1973) found that c y s t e i n e . A n o t h e r reason w a s that m e t h i o n i n e exerts its major effect on c e p h a l o s p o r i n C synthesis w h e n a d d e d d u r i n g g r o w t h . O n e of the m o r e interesting findings c o n c e r n i n g m e t h i o n i n e and n o r l e u c i n e s u p p l e m e n t a t i o n is the m o r p h o l o g i c a l effect that results (Nash and H u b e r 1 9 7 1 . T h e D . Because norleucine has no sulfur. L . as described b e l o w . 1980a). sulfur donation is not the reason for stimulation by methionine. Further suspicion concerning the role of methionine as strictly a sulfur donor c a m e from the observation that. 1973). methionine stimulation of cephalosporin production apparently is not due to sulfur donation. and cysteine ( D r e w and D e m a i n 1975a). Early studies ( r e v i e w e d by D e m a i n 1974) on penicillin Ν formation s h o w e d that neither h o m o c y s t e i n e nor cysteine could replace m e t h i o n i n e . T h i s o b s e r v a t i o n suggests s o m e sort of a regulatory effect such as e n z y m e d e r e p r e s s i o n . C.[ S ] m e t h i o n i n e a p p e a r s to b e incorporated into the antibiotic without dilution by the reverse transsulfuration p a t h w a y : m e t h i o n i n e . h o m o c y s t e i n e .c o n t a i n i n g m e d i a coincides with the t h i c k e n i n g . Results of D r e w and D e m a i n ( 1 9 7 5 b and c) using sulfur a m i n o acid a u x o t r o p h s .

M e t h i o n i n e addition increases p r o d u c t i o n in both parent and m u t a n t but the m u t a n t requires less for optimal p r o d u c t i o n ( 0 . (2) induction of c e p h a l o s p o r i n C synthetases by m e t h i o n i n e . Exploitation of m e t h i o n i n e control has been a c c o m p l i s h e d by the isolation of a s e l e n o m e t h i o n i n e .2 of C. and in the control m e d i u m of 2 .m e t h i o n i n e rather than 4 g/1). acremonium Effectors of Idiolite Biosynthesis 101 s u p p o r t e d the i m p o r t a n c e of e x o g e n o u s m e t h i o n i n e as a regula- tory effector of c e p h a l o s p o r i n biosynthesis and furthermore e m p h a s i z e d the imp o r t a n c e of e n d o g e n o u s m e t h i o n i n e in fermentations c o n d u c t e d without e x o g e n o u s m e t h i o n i n e .5. 3 % g l u c o s e . ( 1 9 8 0 b ) . D r e w and D e m a i n (1975c) had already s h o w n that a m u t a n t b l o c k e d in the reversed transsulfuration p a t h w a y still r e s p o n d e d to m e t h i o n i n e . 6 % s u c r o s e . and (3) catabolite repression of these e n z y m e s by g l u c o s e . or both. in 6 . 1980). T h e y also found that the specific c e p h a l o sporin C p r o d u c t i o n w a s proportional to the intracellular m e t h i o n i n e c o n c e n t r a t i o n . Treichler et al. D e s p i t e this. for e x a m p l e . 7 % g l u c o s e plus 3 . ( 1 9 7 9 ) w e r e m a i n l y in a g r e e m e n t with the a b o v e results of D r e w and D e m a i n . T h e data of M a t s u m u r a et al. cyclase and e x p a n d a s e ( S a w a d a et al. In both the control m e d i u m and the sucrose m e d i u m . It is not k n o w n w h e t h e r this is d u e to an effect on m e t h i o n i n e u p t a k e . T h e intracellular A C V c o n c e n t r a t i o n has b e e n s h o w n to be increased by m e t h i o n i n e ( A l o n s o and L u e n g o 1987). T h e y stated that "it appears that the c l e a v a g e of cystathionine constitutes an essential step in the p r i m a r y m e t a b o l i s m . A t t e m p t s to replace cysteine with m e t h i o n i n e or cystathionine using c r u d e extracts have all been negative (Zhang et al. m e t h i o n i n e b i o s y n t h e s i s . intracellular m e t h i o n i n e and c e p h a l o s p o r i n C p r o d u c t i o n w e r e high. indeed . 5 g/1 D L . is crucial for the m e t h i o n i n e effect. T h e major b l o w to the cystathionine c o n c e p t w a s the finding that cysteine itself is required in the cell-free A C V synthetase reaction ( B a n k o et al. Furthermore. and this reaction m a y also act as an ' i n d u c e r ' of the transfer of the cysteine m o i e t y into the p a t h w a y of s e c o n d a r y m e t a b o l i s m . D r e w and D e m a i n favored m e t h i o n i n e acting as an inducer of o n e or m o r e /3-lactam s y n t h e t a s e s .l y a s e . (1979) felt that cystathionine γ . both w e r e very l o w . ( 1 9 8 0 b ) suggest that o n e effect of the c a r b o n source is exercised via its control of the b u i l d u p of intracellular m e t h i o n i n e prior to antiobiotic sy n t h esi s. unpublished). 1978a) and to a lesser extent.to threefold increase in the e n d o g e n o u s pool of m e t h i o n i n e j u s t prior to c e p h a l o s p o r i n C formation. " H o w e v e r . T h e data of Treichler et al. 1982). T h e s e w o r k e r s noted a transient t w o .. 1986). m e t h i o n i n e has b e e n found to i n d u c e A C V synthetase ( Z h a n g et al. (1981 ) b a s e d on (1) m o r p h o l o g i c a l differentiation to the active f o r m . A kinetic m o d e l of the c e p h a l o s p o r i n C fermentation w a s d e v e l o p e d by M a t s u m u r a et al. swollen h y p h a l fragments. 3 % s u c r o s e . Of particular interest w a s the effect of g r o w t h in 6 . T h e m o d e l w a s tested in a fed-batch culture with feeding of glucose and m e t h i o n i n e and resulted in a 3 0 % increase in c e p h a l o s p o r i n C p r o d u c t i o n .r e s i s t a n t m u t a n t that p r o d u c e s three times m o r e c e p h a l o s p o r i n C than its parent in a sulfate m e d i u m lacking m e t h i o n i n e ( M a t s u m u r a et al. an e n z y m e in the reverse transsulfuration p a t h w a y . the t w o g r o u p s c a m e to s o m e w h a t different c o n c l u s i o n s . in g l u c o s e . A postulation that ties together m e t h i o n i n e stimulation of antibiotic b i o s y n t h e sis with c a r b o n regulation w a s m a d e by M a t s u m u r a et al.

Production of the m y c o t o x i n is stimulated b y increasing concentrations of glucose and the onset of the i d i o p h a s e is . T a b e r 1 9 6 3 . acremonium w a s isolated by Q u e e n e r et al. T h i s c o n c e p t w a s supported by data s h o w i n g stimulation by t r y p t o p h a n an al o g s that w e r e not alkaloid pre c urs or s ( A r c a m o n e et al. A n o t h e r selenomethionine-resistant m u t a n t of C . (1976) that t r y p t o p h a n induces d i m e t h y l a l l y t r y p t o p h a n s y n t h e t a s e . later e n z y m e s appear as a result of coordinate induction by 6- methylsalicylic acid. Induction of a k e y e n z y m e by t r y p t o p h a n w a s postulated by Floss and M o t h e s (1964) and B u ' L o c k and Barr ( 1 9 6 8 ) . 1982).O l m e d o 1976). Several g r o u p s h a v e reported on the stimulation of ergot alkaloid synthesis in Claviceps by the precursor tryptophan (Floss and M o t h e s 1964. It is interesting that c o m p a r t m e n t a t i o n of p h e n y l a l a n i n e exists in this o r g a n i s m so that e x o g e n o u s p h e n y l a l a n i n e m a i n l y g o e s to protein w h e r e a s e n d o g e n o u s p h e n y l a l a n i n e is p r e d o m i n a n t l y incorporated into alkaloids ( N o v e r et al.1 3 has a high intracellular pool of m e t h i o n i n e [5 μmo\/g of D C W ( D C W = dry cell weight)] and cystathionine ( 1 . B u ' L o c k and Barr 1968). A n o t h e r p r e c u r s o r that functions as an inducer is p h e n y l a l a n i n e in p r o d u c t i o n of b e n z o d i a z a p e n e alkaloids by P. 1983). Studies on aflatoxin formation by A. It is interesting that o n c e f o r m e d . V i n i n g and N a i r 1966. D L . P r o d u c t i o n of carotene in Phycomyces blakesleeanus is induced by light. Floss and M o t h e s 1964. it w a s s h o w n by Krupinski et al. parasiticus indicate that a p r o d u c t of g l u c o s e m e t a b o l i s m induces the aflatoxin p a t h w a y . the first being nitrogen (or growth-rate) repressible and the r e m a i n d e r inducible ( G a u c h e r et al. 1 9 6 1 . T h e c o n c e p t of induction of s e c o n d a r y metabolic e n z y m e s prior to idiophase m e t a b o l i s m in the patulin fermentation w a s first suggested by B u ' L o c k et al. 2 μ π ι ο ί cystathionine/g of D C W . A n e v e n m o r e effective inducer of alkaloid biosynthesis is the t r y p t o p h a n a n a l o g u e DL-jS-2-naphthylamine ( R o b b e r s et al. 4 μ π ι ο ΐ / g of D C W ) but n o L-cysteine w a s found in either m u t a n t or parent strain. the first three metabolites of the p a t h w a y (6-methylsalicylic acid. and /3-ionone (Murillo and C e r d a . S o m e 4 h later. and mh y d r o x y b e n y l a l c o h o l ) function as inducers for the rest of the p a t h w a y .N o r l e u c i n e stimulates both strains and again the m u t a n t requires less (1 g/1 instead of 4 g/1). F a c t o r Β is a polar substance with a m o l e c u l a r m a s s of 2 4 2 D a . Finally. that stimulate arthrospore and c e p h a l o s p o r i n C p r o d u c t i o n ( N a t s u m e acremonium and M a r u m o 1984). vitamin A . the first e n z y m e of the p a t h w a y . ( 1 9 6 9 ) . R o b b e r s and Floss 1970) and by timing studies on tryptophan addition (Floss and M o t h e s 1964. T h e parent contains only a trace of m e t h i o n i n e and 0 . T h e effect is eliminated b y c y c l o h e x i m i d e . T h u s it appears that all the e n z y m e s are i d i o p h a s i c . 1979). L-Cysteine d o e s not e n h a n c e p r o d u c t i o n by either strain.Regulation of Secondary Metabolism 102 4 g/1 m e t h i o n i n e is inhibitory to the m u t a n t . G r o o t W a s s i n k and G a u c h e r (1980) later s h o w e d that the initial e n z y m e is formed at the e n d of t r o p h o p h a s e as the result of nitrogen source e x h a u s t i o n . M u t a n t S M R . cyclopium ( L u c k n e r et al. the p r o d u c t of the first e n z y m e . (1984) that p r o d u c e d 1 7 % m o r e c e p h a l o s p o r i n C in a sulfate-based m e d i u m than its parent. T h e m u t a n t takes u p sulfate 5 0 % faster than its parent but n o effect on m e t h i o n i n e u p t a k e w a s o b s e r v e d . T w o synergistic factors (A and B) h a v e b e e n isolated from the filtrate of C . m . V i n i n g 1970). 1977).c r e s o l .

a r a b i n o h e p t u l o s o n a t e 7 . Induction or activation of anthranilate s y n t h a s e . 0 1 h _ 1 . In batch cultures of G. A s e c o n d a r y factor is induction ( G a u c h e r et al. T h e effect w a s not specific for g l u c o s e . and p h o s p h o r u s . Anthranilate synthase is the most limiting e n z y m e of the p a t h w a y and is increased by 1 4 0 % .2 Effectors of Idiolite Biosynthesis 103 a c c o m p a n i e d b y an increased rate of g l u c o s e c o n s u m p t i o n ( A p p l e b a u m and B u c h a n a n 1979). the k e y e v e n t c o u l d be cessation of g r o w t h or low g r o w t h rate. T h e a p p e a r a n c e s of the first and fourth e n z y m e s h a v e b e e n s h o w n to be d u e to d e n o v o biosynthesis of R N A and protein. that i s .5. In contrast to g l u c o s e . it m a y also involve g r o w t h rate. 1983). s u c r o s e . and a c e t a t e . E n z y m e s of ergot alkaloid biosynthesis are synthesized in the idiophase after p h o s p h a t e is depleted from the m e d i u m of certain ergot alkaloid p r o d u c i n g strains. and glycerol ( A b d o l l a h i and B u c h a n a n 1981b).d e o x y . and finally gibberellin is m a d e (70 h ) . . o l e a t e .p h e n y l a l a n i n e and a n t h r a n i l a t e . At this latter dilution r a t e .5 Growth Rate T h e m e c h a n i s m s b y w h i c h idiolite p r o d u c t i o n is frequently d e l a y e d until the e n d of the t r o p h o p h a s e involve repression and inhibition of the e n z y m e s of s e c o n d a r y m e t a b o l i s m d u r i n g g r o w t h (as described in the p r e c e d i n g sections) by sources of c a r b o n . B e c a u s e o t h e r m e a n s of stopping g r o w t h also bring on patulin f o r m a t i o n .i d i o p h a s e shift is the result of g r o w t h cessation at 2 0 . n i t r o g e n . and c h o r i s m a t e m u t a s e o c c u r r e d on addition of the alkaloid c y c l o p e n i n . bikaverin is not p r o d u c e d at all ( B u ' L o c k et al. 5. W h e n p e p t o n e . Inactive w e r e lactose.p h o s p h a t e ( D A H P ) s y n t h a s e . T h e onset of s e c o n d a r y m e t a b o l i s m is b r o u g h t on by g r o w t h cessation c a u s e d by nitrogen d e p l e t i o n . Induction o c c u r r e d w h e n at least 1 g of g l u c o s e per liter w a s a d d e d b e t w e e n 8 and 18 h after inoculation ( W i s e m a n and B u c h a n a n 1987). In patulin b i o s y n t h e s i s by P. 3 . g r o w t h o c c u r s first ( m a x i m u m rate at 18 h ) . T h e batch culture s e q u e n c e a p p e a r s to be c a u s e d by the effect of g r o w t h rate on e x p r e s s i o n of the p a t h w a y g e n e s . T h a t this is not d u e to nitrogen source derepression w a s s h o w n by a d d i n g p e p t o n e to the g l u c o s e r e p l a c e m e n t culture and o b s e r v i n g even better p r o d u c t i o n than in g l u c o s e a l o n e . m a l t o s e . I n d u c t i o n in filamentous fungi a p p e a r s to o c c u r by positive control (Arst 1981). cyclopium ( R o o s and S c h m a u d e r 1989).2.D . In the c h e m o s t a t . and seventh e n z y m e s a p p e a r . T h e s e alkaloids are m a d e from L . 1974). fujikuroi. p e p t o n e as c a r b o n source allows g o o d g r o w t h but n o aflatoxin formation ( A b d o l l a h i and B u c h a n a n 1981a). urticae. citrate. fructose. bikaverin is formed best at a dilution rate of 0 . p y r u v a t e . fourth.g r o w n cells are a d d e d to a r e p l a c e m e n t culture containing g l u c o s e . m a n n o s e . 0 5 h _ 1 w h e r e a s gibberellin is m a d e at 0 . the p r o d u c t of the regulator g e n e is required for transcription of inducible enzymes. lactate. the t r o p h o p h a s e . then b i k a v e r i n is synthesized (at 4 0 h ) . F e e d b a c k induction or activation.2 1 h and the a p p e a r a n c e of the first e n z y m e of the p a t h w a y (6-methylsalicylic acid synthetase) at that s a m e t i m e . an u n u s u a l type of regulation. aflatoxin synthesis o c c u r s . s o r b o s e . H o w e v e r . also active w e r e ribose. A b o u t 4 h later. is exerted on e n z y m e s of a r o m a t i c b i o s y n t h e s i s by b e n z o d i a z a p e n e alkaloids in P. the s e c o n d . raffinose.

and terminal differentiation in m a m m a l i a n cell culture. n o conidiation occurred. and S. c o p p e r . O t h e r investigators h a v e reported stimulatory effects of c a d m i u m . not only G T P .2. iron. c o b a l t . sorghi (Vining 1973). 1976). T h u s on a defined m e d i u m supporting only a slow g r o w t h rate. H o w e v e r . declines in G T P levels are k n o w n to trigger sporulation in Bacillus and yeast. fusiformis. stimulation of cell proliferation in animal cells and yeast by ras proteins requires b i n d i n g of G T P by such proteins. m o l y b d e n u m . 5. crassa causes a m a r k e d decline in G T P p o o l s and in the G T P / A T P ratios. This d o e s not o c c u r on nitrogen deprivation in the nitrogen control m u t a n t nit-2 or on sulfur deprivation in the sulfur control m u t a n t cys-3.104 Regulation of Secondary Metabolism for e x a m p l e .6 Guanine Nucleotides N i t r o g e n or sulfur limitation of N. (1968) with C. t r o p h o p h a s e and idiophase o v e r l a p but with rapid g r o w t h on c o m p l e x m e d i u m . urticae. A l t h o u g h conidia formation by P. t r o p h o p h a s e and idiophase o c c u r at different t i m e s . unlike the other fungi w h i c h n o r m a l l y display a distinct t r o p h o p h a s e and idiophase. several observations d o not fit into a simple nutrient depletion —» low G T P —> high p p G p p —» differentiation h y p o t h e s i s . It has been p r o p o s e d that G T P exerts a general positive control of g r o w t h in living s y s t e m s (Pall and Robertson 1988).3 5.1 OTHER FACTORS Metals In the aflatoxin fermentation. A specific effect of m a n g a n e s e occurs on patulin synthesis by P. and c a l c i u m (for review see M a g g o n et al. It is an open question w h e t h e r these idiophase events are b r o u g h t about strictly by nutrient exhaustion or primarily by low g r o w t h rate b r o u g h t on by p h o s p h a t e e x h a u s t i o n . S e c o n d . b o r o n . purpurea. T h i s fungus usually p r o d u c e s ergot alkaloids during g r o w t h .p h a s e d y n a m i c s w h e n it is g r o w n on a c o m p l e x m e d i u m supporting a high g r o w t h rate. w h e n g r o w n in the p r e s e n c e of the inducer t r y p t o p h a n . m a n g a n e s e . zinc is needed at a concentration greater than that required for g r o w t h to get high production of the m y c o t o x i n ( G u p t a et al. v e r s i c o l o r s A (Failla and N i e h a u s 1986). C. 1977) but these claims h a v e to be carefully evaluated for effects on g r o w t h . C. In support of such a role. paspali can b e forced into t w o . e v e n C. w e r e depleted and n o g u a n o s i n e p e n t a p h o s p h a t e ( p p G p p ) a c c u m u l a tion o c c u r r e d . furthermore. T h e metal m u s t be present during early vegetative g r o w t h ( 2 0 . 1988). 5. paspali. T h a t the g r o w t h rate m a y play a key role is indicated by the w o r k of Brar et al. T h e zinc effect also w a s observed in a strain a c c u m u l a t i n g the i n t e r m e d i a t e . T h e first observation is that all n u c l e o t i d e s . on exhaustion of p h o s p h a t e and a d e c r e a s e in G T P .3 0 h) to exert its stimulation during idiophase (50 h ) . in a high p e p t o n e m e d i u m . T h e effect is strictly on secondary m e t a b o l i s m and involves the p a t h w a y e n z y m e s after . T h e s e data d e m o n s t r a t e that nit-2 and cys-3 control s y s t e m s are not limited to control of c a t a b o l i s m of e x o g e n o u s nitrogen or sulfur sources but affect a w i d e r r a n g e of cellular properties.3. chrysogenum followed e x h a u s t i o n of p h o s phate and a severe d r o p in G T P in a low p e p t o n e m e d i u m ( T s u r u m i et al.

T h e derepression can be b l o c k e d by c y c l o h e x i m i d e or a c t i n o m y c i n D ( N i e h a u s 1989). during the t r e a t m e n t . parasiticus blocked mutant. the cell loses its viral c o m p o n e n t .5 Temperature T e m p e r a t u r e s l o w e r than that supporting o p t i m u m g r o w t h are required for m a x i m u m p r o d u c t i o n of enniatins ( A u d h y a and Russell 1974). O n the other h a n d . for a t e m p e r a t u r e downshift to derepress versicolorin biosynthesis in A. T h e effect a p p e a r s to be d u e to increased chitin synthesis by the m y c e l i a ( E d w a r d s a n d H o 1988). M n uptake o c c u r s p r e d o m i n a n t l y d u r i n g the t r o p h o p h a s e .s t r a n d e d R N A synthesis. Aflatoxin formation is stimulated not only by zinc but also by low t e m p e r a t u r e (Shih and M a r t h 1974a). 1986).4 Cessation of Biosynthesis 105 2+ 6-methylsalicylic acid s y n t h e t a s e . T h e y both are involved in controlling transcription.3. chrysogenum.3. prior incubation with Z n is required.3 Carbon Dioxide Penicillin formation by P. sanbucinum ( A u d h y a and Russell 1974).5.3. 1986). 1977). .s t r a n d e d R N A in A. chrysogenum is inhibited by C 0 2 w h i c h also causes stunting and swelling of m y c e l i a . 5.6 Viruses Viral d o u b l e . higher levels of b r a n c h i n g . flavus interferes with aflatoxin formation ( S c h m i d t et al. 5. 5.i d i o p h a s e shift (Scott et al. T h e effect is on transcription. light stimulates the formation of enniatins by F. that is.2 Oxygen H i g h levels of o x y g e n that p r o v i d e g o o d g r o w t h are inhibitory to the aflatoxin fermentation (Shih and M a r t h 1974b). 5.4 CESSATION OF BIOSYNTHESIS T h e r e are t w o k n o w n r e a s o n s for the cessation of idiolite biosynthesis: (1) irreversible d e c a y of o n e or m o r e e n z y m e s of the antibiotic-synthesizing p a t h w a y and (2) a feedback effect of the a c c u m u l a t e d p r o d u c t .4 Light C o n s i d e r a b l e c o n t r o v e r s y exists c o n c e r n i n g the possible negative effect that light has on the formation of aflatoxins (see Bennett et al.3. A n o n t o x i g e n i c strain harboring this virus is c o n v e r t e d to aflatoxin p r o d u c t i o n by addition of inhibitors of viral d o u b l e . 5.3. S u c h a cell can be c o n v e r t e d b a c k to n o n p r o d u c t i o n by incubation with a similar virus from P. 5. Aflatoxin degradation m a y play a role in this p h e n o m e n o n ( M a g g o n et al. 1981). and l o w e r g r o w t h rates.

1974. Later e n z y m e s . F o r e x a m p l e .c a r o t e n e is inhibited genetically or c h e m i c a l l y .4. T h e half-life w a s increased twelvefold. 1988). F e e d b a c k repression d o e s not a p p e a r to b e important.1 Regulation of Secondary Metabolism Synthetase Decay T h e m a i n c a u s e of cessation of patulin production of P. E l y m o c l a v i n e also inhibits a later e n z y m e . 6 5 % goes for m a i n t e n a n c e and 2 5 % for g r o w t h ( H e r s b a c h et al. C h e n g et al. the r e d u c i n g agent dithiothreitol. h a v e l o n g e r half-lives (17 and 19 h . urticae required a c o m b i n a t i o n of the cofactor N A D P H .106 5. T h e s a m e is p r e s u m a b l y true for c e p h a l o s p o r i n p r o d u c t i o n . T h i s affects alkaloid production b e c a u s e trypt o p h a n is both a precursor and an inducer of alkaloid b i o s y n t h e s i s . 1984). 6-methylsalicylic acid synthetase. w h i c h has an in vivo h a l f .m a x i m a l lifetime of 7 h ( N e w a y and G a u c h e r 1981). In vitro stabilization of 6-methylsalicylic acid synthetase in crude extracts from P. 5. M a n y s e c o n d a r y metabolites inhibit or repress their o w n b i o s y n t h e s i s .4. dimethylallyltryptophan synthetase. Heinstein and Floss 1976. in Pénicillium stoloniferum.O l m e d o 1976). m y c o p h e n o l i c acid ( M u t h and N a s h 1975) limits its o w n synthesis b y inhibiting the final e n z y m e . an O . 0 0 0 mg/1 t o d a y . Yet production of penicillin is still a relatively inefficient process even with c o m m e r c i a l strains of P. helping to raise titers from < 5 mg/1 in 1940 to > 3 0 .C o A ) and m a l o n y l C o A . the substrates a c e t y l . /3-Carotene regulates its o w n synthesis by repression in P. T h i s suggests that in v i v o conformational integrity and proteolysis are important ( L a m et al. c h a n o c l a v i n e . m . p h e n y l m e t h y l sulfonyl fluoride ( P M S F ) . anthranilate synthetase. w h i c h is inhibited b y agroclavine and e l y m o c l a v i n e (Floss et al. T h i s important e n z y m e is inhibited by e l y m o c l a v i n e and c h a n o c l a v i n e ( S c h m a u d e r and G r ö g e r 1976.1 -cyclase (Erge et al. F e e d b a c k inhibition of ergot alkaloid biosynthesis by Claviceps o c c u r s at the first s t e p .c o e n z y m e A ( a c e t y l . A s e c o n d type of feedback inhibition in the ergot alkaloid p a t h w a y involves inhibition of t r y p t o p h a n synthesis at the first e n z y m e of the tryptophan biosynthetic p a t h w a y . M a n n and Floss 1977). and the protease inhibitor.m e t h y t r a n s f e r a s e .5 IMPROVEMENT OF IDIOLITE PRODUCTION Empirical mutation/screening p r o g r a m s h a v e had profound effects o n the p r o d u c tion of penicillin.h y d r o x y b e n z y l alcohol d e h y d r o g e n a s e (the 4th) and i s o e p o x y d o n d e h y d r o g e n a s e (the 7 t h ) . respectively). 5. chrysogenum. urticae is the d e c a y of the first e n z y m e .2 Feedback Regulation T h e role of feedback regulation in controlling secondary m e t a b o l i s m is well k n o w n . T h e g e n e carS is thought to c o d e for a repressor protein b e c a u s e m u t a t i o n s in this g e n e lead to o v e r p r o d u c t i o n of c a r o t e n e . 1980). 1973). This is indicated by the e n o r m o u s a c c u m u l a t i o n of precursors w h e n formation of ß . blakesleeanus (Murillo and C e r d a . O n l y 1 0 % of the c o n s u m e d c a r b o n source in a fermentation e n d s u p as penicillin.

It is thus o b v i o u s that studies on the regulation of s e c o n d a r y m e t a b o l i s m are beneficial for further i m p r o v e m e n t of these i m p o r t a n t fermentation p r o c e s s e s . I m p r o v e m e n t of penicillin p r o d u c t i o n by c o n v e n t i o n a l strain improvement results from both g e n e amplification and increases in g e n e e x p r e s s i o n . (1986) reported that a superior C . C y c l a s e m R N A in B W . O n the o t h e r h a n d . and penicillin Ν w a s d e c r e a s e d by fifteenfold. 1989). acremonium e x p a n d a s e / h y d r o x y l a s e g e n e into the p r o d u c t i o n strain C. acremonium strain p r o d u c e d h i g h e r levels of c y c l a s e and e x p a n d a s e than its ancestral strain and that e x p a n d a s e formation in the superior strain w a s less subject to c a r b o n source r e p r e s s i o n . in flasks.1 9 5 1 (Smith et al. p e n icillin p r o d u c t i o n b e g a n at about 4 0 h . as a c e t o h y d r o x y a c i d s y n t h a s e is the first e n z y m e of valine b i o s y n t h e s i s and valine is n e e d e d as a p r e c u r s o r of penicillin and c e p h a l o s p o r i n in very high a m o u n t s . Improvement of Idlolite Production 107 I m p r o v e m e n t of both titer and c o n v e r s i o n yield has b e e n a c o n s t a n t battle but s u c c e s s h a s b e e n a c c o m p l i s h e d by a c o m b i n a t i o n of genetic and e n v i r o n m e n t a l m a n i p u l a t i o n s . acremo- a c e t o h y d r o x y a c i d s y n t h a s e in a h i g h .4 in- c r e a s e d e x p a n d a s e activity by 8 0 % and c e p h a l o s p o r i n p r o d u c t i o n by 4 7 % and d e c r e a s e d a c c u m u l a t i o n of the i n t e r m e d i a t e . T h e specific activity of the e n z y m e on the basis of cell m a s s w a s a b o u t one-hundred-and-fifty- to t w o . soon after cyclase activity r e a c h e d its peak. T h i s d r o p did not a p p e a r to be d u e to d i s a p p e a r a n c e of m R N A w h i c h w a s at a similar level t h r o u g h o u t the fermentation in both cultures. chrysogenum NRRL-1951 m a k i n g < 1 0 0 μ-g of penicillin V/ml) and an i m p r o v e d strain ( B W . T h e s e c h a n g e s h a v e practical signific a n c e . In 150-1 t a n k s . It is thus clear that increased g e n e d o s a g e is of i m p o r t a n c e in the i m p r o v e m e n t of fungi with respect to p r o d u c t i o n of s e c o n d a r y m e t a b o l i t e s . c e p h a l o s p o r i n C by 1 5 % . M u t a g e n e s i s followed by screening or selection has e m p i r i c a l l y affected e n z y m e regulation as indicated in a n u m b e r of studies.t y p e strain also p e a k e d at 38 h but by 6 2 h w a s at 2 0 % its p e a k level.p r o d u c i n g m u t a n t w a s found to b e nium.t y p e strain (P. it r e a c h e d its m a x i m u m at 38 h and w a s m a i n t a i n e d near that level to the first m e a s u r e d p o i n t . the . chrysogenum strain had t w i c e the a c e t o h y d r o x y a c i d s y n t h a s e activity of an ancestral strain and the e n z y m e in the superior strain w a s d e r e g u l a t e d to valine feedback inhibition.1 8 9 0 w a s 3 2 to 6 4 t i m e s that in N R R L . S h e n et al. chrysogenum strains b e t w e e n c y c l a s e specific activity and penicillin p r o d u c t i o n ability. the w i l d .5. thirtyfold desensitized to valine feedback inhibition but there w a s n o c h a n g e in repression ( M a t s u m u r a and S u z u k i 1986). A l t h o u g h the e x p a n d a s e / h y d r o x y l a s e g e n e is n o r m a l l y found on c h r o m o s o m e II.1 9 5 1 .5 by C. the e x p a n d a s e activity increased b y twofold. acremonium 3 9 4 . ( 1 9 8 5 ) s h o w e d that there w a s a g o o d correlation in four P. Incorporation of a p l a s m i d c o n t a i n i n g the C. R a m o s et al. penicillin N .h u n d r e d . C o m p a r i s o n of the c y c l a s e g e n e d o s a g e b e t w e e n a w i l d . that is.800 ^Lg/ml) s h o w e d that B W . d e a c e t o x y c e p h a l o s p o r i n C d e c r e a s e d by sixfold. In C.f o l d h i g h e r in the high p r o d u c i n g strain and a p p e a r e d to be m o r e stable. s h o w i n g that a s e c o n d factor increases g e n e transcription. G o u l d e n and C h a t t a w a y ( 1 9 6 9 ) s h o w e d that a high p r o d u c i n g P. In both c u l t u r e s . acremoniwn. 8 6 h.1 8 9 0 m a k i n g 1.1 8 9 0 c o n t a i n e d 8 to 16 c y c l a s e g e n e s p e r e a c h o n e in an a u x o t r o p h i c m u t a n t of N R R L . A t t e m p t s to use r e c o m b i n a n t D N A t e c h n i q u e s to increase g e n e d o s a g e in fungi are j u s t b e g i n n i n g but are quite p r o m i s i n g .

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CHAPTER 6 Transformation David B. the only e v i d e n c e p r o v i d e d in this report to distinguish the putative transformants from s p o n t a n e o u s reversion at the inl locus w a s the observation that m a n y of the transformants w e r e meiotically unstable ( M i s h r a and T a t u m 1973). It w a s not until 1979. M a c L e o d . to d e m o n s t r a t e that transformation has occurred o n e m u s t be able to detect a D N A .t y p e (inl ) strain of this o r g a n i s m resulted in an increased r e c o v e r y of inositol-dependent o r g a n i s m s ( M i s h r a and T a t u m 1973). Finkelstein T r a n s f o r m a t i o n m a y b e defined as a m e c h a n i s m of genetic transfer w h e r e b y p u r e D N A extracted from o n e o r g a n i s m is able to induce p e r m a n e n t hereditary c h a n g e s in the cells of a s e c o n d o r g a n i s m to w h i c h it is a d d e d . crassa d o n o r strain that required inositol for g r o w t h only at an 113 . o n e year after the historic report of yeast transformation b y F i n k and his c o l l e a g u e s ( H i n n e n et al. e x p r e s s e d a type III capsular carbohydrate. In this report. I n d e e d . T h u s .d e p e n d e n t alteration in s o m e p h e n o t y p e of the recipient o r g a n i s m . A l t h o u g h it is quite easy to accept these results in retrospect. M i s h r a and T a t u m d e m o n s t r a t e d that e x p o s u r e of an inositolrequiring m u t a n t (inl) of the filamentous fungus Neurospora crassa to D N A isolated + from a w i l d . that M i s h r a w a s to p r o v i d e the first u n e q u i v o c a l proof for the transformation of a filamentous fungus by d e m o n s t r a t i n g that transfer of D N A from a N. and M c C a r t y (1944) proof for bacterial transformation (as o p p o s e d to simple reversion of the m u t a n t p h e n o t y p e of the recipient o r g a n i s m ) w a s p r o v i d e d by the fact that the transformed Pneumococcus. T h e first report suggesting transformation of a filamentous fungus a p p e a r e d in 1973. in the classic e x p e r i m e n t of A v e r y . derived from an o r g a n i s m that originally e x p r e s s e d a type II c a p s u l a r c a r b o h y d r a t e . 1978). u n e q u i v o c a l proof for transformation w a s lacking.

large d a y . A l t h o u g h a " n o n r e v e r t i n g " d o u b l e m u t a n t of yeast w a s e m p l o y e d as a recipient. it is clear that the proportion of c o m p e t e n t cells in any culture is p r o b a b l y quite small.d a y variations are often o b s e r v e d in the n u m b e r of transfor- . O n c e c o m p e t e n t cells h a v e been o b t a i n e d . W i t h the filamentous fungi. it w a s b e c a u s e of the use of r e c o m b i n a n t D N A that the first reported transformation of the fungus Saccharomyces cerevisiae w a s i m m e d i a t e l y accepted ( H i n n e n et al. B a s e d on the transformation frequencies that h a v e been a c h i e v e d with these o r g a n i s m s . I n d e e d . it is not surprising that it has b e e n impossible to define a single transformation protocol that will w o r k with all o r g a n i s m s . In early e x p e r i m e n t s with p r o c a r y o t e s .t o . Interestingly. the cells are g r o w n u n d e r a p r e s s u r e such that only those cells that express the gene(s) e n c o d e d b y the transforming D N A will be p r o p a g a t e d . transformants p r o b a b l y w o u l d not h a v e b e e n detected. Since the first reported transformation of N. it w a s appreciated that cells n e e d e d to be in a "reactive p h a s e " to " r e s p o n d to the transforming s t i m u l u s " (Avery et al. T h e ability to detect a foreign D N A s e q u e n c e (that is. 1944). E v e n w h e n using a routine p r o t o c o l . which allow the application of powerful selection t e c h n i q u e s . Finally. A n u m b e r of different techniques that h a v e been successfully used for this p u r p o s e are discussed b e l o w . the carrier portion of the p l a s m i d vector) by hybridization allowed an u n e q u i v o c a l d e m o n s t r a t i o n that transformation of this o r g a n i s m had occurred. the next step in a transformation protocol is to add D N A and induce the c o m p e t e n t cells to take u p introduced nucleic acid. T h e ability to isolate specific s e g m e n t s of D N A and m a n i p u l a t e t h e m in vitro has a l l o w e d the construction of plasmid vectors w h i c h h a v e been the key to the d e v e l o p m e n t of transformation p r o c e d u r e s for such a w i d e variety of organisms.114 Transformation elevated t e m p e r a t u r e could confer a temperature-sensitive inositol r e q u i r e m e n t to the inl transformation recipient (Mishra 1979). 1978). it is often necessary to modify a " s t a n d a r d " protocol to transform different wild-type strains of the s a m e species. W i t h such a diversity of filamentous fungi. to detect rare fungal transformants.1 TRANSFORMATION TECHNIQUES AND PROPERTIES OF TRANSFORMANTS T o transform a filamentous fungus it is first necessary to cultivate and m a n i p u l a t e it so that the o r g a n i s m is c o m p e t e n t to take u p D N A . it has b e e n necessary to d e v e l o p selective m a r k e r s . G i v e n that the transformation frequency of filamentous fungi is often not m u c h higher than the reversion rate of the average m u t a t i o n . crassa with a r e c o m b i n a n t m o l e cule ( C a s e et al. it has not been possible to define the portion of cells (or spheroplasts) that are c o m p e t e n t to receive D N A . to detect transformed o r g a n i s m s . 6. 1979) m o r e than 5 0 different species of filamentous fungi h a v e b e e n t r a n s f o r m e d . the transformation frequency w a s so low in this e x p e r i m e n t that had unfractionated c h r o m o s o m a l D N A (as o p p o s e d to a c l o n e d gene) b e e n used as the d o n o r nucleic acid.

1987). 1978). W i t h s o m e fungi. B e c a u s e these e n z y m e s are not m a r k e t e d specifically for spheroplast preparation o n e should not b e surprised that significant lot-to-lot variation has often b e e n noted in the ability of these preparations to yield viable spheroplasts. this allows o n e to w o r k with aconidial o r g a n i s m s and also p r o v i d e s a r e a d y supply of starting material ( B u x t o n and Radford 1984).6.g l u c u r o n i d a s e ( S i g m a ) ] h a v e b e e n u s e d b y s o m e . Alternatively.1 Transformation Techniques and Properties of Transformants 115 m a n t s r e c o v e r e d from m a n y fungi. yeast cells are first c o n v e r t e d to spheroplasts (that is. the age of the fungal culture can affect the quality of the spheroplasts (Barrett et al. A variety of e n z y m e s h a v e b e e n utilized to prepare fungal spheroplasts that are c a p a b l e of r e g e n e r a t i n g their cell w a l l s . 1989). 1989). 1979. m a n y w o r k e r s h a v e achieved success with N o v o z y m e 2 3 4 ( N o v o ) . the application of a slight o s m o t i c . Spheroplasts h a v e also b e e n p r e p a r e d from h y p h a l cultures. It has b e e n s u g g e s t e d that g e r m i n a t e d conidia give rise to m o r e regenerable spheroplasts of Schizophyllum commune (Specht et al. the e x o g e n o u s D N A is a d d e d and transformation is then effected by the addition of the fusogen p o l y e t h y l e n e glycol ( P E G ) ( H i n n e n et al. T i l b u r n et al. E x c e p t w h e n electroporation is used (see b e l o w ) . sorbitol. A s spheroplasts are o s m o t i c a l l y fragile. V o l l m e r and Y a n o f s k y 1986). 1988). In addition to the extent of e x p o s u r e to the lytic e n z y m e s . A l t h o u g h spheroplasts from h y p h a l cultures are quite variable in size. a m u l t i . 1986.e n z y m e extract of Trichoderma. m a g n e s i u m sulfate. and mannitol h a v e all b e e n utilized to stabilize Aspergillus spheroplasts during transformation (Ballance et al. 1 9 8 3 . for e x a m p l e . After r e s u s p e n d i n g the spheroplasts in an osmotically supporting solution in the p r e s e n c e of C a C l 2 . addition of heparin or s p e r m i d i n e to the D N A prior to addition to the spheroplasts h a s b e e n reported to i m p r o v e transformation frequency ( C a s e et al. T h e o s m o t i c strength of the spheroplast solution can often h a v e a d r a m a t i c impact on transformation frequency. A w i d e variety of c o m p o u n d s are suitable for this p u r p o s e . I n d e e d . scientists w o r k i n g a single o r g a n i s m often differ as to the best transformation protocol for the o r g a n i s m .1 Transformation of Spheroplasts M o s t reported p r o t o c o l s for the transformation of filamentous fungi h a v e followed the basic principles first d e v i s e d for the transformation of the yeast Saccharomyces cerevisiae. o s m o t i c a l l y fragile cells that are lacking their cell wall) by e n z y m e treatment. they m a y be fractionated b y centrifugation to enrich for those that are transformation c o m p e t e n t (Skatrud et al. B u x t o n and Radford 1984. s o m e o s m o t i c support is required to p r e v e n t lysis. 1987). K C l . A l t h o u g h the e n z y m e of c h o i c e differs for different o r g a n i s m s . T h u s .1. Finkelstein et al. 1 9 8 3 . fungal spheroplasts are usually r e s u s p e n d e d in a solution c o n t a i n i n g C a C l 2 before addition of the e x o g e n o u s D N A a n d P E G . W a r d et al. 6. A d d i t i o n of the n u c l e a s e inhibitor aurintricarboxylic acid at this point in the transformation has also b e e n reported to increase transformation frequencies ( R a m o n et al. snail gut e n z y m e s [ c o m m e r c i a l l y available as G l u s u l a s e ( E n d o ) or ^ . In this p r o c e d u r e .

relative to the c h e m i c a l transformation of spheroplasts with P E G and C a C l 2 ( T h o m a s and K e n n e r l y 1989. the final step in a transformation protocol is to p r o v i d e a selective g r o w t h condition such that only transformed o r g a n i s m s will s u r v i v e . Similarly.2 Electroporation Electroporation has also been e m p l o y e d to facilitate the uptake of the transforming p l a s m i d D N A . niger and Fusarium oxysporium spheroplasts ( W a r d et al. 1990). introduction of the D N A with a c o n c o m i t a n t o s m o t i c shock d o u b l e d the transformation frequency of the b a s i d i o m y c e t e S.f o l d . 6.3 Transformation of Intact Cells U s i n g yeast. respectively. 1988 and 1989. A s noted a b o v e . can negatively affect transformation frequency. h o w e v e r . W h e n transforming s p h e r o p l a s t s .h u n d r e d . such as L i . given a high e n o u g h initial o s m o t i c strength. T h u s . A l t h o u g h it d o e s not increase the transformation frequency. L a n g i n et al.1. W h e r e a s the use of a high-voltage electric pulse allows the h i g h efficiency transformation of intact yeast cells ( M e i l h o c et al. electroporation has thus far b e e n s h o w n to w o r k only for the filamentous fungi w h e n spheroplasts are u s e d . A k i n s and L a m b o w i t z (1985) achieved their highest transformation frequencies of N. o n e is faced with the additional requirem e n t of p r o v i d i n g conditions that will allow the spheroplasts to regenerate their cell w a l l s .116 Transformation shock prior to the addition of P E G appears to i m p r o v e transformation frequency for a n u m b e r of fungi. 1990). A l t h o u g h it is possible to allow spheroplasts to regenerate before applying a selection to distinguish transformed from nontransformed o r g a n i s m s . p r e s u m a b l y by e n h a n c i n g the u p t a k e of the added nucleic acid. these t w o steps are m o s t often carried out simultaneously by adding o s m o t i c support to the selective agar m e d i u m . commune (Specht et al. 1990).1. the solid g r o w t h m e d i u m in w h i c h the spheroplasts are allowed to regenerate is generally s u p p l e m e n t e d with an o s m o t i c support. It has generally b e e n o b s e r v e d that the best regeneration o c c u r s w h e n spheroplasts are e m b e d d e d in (rather than spread on top of) o s m o t i c a l ly supported plating agar. 1988). 6. O a k l e y et al. T o obtain . crassa w h e n spheroplasts w e r e diluted with water at the t i m e of D N A addition. transformation efficiency did not increase by greater than a factor of t h r e e . (1983) w e r e the first to d e m o n s t r a t e that intact fungi c o u l d b e + transformed if they w e r e first e x p o s e d to alkali c a t i o n s . L o w e r i n g the o s m o t i c strength too m u c h . G o l d m a n et al. B e c a u s e spheroplasts are osmotically fragile. electroporation p r o v i d e s an alternative to the use of P E G and C a C l 2 for the transformation of both A. ( 1 9 8 7 ) found that w h e n they added o s m o t i c support to their P E G solution (to p r e v e n t spheroplast lysis) the transformation frequency of Aspergillus nidulans increased b y o n e . Ito et al. W h e n P E G w a s added either before or after electroporation of Trichoderma harzianum and Gliocladium vir ens. h o w e v e r .

as spheroplasts are not u s e d . 1984). In a d d i t i o n . Treatment with lithium acetate has since been used to transform a number of filamentous fungi such as Mycosphaerella spp. (1987) found the frequency of lithium acetate m e d i a t e d transform a n t s w a s only 1/1.6. At the t i m e of the first reported use of lithium acetate with N. crassa could be a c h i e v e d with both circular and linear D N A . with the b a s i d i o m y c e t e Coprinus B i n n i n g e r et al. it is not necessary to e m b e d the regenerating spheroplasts in agar. (Dickman et al. and Colletotrichum capsici (Marek et al. the transformation frequency achieved w a s r o u g h l y equivalent to that obtained with spheroplasts ( D h a w a l e et al. w h e n using equal a m o u n t s of starting t i s s u e . with only m i n o r m o d i f i c a t i o n s . pisi). . D i c k m a n (1988) noted that. transformation of N. crassa. W i t h Fusarium solani f. 1989). phaseoli and f. B y e l i m i n a t i n g the t i m e a n d effort of spheroplast formation. it h a s p r o v i d e d a f r a m e w o r k for the analysis of the transformation p r o c e s s in the filamentous fungi. a brief heat s h o c k stimulated the u p t a k e of the p l a s m i d D N A . protoplasts of C . T h u s . w h e r e few transformants are n e e d e d . trifolii yielded m o r e transformants than lithium acetate t r e a t m e n t of intact cells. Nectria haematococca (anamorph: Fusarium solani f. although the detailed features of the yeast transform a t i o n p a r a d i g m are not precisely duplicated in filamentous fungal t r a n s f o r m a n t s . s p . the first transformation of the various filamentous fungal species (with the notable e x c e p t i o n of the m u c o r a c e o u s fungi) w a s the result of p l a s m i d integration. T h u s . 1989.2 Vector Integration 117 transformation by this p r o c e d u r e it w a s also necessary to treat the cells with P E G . W i t h this m e t h o d . Similarly. T h e lithium acetate protocol has also been successfully adapted to transform basidiomycetes. Since that t i m e . the lithium acetate m e t h o d p r o v i d e s a m o r e rapid and simpler m e t h o d for transformation of fungi. Indeed. but n u m e r o u s separate transformations m u s t b e p e r f o r m e d . sp. D h a w a l e et al. in contrast to the w o r k with yeast. 1989). 6. crassa.000 of that obtainable from spheroplasts. I n d e e d . to transform g e r m i n a t e d conidia of N. 1989). H o w e v e r . Soliday et al. F u r t h e r m o r e . phaseoli. (1984) w e r e able to apply this p r o t o c o l . it w a s not u n r e a s o n able to predict that this yeast m i g h t serve as a m o d e l for the b e h a v i o r of the filamentous fungi. the lithium acetate protocol is favored ( D i c k m a n 1988). lithium acetate treatment yielded a similar n u m b e r of transformants as the use of spheroplasts ( M a r e k et al. sp. j u s t as o b s e r v e d with yeast. Colletotrichum trifolii (Dickman 1988). h o w e v e r . the dimorphic fungus Histoplasma capsulatum (Worsham and G o l d m a n 1990). the lithium acetate protocol is five times as efficient as a spheroplast protocol for transformation of Ustilago violacea (Bej and Perlin 1989).2 VECTOR INTEGRATION G i v e n the fact that Saccharomyces cerevisiae is an a s c o m y c e t e . the spheroplast transformation frequency for N. crassa h a s greatly increased ( A k i n s and L a m b o w i t z 1985). B y w a y of contrast. it h a s not b e e n easy to construct vectors that can replicate a u t o n o m o u s l y in the filamentous fungi.

2 Linearizing Vectors to Increase Transformation Efficiency W i t h yeast.2. the (oft times low) level of sitespecific p l a s m i d integration is greater than w o u l d be expected if the integration event w e r e purely r a n d o m .W e a v e r et al. T h e p r e s e n c e of t a n d e m repeats suggests that p l a s m i d s m a y form m u l t i m e r s before integration. the site of integration is dictated entirely by c r o s s o v e r at regions of h o m o l o g y shared b e t w e e n the i n c o m i n g p l a s m i d and the yeast g e n o m e ( O r r .118 6. Binninger et al. O n e often finds transformants in w h i c h t a n d e m repeats of the p l a s m i d h a v e integrated into the fungal g e n o m e ( W e r n a r s et al. organisms carrying plasmid integrated at a nonhomologous site are frequently recovered when transforming filamentous ascomycetes and basidiomycetes (Wernars et al. although the addition of repetitive D N A to filamentous fungal vectors generally has no effect on transformation frequency (Tilbum et al. the efficiency of plasmid integration can be increased as m u c h as one-thousand-fold if the vector is cleaved within a region that shares h o m o l o g y with . T h i s observation also supports the notion that filamentous fungi can r e c o g n i z e h o m o l o g o u s D N A s e q u e n c e s . By contrast. as the sequences at the junction between the integrated plasmid and the chromosome share little homology (Razanamparany and Bégueret 1988). D e s p i t e the fact that s e q u e n c e h o m o l o g y with the g e n o m e is not essential for p l a s m i d integration in the filamentous fungi. H a m e r and T i m b e r l a k e (1987) w e r e able to select for directed p l a s m i d integration into the A. suggesting that the fungi can r e c o g n i z e h o m o l o g y b e t w e e n D N A s e q u e n c e s . 1985. h o m o l o g o u s r e c o m b i n a t i o n m a y b e favored o v e r r a n d o m crossover for the integration of additional p l a s m i d s . Skatrud et al. 1983). This ectopic integration is not due to homologous recombination. 1990). Addition of homologous r D N A sequences to a vector improved the transformation frequency of this organism twenty fold (Tsuge et al.2. o n c e the appropriate e n z y m e s are situated at a site of p l a s m i d integration.1 Transformation Site of Plasmid Integration In yeast transformation. 1983. 1987). Indeed. F o r e x a m p l e . 1985). 6. T h u s . w h e n plasmid integration o c c u r s . 1983). T h e fact that vector integration into the filamentous fungal g e n o m e d o e s not require s e q u e n c e h o m o l o g y offers the possibility of using vectors that h a v e been d e v e l o p e d for o n e o r g a n i s m to transform h e t e r o l o g o u s fungi (Ballance et al. the transformation frequency of a yeast integrative vector is increased by addition of r i b o s o m a l D N A ( r D N A ) as a c o n s e q u e n c e of increasing the g e n o m i c target for h o m o l o g o u s r e c o m b i n a t i o n (Szostak and W u 1979). nidulans argB g e n e by using a vector that contained a disrupted argB g e n e that could c o m p l e m e n t the c h r o m o s o m a l argB mutation of the transformation recipient only if site-specific integration o c c u r r e d . exceptions have been noted with the phytopathogenic fungus Aiternana alternata. T h e relative level of nonhomologous integration versus integration at a site of homology in the filamentous fungi depends on both the gene being utilized as well as the specific recipient strain that is employed (Kim and Marzluf 1988). 1987). Alternatively. It is possible to design strategies for the filamentous fungi w h e r e only cells carrying p l a s m i d s that h a v e integrated in a site-specific fashion will g r o w .

it has still b e e n possible to adapt the yeast m e t h o d s to these o r g a n i s m s . oryzae b y cutting a vector c o n t a i n i n g the h o m o l o g o u s pyrG g e n e . 1987). transformation efficiency w a s i m p r o v e d t w o . in studies with the Aspergilli W e r n a r s et al. linearization of the p l a s m i d will " d i r e c t " it to integrate at the locus h o m o l o g o u s to the cut s e q u e n c e s . S u c h t w o . 1989).s t e p p r o c e s s is a directed alteration in the yeast g e n o m e ( S c h e r e r and D a v i s 1979).s t e p r e p l a c e m e n t s h a v e also b e e n carried out with Podospora anserina. Interestingly. W h e n such an integrated p l a s m i d is s p o n t a n e o u s l y excised b y a c r o s s o v e r b e t w e e n the duplicated D N A s e q u e n c e s . n o n t a n d e m D N A duplication b r a c k e t i n g the integrated n o n h o m o l o g o u s p l a s m i d s e q u e n c e .R a y n a l et al. a portion of the original p l a s m i d D N A (that is. Defined c h r o m o s o m a l disruptions can b e obtained by p l a s m i d integration if the vector carries an internal s e g m e n t of the g e n e to be targeted (Shortle et al. T h e s e t e c h n i q u e s are predicted o n the notion that p l a s m i d integration will o c c u r via c r o s s o v e r at regions of h o m o l o g y shared b e t w e e n the i n c o m i n g p l a s m i d and the g e n o m e (Shortle et al. I n d e e d . F o r e x a m p l e . A s with the question of ectopic integration discussed earlier. the region located b e t w e e n the " i n c o m i n g " and " o u t g o i n g " c r o s s o v e r sites) will b e retained by the c h r o m o s o m e .2. if a yeast vector contains s e q u e n c e s h o m o l o g o u s to different c h r o m o s o m a l loci.s t e p g e n e r e p l a c e m e n t s h a v e b e e n carried out with filamentous fungi. .J a c o b s et al. W i t h Cephalosporium acremonium. W h e n yeast is transformed with a h o m o l o g o u s cloned g e n e inserted in a v e c t o r . nidulans with a D N A s e g m e n t deleted for a portion of the SpoCl C l . T w o .W e a v e r et al. the impact of p l a s m i d linearization is d e p e n d e n t o n both the D N A and the specific strain being e m p l o y e d .2 Vector Integration 119 the g e n o m e ( O r r .6. 1985).C c o d i n g s e q u e n c e . W h e r e this retained region contains a m u t a t i o n (or other modified s e q u e n c e ) . the h o m o l o g o u s r e c o m b i n a t i o n leading to p l a s m i d loss o c c u r r e d at a relatively high frequency ( > 1 % ) during meiosis of this h o m o t h a l l i c o r g a n i s m (Miller et al. 1982). (1989) o b s e r v e d a tenfold increase in transformation of A. integration by c r o s s o v e r b e t w e e n the p l a s m i d and the h o m o l o g o u s region of the g e n o m e will yield a transformant h a r b o r i n g a direct. w h e r e a s de R u i t e r . 1982. the result of this t w o . Directed integration w a s i m p r o v e d by the use of c o s m i d vectors with this o r g a n i s m ( C o p p i n .to threefold by cutting a vector within a s e q u e n c e of h o m o l o g y (Skatrud et al. T h i s result suggests that the generation of linear D N A e n d s is the rate-limiting step for p l a s m i d integration in this o r g a n i s m . it has b e e n possible to use this t e c h n i q u e to replace the wild-type SpoCl region of A. A l t h o u g h the frequency of h o m o l o g o u s p l a s m i d integration is not as high with the filamentous fungi as with yeast. (1985) found n o increase in transformation of A . cerevisiae.3 Gene Disruption and Gene Replacement T e c h n i q u e s for u s i n g transformation to replace or disrupt g e n e s w e r e first d e v e l o p e d with the yeast S. A t t e m p t s to increase the transformation frequency of the filamentous fungi by u s i n g linearized p l a s m i d s has m e t with only limited s u c c e s s . 6. 1981). nidulans by cleaving a v e c t o r within the h o m o l o g o u s amdS g e n e . F o r e x a m p l e . Rothstein 1983).

the Ν. This technique is predicated on the notion that a g e n e fragment that is incapable of c o m p l e m e n t a t i o n can rescue a c h r o m o s o m a l mutation if it spans the g e n e lesion.d e l e t i o n mutation) is to create t w o i n c o m p l e t e copies of the target g e n e . W h e r e a d o u b l e c r o s s o v e r occurs b e t w e e n an i n c o m i n g p l a s m i d and a c h r o m o s o m e . 1989). the c o n s e q u e n c e of integrating a p l a s m i d . r e p l a c e m e n t of a m u t a n t trpC g e n e with the wild-type g e n e occurred in only o n e of sixteen t r a n s f o r m a n t s . T o i m p r o v e g e n e targeting in a one-step g e n e r e p l a c e m e n t .s t e p g e n e r e p l a c e m e n t can b e used to create specific m u t a t i o n s in yeast. . b e c a u s e it can o c c u r only via an h o m o l o g o u s integration (or g e n e c o n v e r s i o n ) e v e n t . W i t h A. nidulans ( M a y et al. 1985). nidulans. T h e efficiency of o n e . it is simple to select positively for transformants carrying the disrupted g e n e (Rothstein 1983). separated by the integrated n o n h o m o l o g o u s vector s e q u e n c e s .120 Transformation Integration by a single c r o s s o v e r b e t w e e n the internal g e n e region and the 1 h o m o l o g o u s region of the g e n o m e will result in the fusion of the 3 deleted portion 1 of the i n c o m i n g g e n e to the 5 ' end of the c h r o m o s o m a l locus (to create a 3 deletion mutation) and the fusion of the 5 ' deleted portion of the i n c o m i n g g e n e to the r e m a i n d e r of the c h r o m o s o m a l g e n e (to create a 5 ' deletion m u t a t i o n ) . o n e . and e v e n with different targets in the s a m e o r g a n i s m . W h e r e the g e n e has been disrupted by insertion of a selectable m a r k e r . H e n c e .b o r n e internal g e n e fragment (that is. w h e r e a s the efficiency of r e p l a c e m e n t of wild-type argB g e n e with a disrupted (by insertion of the trpC gene) argB g e n e w a s a p p r o x i m a t e l y 3 0 % (Miller et al. may dis pyr6 g e n e could b e replaced with a disrupted (by insertion of the h y g r o m y c i n phosphotransferase gene) pyr6 g e n e with an efficiency of 7 0 % ( K r o n s t a d et al. such a o n e . the b a c k g r o u n d of transformants (due to p l a s m i d integration at h e t e r o l o g o u s sites) normally o b s e r v e d in a g e n e targeting e x p e r i m e n t will be e l i m i n a t e d . Variability is seen from o r g a n i s m to o r g a n i s m . S u c h t a n d e m null mutations h a v e been exploited to identify the c l o n e d ßtubulin g e n e that e n c o d e s the /33-tubulin (the tubC g e n e product) of A. M a r k e r rescue occurs at a m u c h l o w e r frequency than that generally observed for transformation by p l a s m i d integration. a d o u b l e .W e a v e r et al. although U. B e c a u s e free D N A e n d s are r e c o m b i n o g e n i c in yeast and stimulate r e c o m b i n a t i o n by interacting directly with h o m o l o g o u s s e q u e n c e s in the g e n o m e ( O r r .s t e p g e n e r e p l a c e m e n t with the filamentous fungi is limited b y the fact that illegitimate plasmid integration is often m o r e c o m m o n than h o m o l o g o u s p l a s m i d integration. If the i n c o m i n g D N A carries a disrupted g e n e . 1985).s t e p g e n e r e p l a c e m e n t can be performed efficiently in this o r g a n i s m w h e n linear D N A is used for transformation (Rothstein 1983). A s a result. 1981). the t e c h n i q u e of m a r k e r rescue m a y be e m p l o y e d . crassa trpC g e n e w a s replaced in only 1 0 % of cells transformed by a linear piece of D N A containing a disrupted (by insertion of the qa2 gene) trpC g e n e (Paietta and M a r z l u f 1985). the s e q u e n c e s b e t w e e n the c r o s s o v e r sites are evicted from the c h r o m o s o m e and replaced with the c o r r e s p o n d i n g plasmid s e q u e n c e s . Similarly. T h u s . proof that a cloned Ustilago may dis D N A fragment e n c o d e d the LEU] g e n e of this o r g a n i s m w a s p r o v i d e d by disrupting the g e n e with an internal fragment of the cloned D N A ( F o t h e r i n g h a m and H o l l o m a n 1989).

6.2

Vector Integration

121

T h u s , by introducing a 3 ' deleted am g e n e into a host that carries a point mutation in
c o d o n five of the am g e n e o n e can select for only those transformants that are the
result of h o m o l o g o u s r e c o m b i n a t i o n at the N. crassa am g e n e . B y using this
strategy it w a s possible to e x a m i n e the effect of modifying the am p r o m o t e r without
c o n c e r n for any possible u n t o w a r d effects that might h a v e b e e n c a u s e d by
n o n h o m o l o g o u s g e n e integration (Frederick and K i n s e y 1990). M a r k e r r e s c u e has
also b e e n d e m o n s t r a t e d in the b a s i d i o m y c e t e U. maydis ( F o t h e r i n g h a m and H o l l o m a n 1989).
F i n a l l y , it should be noted that g e n e s can be replaced in a cotransformation
e x p e r i m e n t . F o r e x a m p l e , it w a s possible to replace both the A. nidulans and A.
niger trpC g e n e s with lacZ fusions by introducing E. coli lacZ c o d i n g s e q u e n c e s ,
e m b e d d e d within the h o m o l o g o u s fungal trpC g e n e , into the respective fungi in
cotransformation e x p e r i m e n t s . W i t h A. nidulans, t w o t r y p t o p h a n a u x o t r o p h s w e r e
+
found a m o n g 150 L a c Z transformants ( W e r n a r s et al. 1987). U s i n g the s a m e
+
p r o t o c o l , n o t r y p t o p h a n a u x o t r o p h s w e r e found a m o n g 100 L a c Z transformants of
A. niger ( G o s s e n et al. 1989). T o o v e r c o m e the low targeting to the trpC locus in
this o r g a n i s m a variation of the m a r k e r rescue t e c h n i q u e w a s e m p l o y e d . T h e authors
deleted the e n d of the trpC p r o m o t e r from the D N A fragment carrying the lacZ
s e q u e n c e s . A s a c o n s e q u e n c e h o m o l o g o u s integration at the resident trpC locus w a s
required for the ß - g a l a c t o s i d a s e g e n e to be e x p r e s s e d . A l t h o u g h only nine of the
6 , 1 5 0 e x a m i n e d A. niger transformants e x p r e s s e d /3-galactosidase, eight of these
had disrupted the c h r o m o s o m a l trpC g e n e ( G o o s e n et al. 1989).

6.2.4

Mitotic Stability of Transformants

W i t h virtually all filamentous fungi, w h e n transformation occurs by integration
m o s t ( n o n a b o r t i v e ) transformants are mitotically stable. D u n n e and O a k l e y (1988)
w e r e able to m e a s u r e precisely the frequency of vector loss by exploiting the fact
that the A. nidulans benAll
(benomyl-resistant /3-tubulin) m u t a t i o n is r e c e s s i v e .
T h e s e w o r k e r s transformed an A. nidulans pyrG benA22 d o u b l e m u t a n t with a
vector c a r r y i n g both the wild-type A. nidulans benA g e n e as well as the N. crassa
pyr4 g e n e b y selection for p y r i m i d i n e p r o t o t r o p h s . O w i n g to the recessivity of the
benA22 m u t a t i o n , the resulting transformants w e r e sensitive to b e n o m y l . U s i n g
transformants w h e r e p l a s m i d had integrated by h o m o l o g o u s r e c o m b i n a t i o n at the
benA l o c u s , it w a s possible to quantitate the loss of the integrated p l a s m i d b y
c o u n t i n g the n u m b e r of b e n o m y l - r e s i s t a n t p r o g e n y . T h e frequency of p l a s m i d loss
4
d e t e r m i n e d this w a y w a s a p p r o x i m a t e l y 2 x 1 0 ~ .
In a practical test of the mitotic stability of fungal t r a n s f o r m a n t s , Finkelstein et
al. (1989) subjected a n u m b e r of A. niger t r a n s f o r m a n t s , carrying extra copies of the
g l u c o a m y l a s e g e n e , to eight serial conidial transfers in the a b s e n c e of selective
p r e s s u r e . At the e n d of this p e r i o d , n o modification could be detected in either g e n e
d o s a g e or in e n z y m e yield. T h e implication of such mitotic stability is that r e c o m binant filamentous fungi can b e g r o w n at p r o d u c t i o n scale without the need to apply
a selective p r e s s u r e to maintain the transformed p h e n o t y p e (Skatrud et al. 1989).

122

6.2.5

Transformation

Meiotic Instability of Transformants

Studies to date indicate that transformed b a s i d i o m y c e t e s are meiotically
a b s e n c e of selective pressure ( M u h o z - R i v a s et al. 1986b; B i n n i n g e r
Alic et al. 1989). W i t h transformed a s c o m y c e t e s , h o w e v e r , meiotic
pears to b e the e x c e p t i o n ( L e C h e v a n t o n et al. 1989) rather than the rule
U p s h a l l 1986).

stable in the
et al. 1987;
stability a p ( C a s e 1986;

It h a s b e e n noted in a p r e c e d i n g section that the first reported transformation of
N. crassa used c h r o m o s o m a l D N A rather than a r e c o m b i n a n t p l a s m i d . T h e only
feature by w h i c h the transformed o r g a n i s m s could b e distinguished from n o n t r a n s formed o r g a n i s m s w a s their meiotic instability ( M i s h r a and T a t u m 1973). A likely
e x p l a n a t i o n for this o b s e r v e d meiotic instability is suggested b y w o r k of Selker a n d
his c o l l e a g u e s , w h o h a v e d e m o n s t r a t e d that duplicated s e q u e n c e s ( w h e t h e r linked or
unlinked) in Neurospora
are modified premeiotically by both methylation and point
m u t a t i o n s (Selker et al. 1987; Selker and Garrett 1988; G r a y b u r n and Selker 1989).
T h i s p h e n o m e n o n has b e e n given the a c r o n y m R I P (initially standing for r e a r r a n g e m e n t s that are induced p r e m e i o t i c a l l y , and later for r e p e a t - m d u c e d p o i n t m u t a t i o n ) .
A p h e n o m e n o n similar to R I P has been o b s e r v e d with Ascobolus
immer sus.
A l t h o u g h m o s t transformants of this o r g a n i s m are mitotically stable, the only
meiotically stable met2 transformants are those that d o not carry a g e n e duplication
(that is, transformation by g e n e conversion or a d o u b l e crossover g e n e r e p l a c e m e n t
event at the n o r m a l met! locus; F a u g e r o n et al. 1989). U n l i k e the R I P p h e n o m e n o n ,
duplicated s e q u e n c e s in Ascobolus
are inactivated by methylation only ( G o y o n and
F a u g e r o n 1989). F u r t h e r m o r e , the inactivated g e n e s revert s p o n t a n e o u s l y to an
active state after a n u m b e r of mitotic divisions. B y e x a m i n a t i o n of the b e h a v i o r of
strains carrying differing doses of an ectopic g e n e it w a s possible to d e d u c e that
g e n e inactivation required premeiotic pairing of the repeated s e q u e n c e s . A s each
g e n e c o p y can u n d e r g o successive cycles of pairing (that is, o n c e m e t h y l a t e d , a
g e n e c o p y can pair with an as yet u n m e t h y l a t e d gene) it is possible to inactivate an
u n e v e n n u m b e r of copies in a single meiosis (Faugeron et al. 1990).
W i t h Podospora
anserina transformants, meiotic instability is o b s e r v e d only
w h e n the vector has integrated via crossover with a h o m o l o g o u s region of the
g e n o m e to yield a duplicated s e q u e n c e separated by the u n i q u e vector s e q u e n c e s .
M e i o t i c instability in such transformants appears to b e d u e to a reversal of the initial
integration event; that is, i n t r a c h r o m o s o m e r e c o m b i n a t i o n b e t w e e n the duplicated
s e q u e n c e s results in the excision of o n e of the repeated s e q u e n c e s as well as the
intervening u n i q u e vector s e q u e n c e s (Picard et al. 1987; C o p p i n - R a y n a l et al.
1989).

6.3

AUTONOMOUSLY REPLICATING VECTORS

W i t h a few notable e x c e p t i o n s , following transformation of the filamentous fungi
vector s e q u e n c e s are m a i n t a i n e d by integration into the host g e n o m e . B u x t o n and
Radford (1984) h a v e argued that, o w i n g to the coenocytic structure of the filamentous fungi, it should be m o r e difficult to maintain an a u t o n o m o u s l y replicating

6.3

Autonomously Replicating Vectors

123

p l a s m i d in these o r g a n i s m s than in a b u d d i n g o r g a n i s m such as yeast. A s a
c o n s e q u e n c e of c y t o p l a s m i c m i x i n g , o n e c a n n o t select directly for an individual
n u c l e u s in a c o e n o c y t i c o r g a n i s m b e c a u s e nuclei that lose the transforming g e n e can
b e m a i n t a i n e d b y the p r e s e n c e of a small n u m b e r of nuclei that still maintain the
g e n e . B y contrast, if a single cell of a uninucleate b u d d i n g o r g a n i s m , such as the
yeast S. cerevisiae,
w e r e to lose a p l a s m i d containing a required g e n e , this cell
w o u l d b e at a clear d i s a d v a n t a g e and w o u l d be lost from the p o p u l a t i o n . H e n c e ,
a l t h o u g h o n e can maintain a g r o w i n g population of yeast containing a required
a u t o n o m o u s p l a s m i d that is lost with a probability of 5 0 % p e r cell division, the
addition of such a p l a s m i d to a filamentous fungus is likely to result in an abortive
transformation. A s a c o n s e q u e n c e , m u c h effort has been e x p e n d e d in attempts to
d e v e l o p a u t o n o m o u s p l a s m i d s for the filamentous fungi. O n c e a g a i n , these efforts
h a v e been g u i d e d b y the yeast p a r a d i g m .

6.3.1

Use of Endogenous Plasmids

Nuclei of m a n y strains of the yeast S. cerevisiae contain a 2 - μ π ι plasmid that can be
used for the construction of vectors that can replicate autonomously in this organism
(Beggs 1978; Broach 1983). As nuclear plasmids are not c o m m o n a m o n g the filamentous fungi, Stohl and Lambowitz (1983) attempted to exploit a mitochondrial plasmid
from Neurospora
intermedia
strain P-405 Labelle to construct an autonomously
replicating vector for N. crassa. Although the vector they constructed appeared to
replicate autonomously when introduced into N. crassa, deletion of the mitochondrial
plasmid sequences (by passage through the fungus) did not interfere with the apparently
autonomous nature of the vector, thus suggesting that the putative replication origin was
being furnished by sequences other than those of the mitochondrial plasmid (Stohl and
Lambowitz 1983; Stohl et al. 1984). Although Stohl and Lambowitz (1983) demonstrated that the transformation efficiency of these putative autonomously replicating
vectors was higher than for integrative vectors, this observation has not been confirmed
by others (Buxton and Radford 1984).
L i n e a r p l a s m i d s are found in a n u m b e r of filamentous fungi ( M e i n h a r d t et al.
1990). H y p o t h e s i z i n g that the terminal inverted repeats (TIR) o n such p l a s m i d s
m i g h t contain the p l a s m i d replication origin, S u m a c and L e o n g (1989) placed the
T I R from a Nectria
haematococca
linear p l a s m i d onto a vector carrying the
h y g r o m y c i n p h o s p h o t r a n s f e r a s e g e n e linked to a i / . may dis p r o m o t e r . W h e n the
circular form of this vector w a s introduced into U. maydis, it w a s unstable u n d e r
n o n s e l e c t i v e g r o w t h c o n d i t i o n s , suggesting that it w a s replicating a u t o n o m o u s l y
and had not integrated into the host g e n o m e . A u t o n o m o u s p l a s m i d replication w a s
c o n f i r m e d b y a S o u t h e r n blot of D N A obtained from the fungal transformants w h i c h
revealed the p r e s e n c e of only circular p l a s m i d D N A .

6.3.2

Isolation of Autonomously Replicating Sequences
(ARS)

B e c a u s e p l a s m i d integration a p p e a r s to be the rate-limiting step to yeast transformation, it has b e e n possible to identify s e q u e n c e s that can function as replication

124

Transformation

origins in this o r g a n i s m by selecting for vectors that transform yeast at a high
frequency ( S t i n c h c o m b et al. 1979). I n d e e d , b y using such a selection it w a s
possible to isolate s e q u e n c e s that functioned as A R S s in yeast from a n u m b e r of
o r g a n i s m s , including/V. crassa ( S t i n c h c o m b et al. 1980). Unfortunately, w h e n this
Neurospora
A R S w a s placed on a Neurospora
integrating vector, the resulting
p l a s m i d w a s u n a b l e to replicate a u t o n o m o u s l y in this filamentous fungus ( C a s e
1982).
Putative A. nidulans D N A origins of replication, w h i c h function as A R S s in
y e a s t , d o not yield a u t o n o m o u s replication w h e n reintroduced into A.
nidulans
(Tilburn et al. 1 9 8 3 ; Ballance and T u r n e r 1985). Despite this fact, o n e A R S , t e r m e d
ansl, i m p r o v e d the transformation efficiency of both A. nidulans and
Pénicillium
chrysogenum
(Ballance and T u r n e r 1985; Cantoral et al. 1987). T h e d e g r e e of
transformation e n h a n c e m e n t o b s e r v e d with the ansl s e q u e n c e d e p e n d s on the
specific vector construction. C u r i o u s l y , transformation e n h a n c e m e n t is also o b served w h e n the ansl s e q u e n c e is supplied in trans (for e x a m p l e , in a cotransformation e x p e r i m e n t ) , suggesting that multiple p l a s m i d s m a y r e c o m b i n e before integrating into the host g e n o m e (Cullen et al. 1987b).
T h e only reports of s e q u e n c e s isolated as A R S s in yeast being able to support
a u t o n o m o u s replication in a filamentous fungus h a v e involved the z y g o m y c e t e
Phy corny ces blakesleeanus
(Revuelta and J a y a r a m 1986; Suarez and E s l a v a 1988).
Interestingly, a u t o n o m o u s p l a s m i d replication appears to be the preferred m o d e of
D N A p r o p a g a t i o n in this and related m u c o r a c e o u s fungi, as the initial vectors used
to transform both Absidia glauca and Mucor circinelloides
also replicated a u t o n o m o u s l y (van H e e s w i j c k 1986; W ö s t e m e y e r et al. 1987). T h e A R S s e q u e n c e s on
these vectors w e r e fortuitously contained on the fungal D N A s e g m e n t s that carried
the selective m a r k e r s . T h e Mucor A R S s e q u e n c e did not function as a replication
origin w h e n introduced into yeast, despite the fact that the Mucor leuA g e n e could
c o m p l e m e n t a h o m o l o g o u s yeast leul mutation ( R o n c e r o et al. 1989).
A s an alternative a p p r o a c h to identifying A R S s e q u e n c e s , g e n e b a n k s h a v e
b e e n screened in filamentous fungi to select directly p l a s m i d s h a v i n g an increased
transformation efficiency. U s i n g this m e t h o d it w a s possible to isolate transformation e n h a n c i n g s e q u e n c e s from the a s c o m y c e t e N. crassa ( B u x t o n and Radford
1984). Vectors carrying these s e q u e n c e s integrate into the host g e n o m e and are not
stably m a i n t a i n e d as a u t o n o m o u s l y replicating p l a s m i d s . A s the abortive transform a n t s obtained w h e n using these vectors w e r e larger in size than those o b s e r v e d
w h e n using a typical integrating vector, the transformation e n h a n c e m e n t m a y h a v e
b e e n , in part, d u e to a u t o n o m o u s replication of the p l a s m i d s within the fungus
( B u x t o n and Radford 1984). Interestingly, G r a n t et al. (1984) o b s e r v e d that a vector
carrying the N. crassa am g e n e w a s both mitotically and meiotically u n s t a b l e . A s
Southern blot analysis s h o w e d the presence of higher m o l e c u l a r weight D N A , these
w o r k e r s suggested that the a u t o n o m o u s l y replicating form of the vector might be a
plasmid multimer.
T h e only successful isolation of an a u t o n o m o u s l y replicating p l a s m i d by shotg u n screening for A R S function directly in a filamentous fungus to date has c o m e
with the b a s i d i o m y c e t e U. may dis ( T s u k e d a et al. 1988). It should be noted that the

6.3

Autonomously Replicating Vectors

125

A R S selection u s e d a haploid strain of this o r g a n i s m . B e c a u s e haploid U. maydis
divides by b u d d i n g (to p r o d u c e yeast-like colonies) and is filamentous only w h e n
dikaryotic ( S c h u l z et al. 1990), it is not k n o w n w h e t h e r this A R S s e q u e n c e can
function efficiently e n o u g h to allow the m a i n t e n a n c e of an a u t o n o m o u s p l a s m i d
d u r i n g filamentous g r o w t h . D e s p i t e h a v i n g b e e n isolated from a b u d d i n g o r g a n i s m
and h a v i n g similar s e q u e n c e s to yeast A R S s , the Ustilago A R S did not function as
an a u t o n o m o u s l y replicating s e q u e n c e w h e n introduced into S.
cerevisiae.

6.3.3

Linear Vectors

T e l o m e r e s , the e n d s of eucaryotic c h r o m o s o m e s , h a v e a specialized structure that
protects t h e m from r e c o m b i n a t i o n (reviewed in B l a c k b u r n and Szostak 1984). T h e
addition of t e l o m e r e s to the e n d s of a linearized yeast vector p r e v e n t s it from
integrating into the g e n o m e . Surprisingly, the e n d s of the linear r D N A p l a s m i d of
Tetrahymena
thermophila
can function as stable c h r o m o s o m e e n d s in yeast (Szostak
and B l a c k b u r n 1982). W h e n introduced into yeast, the Tetrahymena
thermophila
t e l o m e r e u n d e r g o e s the s a m e yeast-specific e n d addition as d o n o r m a l yeast c h r o m o s o m a l t e l o m e r e s ( S h a m p a y et al. 1984).
U s i n g the s a m e strategy, Perrot et al. (1987) w e r e able to convert an integrating
Podospora anserina vector into an a u t o n o m o u s l y replicating linear vector b y a d d i n g
the e n d s of the linear Tetrahymena
thermophila
r D N A p l a s m i d to the e n d s of the
linearized vector. W h e n the vector w a s introduced into P. anserina, o n e half of the
resulting transformants had stably integrated the vector in a form that had deleted
the Tetrahymena
t e l o m e r e s . T h e r e m a i n i n g transformants, h o w e v e r , w e r e unstable
a n d carried the vector as an a u t o n o m o u s linear m o l e c u l e . U n d e r selective c o n d i t i o n s
only o n e linear m o l e c u l e w a s found p e r five to ten nuclei. G i v e n the c o e n o c y t i c
nature of this o r g a n i s m and the selective m a r k e r e m p l o y e d (orotidylic acid
p y r o p h o s p h o r y l a s e ) , it is not surprising that this low g e n e d o s a g e could supply
sufficient e n z y m e activity ( 2 0 - 2 5 % of the wild-type o r g a n i s m ) to allow selective
g r o w t h . T h e linear p l a s m i d w a s rapidly lost u n d e r nonselective c o n d i t i o n s . T h e
source of the A R S o n this vector w a s not d e t e r m i n e d .
A putative Fusarium oxysporum t e l o m e r e w a s fortuitously isolated d u r i n g w o r k
that w a s a i m e d at i m p r o v i n g vector efficiency by adding s e q u e n c e s from a F.
oxysporum
f. s p . raphani linear p l a s m i d to a transforming vector (Powell and
Kistler 1990). W h e n the vector, p F T l , w a s introduced into F. oxysporum
f. s p .
lycopersici
7 3 s o m e of the transformants carried an unintegrated r e a r r a n g e d linear
form of the vector. A l t h o u g h this rearranged vector could b e m a i n t a i n e d at a c o p y
n u m b e r of 10 to 5 0 p e r g e n o m e u n d e r selective c o n d i t i o n s , it w a s lost w h e n the
o r g a n i s m w a s cultivated u n d e r nonselective conditions (Powell and Kistler 1990;
Kistler, p e r s o n a l c o m m u n i c a t i o n ) . T h e rearranged linear vector, w h i c h h a d
apparently p i c k e d u p Fusarium
t e l o m e r e s at e a c h e n d , could be circularized and
r e c o v e r e d in E. coli b y sequentially treating D N A (isolated from the transformant)
with S I n u c l e a s e , the K l e n o w fragment of D N A p o l y m e r a s e , and ligase prior to
bacterial transformation. T h e r e c o v e r e d (circular) vector, p F O L T 4 R 4 , w a s c o n verted into an a u t o n o m o u s l y replicating linear species w h e n reintroduced into F.

126

Transformation

oxysporum.
T h e A R S activity of the vector is located near p l a s m i d t e r m i n i . S e q u e n c e s similar to the A R S are found on all F. oxysporum
chromosomes resolved
by c o n t o u r - c l a m p e d h o m o g e n e o u s electric field ( C H E F ) electrophoresis (Powell
and Kistler 1990; Kistler, personal c o m m u n i c a t i o n ) .
T h e vector p F O L T 4 R 4 can also transform other Fusarium s p e c i e s , as well as
Nectria haematococca
and Cryphonectria
parasitica,
one-thousand-fold m o r e efficiently than a c o m p a r a b l e vector lacking the putative t e l o m e r e s e q u e n c e s . W h e n
introduced into these fungi, p F O L T 4 R 4 is converted to an a u t o n o m o u s l y replicating
linear form (Powell and Kistler 1990). T h e host r a n g e for the p r o m o t i o n of
a u t o n o m o u s replication and e n h a n c e m e n t of transformation by the putative Fusarium t e l o m e r e is limited, h o w e v e r , as neither property is exhibited w h e n the vector is
i n t r o d u c e d into Aspergillus
(Kistler, personal c o m m u n i c a t i o n ) .

6.4

SELECTABLE MARKERS FOR USE WITH
WILD-TYPE ORGANISMS

B e c a u s e only a limited n u m b e r of filamentous fungi h a v e been the subject of genetic
studies, specific c o m p l e m e n t a b l e m u t a n t s are often not available to b e used as host
o r g a n i s m s for transformation. T o o v e r c o m e this s h o r t c o m i n g a n u m b e r of selective
m a r k e r s that can be used to transform wild-type fungi h a v e b e e n d e v e l o p e d .

6.4.1

Acetamidase

Aspergillus
nidulans can utilize a c e t a m i d e as a sole nitrogen or carbon source o w i n g
to the p r e s e n c e of an a c e t a m i d a s e (amdS) g e n e . This amdS g e n e is u n i q u e insofar as
it is the only n o n m u t a n t g e n e to date that has been used as a m a r k e r to select
transformants of both h o m o l o g o u s and heterologous wild-type filamentous fungi.
T h e A. nidulans amdS g e n e has been isolated ( H y n e s et al. 1983) and seque n c e d (Corrick et al. 1987). A l t h o u g h the cloned g e n e w a s initially used as a
selectable m a r k e r for the transformation of an amdS~ m u t a n t of A. nidulans it w a s
later s h o w n that the g e n e could be e m p l o y e d to select transformants of the w i l d - t y p e
o r g a n i s m (Tilburn et al. 1983; Kelly and H y n e s 1987).
T h e m a i n difficulty in using the a c e t a m i d a s e selection is that, e v e n with amdS~
m u t a n t s of A. nidulans, b a c k g r o u n d g r o w t h of nontransformed cells on m e d i u m that
contains a c e t a m i d e as a sole nitrogen source is often strong e n o u g h to interfere with
the selection of transformants. This residual g r o w t h of amdS~ m u t a n t s , w h i c h is
p r e s u m a b l y d u e to either degradation of the a c e t a m i d e by nonspecific a m i d a s e s or to
impurities in the agar, m a y be eliminated by inclusion of 1 2 . 5 - 1 5 m M C s C l to the
selection m e d i u m (Tilburn et al. 1983). I n d e e d , addition of C s C l to the selection
m e d i u m is sufficient to allow the amdS g e n e to b e used as a selectable m a r k e r for
transformation of wild-type A. nidulans (Kelly and H y n e s 1987).
In addition to A. nidulans,
the amdS g e n e has also been used as a selectable
m a r k e r for transformation of heterologous filamentous fungi w h i c h naturally lack
this g e n e (Table 6 - 1 ) . B e c a u s e selection of amdS function requires g r o w t h on m e d i a

6.4

Selectable Markers for Use with Wild-Type Organisms

127

TABLE 6-1 Filamentous Fungi Transformed with the
A. nidulans amdS Gene
Organism

Reference

Ascobolus

immersus

Faugeron et al. 1990

Aspergillus

ficuum

Mullaney et al. 1988

A.

nidulans

Tilburn et al. 1983

A. niger

Kelley and Hynes 1985

A oryzae

Christensen et al. 1988
1

Cochliobolus

heterostrophus

Glomerella cingulata f. sp.
Pénicillium
P.

chrysogenum

nalgiovense

Trichoderma

reesei

Turgeon et al. 1985
phaseoli

2

Rodriguez and Yoder 1987
Ben and Turner 1987
Geisen and Leistner 1989
Penttilä et al. 1987

'Anamorph: Helminthosporium maydis = Bipolaris
Anamorph: Colletotrichum
lindemuthianium.

2

maydis.

c o n t a i n i n g a c e t a m i d e as a sole nitrogen s o u r c e , use of this g e n e as a selective
m a r k e r i s , in part, limited to those o r g a n i s m s that can be p r o p a g a t e d on a defined
medium.
In all r e p o r t s w h e r e amdS selection h a s b e e n u s e d , C s C l h a s b e e n a d d e d t o the
selection m e d i u m . W h e n C s C l is used in this fashion as a selection aid, the o s m o t i c
support in the regeneration m e d i u m c a n n o t utilize K C l , as the addition of high
levels of this m o n o v a l e n t cation interferes with the selection (Tilburn et al. 1983).

6.4.2

Benomyl Resistance

D e s p i t e the fact that resistance to the fungicide b e n o m y l [methyl 1,2-benimidazole
c a r b a m a t e ( M B C ) ] is usually r e c e s s i v e , it has b e e n possible to use c l o n e d b e n o m y l resistance-conferring jß-tubulin g e n e s as selective m a r k e r s for the transformation of
a variety of filamentous fungi. A s m a n y fungi are sensitive to b e n o m y l , the strategy
first e m p l o y e d to d e v e l o p this selectable m a r k e r required that b e n o m y l - r e s i s t a n t
m u t a n t s b e isolated prior to the c l o n i n g of the resistance g e n e .
Initial studies suggested that b e n o m y l resistance w a s d u e to an alteration in the
jß-tubulin of A. nidulans (Sheir-Neiss et al. 1978). W h e n the c h i c k e n jß-tubulin g e n e
w a s used as a p r o b e to isolate the A. nidulans c o g n a t e , t w o g e n e s w e r e identified; in
addition to the benA g e n e , w h i c h e n c o d e s the jß-tubulin w h i c h is modified in
b e n o m y l - r e s i s t a n t m u t a n t s , a s e c o n d (single c o p y ) jß-tubulin g e n e (tubC, w h i c h
functions primarily d u r i n g the p r o c e s s of sporulation) w a s also isolated ( M a y et al.
1985 and 1987; W e a t h e r b e e et al. 1985; see M o r r i s 1986 for r e v i e w ) .
A l t h o u g h t w o jß-tubulin g e n e s h a v e also been c l o n e d from
Colletotrichum
graminicola
( P a n a c c i o n e et al. 1988; P a n a c c i o n e and H a n a u 1990), not all filamentous fungi h a v e multiple jß-tubulin g e n e s . F o r e x a m p l e , in the c l o n i n g of b e n o m y l -

128

Transformation

resistant genes from Ν. crassa, Leptosphaeria nodorum, as well as A. niger only
single-copy jί-tubulin genes were identified (Orbach et al. 1986; Cooley and Caten
1989; J. Rambosek and D. Finkelstein, unpublished).
A sequence comparison between the wild-type and the benomyl-resistant
Bml511(r) alleles of the N. crassa jί-tubulin gene revealed that benomyl resistance
was attributable to a change from phenylalanine to tyrosine at amino acid residue
167 of the encoded protein (Orbach et al. 1986). Benomyl-resistant transformants
could be selected when the cloned Bml511(r) allele was introduced into a wild-type
strain of N. crassa, thus demonstrating the utility of this cloned gene as a dominant
selectable benomyl resistance marker. In light of the demonstration that a chickenyeast chimeric jί-tubulin can be incorporated into mouse microtubules in vivo
(Bond et al. 1986), it is not surprising that the cloned N. crassa benomyl-resistant
jί-tubulin has also been shown to confer benomyl resistance when expressed in a
variety of filamentous fungi (Table 6-2).
In A. niger, approximately 95% of all benomyl-resistant mutants are recessive
(Finkelstein et al. 1989). Thus, if one desires to clone a benomyl resistance gene
from a specific organism for use as an homologous selective marker, before
choosing a mutant for gene isolation it is probably worthwhile carrying out a test of
dominance (if this is possible with the organism in question). Alternatively, given
the high degree of evolutionary conservation of jί-tubulin, it is also possible to
TABLE 6-2

Filamentous Fungi Transformed to Benomyl Resistance

Organism

Gene Source

Aspergillus
A.

niger

A.

niger

nidulans

Colletotrichum
C.

graminicola

graminicola

C. trifolii
1

Cryphonectria

parasitica

Gaeumannomyces
Leptosphaeria
Metarhizium

graminis
2

nodorum
anisopliae

Reference

N. crassa

Orbach et al. 1986

N. crassa

Orbach et al. 1986

A. niger

Rambosek and Finkelstein, unpublished

N. crassa

Panaccione et al. 1988

C. graminicola

Panaccione et al. 1988

N. crassa

Dickman 1988

Ν •crassa

Churchill et al. 1990

Ν. crassa

Henson et al. 1988

L. nodorum

Cooley and Caten 1989

A. nidulans

Goettel et al. 1990

Neurospora

crassa

N. crassa

Orbach et al. 1986

Pénicillium

chrysogenum

A. niger

Rambosek and Finkelstein, unpublished

P. chrysogenum

Soliday, personal communication

N. crassa

Fernβndez-Larrea and Stahl 1989

N. crassa

Blakemore et al. 1989

P.

chrysogenum

Podospora

anserina

Pseudocercosporella
trichoides

herpo-

'Synonym: Endothia
Anamorph: Septoria

parasitica.
nodorum.

2

6.4

Selectable Markers for Use with Wild-Type Organisms

129

introduce a previously characterized b e n o m y l - r e s i s t a n t mutation into a n e w species
by site-directed m u t a g e n e s i s ( C . L . S o l i d a y , personal c o m m u n i c a t i o n ) .
A l t h o u g h m u l t i c o p y transformants of the N. crassa b e n o m y l - r e s i s t a n t /3-tubulin
g e n e h a v e b e e n found in Podospora
anserina ( F e r n â n d e z - L a r r e a and Stahl 1989),
o v e r e x p r e s s i o n of ß - t u b u l i n seriously inhibits g r o w t h of A. nidulans and is lethal to
the yeast S. cerevisiae
( W a r i n g et al. 1989; B u r k e et al. 1989). T h u s w h e n using
b e n o m y l resistance as a selection w h e r e the e x p e r i m e n t a l goal is m u l t i c o p y g e n e
integration, it is advisable to place the selectable m a r k e r on a separate vector and
employ a cotransformation.

6.4.3

Oligomycin Resistance

T h e respiratory p o i s o n o l i g o m y c i n exerts its toxicity by interacting with subunits 6
+
and 9 of the F 0 portion of the m i t o c h o n d r i a l H - A T P synthase c o m p l e x in fungi
( N a g l e y 1988). A s resistance to o l i g o m y c i n is often a (semi-) d o m i n a n t trait in
Aspergillus,
it has been possible to use a cloned o l i g o m y c i n resistance g e n e as a
d o m i n a n t m a r k e r for selecting transformants of wild-type o r g a n i s m s . T h e (nuclear)
g e n e e n c o d i n g the subunit 9 protein of the mitochondrial A T P synthase c o m p l e x has
b e e n c l o n e d from an oligomycin-resistant m u t a n t (oliC31)
of A. nidulans
by
cross-hybridization to the N. crassa c o g n a t e g e n e ( W a r d et al. 1986). W h e n the
c l o n e d A. nidulans g e n e w a s introduced into a wild-type strain of A.
nidulans,
o l i g o m y c i n - r e s i s t a n t transformants w e r e recovered with the s a m e frequency as for
selection using a h e t e r o l o g o u s g e n e (the pyr4 g e n e of N.
crassa).
T h e oligomycin-resistant p h e n o t y p e of A. nidulans transformants d e p e n d s on
the selection m e t h o d that is e m p l o y e d ( W a r d et al. 1986). Direct selection for
o l i g o m y c i n resistance yields primarily transformants that are the result of g e n e
r e p l a c e m e n t (or integration followed by g e n e conversion) at the resident oliC l o c u s .
T h e s e transformants are as resistant to o l i g o m y c i n as the original oliC31 m u t a n t . If,
h o w e v e r , selection for o l i g o m y c i n resistance is m a d e after a p r i m a r y selection for
a n o t h e r m a r k e r , m o s t of the resulting transformants are less resistant to o l i g o m y c i n
than the original oliC31 m u t a n t . S u c h semiresistant transformants exhibit t w o
different p h e n o t y p e s . T h o s e that h a v e arisen from plasmid integration at the oliC
locus yield fully resistant sectors (due to excision or c o n v e r s i o n of the wild-type
locus) w h e n cultivated on plates containing o l i g o m y c i n , w h e r e a s transformants that
h a v e arisen from p l a s m i d integration at a h e t e r o l o g o u s site rarely give rise to such
fully resistant p r o g e n y ( W a r d et al. 1986).
A s the A. nidulans oliC g e n e a p p e a r s not to function across species lines, it has
b e e n necessary to isolate the h o m o l o g o u s g e n e from oligomycin-resistant m u t a n t s
of A. niger and Pénicillium
chrysogenum
(by cross-species hybridization) to use
o l i g o m y c i n resistance as a selectable m a r k e r for these o r g a n i s m s ( W a r d et al. 1988;
Bull et al. 1988). T h e question of w h e t h e r this strategy can be e x t e n d e d to other
filamentous fungi is c o m p l i c a t e d b y the uncertainty as to the location of the subunit
9 g e n e in these o r g a n i s m s . Putative g e n e s for subunit 9 h a v e b e e n identified on the
m i t o c h o n d r i a l g e n o m e of other fungi (Borkhardt et al. 1988; B r u n s et al. 1988). A s

130

Transformation

it is well d o c u m e n t e d that the functional subunit 9 g e n e of the yeast S. cerevisiae is
m i t o c h o n d r i a l (see Dujohn 1981 for r e v i e w ) , o n e cannot rule out this possibility in
other o r g a n i s m s , despite the fact that such putative mitochondrial g e n e s are n o n functional in Aspergillus
and Neurospora
(reviewed in G r o s s m a n and H u d s p e t h
1985).

6.4.4

Hygromycin Resistance

Of those filamentous fungi transformed to d a t e , a majority h a v e b e e n transformed
(at least o n e time) via the use of selection for h y g r o m y c i n Β resistance. O n e m i g h t
thus say that h y g r o m y c i n resistance represents to filamentous fungal c l o n i n g vectors
w h a t ampicillin resistance has m e a n t to Escherichia
coli cloning v e c t o r s .
H y g r o m y c i n B , an aminocyclitol antibiotic p r o d u c e d by Streptomyces
hygroscopicus,
is an inhibitor of translation that has a b r o a d - s p e c t r u m activity against
both procaryotic and eucaryotic cells (Pettinger et al. 1953; G o n z a l e z et al. 1978).
P l a s m i d p J R 2 2 5 of E. coli strain W 6 7 7 , the original source of the h y g r o m y c i n
resistance g e n e , e n c o d e s a phosphotransferase (hph) that inactivates h y g r o m y c i n Β
by p h o s p h o r y l a t i n g the h y d r o x y l on the 4 position of the cyclitol ring ( D a v i e s and
O ' C o n n e r 1978; R a o et al. 1983). T h e g e n e s e q u e n c e predicts a m o l e c u l a r m a s s of
3 9 , 0 0 0 D a for the phosphotransferase (Gritz and D a v i e s 1983; K a s t n e r et al. 1983).
T h e cloned g e n e is c o m m e r c i a l l y available.
O w i n g to the fact that six A T G triplets are located b e t w e e n a c o n v e n i e n t
restriction site and the translational start c o d o n of the hph g e n e , a n u m b e r of
different strategies h a v e been e m p l o y e d to engineer its expression in filamentous
fungi. U s i n g site-directed m u t a g e n e s i s , a Cla\ restriction site w a s placed imm e d i a t e l y u p s t r e a m from the start of translation. W h e n this modified g e n e w a s
spliced b e t w e e n the A. nidulans trpC p r o m o t e r and terminator, the resulting vector
could transform A. nidulans
(Cullen et al. 1987a). Vectors carrying this g e n e
construct h a v e been deposited in the Fungal G e n e t i c s Stock C e n t e r ( D e p a r t m e n t of
M i c r o b i o l o g y , University of K a n s a s M e d i c a l C e n t e r , K a n s a s C i t y , K a n s a s 6 6 1 0 3 ) .
In an alternate c o n s t r u c t i o n , Bal3l w a s used to delete all but o n e of the u p s t r e a m
A T G triplets (Gritz and D a v i e s 1983). Insertion of this modified g e n e b e t w e e n the
p r o m o t e r and terminator of a m e m b e r of the U. maydis hsp70 g e n e family creates a
vector that can transform this o r g a n i s m ( S . A . L e o n g , personal c o m m u n i c a t i o n ;
W a n g et al. 1988).
A n alternate m e t h o d for r e m o v i n g u p s t r e a m A T G s e q u e n c e s has b e e n to create
translational fusions. T h e feasibility of such an a p p r o a c h for the hph g e n e w a s
d e m o n s t r a t e d with the observation that r e p l a c e m e n t of the first three c o d o n s of this
g e n e with h e t e r o l o g o u s c o d i n g s e q u e n c e s did not result in loss of p h o s p h o t r a n s ferase activity in either E. coli or yeast (Kaster et al. 1983 and 1984). F u s i o n of the
truncated g e n e directly to the A T G start c o d o n of either the A. nidulans glycerald e h y d e - 3 - p h o s p h a t e d e h y d r o g e n a s e (gpd) g e n e or the Nectria haematococca
cutinase g e n e creates vectors that can transform these respective fungi (Punt et al. 1987;
Soliday et al. 1989).
O w i n g to the e x t r e m e l y efficient r e m o v a l of h y g r o m y c i n - s e n s i t i v e o r g a n i s m s

f r e q u e n c y transformation of U. Barrett et al. O n c e r e c o v e r e d . (1990) w e r e able to transform the m y c o r r h i z a l b a s i d i o m y c e t e Laccaria laccata. t e r m e d APH(3 )-\ and APH(3 )-II. H o w e v e r . 1987). Pefialva et . can be detoxified by the action of a m i n o g l y c o s i d e p h o s p h o t r a n s ferase e n z y m e s . 1978). w h i c h p h o s p h o r y l a t e the antibiotic at the 3 ' position ( H a a s and D o w d i n g 1975). G e n e s e n c o d i n g t w o different a m i n o g l y c o s i d e p h o s p h o t r a n s y } ferases. using a vector c o n t a i n i n g the A. respectively (Courvalin et al.6. N o t surprisingly. hyg r o m y c i n . may dis (Smith et al.4. h a v e both been s e q u e n c e d . In a s h o t g u n selection using a b a n k of r a n d o m Cochliobolus heterostrophus D N A s e q u e n c e s inserted adjacent to the truncated hph g e n e .3 . a vector created by fusing the A.r e s i s t a n t Cochliobolus transformants w e r e recovered at a frequency of 6 0 . as well as related a m i n o g l y c o s i d e s . 0 2 to 0 . 0 4 c o l o n i e s per m i c r o g r a m of D N A per 1 0 viable protoplasts ( T u r g e o n et al. such as n e o m y c i n or k a n a m y c i n .5 Aminoglycoside Resistance T h e a m i n o g l y c o s i d e antibiotic G 4 1 8 inhibits protein synthesis in both procaryotic and e u c a r y o t i c cells ( J i m e n e z and D a v i e s 1980). a vector containing a Cochliobolus p r o m o t e r fused to the hph g e n e has been used to transform the h e m i b a s i d i o m y c e t e Ustilago violacea (Bej and Perlin 1989). 1989). and each e n c o d e s a protein h a v i n g m o l e c u l a r m a s s of = 3 0 K D a ( O k a et al. niger g l u c o a m y l a s e p r o m o t e r to the hph g e n e can also be used to transform U. 1982). G 4 1 8 can be replaced with less e x p e n s i v e a m i n o g l y c o s i d e s . h a v e been identified o n t r a n s p o s o n s T n 9 0 3 a n d T n 5 . L o w . T h i s c o m p o u n d . the truncated hph g e n e can also b e used as a p r o b e vector to isolate fungal p r o m o t e r s . w h i c h are c o m m e r c i a l l y a v a i l a b l e . T h e s e g e n e s .p h o s p h a t e d e h y d r o g e n a s e p r o m o t e r fused to the hph g e n e . B e c k et al.4 Selectable Markers for Use with Wild-Type Organisms 131 d u r i n g selection. attempts to e x p r e s s the unmodified ΑΡΗ g e n e s in filamentous fungi h a v e m e t with only limited s u c c e s s . In e x p e r i m e n t s with m o r e distantly related fungi. Cephalosporium acremonium. A r n a u et al. 1988). Similarly.3 ) . these g e n e fusions could be used to transform Cochliobolus at a high frequency. P r o m o t e r s from the a s c o m y c e t e s Aspergillus and Cochliobolus w o r k in a w i d e variety of a s c o m y c e t e s (as well as related fungi imperfecti). G 4 1 8 resistance has b e e n e m p l o y e d as a selective m a r k e r with only a limited n u m b e r of filamentous fungi d u e to the high resistance of m a n y fungi to this antibiotic as well as a lot-to-lot variability in antifungal activity of this c o m p o u n d (Finkelstein et al. 1990). 6. I n d e e d . w h e n selecting for ΑΡΗ function in s o m e z y g o m y c e t e s ( W ö s t m e y e r et al. h e t e r o l o g o u s p r o m o t e r s h a v e b e e n used m o r e often than h o m o l o g o u s p r o m o t e r s with this selection s y s t e m (Table 6 . and Ρ hy corny ce s blakesleeanus was o b s e r v e d w h e n using the u n m o d i f i e d ΑΡΗ g e n e of T n 9 0 3 ( B a n k s 1983. may dis. 1987. T h e functional p r o m o t e r s (fused to the h y g r o m y c i n p h o s p h o t r a n s f e r a s e g e n e ) could b e r e c o v e r e d by screening a g e n e b a n k (in a λ p h a g e vector) constructed with D N A from the h y g r o m y c i n resistant Cochliobolus transformants. T h e p r e s e n c e of a h o m o l o g o u s p r o m o t e r is not required to effect expression of the hph g e n e in a given filamentous fungus. 1 9 8 1 . Finally. nidulans g l y c e r a l d e h y d e .

nidulans trpC Tsuge et al. 1989 Churchill Churchill Churchill Churchill Churchill et et et et et al. 1985 PGK Cullen et al. parasitica cutinase Soliday et al. 1987 Kiick et al. nidulans trpC A. trifolii Cryphonectria 2. ficuum A. nidulans nidulans Cephalosporium nium acremo- Cochliobolus heterostrophus* Nectria haematococca Ustilago maydis 2 gpd Punt et al. 1990 A. Turgeon et al. personal communication C. Colletotrichum A. C. heterostrophus Cullen et al. A. 1989 hsp70 Wang et al. 1990 A. al. 1987 C. nidulans gpd Oliver et al. nidulans trpC Kistler and Benny 1988 C. 1987 C. 1987a IPNS Skatrud et al. nidulans gpd Barrett et al. nidulans trpC C. A. nidulans gpd C. Cochliobolus Huang et al. nidulans gpd C. C. 1989 Alternaria alternata giganteus A. 1987 cutinase Soliday et al. 1990 1990 1990 1990 1990 Curvularia lunata A. al. heterostrophus Dickman 1988 C. al. 1988 A. heterostrophus Dickman. nidulans gpd Mullaney et al. 1987 Unidentified Turgeon et al. 1990 A. acremonium heterostrophus nidulans trpC nidulans gpd maydis hsp70 graminicola C.132 Transformation TABLE 6-3 Filamentous Fungi Transformed to Hygromycin Resistance Organism Homologous Promoter Aspergillus A. 1987 trpC Cullen et al. heterostrophus Thomas and Kenerley 1989 C. 1987 Nectria Fulvia haematococca fulva 4 Fusarium oxysporum Gibberella fujikuroi 5 Gliocladium Glomerella Laccaria 2 vir ens cingulata 6 laccata Leptosphaeria maculans 1 heterostrophus A. nidulans gpd Osiewacz and Weber 1989 Furarium sporotrichioides A. 1987a C. cerevisiae Queener et al. 1989 S. al. A. 1988 Heterologous Organism Reference Promoter Reference A. 1987a Turgeon et al. U. heterostrophus Farman and Oliver 1988 Turgeon et al. nidulans trpC Cullen et al. 1987 . heterostrophus Rodriguez and Yoder 1987 A. caps ici Ν. 1987 A. 1987a A. acremonium Punt et al. nidulans trpC sp. heterostrophus Turgeon et al. haematococca C. niger Botryotinia squamosa Cephalosporium nium acremo- sp. nidulans gpd Wnendt et al.

10 Formerly: Ceratocystis ulmi. chrysogenum w a s a c h i e v e d with a vector e m p l o y i n g the am p r o m o t e r of Neurospora crassa to drive e x p r e s s i o n of the APHII g e n e (Finkelstein et al. 3 Synonym: Endothia parasitica. H o w e v e r . 1989 Stäben et al. 1990 cutinase N. 7 Synonym: Phoma Ungarn. Churchill et al. ep. nidulans gpd Leung et al. It w a s c l a i m e d that transformants a p p e a r e d m o r e rapidly (Penalva et al. Although Achlya ambisexualis could be transformed with a vector that contained the S V 4 0 promoter fused to the APHU gene. nidulans trpC Stäben et al. chrysogenum IPNS Royer et al. T o i m p r o v e the function of the ΑΡΗ g e n e s in fungi. 6 Anamorph: Colletotrichum lindemuthianium. a n u m b e r of defined fungal p r o m o t e r s h a v e b e e n utilized. nidulans gpd Blakemore et al. niger.6. nidulans gpd spp. A. S o m e success has been achieved in using nonfungal promoters to drive the expression of the T n 5 phosphotransferase in fungi. 1990 A. A Cauliflower mosaic virus 35S promoter-driven APHII gene has been used to transform Fusarium solani f. haematococca 10 Reference Cooley et al. and P. 1987). 1988 A. 1989. 1989 P. awamori. 1990 Ustilago C. Northern blot analysis suggested that this promoter was nonfunctional (Manavathu et al. 8 Anamorph: Septoria nodorum. 1989). 1990). . 1990 Ustilago may dis A niger glucoamylase Smith et al. 9 Anamorph: Pyricularia oryzae. 2 133 maydis. 1989 A. 4 Synonym: Cladosporium fulvum. phaseoli and Cryphonectria parasitica (Marek et al. nidulans gpd A. acremonium.4 TABLE 6 . Mycosphaerella Dickman et al. 1 9 8 5 . S i m i l a r l y . W h i l e splicing the yeast ADC1 p r o m o t e r to the ΑΡΗ g e n e did not i m p r o v e transformation frequency for C. S u a r e z a n d E s l a v a 1988). blakesleeanus with an u n m o d i f i e d ΑΡΗ g e n e . R e v u e i t a and J a y a r a m (1986) h a v e transformed P. 1989 A nidulans gpd Goldman et al. 5 Anamorph: Fusarium moniliforme. T r a n s f o r m a t i o n of A. it is possible that a Phy corny ces s e q u e n c e (isolated as an A R S e l e m e n t in yeast) on the vector w a s fortuitously functioning as a p r o m o t e r .3 Selectable Markers for Use with Wild-Type Organisms (continued) Organism Heterologous Leptosphaeria nodorum* Magnaporthe grisea 9 Neurospora crassa Ophiostoma ulmi Promoter A. 1988). it w a s possible to transform the z y g o m y c e t e Absidia glauca by splicing the p r o m o t e r from an h o m o l o g o u s actin g e n e to the APHII g e n e ( W ö s t m e y e r et al. Primer extension analysis of m R N A isolated from one Fusarium transformant showed transcription initiation occurring within 10 to 30 bases of the native start site for this promoter (Marek et al. 1987). al. heterostrophus Bej and Perlin 1989 Pseudocercosporella trichoides Trichoderma herpo- harzianum violacea 'Anamorph: Helminthosporium maydis = Bipolaris Anamorph: Fusarium solani. 1985).

1988 Katz and Hynes 1989 N.t o o n e ratio. crassa am Austin et al. 6. G a t i g n o l et al.134 Transformation T h e APHIl g e n e has also been used to isolate fungal p r o m o t e r s . nidulans gpd N. nidulans trpC van Engelenburg et al. nidulans trpC A. blakesleeanus prom o t e r s b y selecting for expression in E. 0 0 0 transformants p e r m i c r o g r a m ( A r n a u et al. 1989 Mattern et al. 1988). A s with other procaryotic s e q u e n c e s . 1988). and from Streptoalloteichus hindustanus (Gatignol et al. the drug can n o longer cleave D N A (Gatignol et al. coli transposon T n 5 (Collis and Hall 1985). they can be readily destroyed at acidic and at basic p H . 1982. O n c e b o u n d . a p r o m o t e r . from the staphylococcal p l a s m i d p U B H O ( S e m o n et al. 1990 Pénicillium chrysogenum A. B l e o m y c i n is currently used clinically as an antitumor agent. nidulans gpd Kolar et al. coli. the former c o m p o u n d a p p e a r s to b e preferred by those w o r k i n g with filamentous fungi. 1988 A.4 ) . 1989 Neurospora crassa N. 1988 Trichoderma reesei . W h e n p r o d u c e d in E. 1987). the S. T h u s . terreus Claviceps purpurea Promoter Reference A. blakesleeanus to k a n a m y c i n resist a n c e . crassa am Katz and Hynes 1989 A. hindustanus protein g e n e product has been s h o w n to bind reversibly to b l e o m y c i n in a o n e . crassa am van Engelenburg et al. coli. water-soluble g l y c o p e p t i d e antibiotics that kill both eucaryotic and p r o caryotic cells at low concentrations by b r e a k i n g D N A at specific sites ( K r o s s et al. crassa am van Engelenburg et al.6 Bleomycin Resistance R e c e n t l y the g e n e e n c o d i n g b l e o m y c i n resistance (ble) has b e e n used as a d o m i n a n t selectable m a r k e r for filamentous fungi (Table 6 . all (34 i n d e p e n d e n t ) p l a s m i d isolates could be used to transform P. A l t h o u g h both p h l e o m y c i n and b l e o m y c i n a p p e a r to function equally well as agents for selection of t r a n s f o r m a n t s . It is thus p r e s u m e d that all three genes confer resistance to b l e o m y c i n (as well as p h l e o m y c i n ) b y such drug sequestration. R e m a r k a b l y .d e l e t e d APHII g e n e w a s used to identify putative P. nidulans trpC A. nidulans gpd Durand et al. B l e o m y c i n and p h l e o m y c i n are related. 1988 Katz and Hynes 1989 A. 1989 Mattern et al. the genes e n c o d i n g the b l e o m y c i n b i n d i n g proteins m u s t be spliced to appropriate p r o m o t e r s before they can be e x p r e s s e d in TABLE 6-^4 Filamentous Fungi Transformed to Bleomycin Resistance Organism Aspergillus nidulans A. H o m o l o g o u s g e n e s e n c o d i n g b l e o m y c i n resistance h a v e b e e n identified and characterized from the E.4. A l t h o u g h both b l e o m y c i n and p h l e o m y c i n are quite t o x i c . 1988). 1987). niger A. at frequencies r a n g i n g from 2 0 to 3 . nidulans gpd N.

B y transforming such a m u t a n t using a g e n e o b t a i n e d from a wild-type o r g a n i s m as a selectable m a r k e r . o n e m u s t then obtain the appropriate wild-type g e n e for use as a selective agent to allow o n e to distinguish transformed from n o n t r a n s f o r m e d organism. Clutterbuck 1974). c o n c e r n s with obtaining regulatory approval for a g i v e n r e c o m b i n a n t fungus m a y m a k e it advisable for o n e to use a fungal m u t a n t as a transformation recipient. T h i s c h a r a c terization is required b e c a u s e m u t a t i o n s in different genes can often s h o w the s a m e gross p h e n o t y p e (for e x a m p l e . that are not normally carried by the wild-type organism.5 SELECTABLE MARKERS FOR USE WITH MUTANT HOSTS Selective m a r k e r s are used to distinguish transformed from n o n t r a n s f o r m e d o r g a n i s m s . such e n g i n e e r e d o r g a n i s m s will carry n o features. A u x o t r o p h i c m u t a n t s can b e selected by replica plating individual o r g a n i s m s o n t o rich and m i n i m a l m e d i u m . such as drug resistance or an ability to g r o w in a different ecological n i c h e . 6. O n c e the particular lesion has b e e n c h a r a c t e r i z e d . 1987). o n e can enrich for a u x o t r o p h i c m u t a n t s prior to plating by using filtration to separate the n o n g e r m i n a t e d spores from g e r m i n a t e d prototrophic spores that put out h y p h a e ( D a v i s and D e S e r r e s 1970). A l t h o u g h all w o r k to date with filamentous fungi h a s used p r o m o t e r s that d o not contain c o d i n g s e q u e n c e s . First. suitably e n g i n e e r e d for the addition of fungal p r o m o t e r s . 6. niger by standard filtration e n r i c h m e n t m e t h o d s ( G o o s e n et al. Toulouse. T h i s difficulty is p r e s u m a b l y d u e in part to the fact that trpC m u t a n t s of a n u m b e r of fungi g r o w and sporulate p o o r l y . A l t h o u g h the p r e v i o u s section has described a series of m a r k e r s that m a y b e used to transform w i l d .6. O t h e r than the desired c h a n g e that o n e is i n t r o d u c i n g . A l t e r n a t i v e l y .5. France). T o cite but o n e e x a m p l e : it h a s not b e e n p o s s i b l e to isolate a trpC m u t a n t of ultraviolet (UV)-irradiated A. the T n 5 protein can apparently tolerate additional a m i n o acid residues at its a m i n o t e r m i n u s (Gatignol et al. T w o o b v i o u s prerequisites m u s t be met before o n e can transform a m u t a n t o r g a n i s m . m u t a t i o n s in at least ten different g e n e s can result in histidine a u x o t r o p h y in A. nidulans. V e c t o r s c o n t a i n i n g both the T n 5 g e n e and the S.t y p e o r g a n i s m s .5 Selectable Markers for Use with Mutant Hosts 135 filamentous fungi. o n e effectively recreates the w i l d . e v e n w h e n s u p p l e m e n t e d with high levels of t r y p t o p h a n or indole .1 Isolation and Characterization of Auxotrophic Mutants F o r a general description of t e c h n i q u e s used for m u t a g e n e s i s the reader is referred to C h a p t e r 3 b y R o w l a n d s in this v o l u m e . are available c o m m e r c i a l l y ( C a y l a . T h e frequency at w h i c h a given mutation will arise is a function of both the starting o r g a n i s m and the m u t a g e n b e i n g e m p l o y e d . hindustanus g e n e . 1989).t y p e o r g a n i s m . b e c a u s e spores carrying a u x o t r o p h i c m u t a t i o n s d o not g e r m i n a t e in m i n i m a l m e d i a . o n e m u s t isolate and characterize a m u t a n t o r g a n i s m .

nidulans John and Peberdy 1984 A. w h e r e intermediates are not c o m m e r c i a l l y available or e n z y m e assays are quite difficult. nidulans O C T a s e (argB) g e n e . 1987 Neurospora crassa A. 1990). 1987 . niger Penttilä et al. einer eus trp2 g e n e . niger A. 1987 A. niger Buxton et al. F o r other m u t a n t s . 1989. nidulans Berka et al. B y contrast. in the a b s e n c e of other available information. along with the early availability of the c l o n e d A.5 ) . T h e ease of isolating and characterizing O C T a s e m u t a n t s . cells carrying a m u t a t i o n in ornithine carbamoyltransferase ( O C T a s e . oryzae A. w h i c h catalyzes the cond e n s a t i o n of ornithine and c a r b a m o y l p h o s p h a t e to form citrulline in the arginine biosynthetic p a t h w a y ) m a y be conveniently distinguished from other arginine a u x o trophs by their ability to g r o w on m i n i m a l m e d i a s u p p l e m e n t e d with citrulline. 1989). 1990). 1985 A. A s a first s t e p . 1987 1 'Anamorph: Pyricularia oryzae. nidulans A.m u t a g e n i z e d colonies of the b a s i d i o m y c e t e Coprinus bilanatus readily yield the c o r r e s p o n d i n g trpl m u tants (and can be transformed with the C. and v i t a m i n s to identify the g r o w t h r e q u i r e m e n t . such as those in the purine biosynthetic p a t h w a y . nidulans Weiss et al. T h u s . has led to the use of this m a r k e r for the transformation of a variety of fungi (Table 6 . O n c e a u x o t r o p h i c m u t a n t s h a v e b e e n isolated it is desirable to characterize the specific lesion so that o n e can d e t e r m i n e the correct g e n e to use for transformation. Reference Buxton et al. nidulans Buxton et al. G o o s e n et al. nidulans Parsons et al. 1987). nidulans Gomi et al. nidulans A. niger A. T h e specific lesion in a biosynthetic p a t h w a y leading to specific a u x o t r o p h y can then be identified either b y e n z y m e assays or by testing for a g r o w t h r e s p o n s e w h e n the m u t a n t is plated on various pathway intermediates. G o l d and his colleagues successfully transformed t w o different uncharacterized adenine m u t a n t s of Phanerochaete chrysosporium with cloned Schizophyllum commune ADE g e n e s (Alic et al. 1987 Magnaporthe grisea A.136 Transformation (Picknett et al. 1987. B u r r o w s et al. but not with ornithine ( B u x t o n et al. TABLE 6-5 Filamentous Fungi Transformed with the OCTase Gene Organism Gene Source Aspergillus awamori A. the test of g r o w t h p h e n o t y p e can often u n a m b i g u o u s l y define the lesion. purines and p y r i m i d i n e s . the m u t a n t is generally plated on a matrix of m i n i m a l m e d i u m plates c o n t a i n i n g o v e r l a p p i n g pools of various a m i n o a c i d s . 1990 A. U V . o n e m u s t resort to other techniques to characterize m u t a n t s . F o r e x a m p l e . 1985. Penttilä et al. nidulans A. 1985 Trichoderma reesei A. F o r p a t h w a y s w h e r e intermediates are readily available.

A s noted a b o v e .c o e n z y m e A ( a c e t y l . H e noted that resistance to toxic a n a l o g s of s o m e metabolites could b e used to p r o v i d e a positive selection for loss of g e n e function. three fac g e n e s with o n e e n c o d i n g a c e t y l .C o A synthetase) for the b a s i d i o m y c e t e Coprinus lagopus (Casselton and Casselton 1974).2 Counterselectable Genes as Selectable Markers W i t h filamentous fungi. T h e situation is quite similar (that is. counterselectable g e n e s can b e uniquely exploited to select positively for o r g a n i s m s that h a v e lost their integrated D N A .C o A ) synthetase ( R o m a n o and K o r n b e r g 1969). nidulans e n c o d e s a c e t y l . T h u s . T h e ability to select a mutation positively in a counterselectable g e n e eases the task of identifying a suitable host w h e n o n e desires to use m u t a n t c o m p l e m e n t a t i o n for the selection of transformed o r g a n i s m s . F o u r separate acetate nonutilizing fluoroacetate- . T h u s . T h e U. 5 . fluoroacetete-sensitive revertants can b e positively selected by screening for o r g a n i s m s able to utilize acetate as a sole c a r b o n s o u r c e . T h e rationale for the m a n i p u l a t i o n of counterselectable g e n e s w a s first e x plicitly described by Apirion ( 1 9 6 2 ) . it is c o n v e r t e d in vivo to fluorocitrate. crassa ade m u t a n t (Alic et al. Although fluoroacetate (which w a s originally identified as the toxic c o m p o n e n t of the plant Dichapetalum cymosum) inhibits n o e n z y m e s itself. W h e r e the analog is itself n o n t o x i c . m u t a t i o n s in three g e n e s also resulted in an inability to g r o w on acetate as a sole carbon source (Apirion 1965). Resistance to toxic c o m p o u n d s can b e conferred in different w a y s . commune D N A to transform a characterized N. resistance can also o c c u r by p r e v e n t i n g the c o m p o u n d ' s conversion to its toxic form. o n e must often screen a large n u m b e r of m u t a n t s to find a suitable c o m p l e m e n t a b l e a u x o t r o p h . by screening for fluoroacetate-resistant m u t a n t s (a positive selection) o n e can isolate acetate nonutilizers. crassa acu5 g e n e ( H a r g r e a v e s and T u r n e r 1989). o n c e a transformant h a s b e e n o b t a i n e d . Similarly. g e n e s for w h i c h o n e can simply select for a b s e n c e of function (that is.5 Selectable Markers for Use with Mutant Hosts 137 T h e specific lesion of o n e of the m u t a t i o n s w a s then defined by successfully using the s a m e S. o n e that utilizes a n o n m u t a n t h o m o l o g o u s g e n e as a selective m a r k e r ) is the lack of a suitable m u t a n t o r g a n i s m to receive the D N A .5. counterselectable genes) can offer significant a d v a n t a g e s for both genetic and r e c o m b i n a n t D N A studies. nidulans m u t a n t s that w e r e resistant to fluoroacetate. 1990).C o A synthetase g e n e has b e e n successfully e m p l o y e d as a selectable m a r k e r for transformation of the b a s i d i o m y c e t e U. 6. A m o n g the A. 1 F l u o r o a c e t a t e R e s i s t a n c e . 2 . nidulans. 6 . T h e a c e t y l .6. a potent inhibitor of aconitase (Peters 1957). O n e o b v i o u s m e c h a n i s m is to p r e v e n t the analog from reaching the site of its toxic activity b y inhibiting its u p t a k e . maydis. maydis acuA g e n e w a s isolated by p r o b i n g a g e n e b a n k with the c o g n a t e N. the factor that most often limits the d e v e l o p m e n t of an entirely h o m o l o g o u s transformation system (that is. It w a s subsequently s h o w n that the facA g e n e of A. F u r t h e r m o r e . Apirion (1962) exploited fluoroacetate resistance for selecting both forward and b a c k mutations in A.

O M P p p a s e and/or O M P d e c a s e m u t a n t s are found as a subset of the 5-FOA-resistant o r g a n i s m s . G o o s e n et al.F O A resistant o r g a n i s m s . 1988. 1987. a m o n g the m a n y 5-FOA-resistant m u tants isolated from Podospora anserina and Histoplasma capsulatum only O M P p p a s e m u t a n t s (and uridine prototrophs) w e r e identified ( R a z a n a m p a r a n y and B é gueret 1986. nidulans acuD (isocitrate lyase) g e n e . A l t h o u g h the p o w e r of a positive selection should. A l t h o u g h e n z y m e assays h a v e b e e n used to identify O M P d e c a s e and O M P p p a s e mutants prior to transformation (Diez et al. it is possible to avoid e n z y m o l o g y by successfully transforming a specific 5-FOA-resistant mutant with a cloned g e n e ( V a n H a r t i n g s veldt et al. If a n o n c a r b o n o s m o t i c u m can be d e v e l o p e d for the regeneration of the transformed spheroplasts [as has been e m p l o y e d for direct selection of the A.p h o s p h a t e p y r o p h o s p h o r y l a s e ( O M P p p a s e ) or orotidine-5 ' p h o s p h a t e d e c a r b o x y l a s e ( O M P d e c a s e ) activity are resistant to 5 .138 Transformation resistant m u t a n t s of U. 1987. 1987). In p r a c t i c e . as uracil a u x o t r o p h s lacking either orotidine-5 ' . In both cases a cotransformation w a s p e r f o r m e d . nidulans (facA) by differential hybridization to c o m p l e m e n t a r y D N A ( c D N A ) probes ( T h o m a s et al. W o r s h a m and G o l d m a n 1988).n e g a t i v e m u t a n t s . g e n e s for a c e t y l . 1987).U M P . rather than attempting to select directly for a c e t y l . 1984). 5 . the frequency of uridine a u x o t r o p h s a m o n g 5 .C o A synthetase function. S a n d e m a n and H y n e s 1989). F o r e x a m p l e . W o r s h a m and G o l d m a n 1988). maydis were successfully transformed with the c l o n e d U. 2 . A s the o s m o t i c support used in the transformation (sorbitol) allowed a limited g r o w t h of the untransformed ( m u tant) h o s t s . A m o n g the a s c o m y c e t e s .C o A synthetase function in the transformants. Ballance and T u r n e r 1986] it should be possible to select directly for a c e t y l .C o A synthetase h a v e b e e n c l o n e d from N. 6 .and O M P p p a s e . T h e s e cloned g e n e s w e r e subsequently reintroduced into appropriate m u t a n t s of the h o m o l o g o u s o r g a n i s m s . may dis acuA g e n e . crassa (acu5) and A. t w o a p p r o a c h e s m a y be used to d e t e r m i n e the specific e n z y m a t ic lesion of these m u t a n t s .F O A ) R e s i s t a n c e .F O A p r o v i d e s a basis for using s o m e p y r i m i d i n e biosynthetic g e n e s as selective m a r k e r s for transformation of the filamentous fungi.f l u o r o . O n c e the p y r i m i d i n e a u x o t r o p h s h a v e been identified a m o n g the 5 .F O A ( B o e k e et al. in principle. allow the identification of the desired m u t a n t s without m u t a g e n e s i s . 2 5-FIuoro-Orotic A c i d ( 5 . d e m o n s t r a t i n g at the s a m e time that these fluoroacetate-resistant m u t a t i o n s w e r e indeed located in the facA g e n e . . T h e toxic uracil a n a l o g 5 .F O A resistant cells is increased greatly following treatment with m u t a g e n s (Diez et al. It should be appreciated that not all fungi yield both O M P d e c a s e . the initial step in the isolation of specific p y r i m i d i n e a u x o t r o p h s is the selection of 5-FOA-resistant m u t a n t s . it w a s necessary to allow the transformed spheroplasts to r e g e n e r a t e in the a b s e n c e of selection prior to replica plating o n t o acetate m e d i a (lacking o s m o t i c support) to select the acetate-utilizing transformants ( H a r g r e a v e s and T u r ner 1989). T h e toxicity of this c o m p o u n d a p p e a r s to result from its c o n v e r s i o n to 5 .

1983 1 N.o r o t o a s e m u t a n t s are not 5 . anserina Bégueret et al. respectively ( B u x t o n and Radford 1983. B a n k s and T a y l o r 1988). niger A. ( A l t h o u g h d i h y d r o . ) S o m e of these fungal g e n e s h a v e since b e e n e m p l o y e d as p r o b e s to isolate c o g n a t e s from additional fungi ( V a n Hartings veldt et al. 1983). chrysogenum Cantoral et al.o r o t a s e w e r e initially isolated by c o m p l e m e n t a tion of E. crassa pyr4 ( O M P d e c a s e ) g e n e could c o m p l e m e n t the c o r r e s p o n d i n g A. . oryzae Mattern et al. oryzae de Ruiter-Jacobs et al. niger Van Hartings veldt et al.6. 1984. G o o s e n et al. nidulans pyrG m u t a t i o n w a s the first e x a m p l e that g e n e s from filamentous fungi w e r e c a p a b l e of functioning across species lines (Ballance et al. 1987 A. niger Goosen et al. 1987 N. 1989 Not tested in fungi Vian and Penalva 1990 P. crassa P. commune Froeliger et al. nidulans Oakley et al. U s e of specific p y r i m i d i n e biosynthetic g e n e s is not limited to the transformation of h o m o l o g o u s o r g a n i s m s (Table 6 . L e C h e v a n t o n and L e b l o n 1989). pyrE. 1987. niger A. A. 1987 Van Hartingsveldt et al. macrospora Le Chevanton et al. oryzae A. O M P p p a s e .F O A resistant. coli pyrF. anserina anserina Sordaria macrospora P. may dis 'Available OMPdecase mutants were too leaky to allow direct selection. 1987. 1989 U. 1984 Histoplasma Worsham and Goldman 1990 capsulatum S. 1989 Cephalosporium nium Pénicillium acremo- chrysogenum Schizophyllum commune Ustilago may dis OMPppase Podospora P. 1987 U. I n d e e d . awamori Ward et al. and pyrC m u t a n t s . 1987 A. niger A. 1988 5. may dis Kronstad et al. 1987 A. crassa Buxton and Radford 1983 N. oryzae A. TABLE 6-6 Filamentous Fungi Transformed with Pyrimidine Biosynthetic Genes Organism Gene Source Reference OMPdecase Neurospora crassa A. 1987 N. nidulans Ballance et al. B é g u e r e t et al. niger de Ruiter-Jacobs et al. they are listed here for c o m p l e t e n e s s . crassa crassa Aspergillus nidulans A. 1989 A. 1989 A. chrysogenum Cantoral et al.6 ) . and d i h y d r o .5 Selectable Markers for Use with Mutant Hosts 139 F i l a m e n t o u s fungal g e n e s e n c o d i n g the p y r i m i d i n e biosynthetic e n z y m e s O M P d e c a s e . maydis Banks and Taylor 1988 Dihydro-orotase U. the d e m o n s t r a t i o n that a c l o n e d N.

Breton a n d Surdin-Kerjan 1 9 7 7 . L y d i a t e et al. 5 . chlorate resistance varies with both t h e strain . c o n t a i n s a m o l y b d e n u m cofactor that is also a cofactor for the e n z y m e xanthine d e h y d r o g e n a s e . T h u s . nitrate r e d u c t a s e . there is n o reason to believe that selenate resistance should not h a v e the s a m e w i d e s p r e a d utility as the 5 . 1988). selenate resistance h a s b e e n used t o positively select A T P sulfurylase m u t a n t s in o r g a n i s m s as d i v e r s e as Saccharomyces a n d Streptomyces (Arst 1 9 6 8 .m e t h i o n i n e and Se-cysteine w h i c h are subsequently incorporated into (nonfunctional) proteins (see references in B u x t o n et a l . the sulfate analogs selenate and Chromate are generally toxic (Arst 1968). Cells h a r b o r i n g these different m u t a t i o n s c a n b e differentiated by the fact that sulfate p e r m e a s e m u t a n t s are also Chromate resistant. a d i m e r c o m p o s e d of identical s u b u n i t s . 6. nidulans sC~ m u t a n t . niger. In addition to Aspergillus. (1989) isolated the A . a subclass will b e defective in nitrate r e d u c t a s e . nidulans A T P sulfurylase g e n e b y c o m p l e m e n t a t i o n of an A . Selenate-resistant m u t a n t s c a n b e readily isolated from a w i d e variety of o r g a n i s m s . 1989). Arst 1968) lead to resistance to selenate toxicity. A s m a n y fungi c a n g r o w o n other nitrogen s o u r c e s . the nitrate assimilation p a t h w a y for nitrogen m e t a b o l i s m is often dispensible. Selenate toxicity is p r e s u m a b l y t h e result of its m e t a b o l i s m to S e . M u t a t i o n s that result in the loss of either sulfate p e r m e a s e o r A T P sulfurylase (the first step in sulfate m e t a b o l i s m inside t h e cell. m u t a t i o n s in the genes e n c o d i n g the e n z y m e s necessary for t h e biosynthesis of this m o l y b d e n u m cofactor will also give rise to chlorate-resistant o r g a n i s m s . a m o n g m u t a n t s selected for the p h e n o t y p e of chlorate resistance. In all c a s e s . B u x t o n et al. final confirmation for loss of nitrate reductase activity c a n b e a c c o m p l i s h e d b y a simple plate assay ( T o m s e t t a n d Garrett 1980). This m u t a n t subclass c a n b e readily distinguished from other chlorate-resistant m u t a n t s b y a s i m p l e g r o w t h test ( C o v e 1976). these selenate-resistant m u t a n t s h a v e lost t h e ability t o utilize sulfate as sole sulfur s o u r c e . In o r g a n i s m s capable of using sulfate as a sole sulfur s o u r c e . w h e r e a s A T P sulfurylase m u t a n t s retain their sensitivity to Chromate. a highly reactive ( a n d toxic) s u b s t a n c e . T h e principle of positively selecting for nitrase reductase m u t a n t s is b a s e d o n the ability of this e n z y m e to catalyze t h e conversion of chlorate to chlorite. T h e use of the nitrate reductase g e n e as a selective m a r k e r for fungal transformation h a s recently b e e n exploited.140 Transformation 6 .2.F O A s y s t e m described a b o v e . This cloned g e n e w a s also able t o c o m p l e m e n t a selenate-resistant. S u c h m u t a n t s c a n b e readily distinguished from those in the nitrate r e d u c t a s e structural g e n e b y t h e failure of the former t o g r o w on m e d i u m c o n t a i n i n g h y p o x a n t h i n e as a sole nitrogen s o u r c e . 3 S e l e n a t e R e s i s t a n c e . A l t h o u g h this selection system h a s b e e n applied only to the Aspergilli. 2 . A s required.4 C h l o r a t e R e s i s t a n c e . In fungi. T h u s .5. chromate-sensitive m u t a n t of A . This selection can b e used with o r g a n i s m s that are capable of utilizing nitrate as their sole source of nitrogen. W i t h regard to m u t a n t selection.

parasiticus. coli Mutants T h e fact that it w a s possible to transform E. niger and A. nidulans nitrate r e d u c t a s e (niaD) p l e m e n t a t i o n of a niaD g e n e w a s c l o n e d by s h o t g u n c o m - m u t a n t ( M a l a r d i e r et al. 1990). chry- ( W h i t e h e a d et al. 1990). viz. 1989a and b . 6. Aphanocla- F. melonis Pyricularia bassiana. and ( Mar lar d ier et al. Beauveria (f. B e c a u s e c o m p l e m e n t a t i o n of a defined a u x o t r o p h i c mutation e n d o w s an o r g a n i s m with a selectable g r o w t h p h e n o t y p e . oryzae niger 6. W h e r e appropriate m u t a n t s are not a v a i l a b l e . W h i t e h e a d et al. p r o m o t e r s from c l o n e d fungal g e n e s can b e used to drive the e x p r e s s i o n of other g e n e s that can furnish a selectable g r o w t h p h e n o t y p e (such as antibiotic resistance) to a wild-type o r g a n i s m . haematococca. niaD g e n e has b e e n used as a hybridization p r o b e to isolate the niaD g e n e from A. s p . oryzae T h e A. s p . attempt to c l o n e fungal g e n e s by c o m p l e m e n t a t i o n of suitable E. oryzae ( U n k l e s et al. A l t h o u g h at the t i m e there w a s n o c o m p e l l i n g reason to believe that fungal p r o m o t - . C.6.6 Gene Isolation 141 used and the alternate nitrogen source in the selection m e d i u m . lindemuthianum. 1 to 0 . acremonium. sogenum GENE ISOLATION It g o e s w i t h o u t saying that o n e m u s t first isolate a g e n e before o n e can r e i n t r o d u c e it into a n e w o r g a n i s m .6. 1989). F o r a discussion of other standard g e n e isolation strategies and m e t h o d s (such as reverse translation of a m i n o acid s e q u e n c e for o l i g o n u c l e o t i d e p r o b e design and the u s e of antibody p r o b e s ) the r e a d e r is referred to any basic m o l e c u l a r b i o l o g y textbook. oxysporum P. A l t h o u g h e a c h of these c l o n e d niaD g e n e s has b e e n used to d e v e l o p a homologous transformation s y s t e m for the respective o r g a n i s m s . the c l o n e d niaD g e n e s of A. T h e A. as well as A. nidulans N. T h e discussion that follows is limited to a description of those t e c h n i q u e s that h a v e b e e n m o s t widely used for the isolation of filamentous fungal g e n e s . 1989). M u c h of the initial effort in isolating fungal g e n e s w a s directed t o w a r d using these g e n e s for the d e v e l o p m e n t of transformation s y s t e m s for these o r g a n i s m s . coli m u t a n t s . and A. 1989. coli years before such p r o c e d u r e s w e r e d e v e l o p e d for e u c a r y o t e s p r o v i d e d an early strategy for isolation of fungal g e n e s . 1989).1 Isolation of Fungal Genes by Complementation of E. 1990. 5 M ( 1 . ly coper sisci). C o n c e n t r a t i o n s of KCIO3 r a n g i n g from 0 . 1989) and the g e n e has b e e n s e q u e n c e d ( J o h n s t o n e et al. D a b o u s s i et al. H o r n g et al. A p p r o p r i a t e m u t a n t s often can be isolated without m u t a g e n e s i s b y plating spores on a plate of m i n i m a l m e d i u m (Klittich and Leslie 1988. In addition to the h o m o l o g o u s h o st .6 h a v e also been used to transform niaD m u t a n t s of P. D a b o u s s i et al.5 % wt/wt) h a v e been used with various fungi. this c l o n e d g e n e has b e e n used as a selectable m a r k e r to transform niaD m u t a n t s of dium album. caseicolum. in principle a c l o n e d fungal g e n e can serve as a selectable m a r k e r for transforming a suitably m u t a n t transformation recipient. Colletotrichum and f.

and with the d e v e l o p m e n t of m e t h o d s to isolate fungal g e n e s b y c o m p l e m e n t a t i o n of fungal m u t a n t s . S u b s e q u e n t s e q u e n c e analysis of the c l o n e d g e n e revealed that it lacked any introns (Upshall et al. nidulans. the cloned S. coli m u t a n t s (Ratzkin and C a r b o n 1977. nidulans argB [ornithine carbamoyltransferase ( O C T a s e ) ] g e n e . 1984). T u r g e o n et al. S u c h successes inevitably led others to exercise this strategy to exploit their favorite fungus. cerevisiae arg3 mutation (Berse et al. e v e n t h o u g h the C . the g e n e s that w e r e used for the first transformation of both yeast and N. T h e first filamentous fungal g e n e to be cloned by selection for e x p r e s s i o n in y e a s t . w a s also isolated by c o m p l e m e n t a t i o n of an E.R i v a s et al. F o r e x a m p l e . B e c a u s e cloned yeast g e n e s w e r e available at that t i m e .n e g a t i v e bacteria.142 Transformation ers w o u l d be e x p r e s s e d in Ε. commune g e n e w a s used to d e v e l o p the first transformation system for this o r g a n i s m ( M u h o z . 1986a.6. B a l l a n c e et al. crassa w a s the only filamentous fungus that had b e e n transformed. L i k e w i s e . A s further e x p e r i m e n t a t i o n revealed that m a n y filamentous fungal g e n e s c o n tain introns. coli as an expression o r g a n i s m for u n m o d i f i e d fungal g e n e s has fallen from favor. 1983). cerevisiae in the early 1980s. studies w e r e d e s i g n e d to ask w h e t h e r these genes could function w h e n introduced into filamentous fungi. LEU2. 1986). V a p n i c k et al. URA3. crassa ( C a s e 1982). coli (pyrF) m u t a n t ( B u x t o n a n d Radford 1 9 8 3 . the first filamentous fungal g e n e s h o w n to function in a h e t e r o l o g o u s filamentous fungus. A s yeast is an a s c o m y c e t e . it w a s h o p e d that this o r g a n i s m m i g h t serve as a host for the expression of filamentous fungal g e n e s in m u c h the s a m e w a y as E.g l u c o s i d a s e (Penttilä et al. T h e s e e x p e r i m e n t s revealed that the cloned yeast TRP1. niger ß . crassa pyr4 g e n e . the g e n e could not b e exploited as a selectable m a r k e r for the transformation of Cochliobolus o w i n g to the lack of a suitable t r y p t o p h a n a u x o t r o p h . s o m e u n m o d i f i e d filamentous fungal genes are e x p r e s s e d w h e n int r o d u c e d into yeast. the use of E. led to attempts to clone filamentous fungal g e n e s by e x p r e s s i o n in yeast. w a s identified in a shotgun g e n e library by its ability to c o m p l e m e n t the c o r r e s p o n d i n g S.R i v a s et al. coli trpC ( p h o s p h o r i b o s y l a n thranilate isomerase) m u t a n t s allowed the isolation of the c o r r e s p o n d i n g g e n e s from fungi as diverse as Schizophillum commune and Cochliobolus heterostrophus ( M u h o z . crassa qa-2 gene) into suitable d o u b l e m u t a n t s of N. crassa w e r e initially isolated by c o m p l e m e n t a t i o n of E. commune w a s available.2 Isolation of Fungal Genes by Expression in Yeast R a p i d m e t h o d o l o g i c a l a d v a n c e s for the m o l e c u l a r m a n i p u l a t i o n of the yeast S. the N. 1986). coli} this strategy has had its s u c c e s s e s . heterostrophus TRP1 g e n e could function in A. A l t h o u g h the Aspergillus g e n e w a s not expressed strongly e n o u g h to allow . I n d e e d . coli has served as a host for expression of g e n e s from g r a m . 1986b). A s the appropriate trpl m u t a n t of S. 6. at a time w h e n N. T h e only other report of the selection of a filamentous fungal g e n e b y s c r e e n i n g for e x p r e s s i o n in yeast w a s that of a putative A. the A. the availability of E. and HIS3 g e n e s did not express w h e n introduced (on a vector containing the N. O n the other h a n d . D e s p i t e this result. 1977). 1983).

W h e n the g e n e . O n e strategy to o v e r c o m e the inability of yeast to r e c o g n i z e fungal p r o m o t e r s or correctly excise fungal introns is to e x p r e s s c l o n e d c D N A s in yeast. Penttilä et al. 1988). only poorly e x c i s e filamentous fungal introns ( W o u d t et al. g e n e expression w a s sufficient to allow selection of the desired transformant by screening for c l e a v a g e of the c h r o m o g e n i c substrate X . A Phy corny ces blakesleeanus TRP1 g e n e . has also b e e n s h o w n to c o m p l e m e n t a yeast trpl m u t a n t ( R e v u e l t a and J a y a r a m 1987). (1984) failed to isolate g e n e s that could c o m p l e m e n t a n u m b e r of yeast m u t a t i o n s (leu2 and ura3) w h e n using the s a m e c o s m i d g e n e b a n k that had b e e n e m p l o y e d to isolate the putative A. W h e n these cells w e r e e x a m i n e d .6. Similar vectors for e x p r e s s i n g c D N A in yeast are n o w c o m m e r c i a l l y available. the p r o m o t e r of the c l o n e d C . 1985 and 1986.G l u ( w h i c h gives rise to blue c o l o n i e s ) .6. initially isolated by c o m p l e m e n t a t i o n of an E. Initial success in this area has c o m e w h e n using g e n e s that e n c o d e highly c o n s e r v e d p r o t e i n s .t u b u l i n g e n e w a s a c c o m plished by p r o b i n g an A . 1986). w h i c h w a s also initially isolated by c o m p l e m e n t a t i o n of an E. K r o n s t a d et al. B y contrast. 1984. In a n a l o g y with the c D N A e x p r e s s i o n vector of O k a y a m a and Berg ( 1 9 8 2 ) . M c K n i g h t and M c C o n a u g h y ( 1 9 8 3 ) h a v e constructed the vector p M A C 5 6 1 . heterostrophus TRP1 g e n e w a s nonfunctional in yeast. M c K n i g h t et al. 1989). D N A s e q u e n c i n g revealed the p r e s e n c e of introns within both the A. at best. 1985). maydis ( M c K n i g h t et al. cerevisiae) genes have p r o v e n to be a g o o d r e s o u r c e for the isolation of c o g n a t e g e n e s from filamentous .3 Isolation of Fungal Genes by Cross-Species Hybridization O n c e a g e n e has b e e n c l o n e d it can often be used as a p r o b e for the isolation of a c o g n a t e g e n e from a h e t e r o l o g o u s o r g a n i s m . at best.6 Gene Isolation 143 yeast transformants to g r o w on c e l l o b i o s e . T h e s e w o r k e r s also w e r e u n a b l e to select yeast transformants expressing a fungal /3-galactosidase (by g r o w t h o n lactose) or g l u c o a m y l a s e (by g r o w t h on starch). After the g e n e s w e r e s u b s e q u e n t l y isolated by other m e t h o d s . T h e s e studies also d e m o n s t r a t e d that. yeast (S. M o r e direct studies h a v e also d e m o n s t r a t e d that yeast c a n . niger g l u c o a m y l a s e and pyrG (equivalent to the yeast URA3) g e n e s (Boel et al. 1985. b y m a k i n g use of previously c l o n e d g e n e s it has b e e n possible to isolate a n u m b e r of filamentous fungal g e n e s . coli m u t a n t . 1985. niger jS-glucosidase g e n e . w a s introduced into y e a s t . W i l s o n et al. the initial isolation of a fungal ß . F o r e x a m p l e . D e s p i t e the differences in b a s e c o m p o s i t i o n . 1987). coli m u t a n t . nidulans a n d U. nidulans g e n e b a n k with a c h i c k e n jß-tubulin g e n e p r o b e ( M a y et al. 6. Innis et al. it w a s 1 d i s c o v e r e d that the Cochliobolus D N A had u n d e r g o n e a deletion of the 5 e n d of the g e n e ( T u r g e o n et al. T h u s . yeast only poorly r e c o g n i z e filamentous fungal p r o m o t e r s . A similar vector ( p Y c D E 8 ) has b e e n successfully e m p l o y e d to identify and isolate a n u m b e r of g e n e s from both A. w h i c h allows the oriented c l o n i n g of c D N A s b e t w e e n the yeast ADC1 (alcohol d e h y d r o g e n a s e ) p r o m o t e r and CYC1 ( c y t o c h r o m e c) terminator. Yeast cells that e x p r e s s e d the foreign g e n e could h o w e v e r b e r e c o v e r e d o n selection. n o e x p r e s s i o n w a s d e t e c t e d .

Isolation of g e n e s by cross-hybridization with yeast p r o b e s is not.6. the Coprinus einer eus trpl (tryptophan synthetase) g e n e w a s isolated by hybridization to a yeast TRP5 p r o b e ( S k r z y n i a et al.144 Transformation fungi. G e n e s from a s c o m y c e t e s (and related fungi imperfecti) h a v e also b e e n used to isolate c o g n a t e s from b a s i d i o m y c e t e s . limited to g e n e s e n c o d i n g the highly c o n s e r v e d glycolytic e n z y m e s . A m o n g the fungal g e n e s so isolated h a v e been a n u m b e r of g e n e s e n c o d i n g glycolytic e n z y m e s ( C l e m e n t s and Roberts 1985. 1988. 1989). de R u i t e r . T h i s h o m o l o g y w a s sufficient to allow G o y o n et al. chrysogenum by hybridization to fungal g e n e p r o b e s ( V a n Hartingsveldt et al. may dis ( H a r g r e a v e s and T u r n e r 1989). (1988) to clone the MET2 ( h o m o s e r i n e O-transacetylase) g e n e of A. 1988). Cantoral et al. o n e is m o r e likely to r e c o v e r s p o n t a n e o u s r e v e n a n t s at a m u c h higher frequency than the desired transformant. 1987). as expression in a h o m o l o g o u s o r g a n i s m should eliminate p r o b l e m s relating to the ability of the recipient o r g a n i s m to express the desired g e n e . Yeast g e n e s h a v e also been used as hybridization p r o b e s for the isolation of g e n e s from b a s i d i o m y c e t e s . F o r e x a m p l e .J a c o b s et al. 6. Bull et al. U n f o r t u n a t e l y . genes from the filamentous fungi h a v e also b e e n used as p r o b e s for g e n e isolation. F o r e x a m p l e . nidulans has been used to isolate the c o g n a t e (acu7) g e n e of the b a s i d i o m y c e t e Coprinus einer eus (Mellon et al. cerevisiae. the frequency of transformation of m a n y filamentous fungi is often not significantly higher than the reversion rate of the desired m u t a t i o n . de Graaff et al. 1987. For e x a m p l e . the cloned acu5 ( a c e t y l . immersus by heterologous hybridization with the MET2 g e n e of S. Punt et al. G o o s e n et al. A t t e m p t i n g to c l o n e b y m u t a n t c o m p l e m e n t a t i o n in such a s y s t e m . A l t h o u g h s e q u e n c e c o m p a r i s o n of the yeast and Ascobolus immersus h o m o s e r i n e O-transacetylase g e n e suggested the p r e s e n c e of an intron in the g e n e from Ascobolus. 1987. 1988).4 Isolation of Fungal Genes by Complementation in Homologous Filamentous Fungi C l o n i n g by m u t a n t c o m p l e m e n t a t i o n of a h o m o l o g o u s transformation recipient w o u l d a p p e a r to offer the most straightforward m e t h o d for g e n e isolation. the A T P a s e subunit 9 g e n e s of t w o Aspergilli as well as P. L e C h e v a n t o n and L e b l o n (1989) cloned the ura5 g e n e (orotate p h o s p h o r i b o s y l transferase) from the a s c o m y c e t e Sordaria macrospora by cross hybridization with a p r o b e from Podospora anserina.p h o s p h a t e d e c a r b o x y l a s e ) g e n e has b e e n isolated from various Aspergilli as well as P. three r e g i o n s shared 6 7 . L i k e w i s e .C o A synthase) g e n e of Ν . If o n e can already transform the o r g a n i s m in question this p r o b l e m can be o v e r c o m e by . h o w e v e r . 1989). crassa w a s used as a p r o b e to isolate the c o g n a t e acuA g e n e of U. chrysogenum w e r e isolated by cross-hybridization with g e n e s from filamentous fungi ( W a r d et al. 1988. A s they h a v e b e c o m e available. the pyrG (orotidine-5 ' . 1989). T h i s g e n e w a s later used to d e v e l o p a transformation system for Ascobolus ( F a u g e r o n et al. Similarly the cloned acuD (isocitrate lyase) g e n e of A. 1986 and 1988.7 6 % h o m o l o g y with the yeast g e n e .

1985). diagnostic of the desired w i l d . A l t h o u g h the transformation frequency of TV.d e p e n d e n t restriction s y s t e m s (Grant et al.t y p e yA g e n e . V o l l m e r and Yanofsky (1986) created an i m p r o v e d c o s m i d vector for sib selection b y including a b e n o m y l . Modifications of the A. crassa is sufficiently high to allow g e n e selection by m u t a n t c o m p l e m e n t a t i o n . the first filamentous fungal g e n e to b e isolated by c o m p l e m e n t a t i o n of a m u t a n t filamentous fungus. coli transformation recipient lacking t w o m e t h y l c y t o s i n e restriction s y s t e m s has a l l o w e d a limited r e c o v e r y of the transformed D N A ( O r b a c h et al. G e n e c l o n i n g by m u t a n t c o m p l e m e n t a t i o n is not limited to the a s c o m y c e t e s . B y screening the trp transformants obtained by transformation with 100 pig of the g e n e b a n k . three g r e e n . Further i m p r o v e m e n t s in D N A r e c o v e r y from this o r g a n i s m are to b e e x p e c t e d from the d e v e l o p m e n t of n e w e r bacterial strains that lack additional m e t h y l a t i o n . crassa. 1987). nidulans transformation protocol i m p r o v e d the transformation frequency to the point w h e r e it is possible to eliminate the fungal selectable m a r k e r from the vector w h e n a t t e m p t i n g to c l o n e b y m u t a n t c o m p l e m e n t a t i o n with this o r g a n i s m ( O a k l e y et al. w e r e r e c o v e r e d (Yelton et al. nidulans trpC g e n e as a + selectable m a r k e r . 1990). nidulans acuD (isocitrate lyase) g e n e from a p l a s m i d g e n e b a n k by using a p l a s m i d vector that e m p l o y e d the N. crassa is p r e s u m a b l y d u e to the fact that the introduced D N A is subjected to a high level of cytosine m e t h y l a t i o n (Bull a n d W o o t t e n 1984).6. It w a s also possible to isolate the A. 1990). coli culture c a r r y i n g a fungal g e n e b a n k is divided into a n u m b e r of p o o l s and D N A isolated from e a c h pool is used to transform a recipient fungus. Pool(s) that give rise to the desired transformant are divided to create n e w ( s u b ) p o o l s . T h e failure to r e c o v e r transforming D N A from N. recovery of c l o n e d D N A from this o r g a n i s m h a s p r o v e d to b e a p r o b l e m . T h e use of an E. H e n c e . nidulans c o g n a t e of the selectable N. T h e g e n e b a n k used in this e x p e r i m e n t w a s constructed in a c o s m i d vector that could carry 35 to 4 0 k b D N A inserts a n d that utilized the A. T o o v e r c o m e this obstacle a screening t e c h n i q u e t e r m e d sib selection has b e e n d e v i s e d . Sib selection has also b e e n used for c l o n i n g g e n e s from Podospora anserina (Turcq et al. neither a high frequency transform a t i o n s y s t e m n o r a very tight m u t a t i o n is required to clone g e n e s b y c o m p l e m e n t a tion. crassa gene) and acuD g e n e s (Ballance and T u r n e r 1986). w a s a c c o m p l i s h e d at a t i m e w h e n the transformation frequency of this o r g a n i s m w a s only 10/mg of D N A . U s i n g such a strategy o n e requires only that the reversion rate of the m u t a t i o n that o n e is attempting to c o m p l e m e n t is l o w e r than the fraction of the g e n e b a n k that contains the desired g e n e .r e s i s t a n t /3-tubulin g e n e that c a n b e used as a selectable m a r k e r in wild-type strains of N. T h e c l o n i n g of the A. nidulans yA g e n e . T h i s transformation and subdivision p r o c e s s is repeated until the final " p o o l " consists of a single p l a s m i d ( A k i n s and L a m b o w i t z 1985). o n e can eliminate the b a c k g r o u n d of n o n t r a n s f o r m e d revertants b y selecting for transformed o r g a n i s m s before screening for c o m p l e m e n t a t i o n of the desired m u t a t i o n . crassa pyr4 g e n e as a selectable m a r k e r and selecting for transformants that s i m u l t a n e o u s l y c o m p l e m e n t e d m u t a t i o n s in both the pyrG (the A. . 1988).s p o r e d c o l o n i e s . In this a p p r o a c h an E.6 Gene Isolation 145 p l a c i n g an appropriate fungal selective m a r k e r on the transforming vector. W i t h such a v e c t o r .

a wild-type strain of A. A l t h o u g h g e n e s h a v e not yet b e e n isolated by cross-species expression in distantly related filamentous fungi. its introduction into the m a i z e p a t h o g e n C. H y n e s 1989). Schizophyllum commune p r o v i d e d the first e x a m p l e of g e n e cloning by c o m p l e m e n t a t i o n of a fungal mutation (Froeliger et al. nidulans it has been possible to identify transformants carrying the A. haematococca (Weltring et al. nidulans. T h e transformation efficiency of this o r g a n i s m w a s sufficiently high (and the m u t a n t s sufficiently tight) that it w a s possible to select directly for the desired transformant without the use of a selectable m a r k e r on the vector to allow a p r e l i m i n a r y selection of transformed cells. maydis and r e c o v e r e d the desired p l a s m i d by restriction and circularization of D N A isolated from the t r a n s f o r m a n t s . K r o n s t a d and L e o n g (1989) cloned a n u m b e r of alleles of the b m a t i n g . M a k i n g use of a c o s m i d carrying a selectable h y g r o m y c i n Β m a r k e r . 1986.146 Transformation A m o n g the b a s i d i o m y c e t e s . heterostrophus d o e s allow the resulting o r g a n i s m to infect peas (Schäfer et al. 1988). 6. a n u m b e r of recent studies h a v e used available cloned genes to test the feasibility of such a g e n e isolation strategy. In a variation on this t h e m e . niger O C T a s e g e n e as well as an A. maydis. nidulans into a plant p a t h o g e n . 1988. B e c a u s e U. ( 1 9 9 0 ) used a p l a s m i d g e n e b a n k to select mating-type b alleles of U. 1988).t y p e locus ( w h i c h regulate d i m o r p h i s m in this fungus) by sib selection. maydis divides like a yeast. a s c o m y c e t e p r o m o t e r s fused to the h y g r o m y c i n p h o s p h o t r a n s ferase (hph) g e n e allow selection for h y g r o m y c i n resistance in the b a s i d i o m y c e t e s . A l t h o u g h expression of the pisatin d e m e t h y l a s e g e n e d o e s not turn A. niger g e n e e n c o d i n g a secreted p h o s p h a t e repressible acid p h o s p h a t a s e ( B u x t o n et al. C l o n e d genes could be r e c o v e r e d in E. nidulans (Gray et al. nidulans (Ballance et al. M a c R a e et al. crassa pyr4 g e n e could be e x p r e s s e d in A. an o r g a n i s m that cannot n o r m a l l y detoxify p h y t o a l e x i n s . 1987). Similarly.5 Isolation of Fungal Genes by Complementation in Heterologous Filamentous Fungi In light of the limited success achieved w h e n attempting to isolate filamentous fungal g e n e s by expression in yeast. B y selection for c o m p l e m e n t a t i o n of appropriate m u t a n t s of A. F o t h e r i n g h a m and H o l l o m a n 1989).6. 1987. it has also b e e n p o s s i b l e to isolate an a u t o n o m o u s l y replicating s e q u e n c e and thus i m p r o v e the c l o n i n g of g e n e s by direct m u t a n t c o m p l e m e n t a t i o n ( T s u k e d a et al. C l o n i n g by selection for g e n e expression is also possible with the h e m i b a s i d i o m y c e t e U. coli after D N A (isolated from the transformants) w a s restricted and circularized in vitro. S c h u l z et al. 1983). G e n e s from both z y g o m y c e t e s (the Rhizomucor miehei aspartyl protease gene) and b a s i d i o m y c e t e s (the Coprinus isocitrate lyase gene) can function w h e n introduced into the a s c o m y c e t e A. w a s used as the " m u t a n t " transformation recipient to clone the phytoalexin-detoxifying pisatin d e m e t h y l a s e g e n e of the p e a p a t h o g e n N. it is not surprising that attempts w e r e m a d e to c l o n e filamentous fungal g e n e s by expression in h e t e r o l o g o u s filamentous fungal hosts after it w a s s h o w n that the N. 1989). T h e a b o v e e x a m p l e s h a v e exploited cross-species g e n e expression for the isolation of g e n e s from closely related o r g a n i s m s .

it w o u l d a p p e a r m o s t r e a s o n a b l e to stay within the s a m e division if o n e is a t t e m p t i n g to clone a fungal g e n e . g e n e function d e c r e a s e d with increasing distance of the g e n e . W h e n similar e x p e r i m e n t s w e r e attempted with the b a s i d i o m y c e t e C. of c o u r s e . A d d i t i o n a l analysis of g e n e expression by N o r t h e r n blot r e v e a l e d that. A s s h o t g u n cloning has b e e n d e m o n s t r a t e d in o r g a n i s m s from m o s t divisions of filamentous fungi (Table 6 . b e appreciated that observation of cross-species g e n e e x p r e s sion d o e s not constitute proof that a fungal p r o m o t e r is functioning " n o r m a l l y " in the recipient s p e c i e s . It will. a diversity of transformation recipients already exists for o n e w h o desires to c l o n e a fungal g e n e by e x p r e s s i o n in a related (but heterologous) o r g a n i s m . although g e n e s from the b a s i d i o m y c e t e s Schizophyllum commune and Phanerochaete chrysosporium w e r e e x p r e s s e d in C .6 Is There an Advantage to Using a Homologous Gene as a Selective Marker for Transformation? W h e n d e v e l o p i n g a transformatiaon s y s t e m for any o r g a n i s m the question invariably arises as to w h e t h e r it is necessary to isolate a h o m o l o g o u s g e n e as a selective m a r k e r . C l e a r l y . 1990). in an apparently unmodified form w a s confirmed by S o u t h e r n blots analysis of the transformants. 1985 Akins and Lambowitz 1985 Turcq et al. introduced by c o t r a n s f o r m a t i o n . cinereus.6 TABLE 6-7 Gene Isolation 147 Shotgun Cloning in Filamentous Fungi Division Organism Reference Zygomycotina Mucor Ascomycotina Aspergillis nidulans Neurospora crassa Podospora anserina Yelton et al. S m i t h et al. may dis. b e c a u s e g r o w t h of transformants w a s proportional to the c o p y n u m b e r of the introduced g e n e . w h e n attempting to transform a previously untransformed species .6.d o n a t i n g o r g a n i s m .) In light of these results. 1990. it w a s c o n c l u d e d that the Coprinus g e n e did not function as well in Aspergillus as it did in its n o r m a l e n v i r o n m e n t . (In the cases w h e r e the g e n e s w e r e not e x p r e s s e d . the p r e s e n c e of the introduced h e t e r o l o g o u s g e n e s .7 ) . 1987 circinelloides Van Heeswijck and Roncero 1984 Kronstad and Leong 1989 Ustilago and Laccaria laccata (Bej and Perlin 1989. I n d e e d .6. as well as g e n e s from the a s c o m y c e t e A. T h u s . w e r e not e x p r e s s e d (Casselton and d e la F u e n t e H e r c e 1989). nidulans. w h e n i n t r o d u c e d into Aspergillus. 6. einer eus as the transformation recipient. the Coprinus g e n e w a s transcribed constitutively and w a s not regulated by acetate in the s a m e fashion as w h e n the g e n e w a s e x p r e s s e d in Coprinus ( H y n e s 1989). 1990 Basidiomycotina Schizophyllum commune Ustilago may dis Froeliger et al. a h y g r o m y c i n resistance g e n e fused to a p r o m o t e r from the ( h e m i ) b a s i d i o m y c e t e U. Barrett et al.

oryzae can function as selectable m a r k e r s w h e n transforming this o r g a n i s m (Mattern et al. crassa pyr4 ( O M P d e c a s e ) g e n e has been used successfully to transform a variety of o r g a n i s m s (see T a b l e 6 . as d o c u m e n t e d e l s e w h e r e in this chapter. Similarly.r e s i s t a n t jß-tubulin g e n e w a s n o better than a h e t e r o l o g o u s h y g r o m y c i n Β phosphotransferase (driven by an Aspergillus p r o m o t e r ) in transforming Leptosphaeria nodorum. it is to be anticipated that in m o s t instances it will not be necessary to isolate a h o m o l o g o u s g e n e (or splice a h o m o l o g o u s p r o m o t e r o n t o a heterologous gene) before a t t e m p t i n g to transform a given fungus. even in studies that h a v e e m p l o y e d c o g n a t e g e n e s . 1987). crassa c o g n a t e .148 Transformation it is logical to use a h o m o l o g o u s selectable m a r k e r . it d o e s not transform Aspergillus oryzae. Casselton and d e la F u e n t e H e r c e (1989) reported that transformation efficiency of Coprinus cinereus by the Schizophyllum commune trpl g e n e w a s a p p r o x i m a t e l y three times that obtained w h e n using the h o m o l o g o u s . niger and A. 1987. S i m i l a r l y . crassa ( W a r d et al. Variable results h a v e also b e e n obtained w h e n c o m p a r i n g t w o unrelated g e n e s . B u x t o n et al.to twentyfold increase in transformation frequency (to a p p r o x i m a t e l y 5 0 transformants per m i c r o g r a m ) w h e n c o m p a r e d to the N. A s a n u m b e r of the filamentous fungi h a v e been transformed with both h o m o l o g o u s and h e t e r o l o g o u s g e n e s .6 ) . the h o m o l o g o u s pyrG g e n e g a v e a ten. 1989). B y contrast. nidulans o l i g o m y c i n resistance m a r k e r w a s n o different than that obtained w h e n e m p l o y i n g the h e t e r o l o g o u s pyr4 g e n e of N. crassa pyr4 g e n e ( W a r d et al. o n e is still left with the intuitive feeling that there m u s t b e s o m e a d v a n t a g e . V a n Hartings veldt et al. L i k e w i s e . 1987. niger O C T a s e g e n e d o e s not transform A. the transformation frequency with the A. F o r e x a m p l e . niger or A. 1988). T h u s . to the use of a h o m o l o g o u s g e n e . C o o l e y and C a t e n (1989) o b s e r v e d that an h o m o l o g o u s b e n o m y l . as this eliminates any question as to w h e t h e r the g e n e will function w h e n introduced into the o r g a n i s m . the c o r r e s p o n d i n g pyrG g e n e s of both A. nidulans by fivefold (to levels a p p r o a c h i n g 2 . n o generalizations can be d r a w n at this t i m e . niger the transformation frequency w h e n using the h o m o l o g o u s o l i g o m y c i n resistance g e n e w a s tenfold lower than that o b t a i n e d with the N. de Ruiter-Jacobs et al. in A. (1987) report that the A. G i v e n that it p r o b a b l y is not necessary to use a h o m o l o g o u s g e n e for transformation of the filamentous fungi. e x c e p t i o n s d o exist. although the N. 1986). as with all generalizations. nidulans g e n e . It should be appreciated that. nidulans at a significantly different frequency than the c o r r e s p o n d i n g A. H o w e v e r . 0 0 0 stable transformants per m i c r o g r a m ) as c o m p a r e d to use of the N. o n e w o u l d h o p e to b e able to p r o v i d e an a n s w e r to this question. O a k l e y et al. equal transformation frequencies of Colletotrichum graminicola w e r e o b s e r v e d with the h o m o l o g o u s or h e t e r o l o g o u s (N. crassa) b e n o m y l resistance g e n e ( P a n a c c i o n e et al. if o n e is available. in t w o separate studies with A. m o r e often than not it h a s b e e n h e t e r o l o g o u s g e n e s that h a v e been used to d e v e l o p transformation s y s t e m s for unstudied species of the filamentous fungi. A m o n g the b a s i d i o m y c e t e s . niger. (1987) reported that use of the h o m o l o g o u s pyrG g e n e increased transformation frequency of A. 1988). H o w e v e r . crassa c o g n a t e ( G o o s e n et al. B y contrast. such as gaining an i m p r o v e m e n t in transformation frequency.

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1982). 1989). Of the other a d v a n t a g e o u s features associated with e n z y m e c a t a l y z e d p r o c e s s e s . O n e of t h e s e . In spite of the o b v i o u s a d v a n t a g e s of the use of e n z y m e s for m a n y synthetic a p p l i c a t i o n s .and stereoselectivity of e n z y m e . and a n o t h e r . h o w e v e r . by the relatively high substrate specificity s h o w n by s o m e e n z y m e s . the r e q u i r e m e n t for w a t e r solubility of the substrate.1 THE SCOPE OF FUNGAL BIOCONVERSIONS T h e use of e n z y m e s as reagents to carry out specific reactions in the preparation of o r g a n i c c o m p o u n d s has recently achieved the d e g r e e of respectability long associated with m o r e c o n v e n t i o n a l m e t h o d s of organic synthesis.c a t a l y z e d reactions that are the crucial factors in a decision to use an e n z y m e in preference to a c h e m i c a l r e a g e n t . Holland 7. T h e a d v a n t a g e s of carrying out a reaction b y e n z y m a t i c m e t h o d s are m a n y : in m o s t a p p l i c a t i o n s . it is the r e g i o . In m a n y synthetic applications w h e r e decisions are b a s e d on criteria such as yield. the ability to perform reactions at close to neutral p H and r o o m t e m p e r a t u r e . has recently b e e n a d d r e s s e d by the w o r k of K l i b a n o v and others o n the use of e n z y m e s in n o n a q u e o u s solvents (Sakuri et al. and b y the 157 . n e v e r t h e l e s s . are u n d o u b t e d l y the m o s t significant. and the e n o r m o u s rate e n h a n c e m e n t s . there are still limitations to their general u s e . scale. has b e e n solved by the d e v e l o p m e n t of recycling m e t h o d s for these e x p e n s i v e materials (Hirschbein et al. and selectivity. 1 9 8 8 ) .CHAPTER 7 Bioconversions Herbert L. T h e use of isolated e n z y m e s in o r g a n i c chemistry is often limited. the r e q u i r e m e n t of s o m e e n z y m e s for stoichiometric quantities of cofactors. an e n z y m e is often the reagent of c h o i c e (Davies et al.

R o s a z z a and Duffel 1986). T h i s p r o c e s s . steroids and aromatic c o m p o u n d s . dating only from the 1950s. alt h o u g h it is possible to distinguish. e n v i r o n m e n t a l pollutants (Chakrabarty 1982). and reduction p r o c e s s e s . but the m o s t c o m m o n . for e x a m p l e . L . and n o w includes e x a m p l e s of h y d r o l y t i c . O t h e r r e v i e w s h a v e focussed on substrate groups such as steroids ( S m i t h . alkaloids (Holland 1 9 8 1 . Batt 1989).1. Since that t i m e . M a d e s c l a i r e 1988. w h i c h e n a b l e s the c h e m i s t to take a d v a n t a g e of the desirable properties of e n z y m e s w i t h o u t the necessity of their isolation or the provision of cofactors or a cofactor recycling s y s t e m .158 Bioconversions small n u m b e r of e n z y m e s with broad substrate specificity that are c o m m e r c i a l l y available in purified form. Pratt 1989. the r a n g e of reactions that can b e efficiently carried out by fungal b i o c o n v e r s i o n has been e x p a n d e d e n o r m o u s l y . N o t all of these reactions o c c u r with every substrate. often a c c o m p a n y other p r o c e s s e s such as h y d r o x y lation and B a e y e r . D a v i e s et al.1 for t w o c o m m o n substrate g r o u p s . or are c o n t a i n e d as part of general r e v i e w s on the application of e n z y m a t i c reactions to o r g a n i c s y n t h e sis (Jones 1986. 1989) or general r e v i e w s of other p r o c e s s e s (Torrey 1983. L. steroid C . w h i c h c o v e r the entire r a n g e of substrates subjected to fungal b i o c o n v e r s i o n d u r i n g the first three d e c a d e s of its d e v e l o p m e n t .6 β hydroxylation (Holland 1984). either a d d e d to its g r o w t h m e d i u m or simply b r o u g h t into contact with the m i c r o o r g a n i s m in a mutually compatible environment. T h e m o s t frequently o b s e r v e d reactions of fungal b i o c o n v e r s i o n are s u m m a rized in Figure 7 . 1974.V i l l i g e r oxidation and d e h y d r o g e n a t i o n by selection of the appropriate fungus for b i o c o n v e r s i o n . it m a y not a l w a y s be possible to select fungi that will h y d r o x y late a steroid ester without s o m e degree of ester h y d r o l y s i s . Y a m a d a and S h i m i z u 1988. T h u s . is the use of intact m i c r o o r g a n i s m s as a source of e n z y m e activity. steroid h y d r o x y l a t i o n from B a e y e r . t e r m e d biotransformation or b i o c o n v e r s i o n . Butt and R o b e r t s 1986 and 1987. A n alternative a p p r o a c h to the use of isolated e n z y m e s . and alcohol d e h y d r o g e n a s e activity ( H u m m e l and K u l a 1989). uses a g r o w i n g or resting culture of a b a c t e r i u m or fungus to m e t a b o l i z e an organic substrate. T h e s e reactions and others are included in e n c y c l o p e d i c r e v i e w s b y C h a r n e y and H e r z o g (1967) and Kieslich ( 1 9 7 6 and 1984). H o l l a n d 1982). 7. and bioactive c o m p o u n d s ( R o s a z z a 1982).1 The Reactions of Fungal Bioconversion F u n g a l b i o c o n v e r s i o n of organic c o m p o u n d s is by n o m e a n s a n e w t e c h n i q u e — controlled fermentation to p r o d u c e ethanol has a venerable h i s t o r y — b u t its use as a synthetic tool in organic c h e m i s t r y is m o r e recent. . on single b i o c o n v e r s i o n reactions such as h a l o g e n m e t a b o lism ( N e i d e l m a n and Geigert 1986). the use of fungi to introduce h y d r o x y 1 g r o u p s into the steroid skeleton w a s d e v e l o p e d by the p h a r m a c e u t i c a l industry as part of a c o m m e r c i a l synthesis of corticosteroids. sulfides ( H o l l a n d 1988). the hydrolytic and carbonyl-alcohol redox r e a c t i o n s . In that d e c a d e . o x i d a t i o n . c o n d e n s a t i o n . or sulfoxidize a ketosulfide without reduction of the carbonyl g r o u p .V i l l i g e r oxidation.

a p r o c e s s that can readily be p e r f o r m e d on h u n d r e d . the e n z y m e s r e s p o n s i b l e for hydroxylation at saturated or aromatic . 1989).2 The Enzymes of Fungal Bioconversion T h e ability of s o m e fungi to perform only o n e of the oxidative reactions of Figure 7 . E x a m p l e s of the latter include the use of B a k e r ' s yeast to p r o d u c e chiral s e c o n d a r y alcohols by reduction of /3-ketoesters. and large-scale p r o c e s s e s in w h i c h the ability of a g r o w i n g o r g a n i s m to m a n u f a c t u r e its o w n cofactors such as N A D H or N A D P H obviates the need to p r o v i d e either stoichiometric a m o u n t s of these materials or a c o m p l e x recycling s y s t e m . and can b e a d v a n t a g e o u s in s o m e c a s e s . D a v i e s et al.a l c o h o l i n t e r c o n v e r s i o n s . and reduction of c a r b o n . in contrast to the w i d e s p r e a d o c c u r r e n c e of other reactions such as ester h y d r o l y s i s . and a substantial n u m b e r of e n z y m e s of both categories are c o m m e r c i a l l y available in purified form (Davies et al. In contrast to the ready accessibility of m a n y h y d r o l a s e and alcohol d e h y d r o g e nase e n z y m e s . T h i s has led to their e x t e n s i v e use as isolated e n z y m e s in c h e m i c a l synthesis (Jones 1986). h y d r o l a s e and alcohol d e h y d r o g e n a s e e n z y m e s are relatively well characterized s y s t e m s .2 (Miski and D a v i s 1988).1 The Scope of Fungal Bioconversions 159 2 1 · carbon hydroxylation 4: carbonyl reduction 2: carbonyl reduction 3: Baeyer-Villiger oxidation followed by ester hydrolysis 4: dehydrogenation F I G U R E 7-1 Common reactions of fungal bioconversion.c a r b o n d o u b l e b o n d s or d e h y d r o g e n a t i o n of c a r b o n . is of c o u r s e a reflection of the distribution of the various e n z y m e activities i n v o l v e d . 7.g r a m scales (Jones 1986. E x a m p l e s include the formation of p r o d u c t s that are not available b y isolated e n z y m e m e t h o d o l o g y . sulfoxidation. In g e n e r a l .1.7.1 . B a e y e r . the oxidative e n z y m e s r e s p o n s i ble for h y d r o x y l a t i o n . such as the c o n v e r s i o n of 1 into 2 b y Aspergillus ochraceus s h o w n in F i g u r e 7 . but w h o l e cell b i o c o n v e r s i o n s that e m p l o y these e n z y m e s are still p e r f o r m e d . T h u s although m o s t fungi contain a r a n g e of hydrolytic e n z y m e s c a p a b l e of the hydrolysis of esters and a m i d e s and the alcohol d e h y d r o g e n a s e s r e s p o n s i b l e for c a r b o n y l . 1989).c a r b o n single b o n d s are less c o m m o n .V i l l i g e r o x i d a t i o n .

et al 1 9 8 9 d ) .V i l l i g e r o x i d a t i o n .d e p e n d e n t m o n o . E . ochraceus 1 2 ee 92% yield 24% from rac-1 6 5 F I G U R E 7 . so that these reactions are invariably carried out in a whole-cell b i o c o n v e r s i o n m o d e : fungal e n z y m e s c a p a b l e of carbon hydroxylation h a v e recently b e e n isolated in c r u d e form and identified as c y t o c h r o m e P .2 Typical fungal bioconversion substrates and products. 1977 a n d 1978. o r B a e y e r . t h e nature of the e n z y m e s that catalyze these latter reactions m a y b e inferred b y c o m p a r i s o n with e n z y m e s from m a m m a l i a n o r bacterial sources that catalyze similar p r o c e s s e s a n d h a v e similar cofactor r e q u i r e m e n t s a n d inhibitor r e s p o n s e s . c a r b o n . N e v e r t h e l e s s .c a r b o n Saturation or desaturation are not generally susceptible to isolation. Β a e y e r . C r e s n a r et a l . 1985. sulfoxidation.P l e v n i k . b u t n o cell-free preparations of fungal origin are available for performing e p o x i d a t i o n . o r c a r b o n .V i l l i g e r oxidation reactions. . sulfoxidation.160 Bioconversions COOH (rac)A.o x y g e n a s e s ( B r e s k v a r a n d H u d n i k . Smith Κ .4 5 0 . e p o x i d a t i o n .

stereoselection.c a r b o n d e h y d r o g e n a t i o n s . b i o c o n v e r s i o n is often superior o n g r o u n d s of stereoselection: consider for e x a m p l e the m e t h o d s available for oxidation of the alkaloid glaucine ( 5 . 7. o r p e r o x i d a s e s (Colo n n a et al.7.and stereoselectivity inherent in the activity of most of the oxygenase enzymes discussed above ensures for them. and hence for fungal bioconversion. 1988. h o w e v e r . is p r o b a b l y catalyzed b y a flavin-dependent o x y g e n a s e (Abril et al. L. and it is b y these criteria that fungal b i o c o n v e r s i o n as a m e t h o d of d o i n g organic c h e m i s t r y m u s t b e j u d g e d . reactions catalyzed by these enzymes are a m o n g the most useful to the organic chemist. 0 0 0 fungi available from the w o r l d ' s culture c o l l e c t i o n s . L . from p r i m a r y to tertiary levels. by a n a l o g y with the c o r r e s p o n d i n g bacterial r e a c t i o n s . to functionalize at an unactivated carbon atom in a stereo. see Section 7 . C h e m i c a l oxidants are n o n s t e r e o s e l e c t i v e . Charney and Herzog 1967) has n o parallel in organic chemistry.2 FUNGI AS CHEMICAL REAGENTS T h e selection of a particular reagent in synthetic chemistry is usually b a s e d on several criteria such as availability. T h e other important criterion of availability is. by a n a l o g y with the bacterial e n z y m e c y c l o h e x a n o n e m o n o .o x y g e n a s e s (Holland 1988. exemplified by the conversion of 3 to 4 (Figure 7 . w h e n c h o o s i n g a c h e m i c a l reagent for ester h y d r o l y sis. Figure 7 . there exists an e n o r m o u s b o d y of literature. but b i o c o n v e r s i o n using Fusarium solani A T C C 12823 is specific for the S ( + ) e n a n t i o m e r 5 of substrate. w h e r e a s the n u m b e r of such m e t h o d s applicable to a given reaction is relatively small. c h e m i c a l yield. w h e r e a s sulfoxidation m a y b e p e r f o r m e d by these e n z y m e s .4 5 0 . T h e complexity and membrane-bound nature of fungal cytochrome P-450 and flavin-dependent mono-oxygenase enzymes precludes their isolation in the quantities necessary for preparative bioconversions. 1989). 2 ) .and regiospecific manner. w h e r e a s it is p r o b a b l y true to say that of the m o r e than 1 5 . n a m e l y availability and stereoselection. permitting r e c o v e r y of (7c)-glaucine in quantitative yield ( D a v i s and R o s a z z a 1981).d e p e n d e n t m o n o . and ease of w o r k u p and product purification. C a s h m a n et al. m a y also b e catalyzed b y f l a v o e n z y m e s ( S m i t h .2 . o n e that is often m o r e difficult to m e e t . cost.2 Fungi as Chemical Reagents 161 It m a y thus b e inferred that carbon h y d r o x y l a t i n g e n z y m e s (in addition to that isolated and referred to in the p r e c e d i n g p a r a g r a p h ) and e p o x i d i z i n g e n z y m e s are generally c y t o c h r o m e P .2 ) to d e h y d r o g l a u c i n e . All other things b e i n g equal (which m a y not a l w a y s be so. O n the o n e h a n d . O n e has only limited o p t i o n s . F u n g a l B a e y e r . 1974 and 1984).V i l l i g e r o x i d a t i o n . usually b e c a u s e the desired c h e m i c a l transformation is o n e for w h i c h n o equivalent b i o c o n v e r s i o n s are k n o w n . Nevertheless. 1989): c a r b o n . t w o of these criteria are d o m i n a n t . m o s t are capable of ester hydrolysis by bioconversion! . Similarly.o x y g e n a s e s (Ortiz de M o n t e l l a n o 1 9 8 6 ) .o x y g e n a s e . flavin-depend e n t m o n o . for e x a m p l e . on the c h o i c e of the appropriate c h e m i c a l reagent for a desired synthetic c o n v e r s i o n . In c o m p e t i t i o n with an o t h e r w i s e equivalent c h e m i c a l p r o c e s s . 2 . for example. an established place in the arsenal of methods available to the synthetic chemist. c o n v e n i e n c e of u s e . the ability of hydroxylase e n z y m e s . Blee and S c h u b e r 1989). the regio.

2 ) b y b i o c o n v e r s i o n . availability of the correct m i c r o o r g a n i s m is often limited by the relatively small n u m b e r of cultures w h o s e b i o c o n v e r s i o n potentials h a v e b e e n d e t e r m i n e d by t h o r o u g h screening. together with primary literature references. Kieslich 1976 and 1984. and can b e obtained with . T h e p r o c e d u r e can be carried out in a specialized fermentation a p p a r a t u s . but the extent of screening that m a y be necessary to find the a p p r o p r i a t e m i c r o o r g a n i s m is d a u n t i n g .162 Bioconversions F o r r e a s o n s such as t h e s e . S m i t h . In spite of the availability of this information. as outlined in Figure 7 . 1989). In this e v e n t u a l i t y . either by using fungi that are k n o w n to carry out the s a m e generic reaction [for e x a m p l e . h o w e v e r . the selection of strains for screening can be logically b a s e d . several c o m p e n d i a of fungal and other b i o c o n v e r s i o n s are available for consultation ( C h a r n e y and H e r z o g 1967. has the capacity to handle 10 1-1 E r l e n m e y e r flasks. the catalog of the A m e r i c a n T y p e C u l t u r e C o l l e c t i o n . the only specialized items b e i n g an autoclave for sterilizing g r o w t h m e d i a and a rotary s h a k e r for performing incubations. R o s a z z a 1982. unless a very close parallel exists b e t w e e n a literature p r o c e d u r e and the desired b i o c o n v e r s i o n . N e v e r t h e l e s s . fungi k n o w n to carry out benzylic h y d r o x y l a t i o n h a v e been usefully screened for the ability to h y d r o x y late the anthracyclin precursor 7 at the benzylic position (Figure 7 . 1974. R o t a r y incubators are standard microbiological i t e m s . Rhizopus stolonifer w a s selected as the best fungus for h y d r o x y l a t i o n of p r o g e s t e r o n e at C . Figure 7 . standard laboratory g l a s s w a r e . D a v i e s et al.3 ) (Holland and M a n o h a r a n .2 Procedures for Bioconversion F u n g a l b i o c o n v e r s i o n on a laboratory scale (up to 10 g) is a simple p r o c e d u r e that can b e carried out by a n y o n e with a b a c k g r o u n d in n o r m a l laboratory t e c h n i q u e s .2 ) following the initial d i s c o v ery that this reaction could be performed by Rhizopus arrhizus ( M u r r a y 1976). L. C h i e n and R o s a z z a (1979) screened 2 2 4 m i c r o o r g a n i s m s before selecting Aspergillus alliaceus for the production of 9-hydroxyellipticine ( 6 . the w o r l d ' s largest single depository of fungal strains. T h e former need not b e the c o m p l e x a u t o m a t i c m a c h i n e s used in m i c r o b i o l o g y laboratories: a b e n c h t o p autoclave d e s i g n e d for sterilizing e q u i p m e n t in the catering industry. in those cases for w h i c h such data are available. In this w a y . w h i c h can be obtained for a m o d e s t o u t l a y . g i v e s . In addition to these s o u r c e s . information on the types of c h e m i c a l reactions carried out by e a c h strain. It m a y b e true to say that almost any desired c a r b o n hydroxylation or stereospecific hydrolysis is possible by b i o c o n v e r sion.2. 7. or by selecting fungi with a close t a x o n o m i c relationship to o n e s k n o w n to carry out similar r e a c t i o n s . sufficient in m a n y cases for the biotransformation of 1 g of substrate. for the most part. T h e e q u i p m e n t n e e d e d is. unpublished observations 1990)].4 . but even this noble effort u n c o v e r e d only the tip of an u n k n o w n iceberg. F i g u r e 7 . it m a y b e necessary to carry out s o m e small-scale screening e x p e r i m e n t s . H o l l a n d 1981 and 1988. but the m o s t c o n v e n i e n t m e t h o d for m o s t applications is to perform the fungal g r o w t h and s u b s e q u e n t b i o c o n v e r s i o n of substrate in 1-1 E r l e n m e y e r flasks. L. R o s a z z a and Duffel 1986.l l a (conversion 3 to 4 .

and fitted with a platform for 1-1 E r l e n m e y e r flasks.7. T h e stock c u l t u r e . is used to inoculate a suitable liquid . O n e designed for use at a m b i e n t t e m p e r a t u r e . represents the m o s t e c o n o m i c a l i n v e s t m e n t .2 Fungi as Chemical Reagents 163 CHo _ <C H 3 CH3 HO 12 13 F I G U R E 7-3 Typical fungal bioconversion substrates and products. usually an agar s l o p e . or w i t h o u t thermostatic heating c o n t r o l s . located in a small r o o m that is heated to the required t e m p e r a t u r e (usually b e t w e e n 25 and 2 8 ° C ) .

T h e factors that control the g r o w t h rate of filamentous fungi in a n u m b e r of different laboratory-scale systems h a v e been discussed in detail by B r o w n ( 1 9 8 8 ) . dimethyl f o r m a m i d e or d i m e t h y l sulfoxide).7 d a y s ) . with typical substrate loadings of 2 5 .1 0 0 m g p e r flask. either as a m i c r o n i z e d p o w d e r or in solution in a water-miscible solvent ( 9 5 % e t h a n o l . in a technique k n o w n as r e p l a c e m e n t c u l t u r e . or alternatively. After satisfactory g r o w t h has been a c h i e v e d . t w o options are o p e n : the substrate m a y b e a d d e d directly to the fungal g r o w t h . the fungal g r o w t h can be collected by filtration on a B ü c h n e r funnel or centrifuga- . T h e fungus is then g r o w n on a rotary shaker for an appropriate p e r i o d (usually 3 .164 Bioconversions Stock culture growth in liquid medium add substrate OR filter or centrifuge fungus distilled water add substrate incubation period fungal suspension filter or centrifuge fungus aqueous medium extract extraction -product extract F I G U R E 7-4 Standard procedures for bioconversions with growing or resting fungi. T h e n u m b e r of flasks to b e used is dictated by the quantity of substrate to b e biotransformed. m e d i u m contained in 1-1 E r l e n m e y e r flasks ( 1 5 0 .2 5 0 ml per flask) u n d e r sterile c o n d i t i o n s .

w a r n i n g s w h e r e appropriate are included in the A T C C catalog of fungal strains. and each extracted separately. and other m e t h o d s for long-term storage are available ( S m i t h . T h e d e v e l o p m e n t of i m m o b i l i z e d cell t e c h n o l o g y for b i o c o n v e r s i o n offers the potential a d v a n t a g e s of stabilization of the biocatalyst. C o m m o n l y used c o m p o n e n t s of g r o w t h m e d i a and their effect on fungal b i o c h e m i s t r y are discussed in detail e l s e w h e r e ( S t a n b u r y a n d W h i t a k e r 1984. h i g h .2 Fungi as Chemical Reagents 165 tion using a basket centrifuge. c o m b i n e d if so indicated. for e x a m p l e . D a v i s and R o s a z z a 1981).7. Slaughter 1988. U . M a i n t e n a n c e of a small culture collection requires only a dedicated b e n c h .1 2 m o n t h s being typical. and then resuspended in a volume of distilled water equal to that of the original growth m e d i u m in a series of 1-1 Erlenmeyer flasks. (1968) and Kieslich (1984). J e n n i n g s 1988). and p r o d u c t s isolated by c h r o m a t o g r a p h y or crystallization. H o w e v e r .t o p refrigerator for storage of cultures and a clean area for culture transfers. 1984). this t e c h n o l o g y has not . F u n g a l cultures on an agar slope stored in a sealed tube at 4°C can r e m a i n viable for e x t e n d e d p e r i o d s . thus permitting its c o n t i n u e d use for periods o v e r w h i c h a g r o w i n g or resting culture w o u l d not retain viability. D e p a r t m e n t of A g r i c u l t u r e ) . and the flasks replaced on the rotary shaker. C h i e n and R o s a z z a 1979. Cultures are available at n o m i n a l cost from the A m e r i c a n T y p e Culture Collection or other culture depositories: the A T C C catalogs include cross-listing data so that. S o m e fungi c o m m o n l y used for b i o c o n v e r s i o n s are plant p a t h o g e n s . or by using a c o n t i n u o u s l i q u i d liquid extractor. and very simple p r o d u c t isolation p r o c e d u r e s . T h e substrate is then added as before. and require g o v e r n m e n t p e r m i t s for distribution. O t h e r p r o c e d u r e s for b i o c o n v e r s i o n e m p l o y i n g fungal spore s u s p e n s i o n s o r i m m o b i l i z e d cells can also be used. T h e use of spore s u s p e n s i o n s in b i o c o n v e r s i o n is discussed by V e z i n a et al. D . isabellina N R R L 1757 (Jong and G a n t t 1984). identical with M. G a d d 1988. a literature fungus with an N R R L n u m b e r (Northern R e g i o n a l R e s e a r c h L a b o r a t o r i e s . S . w h e r e a s the m e d i u m extraction m a y be d o n e by separatory funnel (for small v o l u m e s ) . T h e solid g r o w t h m e d i u m and conditions for m a i n t e n a n c e of fungi can usually b e d e t e r m i n e d by consultation of the A T C C catalog and the associated m e d i a h a n d b o o k . or gas c h r o m a t o g r a p h y as a p p r o p r i a t e .p e r f o r m a n c e liquid c h r o m a t o g r a p h y . T h e former offers the a d v a n t a g e that. the fungal g r o w t h and a q u e o u s m e d i u m are separated b y filtration or centrifugation. 2 . Extraction of the fungus is c o n v e n i e n t l y carried out in a small b l e n d e r . o n c e p r e p a r e d . spore s u s p e n s i o n s can b e stable o v e r long periods of t i m e . T h e t w o extracts can then b e e x a m i n e d b y thin-layer c h r o m a t o g r a p h y . usually with d i c h l o r o m e t h a n e . d e p e n d i n g o n the fungus c o n c e r n e d . a l l o w i n g their use in a m a n n e r a n a l o g o u s to that e m p l o y e d for c h e m i c a l catalysts. m a y well b e available from A T C C u n d e r a different accession n u m b e r . After a suitable time period for b i o c o n v e r s i o n (usually 1-7 d a y s ) . for e x a m p l e . T h e liquid m e d i u m to b e used for b i o c o n v e r s i o n is usually d e t e r m i n e d from relevant literature b i o c o n v e r s i o n s . as m a n y e x a m p l e s exist of b i o c o n v e r s i o n s that are d e p e n d e n t o n the p r e s e n c e or a b s e n c e of specified m e d i u m c o m p o n e n t s ( M u r r a y 1976. Mortierella isabellina A T C C 4 2 6 1 3 is. Correct choice of incubation m e d i u m is not a trivial m a t t e r .

dibasic p o t a s s i u m p h o s p h a t e (5 g ) . synthetic i n t e r m e d i a t e s . w h i c h w e r e replaced on the shaker at 28°C for a further 2 4 h. n o w performed by R. 5 7 g. T h e flasks w e r e p l a c e d on a rotary shaker at 28°C overnight without s h a k i n g . isabellina (on 4 % malt agar) w a s used to inoculate 15 1-1 E r l e n m e y e r flasks each containing 2 5 0 ml of an autoclaved m e d i u m c o m p o s e d of g l u c o s e ( 4 0 g ) . A detailed p r o c e d u r e for the production of (R)-( + )-phenyl η-propyl sulfoxide (9) from p h e n y l rc-propyl sulfide (8) (Figure 7 . stolonifer. yeast extract (5 g ) . w h i c h w a s purified by c o l u m n c h r o m a t o g r a p h y o v e r silica gel followed by K u g e l r o h r distillation to give (R)-(+)-phenyl n-propyl sulfoxide ( 0 . T h i s b i o c o n v e r s i o n . In spite of almost four d e c a d e s of d e v e l o p m e n t . T h e fungal g r o w t h w a s collected by centrifugation using an I E C b a s k e t centrifuge. After this time the fungus w a s r e m o v e d by centrifugation.3. (1952) that g r o w i n g cultures of Rhizopus arrhizus w e r e able to introduce the hydroxyl g r o u p at C-11 a into a r a n g e of p r e g n a n e substrates in isolated yields u p to 9 0 % (see Figure 7 . 7. and drug metabolites. has been d e v e l o p e d as part of a c o m m e r c i a l p r o c e s s for the production of 11 α .3 THE PRODUCTS OF FUNGAL BIOCONVERSIONS F u n g a l b i o c o n v e r s i o n s as a m e a n s of performing regio. 7. T h e resulting extract w a s e v a p o r a t e d to give a residue ( 0 . and soya flour (5 g) per liter of distilled water.166 Bioconversions b e e n applied extensively to filamentous fungi. A l t h o u g h b i o c o n v e r s i o n as a m e a n s of d e g r a d i n g steroids had been k n o w n for m a n y d e c a d e s prior to 1950. 8 g ) . A solution of p h e n y l π-propyl sulfide (1 g) in 9 5 % ethanol (30 ml) w a s then added to the flasks (2 ml p e r flask).2 ) . and r e s u s p e n d e d in distilled water (3 1) distributed o v e r 15 1-1 E r l e n m e y e r flasks ( 2 5 0 ml per flask). 5 2 % yield. and by research c h e m i s t s . the d e v e l o p m e n t of fungal b i o c o n v e r s i o n as a general synthetic tool can be said to h a v e b e g u n with the n o w classic discovery by Peterson et al. n e w b i o c o n v e r s i o n reactions and n e w applications for k n o w n reactions are still being d i s c o v e r e d .and stereospecific c h e m i c a l reactions are extensively utilized by the p h a r m a c e u t i c a l and fine c h e m i c a l industries in the p r o d u c t i o n of chiral c o m p o u n d s and other useful m e t a b o l i t e s .1 Production of Pharmaceutical Products by Fungal Bioconversion T h e m o s t d r a m a t i c impact of bioconversion in the p h a r m a c e u t i c a l area has u n d o u b t e d l y b e e n in the production of steroid h o r m o n e s . medicinal c h e m i s t s . and the a q u e o u s m e d i u m extracted with d i c h l o r o m e t h ane in a c o n t i n u o u s extraction apparatus for 3 d a y s . O n e slope of M. and p h a r m a c o l o g i s t s in the synthesis of chiral p r o d u c t s . and then shaken at 180 r p m for 3 d a y s . o t h e r steroids p r o d u c e d c o m m e r c i a l l y by b i o c o n v e r s i o n s h a v e h y d r o x y l g r o u p s at C-11 β . e n a n t i o m e r i c purity > 9 8 % by rotation and chiral nuclear magnetic resonance (NMR) analyses). h a v i n g b e e n used mainly for b a c t e rial s y s t e m s ( L e u e n b e r g e r 1984). s o d i u m chloride (5 g ) .h y d r o x y s t e r o i d s such as 4.3 ) b y b i o c o n v e r s i o n with Mortierella isabellina N R R L 1757 ( A T C C 4 2 6 1 3 ) is presented in the following p a r a g r a p h s .

A l t h o u g h these reactions are m o r e efficiently carried out b y bacteria. T h u s 2/3.e n e . 1984. the p r o d u c t i o n of labeled steroids useful for mechanistic studies with other en1 8 z y m e s . 1989).and base-sensitive prostaglandin Ε ( P G E ) derivatives such as 16 (Figure 7 . Holland et al. the antitubercular anthracyclin 10-dihydrosteffimycin Β (17. has recently b e e n reported (Holland et al.alcohols by Phycomyces blakesleeanus. 4 . A m o n g the s a m p l e s p r o d u c e d in this w a y from p r o g e s t e r o n e (3) are the 9/3. a valuable substrate for investigation of the m e c h a n i s m of action of the h u m a n placental a r o m a t a s e e n z y m e .5 ) from p r o g e s t e r o n e (3). H y d r o x y l a t i o n at virtually any position of the steroid n u c l e u s can b e achieved by selection of the appropriate m i c r o o r g a n i s m ( C h a r n e y and H e r z o g 1967.and 1 4 a . A l t h o u g h the major application of fungal b i o c o n v e r s i o n in the area of antibiotics h a s b e e n in the p r o d u c t i o n of metabolites by oxidative p r o c e s s e s (see 7 .e n e . the 6/3-. L. such as the c o n v e r s i o n of ßsitosterol.5 ) (Sih et al.d i o n e (14. T h e effect of substrate structure on the site of hydroxylation w a s studied extensively by Sir E w a r t J o n e s and c o . u n u s u a l in its ability to p o l y h y d r o x y l a t e steroids. A n o t h e r application of fungal b i o c o n v e r s i o n in the area of steroid c h e m i s t r y .w o r k e r s at Oxford during the late 1960s and 1970s (Holland 1982). c o n v e r s i o n of 10 to 11. respectively. In addition to the c o m m e r c i a l p r o d u c t i o n of steroid h o r m o n e s by fungal b i o t r a n s f o r m a t i o n . 3 .4 . N e w fungal b i o c o n v e r s i o n s of steroidal substrates c o n t i n u e d to be reported.3 .4-dien-3-ones (for e x a m p l e .7. Κ. 1984).d i o n e . O t h e r important b i o c o n v e r s i o n s of steroid substrates in the p r o d u c t i o n of p h a r m a c e u t i c a l s include d e h y d r o g e n a t i o n s of 3-keto-4-enes to g e n e r a t e 1. a t m o s p h e r e e n r i c h e d with O t h e r p h a r m a c e u t i c a l p r o d u c t s w h o s e production on a laboratory scale can benefit from the use of fungal b i o c o n v e r s i o n include prostanoids and antibiotics.5 ) .c ) . F i g u r e 7 .d i o l s by Apiocrea chrysosperma. fungal b i o c o n v e r s i o n s can be e m p l o y e d in s o m e instances ( S m i t h . L .15/3. w h e r e a s fungal b i o c o n v e r s i o n is n o w b e i n g used by w o r k e r s at the Steroid Reference Collection in the United K i n g d o m for the production of o t h e r w i s e rare or u n o b t a i n able h y d r o x y steroids ( S m i t h . 3 . D a v i e s et al. 1975). b i o c o n v e r s i o n using Rhizopus oryzae (arrhizus) p r o v i d e s a mild m e t h o d of h y d r o l y s i s of acid. 1 4 a . F i g u r e 7 . 1974 and 1984. L .. 3 .. F i g u r e 7 . 1 5 a .5 ) can be p r o d u c e d b y b i o c o n v e r s i o n of steffimycin Β using Chaetomium species (Marshall et .4 . w h i l e considerations of the m e c h a n i s m of the reaction can also be helpful in predicting the p r o d u c t of b i o c o n v e r s i o n (Holland 1984.[ 0 ] h y d r o x y testosterone (15.17at r i h y d r o x y p r e g n . 1989). 13 (Figure 7 . is c o n v e n i e n t l y p r e p a r e d b y b i o c o n v e r s i o n of testosterone using Gnomonia fructicola A T C C 11430 u n d e r an 1 8 0 2. et al. 1988 and 1 9 8 9 a . the t e c h n i q u e is also v a l u a b l e in the production of small quantities of specific steroids for other p u r p o s e s . O t h e r applications of fungal b i o c o n v e r sion to prostanoid c h e m i s t r y include the production of chiral synthetic intermediates and the formation of prostaglandin m e t a b o l i t e s . 1 7 .1 6 a ( S m i t h . S m i t h . Martin 1984). (1989) recently d i s c o v e r e d that a strain of Acremonium strictum.3 The Products of Fungal Bioconversions 167 and . 7 a . E . F i g u r e 7 . 1 5 a .15/3. 3 and 7 . In the former a r e a . 2 0 . and 15/3.3 . L. 12 to a n d r o s t . Y o s h i h a m a et al. 1989)..alcohol by Sepedonium ampullosporum. L .3 ) and side c h a i n o x i d a t i o n s of cholesterol and p h y t o s t e r o l s . c o n s i d e r e d in Sections 7 . 4 ) .3 ) . and the 1 6 a . L . p r o d u c e d (inter alia) 7/3.

al.5 Some bioconversion substrates and products of pharmaceutical value.d e a c y l a t i o n reactions o n ß . has led to the isolation and c o m m e r c i a l use of the e n z y m e s r e s p o n s i b l e (Sebek 1984).l a c t a m antibiotics. .168 Bioconversions 14 15 Ο 16 HO OH OH 17 OH CH3 OH P 0 3H 2 >=< Η CH3 — Η 19 OR P 0 3H 2 >V7< Η Ο Η 18 F I G U R E 7 . k n o w n for m a n y years ( S a k a g u c h i and M u r a o 1 9 5 0 ) . and the antibiotic fosfomycin (18) is conveniently prepared by e n a n tioselective epoxidation of the c o r r e s p o n d i n g alkene 19 using Pénicillium spinulosum ( W h i t e et al. 1971). In addition. the ability of fungi to perform N . 1981).

the e n a n t i o m e r of substrate represented b y 21 (Figure 7 . A general relationship b e t w e e n the site of o x y g e n substitution in a steroidal substrate and the position of h y d r o x y l a t i o n of that substrate by fungi. applies to the reduction of k e t o n e s by m a n y m i c r o o r g a n i s m s .3.6 ) . c o n v e n i e n t l y p r o v i d i n g the o p p o s i t e e n a n t i o m e r of p r o d u c t to that o b t a i n e d b y k e t o n e reduction (cf. the application of empirically derived rules for prediction of the o u t c o m e can be useful. T h e prediction of the r e g i o .7. In these c a s e s . E m p i r i c a l rules for the prediction of product stereochemistry b a s e d o n the size of substituents present in the substrate also exist for the hydrolysis b y Rhizopus nigricans (stolonifer) of r a c e m i c aryl alkyl esters (Charton and Ziffer 1987) and cyclic alkyl carbinol esters (Kasai et al. H e r e . Curvularia falcata (Prelog 1964). 1984). 2 Â] a w a y from the o x y g e n (Furstoss et al.2 The Products of Fungal Bioconversions 169 Production of Asymmetric Compounds by Fungal Bioconversion A s d i s c u s s e d earlier in this c h a p t e r . 20. F i g u r e 7 . E x a m p l e s of such predictive rules for hydroxylation b a s e d on a d e d u c e d t o p o g r a p h y of the active site of the e n z y m e s c o n c e r n e d exist for several fungi. Similar empirical rules exist for c a r b o n y l reductions p e r f o r m e d b y yeasts (Davies et al. the e n a n t i o m e r i c purities of the resulting alcohols w e r e highly d e p e n d e n t on the difference in size of the g r o u p s labeled S (small) a n d L (large). In addition to these g u i d e l i n e s .or stereospecific b i o c o n v e r s i o n s . In cases w h e r e the e n a n t i o m e r i c purity of the alcohol formed in this w a y is < 1 0 0 % . the site of h y d r o x y l a t i o n can b e from 3.3 7. unlike P r e l o g ' s rule w h i c h m a y b e applicable to all b i o c o n v e r s i o n s . 1986). such predictions are usually restricted to a single well-studied m i c r o o r g a n i s m . T h e m o s t general of t h e s e .6 ) is the o n e that w a s m o r e rapidly h y d r o l y z e d . F i g u r e 1-6. Beauveria sulfurescens h y d r o x y l a t e s simple cyclic a m i d e substrates such as 22 and 23 (Figure 7 . the rule is n e v e r t h e l e s s still useful in predicting the p r e d o m i n a n t absolute stereochemistry of p r o d u c t . 1989). but are b e y o n d the s c o p e of the present d i s c u s s i o n . as with the k e t o n e r e d u c t i o n s c o v e r e d by P r e l o g ' s r u l e . the p r e v a l e n c e of steroid h y d r o x y l a t i o n s at positions axial to existing carbonyl g r o u p s can be explained on m e c h a n i s t i c g r o u n d s . a l t h o u g h . has b e e n d e d u c e d from an extensive p r o g r a m of b i o c o n v e r sions carried out by the J o n e s g r o u p at Oxford and r e v i e w e d in detail by H o l l a n d ( 1 9 8 2 ) . 1968). with m o r e c o m p l e x cyclic a m i d e s such as 24. F i g u r e 7 . the result of the action on the substrate of m o r e than o n e alcohol d e h y d r o g e n a s e e n z y m e present in the f u n g u s . specifically Calonectria decora. In the ester h y d r o l y s e s . In s e e k i n g to use the standard reactions of fungal b i o c o n v e r s i o n with n e w substrates in the h o p e of a c h i e v i n g r e g i o . T h e rule h a s b e e n d e v e l o p e d further into a d i a m o n d lattice active site m o d e l for o n e fungus.5 Â [from the a m i d e o x y g e n a t o m and with s t e r e o c h e m i s try trans to the a m i d e ( J o h n s o n et al. P r e l o g ' s r u l e .3 to 6 .6 ) at a site 5. the stereochemistry of the resulting alcohol is that s h o w n in 20.or stereochemical o u t c o m e of other b i o c o n v e r s i o n s such as h y d r o x y l a t i o n and sulfoxidation is also possible in s o m e c a s e s .6 (Prelog 1964). it is the ability of b i o c o n v e r s i o n s to control both the r e g i o c h e m i s t r y and stereochemistry of a reaction that is largely r e s p o n s i b l e for their c o n t i n u e d application and for the o n g o i n g nature of the investigation of n e w bioconversion processes.

. and some examples of stereospecific product formation.170 Bioconversions \ 22 23 F I G U R E 7-6 Predictive rules for bioconversions.

6 ( H o l l a n d 1988). O t h e r factors that can affect the r e g i o c h e m i s t r y of h y d r o x y l a t i o n of steroids (and p e r h a p s b y inference of other substrates also) include the activation of allylic positions t o w a r d h y d r o x y l a t i o n . F o r e x a m p l e . and the elegant study by A z e r a d of the microbial B a e y e r . T h i s relationship also holds for o n e strain of Aspergillus niger (Auret et al.7. T h e stereochemistry of oxidation at the sulfur a t o m of a d i s y m m e t r i c sulfide. and a steric suppression of h y d r o x y l a tion adjacent to b u l k y and o t h e r w i s e unreactive substituents such as b r o m i n e ( D a v i e s et al. a c r o n y c i n e .7 ) . individual results can nevertheless b e i m p r e s s i v e .m e m b e r e d lactone 3 0 . 1983). p e r h a p s the m o s t consistent are those d i s c o v e r e d by R o s a z z a and others during investigations into the b i o c o n v e r s i o n of a l k a l o i d s . an active site m o d e l for the h y d r o x y l a t i o n of sesquiterp e n e s by Beauveria sulfurescens has b e e n p r o p o s e d ( L a m a r e et al. but not necessarily for others (Auret et al. F i g u r e 7 . H y d r o x y l a t i o n of aromatic rings is o n e such e x a m p l e : the o b s e r v a t i o n that h y d r o x y l g r o u p s are often introduced at positions ortho or ( m o r e c o m m o n l y ) para to electron d o n a t i n g substituents is best explained in t e r m s of the reaction m e c h a n i s m . R e c e n t l y . 1985 and 1988). w h i c h involves the intermediacy of an arene o x i d e and its r e a r r a n g e m e n t via the N I H shift to give product (31 to 3 2 . 1 9 8 9 . 1987). but m a n y e x a m p l e s of fungal b i o c o n v e r s i o n s exist in w h i c h only regioselectivity is exhibited. p .7 ( O u a z z a n i .C h a h d i et al. Recently reported significant e x a m p l e s of stereospecific b i o c o n v e r s i o n s include the production of c h r y s a n t h e m i c acids b y oxidation of the c o r r e s p o n d i n g p r i m a r y alcohols using Aspergillus ochraceus referred to in Figure 7 . the (R)e n a n t i o m e r of 2 8 can b e r e c o v e r e d from the incubation. 176). f u r t h e r m o r e . Curvularia lunata regioselectively o x i d i z e s the ( S j .6 ) by Pichia miso catalyzed hydrolysis of r a c e m i c starting materials ( O h t a et al. can b e predicted for the fungus Mortierella isabellina. 1974). so that both e n a n t i o m e r i c p r o d u c t s are o b t a i n a b l e via fungal biotransformation. h y d r o x y l a t i o n s of Rauwolfia alkaloids 3 3 (Bellet and V a n T h u o n g 1970 and 1972. L e s m a et al. the c o n v e r s i o n of n a p h t h a l e n e to the 1(5). T h e b i o c o n v e r s i o n of 2 8 is o n e that c o m b i n e s both enantioselectivity and regioselectivity. and c a n n o t b e e x t e n d e d to the sulfoxidation of substituted dithianes or dithiolanes by either Mortier ella or Aspergillus species (Auret et al.e n a n t i o m e r of a r a c e m i c m i x t u r e of 2 8 to give the lactone 2 9 . 1988). F i g u r e 7 . Stabilization of the intermediate cation by the electron d o n a t i n g g r o u p m a y thus control the r e g i o c h e m i s t r y of h y d r o x y l a t i o n (Smith and R o s a z z a 1974). T h e latter r e a r r a n g e s u n d e r the conditions of the incubation to the f i v e . leading to chiral sulfoxide f o r m a t i o n .and s t e r e o c h e m i c a l o u t c o m e of m o s t fungal b i o c o n v e r s i o n s . 1987). retaining the absolute stereochemistry s h o w n . 2(S')-diol 2 6 by Cunninghamella elegans (Cerniglia et al. and c o n v e r t e d b y peracid oxidation to the e n a n t i o m e r of 3 0 . w h i c h p r o d u c e s p r e d o m i n a n t l y the sulfoxides s h o w n as 2 5 . an electronic suppression of h y d r o x y l a t i o n close to sites of fluorine substitution (Holland 1982). 1968).3 The Products of Fungal Bioconversions 171 ( H o l l a n d 1984). the production of chiral c y a n o h y d r i n acetate esters 2 7 (Figure 7 .2 ( M i s k i and D a v i s 1988).V i l l i g e r oxidation of the r a c e m i c c y c l o h e x a n o n e 2 8 s h o w n in F i g u r e 7 . 1983). Of the m a n y e x a m p l e s of aromatic h y d r o x y l a t i o n s of this type (Kieslich 1976). In spite of the lack of a general m o d e l for prediction of the regio.

and regioselective bioconversion by Baeyer-Villiger oxidation. (34) (Betts et al.2 ) (Chien and R o s a z z a 1979) o c c u r p r e d o m i n a n t l y at the sites indicated in F i g u r e 7 . 7.172 Bioconversions OH FIGURE 7-7 (Upper) Stereo.3. and ellipticine (giving product 6.8 . synthon) chirality Production of Chiral Synthetic Intermediates by Fungal Bioconversion cases w h e r e b i o c o n v e r s i o n c a n n o t b e used to p r o d u c e a target m o l e c u l e it can still b e valuable in the production of a chiral intermediate (chiral w h i c h can then b e c o n v e r t e d chemically to the desired target. 1974). or a s y m m e t r y introduced by the b i o c o n v e r s i o n p r o c e s s can b e carried . In this w a y . F i g u r e 7 .3 In t h o s e directly. (Lower) The NIH shift route for arene hydroxylation.

and some chiral synthons (35-38) produced by fungal bioconversion. C o m p o u n d s that can b e m a d e from chiral s y n t h o n s p r o d u c e d from b i o c o n v e r s i o n s include p r o s t a n o i d s . n u c l e o s i d e s . A m o n g the chiral s y n t h o n s for prostaglandins available as a result of fungal b i o c o n v e r s i o n s are the alcohol 35 (Figure 7 . F i g u r e 7 . has elicited m u c h ingenuity on the part of synthetic c h e m i s t s . insecticides.8 ) . a m i n o a c i d s . a p r e c u r s o r of the . e n z y m e inhibitors. p h e r o m o n e s .3 37 The Products of Fungal Bioconversions 173 38 FIGURE 7-8 Regiospecific hydroxylations of alkaloids 33 and 34. t e r p e n e s . antibiotics.7.5 ) . t h r o u g h a synthetic s e q u e n c e and retained in the final product. Prostaglandin synthesis presents a severe stereochemical challenge: the necessity of o b t a i n i n g the correct absolute stereochemistry at four chiral centers in the P G E series. and jß-lactams. for e x a m p l e (see 16.

1975). and the ring s y n t h o n s 3 6 (Figure 7 .c e d r e n e (44) and the related cedrol by Beauveria sulfurescens to give access to a series of d i o l s . R e c e n t e x a m p l e s of the latter application include a route for the p r o d u c t i o n of the potential p h a r m a c e u t i c a l forskolin 4 2 involving hydroxylation by a Scopuloriopsis species of a d i d e o x y a n a l o g u e at carbons 1 and 9 ( N a d k a r n i et al.8 ) . unlike the b i o c o n v e r s i o n of the Np h e n y l c a r b a m a t e esters of geraniol and nerol by A. . 2 . and linalool (48) p r e d o m i n a n t l y at the terminal sites s h o w n in F i g u r e 7 .9 .174 Bioconversions l o w e r prostaglandin side c h a i n . p r o d u c e d by reduction of the c o r r e s p o n d i n g k e t o n e using R. T h e s e conversions involve n o c h a n g e s in chirality. and b y reduction of an α. In the area of prostanoid a n a l o g u e s . A further e x a m p l e of b i o c o n v e r s i o n in the sesq u i t e r p e n e area from the s a m e research g r o u p c o n c e r n s the h y d r o x y l a t i o n of caryolanol (45) by Aspergillus niger. In contrast to earlier reports of acyclic m o n o t e r p e n e bioconversion by bacteria in w h i c h ringclosed and rearranged products w e r e c o m m o n l y o b s e r v e d ( K r a s n o b a j e w 1984). 1975). 1987). T h e s e c o m p o u n d s are precursors of cyclic a n a l o g u e s of g l u t a m i c acid. and 3 8 . l ] h e p t a n e carboxylic acid by Aspergillus awamori p r o d u c e s the h y d r o x y a c i d 4 0 (Figure 7 ./3-unsaturated k e t o n e using Aspergillus niger ( K u r o z u m i et al. w h i c h involved reaction at the distal C = C b o n d to give the (S). 1986). 1986). p r o d u c e d b y reduction of the trione by Dipodascus uninucleatus (Sih et al. 3 7 .5 3 (Figure 7 . arrhizus in the case of 5 1 . 1987). 1989). . convertible by chemical m e a n s into the j a s m a n e d e rivative 4 1 ( Y a m a z a k i and M a e d a 1985). 1989). formed by hydroxylation of the parent α. geraniol (47). during w h i c h the ester g r o u p survived intact and a n e w chiral center w a s p r o d u c e d in an e n a n t i o m e r i c excess e x c e e d i n g 9 5 % (Fourneron et al. F u n g a l b i o c o n v e r s i o n in the area of terpene synthesis has been used to p r o d u c e chiral s y n t h o n s that can then be c o n v e r t e d into desirable t e r p e n e s . o n e of w h i c h w a s desirable for its odoriferous properties ( L a m a r e et al. respectively. p r o d u c e d by reduction of the c o r r e s p o n d i n g k e t o n e by Pénicillium decumbens (Sih et al.1 0 ( M a d y a s t h a and M u r t h y 1988). 1973)./3-unsaturated k e t o n e substrate by Geotrichum candidum for 5 2 and 5 3 (Trigalo et al.diols 4 9 and 5 0 . 1985). and to modify readily available terpenes as part of synthetic routes to less c o m m o n or m o r e desirable p r o d u c t s (Krasnobajew 1984). in which the pro-R methyl g r o u p is specifically c o n v e r t e d to h y d r o x y methyl in 2 6 % yield ( L a m a r e et al. 1988).9 ) . Figure 7 .1 0 ) . Butt et al. and w e r e a c c o m p a n i e d by ester h y d r o l y s i s . In the former area. F u n g a l b i o c o n v e r s i o n of m o n o t e r p e n e s as a m e a n s of p r o d u c i n g other terpenes or chiral synthetic intermediates also continues to receive attention. and the h y d r o x y l a t i o n of a . the acid 3 9 . niger. Aspergillus niger is reported to h y d r o x y late the acetates of citronellol ( 4 6 ) . the h y d r o x y l a t i o n of b i c y c l o [ 2 . F u n g a l b i o c o n v e r s i o n has been applied to the synthesis of un-natural a m i n o acids via the chiral synthons 5 1 . obtained from reduction of the c o r r e s p o n d i n g r a c e m i c k e t o n e by M ordere lia ramanniana (Roberts 1986. h y d r o x y l a t i o n of the readily available sesquiterpene d e o x y v u l g a r i n (43) by Aspergillus ochraceus and Rhizopus nigricans as a m e a n s of p r o d u c i n g several e u d e s m a n o l i d e s with different o x y g e n a t i o n patterns (Arias et a l . can b e p r e p a r e d from a route involving hydroxylation of the h y d r o c a r b o n c y c l o h e x y l c y c l o h e x a n e by Cunninghamella (Davies et al.

9 Some fungal hydroxylations of terpenes and related compounds.7.3 The Products of Fungal Bioconversions F I G U R E 7 . 175 .

by a m i d e hydrolysis (Mori and O t s u k a . it should also be n o t e d that hydrolytic e n z y m e s from Aspergillus species are of considerable i m p o r t a n c e in the p r o d u c t i o n of the natural L .a m i n o acids by hydrolysis of r a c e m i c a m i d e p r e cursors (Izumi et al. and have been used to prepare the a m i n o acid 5 4 .176 Bioconversions NK 52 53 54 F I G U R E 7-10 Fungal bioconversions of terpenes and some amino acid precursors. A l t h o u g h not strictly biotransformation p r o c e s s e s . w h i c h in turn are desirable as g l u t a m a t e r e p l a c e m e n t s in e n z y m e m e c h a n i s m studies. a p r e c u r s o r of an oriental hornet p h e r o m o n e . 1978).

A n o t h e r application of fungal b i o c o n v e r s i o n in the area of a s y m m e t r i c synthesis of biologically important m o l e c u l e s is the h y d r o x y l a t i o n of the bicyclic l a c t a m 5 5 b y Beauveria sulfurescens at the position s h o w n in F i g u r e 7 .3 60 The Products of Fungal Bioconversions 177 61 F I G U R E 7-11 Some chiral intermediates available by fungal bioconversion. T h e p r o d u c t can be used in a s i m p l e synthesis of 2 .h y d r o x y .7. 1984 and 1988).3 - . 1985). O t h e r p r o d u c t s with desirable biological properties w h o s e a s y m m e t r i c synthesis can b e a c h i e v e d by routes involving fungal b i o c o n v e r s i o n include the 3 .1 1 . A r c h e l a s et al.d e o x y n u c l e o s i d e a n a l o g u e s (56) ( A r c h e l a s and M o r i n 1984.

Cerniglia et al. was pioneered by Rosazza and Smith (1974. the p r o d u c t of b e n z y l i c h y d r o x y l a t i o n of the c o r r e s p o n d i n g d e o x y substrate b y Pénicillium or Aspergillus species ( K o n d o et al. in the s a m e w a y as the m a m m a l i a n m e t a b o l i s m (Foster et al. 1979. R e c e n t l y reported e x a m p l e s of the former application include the biotransformation of the antic o a g u l a n t s warfarin and p h e n p r o c o u m o n (62) by Aspergillus niger to the k e t o n e s (63). 7. A recent study of the b i o c o n v e r s i o n of the adrenergic agent m e t h o x y p h e n a m i n e (64) by Cunninghamella bainieri (echinulata) found that. 1988). regiochemistry. T h i s finding suggests an extension of the role of fungal b i o c o n v e r s i o n as a m o d e l of m a m m a l i a n m e t a b o l i s m to o n e in w h i c h fungal conversion can b e used to study d r u g interactions during m e t a b o l i s m . Rizzo and Davis 1988).l a c t a m s 6 0 and 6 1 . and potential a n t i t u m o r p o d o p h y l l o t o x i n a n a l o g u e s obtainable from 5 9 (Figure 7 . a product of the reduction of the c o r r e s p o n d i n g r a c e m i c d i k e t o n e b y Aureobasidium pullulans (Hsu et a l . and 1982). often in a chiral s e n s e . .ß .h y d r o x y . 1983). 1989a). T h e s e p r o d u c t s are only m i n o r metabolites in m a m m a l i a n s y s t e m s . and has since been extensively developed. T h e former application. N o t a b l e a m o n g these are the use of fungal b i o c o n v e r s i o n as a m e a n s of production of m a m m a l i a n metabolites of drugs or xenobiotic c o m p o u n d s . and stereochemistry of the reactions concerned. and Foster ( 1 9 8 9 a . In light of the difficulties often associated with the extrapolation of toxicological and metabolic data from animals to m a n (Miyamoto et al. and can also be used in cases w h e r e n o m a m m a l i a n m e t a b o l i c data are available for the prediction of metabolic p a t h w a y s . and the use of fungal b i o c o n v e r s i o n of h y d r o c a r b o n s as a w a y of functionalizing these c h e m i c a l l y unreactive m o l e c u l e s . it is remarkable that fungal bioconversion is able to mimic many mammalian biotransformations in terms of the range. N e w chiral synthons of potential value in the preparation of biologically useful materials include the ( S J . so that fungal b i o c o n v e r s i o n s are valuable in facilitating their isolation and c o m p l e t e characterization ( R i z z o and D a v i s 1988). bainieri w a s also susceptible to the effect of added c o m p o u n d s . the process also has u n i q u e applications in several general a r e a s . notably sparteine and q u i n i d i n e .4 Other Applications of Fungal Bioconversion In addition to the specific applications for fungal b i o c o n v e r s i o n d i s c u s s e d in the p r e c e d i n g section. Cerniglia (for example. and the large range of reaction types associated with mammalian biotransformation (Hawkins 1988).c ) . p r o v i d i n g an alternative to the use of m a m m a l i a n s y s t e m s or c h e m i c a l synthesis for the production of these c o m p o u n d s . . T h e s a m e o r g a n i s m has also b e e n used to study the b i o c o n v e r s i o n of other p h a r m a c e u t i c a l p h e n y l e t h y l a m i n e s (Foster et al. the use of fungi as models of mammalian metabolism. F u n g a l b i o c o n v e r s i o n s h a v e been used to p r o d u c e the p r o d u c t s of m a m m a l i a n m e t a b o l i s m in quantities sufficient for further e x p e r i m e n t a t i o n or for use as analytical s t a n d a r d s . notably by Davis (for example. obtainable from reduction of the ketones by Pichia terricola (Hirai and N a i t o 1989). the rate and extent of b i o c o n v e r s i o n by C .178 Bioconversions methylglutaryl c o e n z y m e A ( H M G C o A ) reductase inhibitor c o m p a c t i n ( 5 7 ) .3. w h i c h can b e prepared via the diol 5 8 .1 1 ) . 1989). in addition to p r o d u c i n g the full r a n g e of m a m m a l i a n m e t a b o l i t e s . 1988).

1990). the p r o d u c t i o n of the 19. 1987 and 1988. F i g u r e 7 . by h y d r o x y l a t i o n of the c o r r e s p o n d i n g r a c e m i c unsubstituted c o m p o u n d s using Rhizopus s p e c i e s .9 ) ( F o n k e n et al. a l t h o u g h b i o c o n v e r s i o n s involving h y d r o c a r b o n substrates h a v e b e e n m o s t frequently carried out with bacteria (Kieslich 1976) and h y d r o x y l a t i o n of unfunctionalized saturated h y d r o c a r b o n s by fungi is quite rare. A n a l o g o u s c h e m i c a l reactions are usually either nonspecific or low-yield p r o c e s s e s . C. F i g u r e 7 . such as the H . 1989c). E v e n for substrates w h o s e fungal b i o c o n v e r s i o n has not b e e n studied.1 3 ) in a m a n n e r a n a l o g o u s to that o b s e r v e d in m a m m a l i a n s y s t e m s ( M c M i l l a n et al. all of w h i c h p r o d u c e the diol 7 0 (Figure 7 . Figure 7 . not only w a s the absolute s t e r e o c h e m i s t r y of h y d r o x y l a t i o n at C . 1988). has b e e n found by C e r n i g l i a and c o . b y contrast.1 9 identical to that reported for the m a m m a lian m e t a b o l i t e s .1 2 .w o r k e r s to b e v a l u a b l e in the production of metabolites from a r a n g e of antihistamines ( H a n s e n et a l .w o r k e r s on the ability of Cunninghamella elegans and other m i c r o o r g a n i s m s to m e t a b o l i z e p o l y c y c l i c a r o m a t i c h y d r o c a r b o n s such as d i m e t h y l b e n z [ a ] a n t h r a c e n e ( 6 9 . In the p r o s t a g l a n d i n area. T h e latter p r o c e s s is an e x a m p l e of another of the m o r e r e m a r k a b l e features of b i o c o n v e r s i o n . In this study. all the reactions o b s e r v e d d u r i n g m a m m a l i a n m e t a b o l i s m (saturated C . and of the β-adrenergic b l o c k i n g drug p r o p r a n o l o l ( 6 5 ) . 1987). F u n g a l h y d r o x y l a t i o n of unsaturated h y d r o c a r b o n s is. N .1 3 ) from c y c l o h e x y l c y c l o h e x a n e (cf.d e a l k y l a t i o n . . suggesting a role for the latter in the prediction of the m e t a b o l i c profile in m a m m a l i a n s y s t e m s . elegans. 1964. 1987) h a v e their counterpart in fungal b i o c o n v e r s i o n . Cerniglia et a l .1 2 ) . O . and a m i d e h y d r o l y s i s .and stereospecific. so that b i o c o n v e r sion is often the m e t h o d of c h o i c e in the p r o d u c t i o n of a s y m m e t r i c alcohols directly from h y d r o c a r b o n s .7. relatively c o m m o n . bainieri to g i v e a m e t a b o l i c profile parallel to that o b s e r v e d in m a m m a l i a n s y s t e m s (Foster et al. 1986).3 The Products of Fungal Bioconversions 179 1 9 8 9 b ) . H o n m a et al.h y d r o x y l a t i o n . ester h y d r o l y s i s . F u n g a l b i o c o n v e r s i o n usually o c c u r s best with h y d r o p h o b i c substrates that already carry o n e or m o r e functional g r o u p s capable of acting as an e n z y m e b i n d i n g site or p r o v i d i n g activation t o w a r d e n z y m i c attack.d e a l k y l a t i o n .1 2 ) . D a v i e s et al. and Sporotrichum sulfur esc ens. w h i c h w a s s u s c e p tible to a r o m a t i c h y d r o x y l a t i o n . aromatic Ch y d r o x y l a t i o n . but nevertheless fungi are c a p a b l e of the o x i d a t i v e m e t a b o l i s m of unactivated h y d r o c a r b o n s . In these cases it is possible that the polarizable π electron density assists . . A n o t h e r Cunninghamella s p e c i e s . Geotrichum lacrispora. indicates a possible role for fungal b i o c o n v e r s i o n in the p r o d u c t i o n of m a m m a l i a n prostaglandin metabolites h y d r o x y l ated at these positions (Holland et al. n a m e l y the activation of a h y d r o c a r b o n substrate by h y d r o x y l a t i o n . and o x i d a t i v e d e g r a d a t i o n b y C . but the r a c e m i c substrates w e r e h y d r o x y l a t e d enantioselectively to g i v e the correct prostanoid absolute configuration depicted in F i g u r e 7 .2 r e c e p t o r antagonist R o x a t i d i n e acetate ( 6 8 . 3 9 . A m o n g the fungi that are k n o w n to be c a p a b l e of this reaction are Cunninghamella blakesleeana. T h e application of fungal b i o c o n v e r s i o n in the metabolic studies of xenobiotic c o m p o u n d s is best exemplified by the w o r k of Cerniglia and c o .and 18-hydroxy p r o s t a n o i d s 6 6 and 6 7 (Figure 7 . the result of w h i c h is usually both r e g i o .

are often o b s e r v e d to give the p r o d u c t s of attack at ally lie positions (Krasnobajew 1984). for e x a m p l e .c e d r e n e by Beauveria sulfurescens at the site indicated in 4 4 . F i g u r e 7 . and also activates adjacent positions t o w a r d attack. w h e r e a s the h y d r o x y 1- .9 . the h y d r o x y l a t i o n of α . has been referred to a b o v e ( L a m a r e et al.180 Bioconversions ο ο F I G U R E 7-12 Drugs and drug metabolites studied by fungal bioconversion. 1987). O n e e x a m p l e . T e r p e n o i d substrates. in b i n d i n g of the substrate to the e n z y m e .

3 74 The Products of Fungal Bioconversions 181 75 \ 76 77 F I G U R E 7-13 Fungal bioconversions of some xenobiotic compounds. jS-pinene (72). B e n z y l i c positions of aromatic h y d r o c a r b o n s can be similarly susceptible to .7. ations of a .1 3 illustrate the i m p o r t a n c e of allylic activation in d e t e r m i n i n g the position of attack u p o n these substrates ( B a t t a c h a r y y a et al. and l i m o n e n e (73) by Aspergillus niger at the sites indicated in F i g u r e 7 . 1960. B a t t a c h a r y y a and G a n a p a t h y 1965).p i n e n e (71).

without providing activation for h y d r o x y l a t i o n ( F o n k e n et al. followed by s p o n t a n e o u s hydrolysis of the resulting hemi-acetal (Holland et al.1 3 . 1988). T h e opportunities offered by m o l e c u l a r genetics for e n z y m e p r o d u c t i o n via c l o n i n g t e c h n i q u e s . 1987).m e t h o x y c a t e c h o l (78) by Aspergillus repens s h o w n in Figure 7 . but are also k n o w n for fungal b i o c o n v e r s i o n s . T h e s e reactions c o m m o n l y o c c u r with bacterial s p e c i e s . M c M i l lan et al. it is still possible to find n e w applications for k n o w n reactions. 1987). 7. 1987). 1989).or r e g i o c h e m i s t r y of reaction M. In addition to the e x a m p l e s discussed a b o v e involving fungal b i o c o n v e r s i o n of n a p h t h a l e n e and the p o l y c y c l i c substrate 6 9 by Cunninghamella species (Cerniglia et al. In addition. 1989c). H y d r o x y l a t i o n of aromatic h y d r o c a r b o n s can also o c c u r by attack on the a r o m a t i c r i n g . for e x a m p l e . biphenyl is c o n v e r t e d to the phenol 7 7 by Aspergillus parasiticus ( C o x and G o l b e c k 1985). other e x a m p l e s such as the h y d r o x y l a t i o n of phenyl c y c l o h e x a n e (76) by Rhizopus arrhizus at the site s h o w n in Figure 7 .182 Bioconversions h y d r o x y l a t i o n : Mortierella isabellina. and formylation of s e c o n d a r y a m i n o g r o u p s (Holland et al.1 4 . hydroxylates a series of s u b strates such as ethyl b e n z e n e (74) and the bicyclic c o m p o u n d s 7 5 (η = 0 and 1). isabellina 79 F I G U R E 7-14 Examples of recently discovered fungal bioconversions.1 4 (Doi et al. and for the selection of the stereo. almost certainly involves the k n o w n benzylic hydroxylation reaction (Holland et al. 1964). . s h o w n in Figure 7 . giving phenolic p r o d u c t s . T h e r e m o v a l of O-benzyl g r o u p s from p h e n o l ethers such as 7 9 by Mortierella isabellina. H o w e v e r . 1983. the fungal d e h y d r o g e n a t i o n of steroids at c a r b o n s 6 and 7 (Smith et al. F i g u r e 7 .4 THE FUTURE OF FUNGAL BIOCONVERSIONS N e w fungal b i o c o n v e r s i o n reactions are still being discovered: recent e x a m p l e s include the regioselective O-methylation of 3 .1 3 are k n o w n in which the aromatic ring apparently acts only as a b i n d i n g site for the e n z y m e . 1988). at the benzylic carbon (Holland et al.

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R o n a l d ( 1 9 8 9 ) . viral. l e u k e m i a . I m m u n e deficiencies c a u s e d by antineoplastic c h e m o t h e r a p y . R y l e y and R a t h m e l l ( 1 9 8 4 ) . effective. a n d . f a r m e r s . G o o t z ( 1 9 9 0 ) . S h e p h a r d ( 1 9 8 7 ) . ( 1 9 8 1 ) . A l t h o u g h the m a i n e m p h a s i s will b e on h u m a n t h e r a p e u t i c s . Cathy S. w h e r e a s fungal infections of plants represent significant losses of agricultural p r o d u c t s . and fungal infections result in the greatest mortality ( M o s k o w i t z et al. and K u h n ( 1 9 8 9 ) . 1985. T h i s c h a p t e r will focus on s o m e of the strategies in the search for antifungal antibiotics as well as n e w or neglected d r u g targets. and safe antifungal d r u g s are n e e d e d . R y l e y et al. Taft. o r g a n t r a n s p l a n t s . congenital defects. of latest c o n c e r n . p o t e n t .CHAPTER! 8 Screening for Antifungal Drugs Claude P. all r e n d e r an i m m u n o c o m p r o m i s e d host susceptible to a large n u m b e r and variety of n e o p l a s t i c . Of t h e s e . bacterial. O m u r a ( 1 9 8 6 ) . plant antifungal c o m p o u n d s and screening m e t h o d s will b e d i s c u s s e d as w e l l . Selitrennikoff T h e r e is general c o n s e n s u s a m o n g r e s e a r c h e r s . This was supported in part by an award from The National Science Foundation (DCB 8818914). H o d g k i n ' s d i s e a s e . R e a d e r s are also directed to a n u m b e r of excellent r e v i e w s and a recent b o o k c o n c e r n i n g various aspects of d r u g discovery and screening: F r o m t l i n g ( 1 9 8 7 ) . clinicians. p h a r m a c e u t i c a l and a g r o c h e m i c a l c o m p a n y e x e c u t i v e s . H o w to find these is yet a n o t h e r matter. viral. and patients that n e w . acquired i m m u n o d e f i c i e n c y s y n d r o m e ( A I D S ) . I m m u n o c o m p r o m i s e d patients p r o v i d e p e r h a p s the greatest c h a l l e n g e to m o d e r n health care delivery. and fungal d i s e a s e s . and Marianne Zugel for their critical comments and helpful suggestions. 189 . p r o t o z o a l . I would like to thank Alison Vigers. bacterial. H u m a n and animal fungal infections p o s e serious m e d i c a l and veterinary i s s u e s . d i a b e t e s .

Rhizomucor sp. Chromomycosis Cladosporium sp. Dei Cas and Vernes 1986. L a C a m e r a et al. Coccidioides. and Khardori 1989. Lobomycosis Loboa loboi Maduromycosis Alle scher ia boydii Madurella sp. are clearly the m o s t c o m m o n ( H o l m b e r g a n d 1 M a y e r 1986). of t h e s e . Paracoccidioidomycosis Paracoccidioides Phycomycosis Absidia sp.1 lists a few of the clinically important fungi and the disease(s) that each c a u s e s . especially disseminated systemic m y c o s e s in imm u n o d e f i c i e n t hosts ( B o d e y and Anaissie 1989). (15) Piedras Trichosporon beigelii Piedraia hortai Tinea versicolor Pityrosporum orbiculare Tinea nigra Cladosporium werneckii Candidiasis (superficial and systemic) Candida albicans. u n d e r certain c o n d i t i o n s . Y o u n g 1986. Mucor sp. 1985. D u r i n g the last three d e c a d e s there has been a d r a m a t i c increase in the frequency of fungal infections. especially albicans. p a t h o g e n i c . M y c o s e s in c o m p r o m i s e d hosts are m a i n l y the result of opportunistic infections by o r g a n i s m s that are n o r m a l l y h a r m l e s s a s y m p t o m a t i c c o m m e n s a l s (Waldorf 1986. and Aspergillus are important causative a g e n t s . Rhizopus sp. F u k a z a w a and K a g a y a 1988) but that can b e .190 Screening for Antifungal Drugs TABLE 8-1 Important Human Pathogenic Fungi and Their Diseases Disease Organism Ringworm Epidermophyton floccosum Trichophyton sp. S p e n c e r and J a c k s o n 1989). Candida s p e c i e s . Species of Candida. Histoplasma. Candidiasis has a large n u m b e r of clinical presentations r a n g i n g from c u t a n e o u s to disseminated systemic infections and includes oral 'There are approximately 140 fungi that are pathogenic to animals (Ajello 1977). Phialophora sp. (21) Microsporum sp. T a b l e 8 . F u r i o and W o r d e l l . . Candida sp Sporotrichosis Sporothrix Coccidiomycosis Coccidioides Cryptococcosis Cryptococcus Histoplasmosis Histoplasma Blastomycosis Blastomyces Aspergillosis Aspergillus Aspergillus schenckii immitis neoformans capsulatum dermatitidis fumigatus sp. brasiliensis Adapted from Shadomy and Mayhill 1982. 1985.

carnii is a very c o m m o n lung p a t h o g e n of A I D S patients) u n d e r s c o r e s the i m p o r t a n c e of fungal infections in i m m u n o c o m p r o m i s e d patients ( E d m a n et al. this m a y c h a n g e as several n e w b r o a d . W a l s h and P i z z o 1988). s e p t i c e m i a . and e n d o c a r d i tis. A m B is the m o s t used systemic antifungal agent. M e d o f f et al. and Espinel-Ingroff and Shadomy 1989. However. 2 This latter point is of little comfort to an immunodeficient patient.s p e c t r u m agents with greater efficacy and safety b e c o m e available for clinical u s e . 1983). 1990). 1 9 8 3 . m e n i n g i t i s . and S C H 3 9 3 0 ( W a l s h and P i z z o 1988. b r o n c h i t i s . 1 9 8 1 .2 Clinically Important Antifungal Drugs and Their Modes of Action Drug Polyenes Amphotericin Β Mode of Action Complex with ergosterol in fungal plasma membrane Nystatin Imidazoles and Triazoles Inhibit ergosterol biosynthesis by blocking C-14 demethylation of lanosterol Miconazole Ketoconazole Itraconazole Fluconazole Clotrimazole Terconazole Econazole 5-Fluorocytosine (flucytosine) Inhibits DNA and RNA syntheses Griseofulvin Interferes with microtubule function Terbinafine. 1988). A m B is very toxic to h u m a n cells and A m B t h e r a p y is fraught with side effects: these include renal dysfunction.Screening for Antifungal Drugs 191 t h r u s h . chills. A list of a few of the antifungal c o m p o u n d s used clinically and their k n o w n (or suspected) m o d e s of action are presented in T a b l e 8 . uveitis. new delivery systems that are less toxic are being developed (see Brajtburg et al. Bossche et al. but m u s t be administered i n t r a v e n o u s l y ) . E v e n after 2 9 years of u s e . T h e apparent m o d e of action of A m B is to c o m p l e x with m e m b r a n e sterols. S a k s e n a et al. TABLE 8 . 1987. In spite of toxicity and p r o b l e m s with 3 f o r m u l a t i o n (it is not orally a c t i v e . however. A m p h o t e r i c i n Β ( A m B . fever. a p o l y e n e ) is still the drug of c h o i c e to treat systemic fungal infections (Medoff et al. S a a g and D i s m u k e s 1988. resulting in m e m b r a n e distortion and 2 l e a k a g e of intracellular c o n t e n t s . Walsh and Pizzo 1988. B o d e y 1988. A m B is an i m m u n o s t i m u l a n t (Ryley et al. 1989). O n a m o r e positive n o t e . i t r a c o n a z o l e . A s h a s b e e n pointed out in other chapters of this v o l u m e as well as in the literature. naftifine Inhibits ergosterol biosynthesis by inhibiting squaline epoxidase From Ryley et al. fluconazole. 1981. t r e a t m e n t of h u m a n m y c o t i c infections is difficult d u e to a lack of effective antifungal antibiotics. In addition.2 . and e v e n cardiac failure. T h e recent finding that Pneumocystis carnii is likely to b e a fungus (P. H o w e v e r . 3 . a s t h m a . h y p o t e n s i o n . gastritis. for e x a m p l e .

O n e of the fundamental c o n c e p t s of antimicrobial c h e m o t h e r a p y is to inhibit a m o l e c u l a r p r o c e s s of a p a t h o g e n that is either lacking in the host or sufficiently different. resulting in the l o w e r i n g or d e s t r u c tion of v a l u e . fungal. and trees are attacked b y fungi (Brent 1984). W a t e r and field pollution with long-lived fungicides has also been reported ( O m u r a et al. 1977). R e s i s t a n c e to widely used agricultural fungicides has e m e r g e d as a significant p r o b l e m ( D e k k e r 1984). explains the o b v i o u s lag b e t w e e n the d e v e l o p m e n t of antifungal versus antibacterial c o m p o u n d s . so that host m e t a b o l i s m will b e minimally affected. Presently available plant fungicides are classified broadly into s y s t e m i c s and n o n s y s t e m i c s . A recent estimate of ~ 3 x 1 0 U S dollars per year has b e e n given for w o r l d w i d e use of plant antifungal c o m p o u n d s . and m a m m a l i a n cells are not that different. protein synthesis differences.4 . plant. 1986). D a m a g e caused by fungi is classified as acute. with the United States. e v e n t h o u g h there is an extensive list of antifungal c o m p o u n d s and formulations available to both the clinician and the farmer. M a n y h u n d r e d fungi [ 1 . for e x a m p l e . horticulturally important flowers. less t o x i c . for e x a m p l e . A list of the m o s t widely used plant fungicides and their m o d e s of action are s h o w n in T a b l e 8 . in part. in transit. arising from p a t h o g e n s p r e s e n t d u r i n g harvest or from infections acquired during transportation or storage (Brent 1984). T a b l e 8 . and are e n v i r o n m e n t a l l y safe are still n e e d e d . A large n u m b e r of differences b e t w e e n p r o k a r y o t e s and e u k a r y o t e s has b e e n exploited to o u r benefit. essentially all c r o p s . p o l y o x i n resistant strains of Alternaria ( M i s a t o et al. s h o w b r o a d e r activity. T h u s .192 Screening for Antifungal Drugs Infection of plants by fungi (either on the v i n e . T h i s dearth of information is e v e n greater c o m p a r i n g plants and fungi! . H o w e v e r . direct damage to p r o d u c e or flowers. and post-harvest damage to p r o d u c e . resistance is d u e to a single g e n e mutation that alters the p a t h o g e n target or alters transport of drug to the target. T h e s e c o m p o u n d s 9 represent significant c o m m e r c i a l p r o d u c t s . W e s t e r n E u r o p e . T h i s . often less effective fungicides. leading to severe d a m a g e or death. n e w antifungals that are m o r e effective. L a c k i n g in the arsenal of fungicides are those that attack root p a t h o g e n s and soil-borne p a t h o g e n s .3 lists a n u m b e r of the important plant p a t h o g e n i c fungi and their d i s e a s e s . All are e u k a r y o t e s and share a great deal of e n z y m a t i c and b i o c h e m i c a l m a c h i n e r y . and the F a r East h a v i n g a m a r k e t share of r o u g h l y 8 0 % ( S h e p h a r d 1987). S y s t e m i c c o m p o u n d s are transported into the plant and distributed by the v a s c u l a r system w h e r e a s n o n s y s t e m i c s r e m a i n w h e r e applied (essentially e q u i v alent to h u m a n topical antifungal c o m p o u n d s ) . R o u g h estimates r a n g e from o n e h u n d r e d billion U S dollars to five times that a m o u n t (Brent 1984). T h i s difficulty is further e x a c e r b a t e d by the surprisingly general lack of information c o n c e r n i n g the differe n c e s in b i o c h e m i s t r y of fungi and m a m m a l i a n cells. Often. chronic. or in storage) represent h u g e yearly losses in productivity and m o n e y . leading to a decrease in productivity of affected p l a n t s . Resistance has required alternate strategies for control such as c r o p rotation and the use of different. 2 8 8 ! (Dei C a s and V e r n e s 1986)] c a u s e a diversity of disease.

Ceratocystis ulmi Verticillium sp. (7) Tilletia sp. F. Botrytis cinerea Fusarium oxysporum Fusarium sp. Phytophthora infestans Endothia parasitica Septoria sp. Erysiphe graminis E. (13) Uromyces fabae U. Helminthosporium sp. Ustilago sp. Cochliobolus sp. Verticillium dahliae Sclerotium sp. 193 . Geotrichum sp. phaseoli Hemileia vastatrix Gymnosporangium sp. Venturia sp. graminis Puccinia sp. coronata P. Rhizoctonia solani Adapted from Brent 1984. Pythium sp. cichoracearum Podosphaera leucortricha Plasmopara viticola Pseudoperonospora cubensis Phytophthora sp.Screening for Antifungal Drugs TABLE 8-3 of Plants Disease Rice blast Rusts Smuts Gray mold Wilts Mildews Rots Blights Other diseases Important Fungal Diseases Organism Pyricularia oryzae Puccinia recondita P. and Shephard 1987. Gibber ella sp. (16!) Sphaerotheca pannosa Rhizopus sp. Agrios 1988. Pénicillium sp. Alternaria sp. Colletotrichum sp. caeruleum Sclerotinia sp.

1 8. a target can b e an intact fungal p a t h o g e n in vitro or in v i v o . and to be lucky. but r a n g e from 3 0 to 100 million U S dollars ( S h e p h a r d 1987. d e v e l o p m e n t . the m a i n source of antimicrobial (both antifungal and antibacterial) c o m p o u n d s has been from soil m i c r o o r g a n i s m s and m a n y screening p r o g r a m s h a v e a c o m p o n e n t that tests fermentation broths of isolated soil m i c r o b e s . to give u n a m b i g u o u s yes/no a n s w e r s . bacteria) are receiving increasing attention and s o m e of these will be discussed later in this chapter. antibacterials. T h e cost of finding and d e v e l o p i n g a n e w drug is very e x p e n s i v e : estimates vary w i d e l y . for o n c e a s a m p l e has b e e n eliminated from further testing. Zineb) Energy production. triforine) Ergosterol biosynthesis Acylalanines (Metalaxyl) RNA polymerase Adapted from Brent 1984 and Lyr and Edlich 1986. Historically. " A s discussed b e l o w . respiration Copper compounds (Bordeaux mixture) Various Mercurials Dicarboximides (Vinclozolin) Lipid peroxidation Carboximides Energy production—succinate dehydrogenase complex Benzimidazoles (Benomyl) Microtubule function Polyoxins. the basic function of a screen is to act as a filter so that a detailed evaluation of a few s a m p l e s from a m u c h larger n u m b e r m a y be carried out.1 ANTIFUNGAL SCREENS General Objectives T h e object of all antimicrobial screens is to provide a d i c h o t o m o u s a n s w e r to the question: Is there s o m e t h i n g in this s a m p l e that warrants further e x a m i n a t i o n ? S a m p l e s can b e r a n d o m c h e m i c a l s . n e w sources of m i c r o o r g a n i s m s (for e x a m p l e . and safety (toxicology) and efficacy . 8. or others. h a l o p h i l e s . H o w e v e r . w h i l e the screen can b e for antifungals. Gilbert 1987). to b e c h e a p and simple to o p e r a t e . A screen tests the effect of a c o m p o u n d (or m o r e likely. e t c . Nikkomycins Chitin synthase inhibitors Triazoles (Triadimefon. p l a n t s .194 Screening for Antifungal Drugs TABLE 8-4 Important Agricultural Fungicides and Their Modes of Action Mode of Action Fungicide Dithiocarbamates (Maneb. the selection of the material to b e tested is crucial. T h e essential e l e m e n t s of a successful screen are the ability to e x a m i n e large n u m b e r s of s a m p l e s (high t h r o u g h p u t ) . T h e correct design of a screen is important for s u c c e s s . or an nondispensible e n z y m e activity or p r o c e s s . t h e r m o p h i l e s . m a r i n e i n v e r t e b r a t e s . fermentation b r o t h s .1. This formula for success includes the right c h o i c e of target and the right source of material to be screened. . it is likely to be forever lost. T h i s includes d i s c o v e r y . plant extracts. a m i x t u r e of c o m p o u n d s containing a small a m o u n t of an " a c t i v e " c o m p o u n d ) o n a " t a r g e t . A s with the selection of targets.

These include carbohydrates.1. for e x a m p l e . 1982.8. G i v e n that e a c h of the e l e m e n t s that c o m p r i s e s an antifungal p r o g r a m is very important for s u c c e s s . e t c . 1985). 8.1 Antifungal Screens 195 tests. r e c o m b i n a n t D N A m e t h o d o l o g y a n d genetic strain e n g i n e e r i n g ( H o p w o o d et al.1. 8 7 6 a n t i b i o t i c s (de S o u z a et al. . ^ h i s has led to the anecdotal. T h e r e is the w i d e s p r e a d belief that n e w sources of materials will bring n e w d r u g s . the S t r e p t o m y c e t a c e a e — 5 4 . at least until patents h a v e b e e n filed. the a v e r a g e t i m e from discovery to m a r k e t is 7 . antifungal. The reader is directed to Nisbet and Porter (1989) and references therein for further information concerning the biosynthesis of these very important secondary metabolites. T h u s . sulfur.and oxygen-containing). 5 Natural products have been classified into nine major families by Berdy (1974). 0 0 0 natural antimicrobial c o m p o u n d s h a v e been isolated from a single family of soil m i c r o o r g a n i s m s . there h a v e b e e n e x t e n s i v e p r o g r a m s to isolate m i c r o o r g a n i s m s from exotic e n v i r o n m e n t s (de S o u z a et al.2. including interspecific a n d intergenic cell fusions. soil s a m p l e s from a n u m b e r of geographical l o c a t i o n s are b r o u g h t b a c k to the laboratory and individual m i c r o o r g a n i s m s isolated [see G o o d f e l l o w and O ' D o n n e l l ( 1 9 8 9 ) for further details c o n c e r n i n g isolation of A c t i n o m y c e t e s . pejorative observation that if a screen is published by a pharmaceutical company. heterocycles (nitrogen-. and aliphatics. it is easy to u n d e r s t a n d w h y m o s t p h a r m a c e u t i c a l and a g r o c h e m i c a l c o m p a n i e s are secretive about the details of their 4 s c r e e n s and their sources of m a t e r i a l s . or from a c o m b i n a t i o n of both (semisynthetic). a n t i m i c r o b i a l . the broths are fractionated and the active c o m p o n e n t isolated and identified [see G o o t z (1990) for sobering details c o n c e r n i n g the isolation of an active c o m p o u n d from a c o m p l e x fermentation b r o t h ] . aromatics. " P u r e c u l t u r e s " are g r o w n in liquid m e d i a and resultant fermentation broths are tested u s i n g a n u m b e r of different screens (usually by m a n y different screening g r o u p s within a c o m p a n y . 6 T y p i c a l l y . T h u s . G o o d f e l l o w and O ' D o n n e l l 1989). alicyclics. C o r r e s p o n d i n g l y .2 Sources of Materials: New Sources—New Drugs A n t i m i c r o b i a l d r u g s h a v e b e e n isolated from nature (natural p r o d u c t s ) . T h e o v e r w h e l m i n g n u m b e r of the greater than 5 . antifungal d i s c o v e r y p r o g r a m s are large c o m m i t m e n t s of t i m e and m o n e y and with l o w rates of s u c c e s s — f e w e r than o n e in 1 5 . has led to only limited success in constructing strains with novel antibiotic p r o d u c i n g capacities. amino acids and peptides. Often the cost of obtaining samples plays a decisive role in the locations. near an industrial effluent. O n c e " a c t i v e " broths are identified. Natural p r o d u c t s h a v e yielded a vast and b e w i l d e r i n g array of unusual and e x t r e m e l y interesting c h e m i c a l structures. lactones and lactams. from synthetic c h e m i c a l s . it probably does not work very well. quinones. ) .9 years (Gilbert 1987). antineoplastic. 6 Samples can either be randomly selected or selected from areas that are stressed. it s e e m s that the m o s t likely s o u r c e of n e w natural p r o d u c t s is g o i n g to be n e w sources of o r g a n i s m s (or testing old o n e s in n e w w a y s ! ) . for example.1 " N e g l e c t e d " S o u r c e s . 0 0 0 c o m p o u n d s r e a c h e s the m a r k e t p l a c e ( M e n n and H e n r i c k 1981). A l t h o u g h introduced with great p r o m i s e and e x p e c t a t i o n s . 8.

R o b e r t s and Selitrennikoff.l i k e activity has also been detected in three other m o n o c o t s and o n e dicot. suggesting that this protein represents a novel class of plant antifungal defense proteins. Figure 8 . intertidal. 1 .196 Screening for Antifungal Drugs 1982). and acidophilic bacteria. p h y t o a l e x i n s ) to proteins (for e x a m p l e . N o t e the zones of inhibition are m u c h larger w h e n z e a m a t i n is a d d e d to d i s k s . algae. 0 0 0 k n o w n plant species h a v e been e x a m i n e d for the p r e s e n c e of antimicrobial products (Balandrin et al. S o m e plant antimicrobial defense proteins h a v e potent antifungal activities (Roberts and Selitrennikoff 1985 and 1988. T h e types of o r g a n i s m s sought and the e n v i r o n m e n t s e x p l o r e d h a v e been limited only by s a m p l i n g and isolation t e c h n i q u e s . 1985. Z e a m a t i n . 2 . 1988. T h e isolation of the relevant genes for plant antifungal proteins will permit their introduction into h e t e r o l o g o u s plants (Shargool 1982. 1986. 1 . R e c e n t l y . A large n u m b e r of successful c o m p o u n d s h a v e b e e n isolated by the screening of r a n d o m synthetic c h e m i c a l s . von Wettstein 1989). chitinases. only a small fraction of the 7 5 0 . H i r a n o and N a g a o 1989).1 presents a standard agar diffusion assay in w h i c h C. T h i s observation (which is not n e w ! ) m a k e s plants a very attractive source of n e w d r u g s . H o w e v e r . p l a n t s . their c o m m e r c i a l potential has not yet been fully d e v e l o p e d . w e h a v e isolated and purified a protein from Zea mays that has potent activity against several important h u m a n and plant fungal p a t h o g e n s . T h e r e is the exciting possibility of having these proteins e x p r e s s e d in different regions of plants at specific times to c o m b a t individual p a t h o g e n s . fungi. and intertidal m a r i n e s o u r c e s . T h e s e include b e n t h i c . plant e n d o c h i t i n a s e s and g l u c a n a s e s inhibit the g r o w t h of a n u m b e r of plant and h u m a n 7 p a t h o g e n i c f u n g i (Boiler 1988. and a n i m a l s . Plants possess a variety of antimicrobial d e f e n s e s — u p to 10% of their dry m a s s ( A b e l s o n 1990). A b e l s o n 1990). m i n i n g holding p o n d s . 2 . 2 P l a n t s . T h e search for n e w sources has also e x t e n d e d to s p o n g e s . halophilic. 1988. 8 . l o w e r i n g the d o s e necessary for fungal-cell killing. 8 . including p r o t e i n s . In addition and p e r h a p s m o r e imp o r t a n t l y . and s e d i m e n t s of all types (estuary. fresh water l a k e s . a seed-localized defense protein could b e m a d e to be e x p r e s s e d in stalks d u r i n g late harvest to c o m b a t Fusarium oxysporum. H i r a n o and N a g a o 1989). M a u c h and Staehelin 1989. including C. F o r e x a m p l e . F o r e x a m p l e . . A l t h o u g h there are a large n u m b e r of plant antifungal c o m p o u n d s . ranging from small m o l e c u l e s (for e x a m p l e . 3 R a n d o m C h e m i c a l s . this protein (zeamatin) acts synergistically with a n u m b e r of k n o w n antifungal d r u g s . M a u c h et al. p e l a g i c . Flavell 1989. g l u c a n a s e s ) . albicans (Roberts et al. and m e t h a n o g e n s . s u b m i t t e d ) . S c h l u m b a u m et al. albicans w a s seeded into the m e d i u m and filter p a p e r disks containing the indicated additions placed on the surface of the m e d i u m . M o s t a g r o c h e m i c a l and o r g a n i c c h e m i c a l c o m p a n i e s h a v e extensive inventories (tens of t h o u s a n d s ) of 7 Mixtures of fungal cell-wall degrading enzymes have been shown to cure mice infected with Aspergillus fumigatus (Davies and Pope 1978). O t h e r scenarios are likely. Selected m i c r o o r g a n i s m s include (but in n o w a y limited to) t h e r m o p h i l i c . e v e n w a s t e w a t e r treatment p l a n t s — s e w a g e sludge).

1 Antifungal Screens 197 F I G U R E 8 . 17 ng.8. 170 ng. disk B. 5 ng. albicans were grown and seeded into molten-agar medium as described by Roberts et al. 170 ng per disk of nikkomycin had only a slight effect on fungal growth in the absence of zeamatin. albicans. In contrast.7 ng) in the (a) absence or (b) presence of 15 /ig/disk purified zeamatin. disk E. (1988). . 50 ng. disk C. Cultures of C. filter paper disks containing nikkomycin (disk A. This concentration of zeamatin has no effect on the growth of agar-medium cultures of C.7 ng per disk nikkomycin and 15 /xg per disk purified zeamatin. Note the sharp. clear zone of growth inhibition with 1. After the medium had solidified.1 Effect of nikkomycin with and without zeamatin on the growth of Candida albicans. 1. disk D.

followed by additional testing. and the material to be tested. ) . 8. M o r e recently. 8 Lead compounds are those that are uncovered in a screen and. A s c o m p u t e r m o d e l s continue to b e c o m e m o r e refined and useful. they will play an increasingly important role in the u n d e r s t a n d i n g of target m o l e c u l e s as well as in the design of n e w antibiotics. The modified compounds are tested to determine their efficacy and potency [see Debono et al. a purified e n z y m e p r e p a r a t i o n ) . Essentially all antibiotic discovery p r o g r a m s h a v e a m o l e c u l a r m o d e l i n g g r o u p as active and important collaborators. in turn. agar dilution. T h i s brute-force a p p r o a c h c i r c u m v e n t s a n u m b e r of difficulties e n c o u n tered with other p r o g r a m s (for e x a m p l e . the ability of c o m p u t e r s to m o d e l accurately the tertiary structure of active c o m p o u n d s has led to the u n d e r s t a n d i n g of the target structure. Often these are included in an antifungal p r o g r a m . Often a screening p r o g r a m will use both types in the initial p h a s e s .m o l e c u l a r . for e x p e r i e n c e has s h o w n that often in vitro and in vivo activity are poorly correlated. T h e r a n d o m screening of synthetic c h e m i c a l s has fallen out of favor (at least in j o u r n a l articles and reviews) and has given w a y to a m o r e rational a p p r o a c h called variously. In vitro screens include a p a t h o g e n g r o w n in liquid or solid m e d i u m or an extract of the o r g a n i s m (for e x a m p l e .4 R a t i o n a l D e s i g n . are frequently used as primary screens. T h i s . based on detailed. In vitro screens are m u c h c h e a p e r and h a v e higher t h r o u g h p u t than in vivo screens a n d . e t c . T h e c o n v e r s e also has b e e n found ( B o y l e et al. Espinel-Ingroff and S h a d o m y 1989). that is. 8.w e i g h t c o m p o u n d s that h a v e a c c u m u l a t e d as a result of synthetic p r o g r a m s of o n e sort or another. The structure is modified by the medicinal chemists to determine which parts of the molecule are important for activity—structural activity relationships (SAR). and to an understanding of the precise structures required for inhibition. H e d i n 1982. but h a v e very p o o r in v i v o activity. has been predictive for further modifications of the lead m o l e c u l e . S c h w i n n and G e i s s b u h l e r 1986). purification of the active c o m p o n e n t from a fermentation b r o t h . s o m e c o m p o u n d s h a v e excellent in vitro activity. an e n z y m e active site. rational or biorational design ( M e n n and H e n r i c k 1 9 8 1 .3 In Vitro Screens A n t i m i c r o b i a l screens are broadly divided into t w o classes: in v i v o and in vitro. Broth dilution involves adding decreasing a m o u n t s of s a m p l e to t u b e s . the infecting o r g a n i s m . flasks. (1988) for an example]. a p l a n t ) . a m o u s e . 1987. or broth dilution tests (Ryley et al. a c r u d e lysate. M o s t in vitro screens that test the sensitivity of a n u m b e r of fungi to s a m p l e s use m e t h o d s k n o w n as agar diffusion. . T h e long-term goal of such p r o g r a m s is to replace r a n d o m screening with testing of d e s i g n e d c o m p o u n d s . three-dimensional k n o w l e d g e of the targets i n v o l v e d . 1 9 8 1 . T h e s e p r o g r a m s take 8 a d v a n t a g e of a lead c o m p o u n d (often obtained by r a n d o m screening!) and i n v o l v e the derivatization and modification of the active c o m p o u n d . provide a novel structure. isolation of pure cultures of o r g a n i s m s .2. l o w .1. but often relies primarily o n serendipity in isolating an active c o m p o u n d .1. In v i v o screens utilize an intact host (for e x a m p l e . h e n c e . Espinel-Ingroff and S h a d o m y 1989). and the material to be tested. hopefully.198 Screening for Antifungal Drugs p u r e . for e x a m p l e .

A g a r diffusion tests m e a s u r e the inhibition of fungal g r o w t h by a d r u g diffusing from a point s o u r c e . and after c o o l i n g . c o m p a r e d to an untreated control. D r u g . T h e lowest a m o u n t of d r u g required to p r e v e n t g r o w t h is called the m i n i m u m inhibitory concentration ( M I C ) . flask. This t e c h n i q u e p e r m i t s the testing of several c o n c e n t r a t i o n s of a s a m p l e or o n e concentration of several s a m p l e s against o n e fungus in a s i m p l e .7 ng. Plates containing R. Note the clear zones of inhibition surrounding each disk.1 Antifungal Screens 199 or microtiter plate wells c o n t a i n i n g liquid m e d i u m with a defined fungal i n o c u l u m . rubra were prepared as described in the legend to Figure 8 . each t u b e .i m p r e g n a t e d filter p a p e r disks are c o m m o n as is boring holes in the agar m e d i u m forming wells w h i c h are filled with d r u g . 170 ng. 17 ng. plates p o u r e d . A g a r dilution tests are nearly the mirror i m a g e of agar diffusion assays in that the d r u g is seeded into the m e d i u m and fungi are inoculated on the surface of the F I G U R E 8-2 Inhibition of growth of Rhodotorula rubra by zeamatin-nikkomycin combinations. disk D. 1. s a m p l e s are p l a c e d at intervals on the surface of the solidified m e d i u m .2 is a typical e x a m p l e . disk C. disk E. F i g u r e 8 . easy to evaluate assay.c o n t a i n i n g solutions. or well is e x a m i n e d for the a m o u n t of fungal g r o w t h . After the molten-agar medium had solidified. T y p i c a l l y . After incubation for a specific t i m e . disks containing 15 μg of zeamatin and the following concentrations of nikkomycin were added: disk A. .1 . 5 ng. After a suitable period of incubat i o n . T h e use of 9 6 .w e l l microtiter plates p e r m i t s the testing of 8 s a m p l e s at 12 c o n c e n t r a t i o n s (including zero) against a single fungus at o n e t i m e . After 48 h of incubation at 37°C. plates are scored by noting the size of the z o n e of inhibition s u r r o u n d i n g e a c h disk or w e l l . disk B. the plate was photographed.8. the fungal i n o c u l u m (spores or h y p h a l fragments) is s e e d e d into m o l t e n agar (45°C) m e d i u m . 50 ng.

After suitable incubation. V a g i n a l m o d e l s are b e g u n by r e m o v i n g the ovaries of n o r m a l female m i c e (for e x a m p l e ) and administering oestradiol several w e e k s later. g u i n e a p i g s . M i c e are b e i n g increasingly used for they require less material than other animals and are c h e a p e r to m a i n t a i n . This m a k e s c o m p a r i s o n s from test to test ( c o m p a n y to c o m p a n y ) nearly i m p o s s i b l e . the ability to screen cheaply and easily large n u m b e r s of s a m p l e s against a n u m b e r of fungi m a k e s these assays very attractive and useful.1 .4 In Vivo Fungal Infection Screens 8.200 Screening for Antifungal Drugs m e d i u m . active.d i r e c t e d assays and will be discussed in Section 8 . animal m o d e l s p r o v i d e the link b e t w e e n the in vitro data and the o u t c o m e of therapy (Espinel-Ingroff and S h a d o m y 1989). 2 .1. T h i s p e r m i t s the testing of a given concentration of a drug against a n u m b e r of different fungi at o n e t i m e . L a b o r a t o r y a n i m a l s are infected with a fungal p a t h o g e n and treated with a test drug to d e t e r m i n e w h e t h e r the infection is c u r e d . these infections are only models that m i m i c the disease state. are often u s e d ) . Interlaboratory differences as great as several t h o u s a n d fold in the M I C of various d r u g s h a v e been found (Galgiani 1987). this situation only a p p o r o x i m a t e s naturally o c c u r r i n g infections and often infections are a s y m p t o m a t i c . 2 . and systemic (including respiratory). E a c h of these screens has its u n i q u e set of advantages and d i s a d v a n t a g e s [the r e a d e r is directed to R y l e y et al. In vitro inhibition screens hopefully h a v e s o m e p r e d i c tive value for the effect of d r u g s in therapeutic u s e .t a r g e t e d or e n z y m e . or not active (Ryley et al. and m o n k e y s (but not all for all m o d e l s ) . Trichophyton s p .1. Results are reported as m a x i m u m activity. topical. Alternatively. 1988. T h e level and rate of healing with drug treatment are c o m p a r e d to untreated c o n t r o l s .4. Intravaginal s a m p l e s are t a k e n at intervals and fungal cells cultured and c o u n t e d . T h u s . 1981) d e p e n d ing o n the n u m b e r of fungal cells r e m a i n i n g after treatment. d o g s . 2 .c o n t a i n i n g c r e a m s or s a m p l e s are orally administered. s a m p l e s m a y b e administered orally. and m e d i u m ( G o r d o n et al. A n i m a l s used include rats. Espinel-Ingroff and S h a d o m y 1989). H e n c e . H o w e v e r . A s w a s noted p r e v i o u s l y . R e c e n t l y . fungal g r o w t h is scored. O t h e r in vitro screens include e n z y m e .1 A n i m a l M o d e l s . S y s t e m i c m o d e l s are initiated by intravenously injecting animals with a fungal . slightly active. Infections are allowed to b e c o m e established and are then treated with topically applied d r u g . m i c e . without clinical p r e sentations. albicans is c o m m o n ) and test s a m p l e s are squirted or a d m i n i s t e r e d as a c r e a m into the vagina daily during the c o u r s e of several to m a n y d a y s . T h e r e are three general animal m o d e l s : vaginal. conditions of incubation. (1981) for details]. this has not a l w a y s been the c a s e — o f t e n only general trends or o u t c o m e s can b e predicted. Topical m o d e l s are b e g u n by scraping the back of test animals with w i r e b r u s h e s or piercing s a w s and inoculating the w o u n d e d area with a d e r m a t o p h y t i c fungus (see T a b l e 8 . A n important difficulty is that these assays h a v e not b e e n well standardized with respect to precise i n o c u l u m . 8. h o w e v e r . r a b b i t s . h o w e v e r . In spite of t h i s . thymic-deficient (nude) m i c e and irradiated animals h a v e b e e n used to m o d e l m o r e closely an i m m u n o c o m p r o m i s e d m a m m a l . A fungal i n o c u l u m is introduced into the vagina ( C . or Microsporum s p .

and application with precise a p p l i c a t o r s — c o n d i t i o n s still very different from the field. Plants to b e used are g r o w n to a certain height in pots in g r e e n h o u s e s and are sprayed with c o m p o u n d — s a m p l e s m a y also b e applied to roots b y d r e n c h i n g pots or b y s o a k i n g pots in a solution of s a m p l e . A c t i v e c o m p o u n d s are evaluated further b y field screening.1 Antifungal Screens 201 inoculum. Whether this impacts only NIH-sponsored research remains to be seen. 10 This has led to calling the entire process of random screening "spray and pray!" 1 'There has evolved a rather impressive nomenclature to describe screens. ( S h e p h a r d 1987). followed shortly by the administration of drug. A screening p r o g r a m m a y h a v e a n u m b e r of individual tests (or screens) that in toto c o m p r i s e a " p r i m a r y s c r e e n .2 P l a n t M o d e l s . for example. b y necessity. 1 . After several d a y s . plant m o d e l s only m i m i c naturally o c c u r r i n g infections for. n o rain. a s a m p l e m a y b e tested in a n u m b e r of different w a y s in vitro against a n u m b e r of p a t h o g e n s 11 and the entire battery of tests is referred to as a " p r i m a r y s c r e e n .4. high t h r o u g h p u t . Respiratory infections are modeled by administering fungal cells [for example. misting with w a t e r . e t c . After suitable incubation t i m e . 9 Animals are sacrificed at various times during the course of testing. There is a recent report of a multi-infection model that incorporates vaginal. plants are scored for the p r e s e n c e of fungal lesions and c o m p a r e d to control p l a n t s . and fungal cells either counted directiy or organs cultured and resultant fungal growth quantified. 8. those of Crpytococcus neoformans or Paracoccidioides brasiliensis (Castaneda et al. T h i s test o c c u r s in a sheltered field. multi-dimensional screening. systemic. either orally or intravenously. A n important consideration of all p r i m a r y screens is that the n u m b e r of falsen e g a t i v e s b e very s m a l l . kidneys and other organs removed. n o s u n . single technique-multiple goal screening.2 % of the initial s a m p l e s for further testing by the h i g h e r o r d e r s c r e e n s . A s is the case for animal m o d e l s .1. and will (should?) generate a n u m b e r of falsep o s i t i v e s — t h e s e are eliminated b y s u b s e q u e n t s c r e e n s .3 for a list of important fungal plant 10 p a t h o g e n s ) using a spray g u n and the infected plants incubated u n d e r g r e e n h o u s e c o n d i t i o n s . then the drug is effective. D r u g s p a s s i n g this test are evaluated u n d e r actual field c o n d i t i o n s .8. P r i m a r y screens usually identify 0 . u n d e r very controlled c o n d i t i o n s including irrigation. plants are inoculated with a fungal spore s u s p e n s i o n (see T a b l e 8 . topical. and a number of other byzantine names. for example. " F o r e x a m p l e . and respiratory infections of individual mice (Ryley and M c G r e g o r 1988). n o w i n d . " Higher 9 order screens are d e s i g n e d to further "filter" c o m p o u n d s b y b e i n g m o r e Recent NIH guidelines state that animal death may not be used as an endpoint. if the test animals survive. 8. while controls die after a specified period of time.5 Primary and Higher Order Screens S c r e e n s are also divided c o n c e p t u a l l y and in practice into primary and higher order ( s e c o n d a r y . e t c . infection and evaluation of the test o c c u r u n d e r very controlled c o n d i t i o n s — g r e e n h o u s e .1. constant t e m p e r a t u r e . a w h o l e o r g a n i s m ) . .b a s e d targets (for e x a m p l e . 1987)] intranasally. ) . P r i m a r y screens are the first set of "filters" against w h i c h c o m p o u n d s will be tested and typically h a v e b r o a d . D r u g s are rated o n a scale of 0 to 4 ( S h e p h a r d 1987). single techniquesingle goal screening.

and g r e e n h o u s e and field staff. microbial ecologists and t a x o n o m i s t s . m o r e laborious assays such as m i c r o s c o p i c e x a m i n a t i o n (see the following section) or e n z y m e . attorneys (patent). E a c h of the c o m p o n e n t s of the m i x t u r e m a y be retested with the p r i m a r y screen or tested against higher order s c r e e n s . and III) and finally. A c o m p o u n d " s u r v i v i n g " in vivo or field screening will b e subjected to further evaluation u n d e r actual field conditions (rather than the m o r e controlled conditions of field s c r e e n i n g ) . the effect of s a m p l e s on an intact test p a t h o g e n is d e t e r m i n e d . c h e m i s t s (separating active m i x t u r e s . toxicologists (in vivo testing).C e l l T a r g e t s . what constitutes a target is of p a r a m o u n t i m p o r t a n c e . In a whole-cell-target s c r e e n .2 8. and to acute and chronic t o x i c o l o g y (for h u m a n or animal u s e ) . 8.2. isolation of large quantities of active material for further testing). After additional testing (which is likely to involve several p r i m a r y s c r e e n s .6 Screening Programs O n c e a s a m p l e has b e e n identified as " a c t i v e " by a p r i m a r y screen.1 W h o l e .2 Types of Targets 8. F o r e x a m p l e . to the m a r k e t p l a c e . F o r e x a m p l e . and its c o m p a r i s o n with k n o w n c o m p o u n d s ) . T h e n .2. m o r e likely b o t h . as the kinds of c o m p o u n d s isolated will be directly related to the target of the screen. and finally.2. a fermentation broth.1.2. for e x a m p l e . E a c h additional set of screens will attempt to d e t e r m i n e the s p e c t r u m of activity of a s a m p l e .t a r g e t e d a s s a y s . structure d e t e r m i n a t i o n . the identification of the active c o m p o u n d . Targets are s o m e w h a t artificially divided into t w o general types: whole cell and enzyme-directed or targeted.202 Screening for Antifungal Drugs specific and selective. 8. a c a n d i d a t e s a m p l e will be tested in vivo (for h u m a n antifungals) or field tested (for plant antifungals). to begin to suggest the m o d e of action. T h e s e c o n d a r y screen will involve m o r e detailed testing using additional. b i o c h e m i s t s . 8.3 s h o w s the flow of an imaginary fermentation broth derived from a soil m i c r o o r g a n i s m containing an active antifungal drug through a screening p r o gram. a s a m p l e is d e t e r m i n e d to h a v e activity in a w h o l e . with luck. its effect on fungal m e t a b o l i s m . in vitro broth dilution screen (the primary screen). a screen provides a yes/no a n s w e r to the question: D o e s this s a m p l e affect a target? O b v i o u s l y . m o l e c u l a r biologists. geneticists. F i g u r e 8 . m o d ification of lead structures.1 TARGETS General Objectives A s m e n t i o n e d in the p r e c e d i n g section. Figure s h o w s a whole-cell in vitro screen using a variant of the filamentous . II.c e l l . on to clinical trials (phases I. T h u s . the c h e m i s t s will isolate the active c o m p o n e n t from p r e s u m a b l y a m i x t u r e of c o m p o u n d s . a screening p r o g r a m is an active and interactive collaboration of m i c r o biologists.

IND and Clinical Trials F I G U R E 8-3 The flow of an active sample through an antifungal screening program. See text for further explanation. Effects other than inhibition of g r o w t h can be detected using whole-cell screens. m o l d Neurospora crassa and several concentrations of an antifungal d r u g . investigational new drug. FDA. c h a n g e s in o s m o t i c sensitivity. SAR • stability « legal φ mode of action screens Φ spectrum of activity φ • MIC EVALUATION In vivo Models Acute and Chronic Toxicology To FDA. Federal Drug Administration. Cilofun12 gin. and MIC. c h a n g e s in h y p h a l m o r p h o l o g y .2 Targets 203 Pure Culture Isolation of Soil Samples Separation of Mixture Molecular Biologists Testing of Isolated Components • comparisons with known drugs structure determinations. inhibition of cell e l o n g a t i o n . structural-activity relationships. IND. h y p h a l 12 Figure 8-1 was also a whole-cell-test using C. For e x a m p l e . This figure diagrammatically depicts the flow of an active sample through an imaginary and simplified antifungal program. albicans as the test pathogen. N o t e that the n o r m a l g r o w t h of the m o l d has been inhibited by the d r u g as e v i d e n c e d b y a z o n e of inhibition a r o u n d each disk.8. SAR. minimum inhibitory concentration. .

W h e n cultures w e r e transferred to a p e r m i s s i v e t e m p e r a t u r e . rather indistinct edges.4 Inhibition of growth of Neurospora crassa by cilofungin. disk C. that is. w e d e v e l o p e d a temperature-sensitive protoplastforming strain of N. resulting in h a z y z o n e s 1 3 of inhibition (Figure 8 . disk B. h a z y .204 Screening for Antifungal Drugs F I G U R E 8 . In this c a s e . w h e n c e l l . 40 μ%). for they m u s t be microscopically o b s e r v e d .p o l y m e r synthesis w a s inhibited b y d r u g treatments or b y m u t a t i o n . particularly to detect cell-wall acting c o m p o u n d s (Gunji 1983). Edges of zones are sharp or fuzzy. formation of p s e u d o p r o t o p l a s t s . disk D. U n d e r certain conditions (37°C and osmotically supported m e d i u m ) . T h e evaluation of the screen w a s the d e t e r m i n a t i o n of the t y p e of z o n e of inhibition. each protoplast regenerated a cell wall and formed a m y c e l i u m . Detecting these latter c h a n g e s suffers from being very labor i n t e n s i v e . hyphal c u r l i n g s . The plate was photographed after 48 h of incubation at 25°C. 20 μ-g. and a b n o r m a l b r a n c h i n g (too m a n y or too few or altered shapes) h a v e b e e n used in the evaluation of whole-cell s c r e e n s . 1981). crassa (a filamentous m o l d ) called os-l (Selitrennikoff et al. this strain g r e w as a p o p u l a t i o n of cells without cell walls (protoplasts). or n o n e . . Cultures of N. 10 /ig. modified b y K i r s c h and Lai 1986).w a l l . apical s w e l l i n g . 1 3Zones of inhibition are described as clear (no growth) or hazy (reduced or altered growth). clear. the target of this screen w a s cell-wall a s s e m b l y (any c o m p o u n d that inhibited any o n e of a n u m b e r of steps essential for wall a s s e m b l y w o u l d b e detected) using a whole-cell assay (Selitrennikoff 1 9 8 3 .5 ) . Filter paper disks containing the following concentrations of Cilofungin were added (disk A. Note that the zones of inhibition have fuzzy. cells c o n t i n u e d to g r o w and divide as p r o toplasts. e v e n at the p e r m i s s i v e t e m p e r a t u r e . H o w e v e r . A n u m b e r of years a g o . 5 μ-g. crassa strain os-l were grown and seeded into molten agar as described by Selitrennikoff (1983).

5 ^ g . Enzyme-targeted screens are in sharp contrast to whole-cell screens. disk C. for it has been reported that yeast and h y p h a l forms of the s a m e o r g a n i s m differ in their sensitivities to antifungal d r u g s ( P l e m pel et al. Enzyme-directed or enzyme-targeted screens have as their target a single e n z y m e activity that is required for pathogen growth or the establishment of pathogen growth. Paracoccidioides brasiliensis. m a n y different ways in which cell death or changes in fungal morphology can occur but m a n y fewer ways to inhibit an e n z y m e . T h e r e f o r e . and C. 1986). 40 μg). albicans (Staebell and Soil 1985. a cell-wall c a r b o h y d r a t e p o l y m e r . recognition of a susceptible leaf. Plates were prepared as described in the legend to Figure 8 ^ and filter paper disks containing the following concentrations of nikkomycin added (disk A. 10 /ig. A s a s i m p l e e x a m p l e . for example. in . M a r e s c a and K o b a y a s h i 1989)] are d i m o r p h i c . the choice of w h i c h m o r p h o l o g i cal t y p e to use in whole-cell assays (or for the starting material for in vitro enz y m e . 8 . that is. and a c o n v e n i e n t e n z y m e a s s a y .6 s h o w s the incorporation of a radioactively labeled substrate into ß .t a r g e t e d assays) is i m p o r t a n t . For example. 20 ^ g . Figure 8 . T h i s usually involves an extract derived from a test fungal o r g a n i s m . u n d e r certain conditions they can g r o w as either yeast or filamentous forms. Note that the zones of inhibition are hazy and faint. S a m p l e s are tested for their ability to inhibit an e n z y m e activity in vitro. 2 . Microscopic examination of cells within the zones of inhibition revealed cells growing without cell walls [see Selitrennikoff (1983) for photograph]. Blastomyces dermatitidis. disk B. 2 . disk D. there are m a n y .8. crassa os-1 by nikkomycin. Several h u m a n p a t h o g e n s [Histoplasma capsulatum. Often screening p r o g r a m s utilize both f o r m s . 2 Enzyme-Directed Targets.2 Targets 205 F I G U R E 8-5 Inhibition of Ν.g l u c a n .

4 μg of each sample (indicated by the different shadings in the figure). but the screens are ingeniously d e s i g n e d to test the effect of s a m p l e s on a single e n z y m e activity. and h a v e less t h r o u g h p u t capacity than whole-cell s c r e e n s . A n e x a m p l e is testing the effect of c o m p o u n d s on an essential e n z y m e activity that is secreted (or the o r g a n i s m can be genetically altered to secrete a n o r m a l l y nonsecreted e n z y m e ) . that is.206 Screening for Antifungal Drugs 80 H F I G U R E 8-6 Inhibition of (1. N o t e that s o m e s a m p l e s are quite effective w h e r e a s others d o not inhibit e n z y m e activity. A s has been pointed out by Nisbet and W e s t l e y ( 1 9 8 6 ) . N o m b e l a et al. m o r e e x p e n s i v e . ( 1 9 8 8 ) for a r e v i e w . Particulate (1. The amounts of glucan were determined and the percent inhibition compared to untreated controls calculated (Taft. Note that one compound inhibits enzyme activity by nearly 80%. T h e test o r g a n i s m is g r o w n in microtiter plates.t a r g e t e d screen based on s a m p l e s inhibiting another cell-wall p o l y m e r biosynthetic e n z y m e .t a r g e t e d screens is fuzzy for there are a n u m b e r of screens that utilize w h o l e cells. T h e use of r e c o m b i n a n t D N A m e t h o d o l o g y to .3) /3-glucan synthase activity was assayed as described by Quigley et al. whereas others have no inhibitory effect.3) j3-glucan synthase activity by samples. unpublished data). cerevisiae containing different and specific genes to screen for d r u g s that are inhibitory for the p a t h o g e n protein. Putative glucan synthase inhibitors were tested for their abilities to inhibit enzyme activity in vitro. chitin s y n t h a s e . T h e recent and exciting d e v e l o p m e n t of being able to clone p a t h o g e n g e n e s into well studied fungi such as Saccharomyces cerevisiae [see M a g e e et al. the p r e s e n c e and a b s e n c e of a n u m b e r of putative inhibitors. a severe limitation in screen d e v e l o p m e n t has b e e n the slow progress of g e n e isolation and c l o n i n g from p a t h o g e n s . has b e e n described ( A d a m s and G o o d a y 1980). and the test evaluated by the a b s e n c e of color.b a s e d screens typically are used in higher order screens for these assays tend to be m o r e labor intensive. A n in vitro e n z y m e . ( 1 9 8 9 ) ] . inhibition of e n z y m e activity. (1988) in the presence of 5. c h r o m o g e n i c substrate and s a m p l e s are a d d e d . T h e distinction b e t w e e n whole-cell screens and e n z y m e . permits using isogeneic strains of S. E n z y m e .

S o m e of these are presently b e i n g used in screening p r o c e d u r e s . are e n c a s e d in a c a r b o h y d r a t e . both filamentous and yeast f o r m s . 8.3 Fungal Biology—Source of New Targets 207 construct specific g e n o t y p e s for screens is b e c o m i n g increasingly c o m m o n — m o s t s c r e e n i n g p r o g r a m s h a v e m o l e c u l a r biologists as integral m e m b e r s .3. N e w targets are d i s c o v e r e d as basic research reveals n e w u n d e r s t a n d i n g s c o n c e r n i n g p a t h o g e n b i o l o g y and h o s t . T h e r e are a n u m b e r of actual and possible targets for antifungal d r u g s . T h e y include • • • • cell-wall a s s e m b l y chitin synthase activity and chitin synthesis g l u c a n synthase activity and g l u c a n synthesis cell-wall p o l y m e r crosslinking • • • • m e m b r a n e sterols m i c r o t u b u l e s .1 Cell Wall Assembly 14 T h e o b v i o u s difference b e t w e e n m a m m a l i a n cells and fungal c e l l s is that fungi. F o r this reason a l o n e . actin filaments. For most fungi each hypha is multinucleate and contains septae with central perforations that permit the migration of cytoplasm. 8. (1989) for a recent c o m p u t e r m o d e l of apical g r o w t h ] . and c y t o p l a s m i c m o t o r s p r o t o n currents topoisomerases. 3 ) .p a t h o g e n interactions. an extracellular m a t r i x ) ." . All h u m a n p a t h o g e n s and m o s t plant p a t h o g e n s contain chitin and ( 1 .8. Y e a s t s form d a u g h t e r cells that e x p a n d and are eventually separated from m o t h e r cells by the highly localized synthesis of a chitin-containing s e p t u m . T h e p h r a s e " n e w s c r e e n s — n e w d r u g s " typifies this v i e w . T h i s distinction is not so tidy for plant cells w h i c h also contain a cell w a l l . and fungi. F u n g a l cell walls h a v e c o m p l e x c o m p o s i t i o n s and structures. T a b l e 8 .c o n t a i n i n g cell wall (formally. a basic research p r o g r a m is (or at least should be) an integral part of a n e w d r u g d i s c o v e r y p r o g r a m .5 lists the c o m p o s i t i o n s of the extracellular matrix of p l a n t s . N o t e that chitin and g l u c a n s are absent from mammalian matrix. H o w e v e r .4 Filamentous fungi are not composed of cells in the usual sense. O t h e r s m a y b e useful for future s c r e e n s . plant cell-wall c o m p o s i t i o n is s o m e w h a t different from that of fungi. see Bartnicki-Garcia et al. H y p h a l g r o w t h in filamentous fungi is polarized such that e x t e n s i o n o c c u r s only at e a c h h y p h a l apex [Wessels ( 1 9 8 8 ) . In contrast. W a l l synthesis occurs in t w o steps: p r i m a r y or initial wall is synthesized directly at h y p h a l a p e x e s w h e r e a s s e c o n d a r y synthesis o c c u r s at r e g i o n s p r o x i m a l to each a p e x . p e o p l e .3 FUNGAL BIOLOGY—SOURCE OF NEW TARGETS T h e search for n e w screens is really a search for novel targets or n e w w a y s to attack an old target. and organelles including nuclei. yeasts contain typically a single nucleus and most resemble single "cells.

etc. Willison and Klein 1982.p l a s m a m e m b r a n e fusion activates e n z y m e activity and d e n o v o c a r b o h y d r a t e p o l y m e r synthesis b e g i n s . 3). (l. McDonald 1988. After p r o c e s s i n g . Unfortunately. /3-linked g l u c a n s in their cell walls [see Bartnicki-Garcia (1968) for a c o m p r e h e n s i v e t a x o n o m i c s c h e m e based on cell-wall c a r b o h y d r a t e c o m p o s i t i o n ] . and Mammalian Extracellular Matrix Organism Composition Fungi Chitin j8-Linked glucans ( 1 . It only r e m a i n s for clever microbiologists and m o l e c u l a r biologists to devise appropriate s c r e e n s . 1988) c o n c e r n i n g the intracellular targeting of e n z y m e s involved in cell-wall biosynthesis is that these e n z y m e s are synthesized in the e n d o p l a s m i c r e t i c u l u m on b o u n d ribosomes. it is likely that s o m e of these control e l e m e n t s are u n i q u e to fungi. and sent by vesicular transport to the G o l g i apparatus for p r o c e s s ing (for e x a m p l e . Fungal. Agrios 1988. fibronectin. Varner and Hood 1988.) Fibrous proteins (collagen. ) . Rouslahti 1988a and b.208 Screening for Antifungal Drugs TABLE 8-5 Compositions of Plant. glycosylation. V e s i c l e . elastin. San-Bias 1982. C a b i b et al. T h e current m o d e l ( W e s s e l s 1984 and 1988.6)-linked α-Linked glucans Chitosan Cellulose Mannans Galactans Proteins and peptides Lipids Plant Cellulose Hemicelluloses Callose Pectins Lignins Suberin Galactans Cutins (hydroxy fatty acid polyesters) Waxes Proteins and peptides 1 Chitin Mammalian Glycoaminoglycans (hyaluronic acid. chrondroitin sulfate. into vesicle m e m branes) and these " c e l l . laminin) Proteoglycans 'Diatoms contain chitin (Herth and Barthlott.w a i r vesicles are sent to h y p h a l a p e x e s w h e r e they fuse with the p l a s m a m e m b r a n e . even the most r u d i m e n t a r y details of cell-wall a s s e m b l y h a v e . e t c . Data from Bartnicki-Garcia 1968. B e c a u s e neither m a m m a l i a n cells nor plant cells contain certain c a r b o h y d r a t e p o l y m e r s . wall-forming e n z y m e s are p a c k a g e d into vesicles (either into the l u m e n or m o r e likely. 1979). Bartnicki-Garcia 1987. This s c h e m e is rich with u n a n s w e r e d questions c o n c e r n i n g control points and n e w targets.

w h e n chitin s y n t h a s e activity is disrupted b y m u t a t i o n or drug t r e a t m e n t . the details of the control of e n z y m e activity and the control of chitinmicrofibril a s s e m b l y into cell walls r e m a i n unexploited n e w targets. and recently C i l o f u n g i n ) . T h i s is particularly d i s a p p o i n t i n g in light of the spectacular success of cell-wall acting antibacterial c o m p o u n d s . 1 .8. 1986. is effective in a C . 15 Chitin is p o l y m e r i z e d by chitin s y n t h a s e (CS) activity using U D P . A l t h o u g h the general features of chitin synthase activity and chitin synthesis are k n o w n . 8 . M u c h basic science is still n e e d e d to be able to d e v i s e useful and effective cell wall s c r e e n s . The first pathway-specific enzyme is a ketol-isomerase that is regulated by phosphorylation-dephosphorylation (Etchebehere and da Costa Maia 1989).G l c N A c as substrate. Chitin s y n t h a s e h a s b e e n reported to b e transported to apical p l a s m a m e m b r a n e s b y transport vesicles k n o w n as " c h i t o s o m e s " ( L e a l . with little h o m o l o g y in the a m i n o . it h a s b e e n s h o w n that C S 1 is not essential for wall p o l y m e r i z a t i o n . Chitin is subsequently crosslinked to other c a r b o h y d r a t e p o l y m e r s and to p e p t i d e s and proteins (see the following section). o n e G l c N A c residue at a t i m e . Chitin s y n t h a s e is a t r a n s m e m b r a n e e n z y m e w h o s e active site for substrate h y d r o l y s i s is c y t o p l a s m i c facing. B y g e n e disruption e x p e r i m e n t s . w h e r e a s C S 2 is required ( B u l a w a et al.3 Fungal Biology—Source of New Targets 209 b e e n very slow in b e i n g elucidated. T h a t there are t w o i n d e p e n d e n t chitin s y n t h a s e e n z y m e activities has only recently been d i s c o v e r e d . R e c e n t l y . T h i s result c a m e as s o m e w h a t of a surprise b e c a u s e there existed a large litany (with apparently little basis in data!) c o n c e r n i n g the use of chitin synthase c o m p e t i t i v e substrate inhibitors 15 UDP-GlcNAc is synthesized from fructose-6-phosphate and glutamine by the Leloir pathway. S i l v e r m a n et al. 1983). 3 . it is surprising that other c o m p o u n d s h a v e not been readily f o r t h c o m i n g . Chitin synthesis is essential for n o r m a l fungal g r o w t h . 1988). other than those b a s e d on glucan s y n t h a s e and chitin s y n t h a s e activities (see the following section).M o r a l e s et al. 1988 a n d refere n c e s t h e r e i n ) . from the c y t o p l a s m i c face of the p l a s m a m e m b r a n e to the e x t r a c y t o p l a s m i c face w h e r e individual c h a i n s h y d r o g e n b o n d to form microfibrils that are incorporated into m a t u r e cell walls ( C a b i b et al. O n c e chitin s y n t h a s e is incorporated into the p l a s m a m e m b r a n e . a p o l y m e r of yV-acetylglucosamine ( G l c N A c ) . 1 C h i t i n S y n t h e t a s e Activity a n d C h i t i n S y n t h e s i s . Chitin synthesis o c c u r s by the vectorial extrusion of chitin p o l y m e r c h a i n s .t e r m i n u s d o m a i n ( S i l v e r m a n 1989). it is activated b y a protease ( U l a n e and C a b i b 1976). is w i d e l y distributed a m o n g fungi. fungal cell death results [see W e s s e l s (1988) a n d references t h e r e i n ] . albicans in v i v o m o d e l ( B e c k e r et al.t e r m i n u s portion of the p r o t e i n s . although the a m o u n t of chitin varies from trace a m o u n t s to — 6 0 % of the dry w e i g h t of the w a l l . p o l y o x i n s and n i k k o m y c i n s . W h e t h e r these vesicles contain C S 1 or C S 2 activity or even if these vesicles are biologically relevant is the subject of intense c o n t r o v e r s y . C h i t i n . . n i k k o m y c i n . A l t h o u g h there are a n u m b e r of isolated e x a m p l e s of cell-wall biosynthesis inhibitors b e i n g used as antifungals (for e x a m p l e . it has b e e n s h o w n that a c o m p e t i t i v e substrate inhibitor. C S 1 and C S 2 s h o w r e g i o n s of high h o m o l o g y in the c a r b o x y . 1988).

Taft and Selitrennikoff 1988 and references therein). s o r b o s e .Screening for Antifungal Drugs 210 16 in v i v o (in a n i m a l s ) .3)0-Glucan s y n t h a s e ( E C 2 . 17 When the level of functioning of glucan synthase is perturbed by mutation or drug treatment. 1988). 1 9 8 5 . H o w e v e r . 1 . 1990). Cilofung i n . inhibitors include Aculeacin A . E c h i n o c a n d i n B . (1. a n d d e v e l o p m e n t for all fungi in 1 w h i c h it h a s been tested. a 5 7 . including trafficking of chitin synthase to the p l a s m a m e m b r a n e a n d its proteolytic activation.k D a peptide h a s been d e t e r m i n e d to b e the substrate binding subunit (Frost et al. ( l . glucan chains h y d r o g e n b o n d spontaneously to form glucan microfibrils that are incorporated b y an u n k n o w n m e c h a n i s m into the cell wall. Plants also contain (1. 1 .b i n d i n g protein c a n activate glucan synthase activity of h o m o l o g o u s a n d h e t e r o l o g o u s fungi (Szaniszlo et al. fungal (l.G l u c a n synthase activity is essential for n o r m a l cell-wall a s s e m b l y . 1990). 3 ) / 3 . 16 Nikkomycins and polyoxins have been extensively used as agricultural fungicides in Europe and the Far East. It is not k n o w n w h e t h e r glucan synthase is transported via its o w n vesicle ( g l u c a n o s o m e ? ) o r co-transported with chitin synthase in c h i t o s o m e s . 3 ) ß . A l t h o u g h there are reports of m u t a n t s lacking glucan synthase activity (Phelps et al. Interestingly. Later. Taft a n d Selitrennikoff 1988). K a n g a n d C a b i b 1986). and Neurospora} E n z y m e activity d o e s not require a divalent metal i o n . 4 . Aspergillus. . P a p u l a c a n d i n B . glucan a n d chitin microfibrils are covalently crosslinked.3)/3-glucan synthase is an integral t r a n s m e m b r a n e protein that vectorially synthesizes glucan. T o m y k n o w l e d g e . C a l l o s e synthase ( E C 2 . t o o little is k n o w n c o n c e r n i n g plant a n d fungal glucan synthase activities to rule out completely e n z y m e activity as a viable target. a G T P . g r o w t h .3)/3-linked g l u c a n . 3 . 3 4 ) catalyzes the polymerization of g l u c o s e using U D P . H o w e v e r . 3 . called callose ( D e l m e r 1987).b i n d i n g protein s e e m s to play an important regulatory role a n d c a n b e dissociated from " c o r e " e n z y m e activity. neither the e n z y m e nor the relevant g e n e has been isolated. does not u s e a lipid-linked i n t e r m e d i a t e . 1 . including Candida. a n d g l u c o n o l a c t o n e (Quigley a n d Selitrennikoff 1984. O n c e external to the p l a s m a m e m b r a n e . T h i s w o u l d b e very interesting a n d useful. o n e glucosyl residue at a t i m e (Jabri et al. T h e r e is current c o n s e n s u s that the targeting of glucan synthase from the e n d o p l a s m i c reticulum to h y p h a l apexes is similar to that of chitin s y n t h a s e . there h a v e not been c o m p r e h e n s i v e studies c o m p a r i n g plant a n d fungal glucan synthase activities to d e t e r m i n e their similarities a n d differences.3)/3-linked linear glucan. not activated b y a protease (see Q u i g l e y et al. 1989). abnormal growth results (Mishra 1977. that i s . This result should e n c o u r a g e n e w research c o n c e r n i n g chitin synthase activity p e r se as well as the regulation of e n z y m e activity. 3 4 ) has been well characterized a n d m o s t recently.G l u c a n S y n t h a s e Activity a n d G l u c a n S y n t h e s i s . the G T P . and activity is not z y m o g e n i c . A s w a s found for chitin s y n t h a s e . /3-linked d i s accharides are activators.g l c as substrate into a (l. 8 . T h e observation that plants contain an e n z y m e activity similar to that of fungi m a k e s glucan synthase a priori a less-than-optimal target for plant antifungal c o m p o u n d s . 2 ( l .

disk C. fumigatus!). E c h i n o c a n din B) h a v e b e e n k n o w n for s o m e t i m e . Note the clear zones of inhibition surrounding each disk [previously reported by Selitrennikoff (1983)]. crassa os-1 were prepared as described in the legend to Figure 8-4. not the least of w h i c h is definitive proof that these c o m p o u n d s act directly on the g l u c a n s y n t h a s e p o l y p e p t i d e rather than on the adjacent m e m b r a n e . F I G U R E 8-7 Papulacandin Β causes lysis of N. Plates were photographed after 48 h of incubation at 25°C. \0 μg. Taft and Selitrennikoff 1988)] suggests that this c o m p o u n d inhibits fungal g r o w t h b y perturbation of the p l a s m a m e m b r a n e . leading to cell d e a t h directly. and w h e t h e r the inhibitors can be c o m m e r c i a l l y useful. 20 μ%. It will be very interesting to d e t e r m i n e the nature of the inhibitors (small m o l e c u l e s . they suffer from a n u m b e r of flaws.7 .3 Fungal Biology—Source of New Targets 211 A l t h o u g h g l u c a n s y n t h a s e inhibitors (for e x a m p l e . disk B. A l t h o u g h the inhibitors h a v e not been isolated and c h a r a c t e r i z e d . T h i s reaffirms that g l u c a n synthase and g l u c a n synthesis are viable targets for antifungal d r u g s . p r o t e i n s ) . Plates containing N. note clear z o n e s on inhibition (Selitrennikoff 1 9 8 3 . P a p u l a c a n d i n B .8. T h e r e is a recent report of the identification of naturally o c c u r r i n g inhibitors of chitin a n d g l u c a n synthase activities from several relatively o b s c u r e fungi that are naturally c a p a b l e (that is. . disk D. Filter paper disks containing the following concentrations of Papulacandin Β were added: disk A. n o n m u t a n t s ) of g r o w t h as protoplasts ( B e a u v a i s and L a t g e 1989). their m e c h a n i s m of action. it is very e n c o u r a g i n g that an E c h i n o c a n d i n Β derivative [ C i l o f u n g i n — a glucan synthase inhibitor (Taft and Selitrennikoff 1988)] is currently in clinical trials with apparent s u c c e s s . 5 μg. 40 μg. T h e o b s e r v a t i o n that P a p u l a c a n d i n c a u s e s lysis of os-1 cells [Figure 8 . E v e n with the a b o v e c a v e a t s . they a p p e a r to h a v e the very interesting property of being able to inhibit h e t e r o l o g o u s s y n t h a s e s (even those of A. crassa os-1 cells.

8. p o l y e n e s . (1983) and G a l e (1984) for further details]. Thus.212 Screening for Antifungal Drugs 8 . T h i s lack of k n o w l e d g e m a k e s p o l y m e r . Antifungal azols and other c o m p o u n d s (for e x a m p l e . search for n e w derivatives. T h i s p r o c e s s r e m a i n s neglected as a possible target for antifungals. to proteins and p e p t i d e s . as treatm e n t s with d r u g s that c o m p e t e for interchain h y d r o g e n b o n d i n g result in a b n o r m a l g r o w t h and m o r p h o l o g y (Elorza et al. This is n o d o u b t the c a u s e of the intense. O v e r a l l . each h o m o p o l y m e r forms h y d r o g e n b o n d s with h o m o l o g o u s chains (Herth 1980. and to other p o l y s a c c h a r i d e s ( W e s s e l s a n d S i e t s m a 1 9 8 1 . ally lamines) inhibit e n z y m e s of the ergosterol biosynthetic p a t h w a y — f o r e x a m p l e . for e x a m p l e .3.C a r b o h y d r a t e P o l y m e r C r o s s l i n k i n g . C-14 d e m e t h y l a s e (see Table 8-2).W a l l . A l m o s t all p h a r m a c e u t i c a l c o m p a n i e s h a v e at least o n e p r o g r a m that involves the search for n e w sterol biosynthesis inhibitors. M a m m a l i a n cells contain cholesterol w h e r e a s fungi contain ergosterol. a z o l e s .p o l y m e r crosslinking attractive n e w areas of research. chitin and glucan chains are covalently c r o s s l i n k e d to e a c h o t h e r . squalene e p o x i d a s e . almost frantic. or take a d v a n t a g e of the difference in b i o s y n t h e s i s . R o n c e r o and D u r a n 1985).2 Membrane Sterols All eukaryotic p l a s m a m e m b r a n e s contain sterols w h i c h aid in regulating m e m b r a n e fluidity. 1982). O n e cursory glance at the literature will affirm that the c h e m i s t s h a v e b e e n very successful! H o w e v e r . This p r e s u m a b l y adds stability to the cell wall and m a y participate in the final m o r p h o g e n i c p r o c e s s . W h e t h e r these reactions are e n z y m e catalyzed or o c c u r spontaneously is not k n o w n . that is. 1988). weakening of the cell wall by the inhibition of chitin or glucan synthase activities or by perturbing the plasma membrane results in cell lysis and death. 1983. these c o m p o u n d s h a v e been e n o r m o u s l y clinically and financially successful. nor is it k n o w n w h e t h e r these reactions are essential for n o r m a l g r o w t h and d e v e l o p m e n t . leading to the notion that e v e r y o n e has their favorite azol. 3 . P o l y e n e antibiotics h a v e been reported to bind directly with ergosterol in fungal 18 m e m b r a n e . T h e formation of fibrils by h y d r o g e n b o n d i n g is essential for n o r m a l cell-wall a s s e m b l y and g r o w t h . leading to m e m b r a n e distortion and l e a k a g e . 1 . s o m e w o r k e r s believe that this area of research is quickly reaching saturation. After chitin and glucan c h a i n s (cellulose as well) are vectorially synthesized to the e x t r a c y t o p l a s m i c face of the p l a s m a m e m b r a n e . Surarit et al. Roberts et al. . for e x a m p l e . D u r i n g cell-wall a s s e m b l y . T h e release of fluconazole and itraconazole should p r o v i d e n e w data c o n c e r n i n g the c o n t i n u e d efficacy of these c o m p o u n d s . resulting in cell death [see Medoff et al. T h i s fortuitous difference has b e e n the focus of intensive study resulting in a very large n u m b e r of antifungals that exploit the difference directly. are not e n z y m e catalyzed. 3 C e l l . A p p a r e n t l y these o c c u r s p o n t a n e o u s l y .p r o t e i n and p o l y m e r . 18 The internal turgor pressure of fungi has been estimated to be 17 atmospheres (Wessels 1984).

S t e b b i n g s 1990). m a n c o z e b . d i n o c a p . and fungal cell cycles (including nuclear division). u n c o u p l e r s . In fungal c e l l s .3. n a b a m . there m u s t exist a c o m p o u n d that inhibits the fungal A T P a s e .5 Topoisomerase II F r o m a n u m b e r of sources it is clear that D N A t o p o l o g y plays a significant role in replication. 1984 and references therein). including griseofulvin. T h e p r o c e s s of using a proton current.3.m y o s i n ) is recent ( W a t t s et al.b a s e d m o t o r s (kinesin. T o p o i s o m e r a s e s introduce D N A . thus forming a proton gradient (inside n e g a t i v e ) . m i c r o t u bules are absent in p r o k a r y o t e s .3. and m a m m a l s s h o w c o n s i d e r a b l e a m i n o acid and D N A s e q u e n c e h o m o l o g y . 1989). Interestingly. the d i s c o v e r y of actin-based a n d m i c r o t u b u l e . 1988. 1985. J a c k s o n and H e a t h . m i n i . 1986. F u n g i c o n s u m e nearly a third of mitochondrially p r o d u c e d A T P to expel p r o t o n s from the c y t o p l a s m . resulting in g r o w t h . T h e m o l e c u l a r dissection of the S. to n e w s c r e e n s . especially that of plant p a t h o g e n s . as it d o e s in m a m m a l i a n . S u r e l y .4 Energy Generation—Mitochondria and Plasma Membrane ATPases Inhibitors of e n e r g y p r o d u c t i o n h a v e b e e n used for a very long t i m e to inhibit fungal g r o w t h . Actin.t r a n s l o c a t i n g . for e x a m p l e .8. T h e s e act to alter fungal mitochondrial function b y a n u m b e r of different m e c h a n i s m s . N a k a m o t o and + + S l a y m a n 1989). p l a s m a m e m b r a n e A T P a s e h a s b e e n studied in detail ( H e n n e s e y and S c a r b o r o u g h 1988. r e c o m b i n a t i o n . c a r b o x a m i d e s . and derivatives. h e n c e .r a t e reduction. the nuclear m e m b r a n e d o e s not dissociate and r e f o r m . It s e e m s likely these actin and m i c r o t u b u l e associated A T P a s e s represent novel targets. T h e p r o t o n . or frog cells. intracellular transport of v e s i c l e s .3 8. c y t o p l a s m i c d y n e i n . p l a n t s . 8. and e v e n c h r o m o s o m e s utilizes m i c r o t u b u l e s and actin and associated m o t o r s . V a n T u i n e n et al. but rather r e m a i n s intact t h r o u g h o u t m i t o s i s — a n " e n d o m i t o s i s " (Heath et al.3 Fungal Biology—Source of New Targets 213 Microtubules. fentin. T h e s e c o m p o u n d s include t h i r a m .and /3-tubulins of fungi. 8. for transport is u n i q u e to fungal a n d plant cells (see H a r o l d 1982). o r g a n e l l e s . Essentially n o t h i n g is k n o w n c o n c e r n i n g the m o l e c u l a r basis for these fundamental differences. a m p h i b i a n . (Harold 1986). T h e s e c o m p a r i s o n s will m o s t likely lead to n e w m o l e c u l e s and activities of interest a n d . b e n z i m i d a z o l e s . a m i n o a c i d s . and Actin-Associated Motors In all e u k a r y o t e s . H o w e v e r . A l t h o u g h the c o m p o n e n t a. and others ( K a a r s Sijpesteijn 1984). plant. Both m i c r o t u b u l e and actin n e t w o r k s are found aligned parallel to h y p h a l axes p r o v i d i n g the structural basis for m o v e m e n t of vesicles ( c h i t o s o m e s ? ) to h y p h a l tips ( R a u d a s k o s k i et al. F u n g a l n u c l e a r division is m o r p h o l o g i c a l l y different from that of plant and m a m m a l i a n cells. rather than a N a / K c u r r e n t . T h e p r o t o n gradient is then used to co-transport n e e d e d nutrients. there exist sufficient functional differences such that tubulins are targets for a n u m b e r of successful antifungals. e t c . cerevisiae cell cycle is p r o v i d i n g an increasingly interesting picture of the similarity and differences b e t w e e n m a m m a lian. Microtubule-Associated Motors. and transcription. s u g a r s .

Microbiol. identify the active c o m p o n e n t . P. 18. Α. 3. fractionate m i x t u r e s . A s has b e e n d e s c r i b e d p r e v i o u s l y . h e n c e . they will take care of your future (and you will live happily ever after)... 229-236.. (1988) J. 157. New York. and Gierz. and Bollinger. British Mycological Society Symposium # 1 3 . Wurtele. N o t a small task! It s e e m s fitting to e n d by recalling the " L a w s of A p p l i e d M i c r o b i o l o g y " as formulated by the late D a v e P e r l m a n ( 1 9 8 0 ) . s h o w that it is nontoxic and e n v i r o n m e n t a l l y safe. G. etc. Adams. 75-78. Bartnicki-Garcia. (1989) Protoplasma 1853. R e c e n t results h a v e s h o w n that a n u m b e r of cytotoxic agents (podophyllotoxin derivatives) are inhibitors of t o p o i s o m e r a s e II (Figgitt et al. (1974). Appl. 7-29. North Am. Tullock. 212-214. P. L. Bartnicki-Garcia. 3. Immunol. Academic Press. Cambridge University Press. .. Microbiol. (1985) Science 228. p r o v e it is novel (and has not been p a t e n t e d ) . J. T h e r e w a r d s and the difficulties in finding n e w c o m p o u n d s are i m m e n s e . E. d e t e r m i n e its in vitro and in v i v o p o t e n c y and efficacy. Microbiol. M. type I and type II. 152. 2. bring it to m a r k e t . d e t e r m i n e its s p e c t r u m of activity. Rev.214 Screening for Antifungal Drugs nicks on o n e strand or both strands. G. Marcus. 513. (1977) Contrib. Bodey. and Latge. F.. a n d finally. et al. and Gooday. Lett. (1968) Annu. 4. Agrios. S. J. Infect. (1989) Arch.. W. Microbiol. J.4 CONCLUDING REMARKS T h e need for i m p r o v e d antifungal drugs is chronic and soon m a y be critical. natural product discovery p r o g r a m s (as well as other p r o g r a m s ) m u s t isolate n e w o r g a n i s m s . Becker. REFERENCES Abelson. Klocke.. G. detect antifungal activities. (1987) in Evolutionary Biology of the Fungi. 46-57. G. Berdy. J. 1989). 309-406. 8. 5. c o m p e t e with established p r o d u c t s .. Balandrin. 22. Adv. Clin. 1. 1154-1160. S.. Beauvias. always right your friend a sensitive partner There are no stupid microorganisms Microorganisms can do anything will Microorganisms are smarter than chemists wiser engineers. M. C. 72. Bartnicki-Garcia. Hergert. Dis. P. S. 632-658. 87-108. (1988) Med. S. T h e r e are clear differences b e t w e e n fungal and m a m m a l i a n t o p o i s o m e r a s e s (and likely other D N A t o p o l o g y altering e n z y m e s as well) so that this e n z y m e m a y be a useful target for antifungals. (1988) Plant Pathology. The microorganism is 2. more energetic If you take care of your microbial friends. (1980) Biotechnol. J.. (1990) Science 247. Ajello.

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B y far the m o s t important contribution leading to the m o d e r n a g e of antibiotic therapy h a s b e e n the d i s c o v e r y of penicillin. Q u e e n e r 1990).1 HISTORICAL PERSPECTIVES O n e of the great a c h i e v e m e n t s of m e d i c a l science is the virtual eradication of m a n y infectious diseases through the use of selectively toxic antimicrobial d r u g s . and address the question of h o w this k n o w l e d g e might b e put to practical u s e .CHAPTER 9 Molecular Biology and Biochemistry of the ß-Lactam Antibiotics John A. B e c a u s e the m o l e c u l a r biological w o r k has b e e n fueled b y the w e a l t h of b i o c h e m i c a l detail c o n c e r n i n g synthesis of these antibiotics. w h o o b s e r v e d g r o w t h inhibition of Staphylococcus aureus b y c o n t a m i n a t i n g 221 . T h e p u r p o s e of this chapter is to m a k e this rapidly e x p a n d i n g k n o w l e d g e available to the reader. Rambosek In the past few years there has b e e n an e x p l o s i v e g r o w t h in the c l o n i n g and analysis of g e n e s involved in the biosynthesis of ß . 9. Penicillin w a s originally described b y F l e m i n g in 1929. In this c h a p t e r I will attempt to r e v i e w w h a t is k n o w n about penicillin and c e p h a l o s porin b i o s y n t h e s i s from both a m o l e c u l a r biological and b i o c h e m i c a l p e r s p e c t i v e .l a c t a m antibiotics (Miller and Ingolia 1989. A c o m p r e h e n s i v e r e v i e w of the e n z y m e s involved in penicillin and c e p h a l o s p o r i n biosynthesis has b e e n published recently (Martin and Liras 1989). the current u n d e r s t a n d i n g of the b i o c h e m i c a l p a t h w a y s and e n z y m e s will b e c o v e r e d .

such as methicillin. c a r b a p e n e m s . is different in the t w o antibiotics. the C P C n u c l e u s . T h e y then w e n t o n to p r o v e its clinical effectiveness in h u m a n trials (Chain et al. they h a v e a b r o a d e r antimicrobial s p e c t r u m and are m o r e resistant to /3-lactamases. the Pénicillium chrysogenum strain N R R L 1 9 5 1 . T h e use of semisynthetics g r e w out of the o b s e r v a t i o n by the d r u g c o m p a n y B e e c h a m that w h e n Pénicillium w a s fermented in the a b s e n c e of side chain p r e c u r s o r s .222 Molecular Biology and Biochemistry of the /3-Lactam Antibiotics colonies of Pénicillium notatum. it elicits allergic r e s p o n s e s in sensitive individuals and its antimicrobial s p e c t r u m is largely limited to the g r a m p o s i t i v e s . 1940. T h i s can m a r k e d l y alter properties of the d r u g . In the late 1930s the search for antimicrobial agents resulted in the r e d i s c o v e r y and s u b s e q u e n t purification of penicillin by a g r o u p at O x f o r d . the sulfur- c o n t a i n i n g r i n g .a m i n o c e p h a l o sporonic acid ( 7 . Early d e r i v a t i v e s . Addition of a c h a r g e d a m i n o g r o u p to the side chain in ampicillin p r o d u c e d an antibiotic that w a s considerably m o r e effective against g r a m . the original observation by F l e m i n g w a s largely forgotten. w a s d i s c o v e r e d in the 1950s and its structure d e t e r m i n e d by 1961 ( A b r a h a m and N e w t o n 1961). A n o t h e r important jS-lactam antibiotic. 1941).n e g a t i v e o r g a n i s m s while retaining m o s t of the p o t e n c y of penicillin G against gram- positives. A b r a h a m et al. w a s isolated at the N R R L laboratory in Peoria. A l t h o u g h these semisynthetic c e p h a l o s p o r i n s are less potent than the penicillins. c l a v a m s . 1 ) . 1943). T h i s success spurred further research and b y 1943 the structure of penicillin w a s solved ( A b r a h a m et al. resistance to ^ . a significant i m p r o v e m e n t o v e r the a p p r o x i m a t e l y 2 U / m l p r o d u c e d b y the P. T h i s strain p r o d u c e d about 100 U / m l of penicillin. the p r o g e n i t o r of future industrial strains. A full d e c a d e w o u l d p a s s . p e o p l e with allergies to penicillin are usually not sensitive to c e p h a l o s p o r i n s b e c a u s e the p r i m a r y i m m u n o g e n .l a c t a m a s e can be achieved by sterically hindering the e n z y m e from attacking the /3-lactam ring. it is not w i t h o u t its limitations. Illinois. T h e s e p r o b l e m s w e r e addressed with the next major a d v a n c e . T h e rather n a r r o w antimicrobial s p e c t r u m of penicillin G w a s also i m p r o v e d using s e m i s y n t h e t i c s . T h e free a m i n o g r o u p of 6 . and m o n o c y c l i c ß- l a c t a m s ( r e v i e w e d in Ο ' S u l l i v a n and Ball 1 9 8 3 ) — h o w e v e r the penicillins and c e p h a l o s p o r i n s r e m a i n the m o s t important clinically. the use of s e m i s y n t h e t i c penicillins.A P A .A P A ) . c o n s i d e r a b l y less potent than penicillin G . It is quite susceptible to /3-lactamases and c a n n o t b e taken orally as it is sensitive to low p H . rather than penicillin. w a s p r o d u c e d . Fo r e x a m p l e . In addition. before the p r o m i s e of this observation w a s realized. cephalosporin C ( C P C ) . A l t h o u g h penicillin G is a very selective and nontoxic antibiotic.A C A ) . h o w e v e r . by adding bulky side ch ai n s to 6 . After unsuccessful attempts to isolate the active c o m p o u n d . D u r i n g this p e r i o d . 6-aminopenicillanic acid ( 6 . A l t h o u g h C P C itself is only a w e a k antibiotic. 7 . In addition. . w e r e . notatum strain identified by F l e m i n g . 5 . S u b s e q u e n t semisynthetics h a v e b e e n d e v e l o p e d that are m o r e potent than methicillin and still resistant to penicillinase.A P A could be c o n d e n s e d with a variety of acyl acids to p r o d u c e penicillins with novel acyl side c h a i n s . has b e e n used to generate a variety of clinically useful semisynthetic c e p h a l o s p o r i n s . F o u r other families of /3-lactams h a v e b e e n d i s c o v e r e d — c e p h a m y c i n s . but still a long w a y from current industrial strains (see Section 9 . h o w e v e r .

a m i n o a d i p i c acid.a m i n o a d i p y l ) .2 Pathway for Synthesis of /3-Lactams 223 A l t h o u g h /3-lactams are n o w 5 0 years o l d . as well as strain i m p r o v e m e n t that has a l l o w e d large scale p r o d u c t i o n .1 Common Steps Penicillin. d i s c o v e r y . unlike fungi.L . T h e e n z y m e catalyzing this e x c h a n g e is 6 .2.a . F o r m a t i o n of this c o m p o u n d . 9.v a l i n e .2. F r o m their initial d i s c o v e r y to the s u b s e q u e n t structure e l u c i d a t i o n s . C P C .9. b i o c h e m i c a l s t u d i e s . the science of /3-lactam antibiotics has b e e n well p l a n n e d and skillfully e x e c u t e d . and L . r e s p e c t i v e l y . r e p r e s e n t i n g the w o r l d ' s m o s t frequently used antibiotics. 9. they are still very i m p o r t a n t clinical t o o l s . T h e s e form penicillin G and penicillin V . L .1 . 9. 9. and c e p h a m y c i n h a v e in c o m m o n the first t w o e n z y m a t i c s t e p s .A P A : A c y l C o A acyltransferase ( A A T ) .a m i n o a d i p y l side chain of I P N to the D .2 Penicillin Biosynthesis Penicillins are p r o d u c e d from I P N b y the action of an e n z y m e that e x c h a n g e s the α .( L . to form isopenicillin Ν ( I P N ) .2.c y s t e i n e . requires lysine a m i n o transferase ( L A T ) activity. T h e current w o r k o n the m o l e c u l a r b i o l o g y follows in these traditions.a m i n o a d i p i c acid. M a n y different a r o m a t i c acid c o e n z y m e A ( C o A ) derivatives serve as substrates in the r e a c t i o n .a . It is w o r t h noting that in b a c t e r i a . T h u s L A T m i g h t also be t h o u g h t of as forming part of the p a t h w a y in b a c t e r i a . and c e p h a m y c i n C are s h o w n in F i g u r e 9 . I P N r e p r e s e n t s the b r a n c h point in the synthesis of penicillins and c e p h a l o s p o r i n s . and will u n d o u b t e d l y p r o v i d e the next major a d v a n c e s in the history of /3-lactam antibiotics.2 PATHWAY FOR SYNTHESIS OF /3-LACTAMS T h e p a t h w a y s for the b i o s y n t h e s i s of penicillin. T h i s e n z y m e c o n v e r t s the L configuration of the α . w h i c h is r e q u i r e d for synthesis of A C V . or c y c l i z a t i o n . c e p h a l o s p o r i n .3 CPC Biosynthesis C P C and c e p h a m y c i n C b i o s y n t h e s i s p r o c e e d from isopenicillin Ν first t h r o u g h penicillin Ν via the action of isopenicillin Ν e p i m e r a s e ( I P N E ) . T h e first is formation of the tripeptide y .a m i n o a d i p y l side c h a i n of I P N for a r o m a t i c a c i d s .c y s t e i n y l . the m o s t c o m m o n b e i n g p h e n y l a c e t i c C o A and p h e n o x y a c e t i c C o A . T h i s reaction is c a t a l y z e d by A C V s y n t h e t a s e . T h e i r u s e can be attributed b o t h to the fact that they c a n b e readily p r o d u c e d in a b u n d a n c e and to the efficacy of the antibiotics. and d e s i g n of s e m i s y n t h e t i c s .D v a l i n e ( A C V ) from the a m i n o acids L . lysine b i o s y n t h e s i s d o e s not p r o c e e d t h r o u g h a p a t h w a y that utilizes α . T h e next c o m m o n reaction is ring c l o s u r e . catalyzed by isopenicillin Ν s y n t h e t a s e ( I P N S ) .

224 Molecular Biology and Biochemistry of the ß-Lactam Antibiotics .

1 9 9 0 ) .h y d r o x y position m e d i a t e d by cephamycin C synthetase. 5 .4 Cephamycin C Biosynthesis T h r e e additional e n z y m e s are r e q u i r e d for p r o d u c t i o n of c e p h a m y c i n C . chrysogenum as well as the a p p r o x i m a t e l y 9 . In Cephalosporium. T h e relatively high Km with α . and the m o l e c u l a r m a s s reported to be 2 2 0 .3 c o n f i g u r a t i o n .h y d r o x y l a t i o n by O C D A C h y d r o x y l a s e and s u b s e q u e n t c o n v e r sion to c e p h a m y c i n C by m e t h y l a t i o n at the 7 . nidulans. In Cephalosporium. or p e p t i d e s y n t h e t a s e s . et al. and s u b s e q u e n t l y h y d r o x y l a t e d to D A C by a s e c o n d ( h y d r o x y l a s e ) . D . 0 2 6 m M . 1990) and A. 1987). and valine 0 . acremonium for the three a m i n o acids are: α .α .a n d . 9.e i g h t e e n . and cephamycin.a m i n o a d i p i c acid 0 . 0 0 0 D a . A s A C V synthetases h a v e n o w b e e n c l o n e d from a variety of /3-lactam p r o d u c e r s ( S m i t h .1 ACV Synthetase A C V s y n t h e t a s e is a l a r g e . O- c a r b a m o y l .f o l d from A . D A C is c o n v e r t e d to C P C b y C P C synthetase (acetyltransferase) w h i c h utilizes acetyl C o A as the acetyl d o n o r .3 ENZYMES OF /3-LACTAM BIOSYNTHESIS 9. it will b e of interest as to w h a t relationship they h a v e to fatty acid s y n t h e t a s e s .k b A C V specific ( S m i t h . D . nidulans (MacCabe et al. 225 Strep- t w o separate e n z y m e s carry out these reactions: penicillin Ν is first e x p a n d e d to d e a c e t o x y c e p h a l o s p o r i n C ( D A O C ) by o n e e n z y m e ( e x p a n d a s e ) . cephalosporin. 9.3.N i e t o et al. D e n a t u r i n g gels s h o w a single p o l y p e p t i d e of similar m o l e c u l a r m a s s . p o l y k e t i d e s y n t h e t a s e s .D A C by transfer of a carbam o y l g r o u p to the 3 position from c a r b a m o y l p h o s p h a t e .a m i n o a d i p i c acid. Enzymes of /3-Lactam Biosynthesis penicillin N is c o n v e r t e d further to d e a c e t y l - c e p h a l o s p o r i n C ( D A C ) by a bifunctional e x p a n d a s e / h y d r o x y l a s e e n z y m e .t r a n s f e r a s e c o n v e r t s D A C to O . T h e reported size is s o m e w h a t s m a l l e r than m i g h t be e x p e c t e d b a s e d on the size of the A C V c o n t a i n i n g D N A fragments from P.9.a m i n o a d i p i c acid. multifunctional e n z y m e that catalyzes the A T P d e p e n d e n t formation of the tripeptide A C V from L . c o u p l e d with the fact F I G U R E 9-1 Biosynthetic pathways for the synthesis of penicillin. and L . 1 7 m M . nidulans (van L i e m p t et al. chrysogenum and A . L . 1990) T h e Km v a l u e s of the e n z y m e from C .a .2. 1989). T h i s h a s led to a p r o p o s e d m o d e l in w h i c h A C V s y n t h e t a s e catalyzes the sequential c o n d e n s a t i o n of the three a m i n o acids in a m a n n e r that is a n a l o g o u s to the t h i o t e m p l a t e b a s e d sequential c o n d e n s a t i o n of acetate units by fatty acid s y n t h e t a s e s . T h e e n z y m e has b e e n purified o n e h u n d r e d . T h i s c o m p o u n d is then the substrate for 7 . 1 9 8 5 ) . s u g g e s t i n g that the active e n z y m e is m o n o m e l i c .c a r b a m o y l . et al. In tomyces. S y n t h e s i s is not inhibited by protein inhibitors ( L o p e z . cysteine 0 . m e s s a g e s from P. indicating a n o n r i b o s o m a l synthesis for the tripeptide.v a l i n e . . 3 4 m M ( B a n k o et al.c y s t e i n e .

I P N S is stimulated b y ferrous ions and a s c o r b a t e . inactivation of the e n z y m e by reagents that covalently modify cysteine residues) suggested a role for sulfhydryl residues(s) in e n z y m e activity.a m i n o a d i p y l side chain of I P N with a r o m a t i c acids activated as C o A derivatives (for e x a m p l e . 9. as either the C y s . P. 1986). 1989a). T h e s e residues w e r e c h a n g e d to serine.226 Molecular Biology and Biochemistry of the /3-Lactam Antibiotics that α .3.2 5 5 residue appeared to be less critical. 9.2 IPNS I P N S catalyzes the s e c o n d reaction c o m m o n to penicillin. in at least o n e case ( H ö n l i n g e r and K u b i c e k 1989). 1988).l a c t a m ring is formed d u r i n g cyclization. 1987a). and C . T h e y m a y be residues that contact or position the substrate without being involved in the actual catalytic process.C o A for p e n G and p e n V . T h e I P N S of C . D u r i n g the reaction. 0 0 0 D a . availability of α . p h e n y l a c e t y l . T h e C y s . W h e t h e r this alteration affects the specific activity of the e n z y m e is currently not k n o w n .1 0 6 m u t a t i o n or the C y s . T h e e n z y m e is a m o n o m e r of a p p r o x i m a t e l y 4 0 . T h e C y s . 1984). I P N is the first c o m p o u n d p r o d u c e d with antibiotic activity. Streptomyces ans (Castro et al. and c e p h a m y c i n b i o s y n t h e s i s . either individually or in c o m b i n a t i o n . lactamduracremonium ( H o l l a n d e r et al.3 AAT T h e final step in penicillin biosynthesis consists of replacing the α . I P N S contains a tyrosine residue at position 1 9 8 . acremonium has been the subject of a structure/function analysis utilizing the c l o n e d I P N S g e n e and site-specific m u t a g e n e s i s ( S a m s o n et al. a p r i m a r y m e t a b o lite w h i c h m i g h t b e e x p e c t e d to be in low supply during s e c o n d a r y m e t a b o l i s m .C o A and p h e n o x y a c e t y l .2 5 5 d o u b l e m u t a n t r e d u c e d the specific activity of the e n z y m e by a b o u t 9 5 % . In the high p r o d u c i n g strain. I P N S has b e e n purified from a w i d e variety of /3-lactam p r o d u c i n g o r g a n i s m s including Streptomyces clavuligerus (Jensen et al. acremonium e n z y m e has t w o cysteine residues at positions 106 and 2 5 5 . chrysogenum ( R a m o s et al. r e s p e c t i v e l y ) .1 0 6 residue a p p e a r e d to be critical for m a x i m a l activity. A C V is cyclized by this e n z y m e in an A T P . A single a m i n o acid difference in the I P N S e n z y m e s of high penicillin p r o d u c ing and low penicillin p r o d u c i n g strains of P. C y s . four h y d r o g e n a t o m s are r e m o v e d from A C V and o n e m o l e c u l e of m o l e c u l a r o x y g e n is u s e d .i n d e p e n d e n t reaction to form I P N . T h u s the cysteine residues a p p e a r to be i m p o r t a n t . w h i c h are c o n s e r v e d a m o n g I P N S e n z y m e s . replacing an isoleucine r e s i d u e . c e p h a l o s p o r i n . 1985). chrysogenum has b e e n reported ( B a r r e d o et al.1 0 6 .3. B i o c h e m i c a l e v i d e n c e (that is. as r e p l a c e m e n t by serine r e d u c e d the specific activity by only 5 0 % . B e c a u s e the ß .a m i n o a d i p i c acid is an intermediate of lysine b i o s y n t h e s i s . for e n z y m a t i c activity.a m i n o a d i p i c acid has b e e n reported to be the limiting factor in penicillin p r o d u c t i o n . T h e exact nature of the reactions catalyzed by A A T is finally b e i n g u n d e r s t o o d in greater . and requires dithiothreitol.a m i n o a d i p i c acid could b e a limiting factor in /3-lactam b i o s y n t h e s i s . T h e C. but not essential. s u g g e s t s that availability of α . Consistent with this.

A P A a n d I P N .s t e p s w i t c h . so the possibility r e m a i n s that the e n z y m e can r e m o v e the a m i n o a d i p y l side chain from C P C and i s o C P C without carrying out the acyltransfer reaction.4 IPN Epimerase I P N represents the b r a n c h point b e t w e e n penicillin and c e p h a l o s p o r i n s y n t h e s i s . T h e f i v e . as it failed to accept C P C . h o w e v e r . clavuligerus (Usui and Y u 1989). T h e A A T purified from P. either o n e or t w o e n z y m e s could b e i n v o l v e d . cultures of P. In the latter c a s e . O n the other h a n d .a m i n o a d i p y l side c h a i n s of penicillin Ν and I P N .A P A . the e n z y m e has b e e n purified from S. consistent with a o n e . chrysogenum ( B a r r e d o et al. 1987. in addition to acyl transfer activity. 1987). and catalyzes an e q u i l i b r i u m isomerization b e t w e e n the D . chrysogenum g r o w n in the a b s e n c e of aromatic side chain precursors are reported to a c c u m u l a t e 6 . isocephalosporin C ( i s o C P C ) . 0 0 0 D a .9. the acyl transfer is reported to o c c u r with a m u c h higher specific activity than the a m i d o l y ase. T h e e p i m e r a s e from C. 1979). acremonium has b e e n reported to be quite u n s t a b l e ( K o n o m i et al.A P A as a substrate).3. or w h e t h e r the a m i n o a d i p y l side chain of I P N w a s first r e m o v e d to form 6 . I P N is c o n v e r t e d to penicillin Ν by I P N e p i m e r a s e .A P A to p e n G . 1990) c l o n e d a n d s e q u e n c e d . 1989c) and A.A P A ( K i t a n o et al. In the p a t h w a y to c e p h a l o s p o r i n and c e p h a m y c i n . suggesting a separate a m i d o l y a s e activity.A P A from I P N . and c o n s e q u e n t l y has not b e e n purified. followed b y acyltransfer of the aromatic side c h a i n . or 7 . T h e existence of an a m i d o l y a s e activity is consistent with the observations that the purified A A T prefers 6 .A C A as substrates (Alvarez et al.5 Fungal Expandase/Hydroxylase and Bacterial Expandase and Hydroxylase Penicillin Ν is c o n v e r t e d to D A O C by the action of the "ring e x p a n d i n g " or " e x p a n d a s e " e n z y m e s . W h e n the activities are assayed separately. 9. but did not d e m o n s t r a t e the c o n v e r s i o n of I P N to 6 .3 Enzymes of ß-Lactam Biosynthesis 227 detail as the e n z y m e h a s b e e n purified (Alvarez et al. nidulans ( M o n t e n e g r o et al. P r e v i o u s reports (Alvarez et al. an a m i d o l y a s e activity c a p a b l e of p r o d u c i n g 6 . H o w e v e r . these e x p e r i m e n t s w e r e not d e s i g n e d to detect a m i d o l y a s e activity. 1987) had s h o w n that the e n z y m e could convert I P N or 6 .A P A . A s cited in Q u e e n e r ( 1 9 9 0 ) .3.C o A d e r i v a t i v e s . T h e e n z y m e is m o n o m e l i c with a m o l e c u l a r m a s s of a p p r o x i m a t e l y 5 0 . preferring 6 . In any e v e n t . A A T is capable of using I P N directly for acyl transfer (although it d o e s so inefficiently.m e m b e r e d ring of penicillin Ν is oxidatively . A point of uncertainty had b e e n w h e t h e r a single e n z y m e catalyzed a direct e x c h a n g e b e t w e e n the I P N a m i n o a d i p y l side chain and the a r o m a t i c a c i d . Q u e e n e r 1990) and the g e n e s from P. 1975). W h i t e m a n and A b r a h a m h a v e n o w s h o w n that A A T p o s s e s s e s . 9. the e v i d e n c e n o w supports a reaction that is carried out in t w o steps b y a single e n z y m e .and L-configurations of the a . chrysogenum is reported to b e quite specific for 6 .A P A to I P N as a substrate for acyl transfer.

a . 9. T h e first step is likely to be addition of a c a r b a m o y l g r o u p from c a r b a m o y l p h o s p h a t e to the h y d r o x y m e t h y l of D A C to form O . 5 0 0 D a ( K o v a c e v i c et al. Both the fungal (Kupka et al. acremonium.6 Acetyltransferase T h e last step in the biosynthesis of C P C is the addition of an acetyl g r o u p from a c e t y l . 1985). T h i s reaction is catalyzed by O-carbamoyltransferase. 1982) expandases show a high degree of substrate specificity. these reactions are carried out b y separate e n z y m e s (Jensen et al. w h e r e a s the bacterial exp a n d a s e is 21 a m i n o acids smaller with a m o l e c u l a r m a s s of a p p r o x i m a t e l y 3 4 . OCDAC-Hydroxylase.a d e n o s y l m e t h i o n i n e as the methyl d o n o r . They will expand penicillin N .h y d r o x y . 9.3. h o w e v e r . and c e p h a m y c i n C s y n t h e t a s e .3. 1983).4 MOLECULAR BIOLOGY OF PENICILLIN AND CEPHALOSPORIN PRODUCING FUNGI T h i s discussion will focus primarily on the m o l e c u l a r biology of /3-lactam p r o d u c tion in P.h y d r o x y l a s e ( O ' S u l l i v a n and A b r a h a m 1980). chrysogenum and C. T h e fungal e x p a n d a s e / h y d r o x y l a s e has a m o l e c u l a r m a s s of 3 6 . T h e e n z y m e has not b e e n purified to h o m o g e n e i t y . as these are the o r g a n i s m s that are used . expandase functions on adipyl-6-APA as well as Att-carboxyphenylacetyl-6-APA (Baldwin 1988).228 Molecular Biology and Biochemistry of the /3-Lactam Antibiotics e x p a n d e d to the s i x . T h e specificity is not absolute.m e m b e r e d cephalosporin ring. however. In fungi. T h e fungal e x p a n d a s e requires A T P ( K u p k a et al. T h i s reaction is catalyzed by an acetyltransferase k n o w n as C P C synthetase. penicillin G. T h e s e e n z y m e s are not as well characterized as m o s t of the e n z y m e s in the fungal p a t h w a y s . In bacteria such as Streptomyces.c a r b a m o y l D A C ( O C D A C ) . but not isopenicillin N . the activity has been d e m o n s t r a t e d to b e present in cell-free extracts of wildtype C . 1987b). 5 9 9 D a ( S a m s o n et al. a bifunctional e x p a n d a s e / h y d r o x y l a s e e n z y m e both e x p a n d s penicillin Ν to D A O C and h y d r o x l a t e s it to D A C . unlike O C D A C . catalyzes the formation of c e p h a m y c i n C b y methylation at the 7-hydroxyl. T h e hydroxylase also shows a high degree of substrate specificity in that it will not tolerate side chains other than the D-a-aminoadipyl side chain of D A O C (Queener and Neuss 1982). in contrast to the bacterial e n z y m e . D A C is not a substrate for O C D A C . O C D A C is c o n v e r t e d to 7 . acremonium and absent in m u t a n t s that a c c u m u l a t e D A C (Fujisawa and K a n z a k i 1975).7 Synthesis of Cephamycin C: 3-Hydroxymethyl-3-emOCa r ba m oy I transferase. or 6-ΑΡΑ. using S . 1983) and the bacterial (Jensen et al.O C D A C b y O C D A C h y d r o x y l a s e . 1989). T h i s reaction p r o b a b l y occurs first b e c a u s e . Thus the enzymes recognize not only the /3-lactam nucleus but also are highly selective with respect to the side chain.C o A to the n e w l y formed h y d r o x y m e t h y l at position 3 . 9. Methyltransferase (Cephamycin C Synthetase) T h r e e additional e n z y m a t i c steps are required for streptomycetes to convert D A C to c e p h a m y c i n C .

h o m o l o g o u s integration also o c c u r s . 1987. T h i s is true e v e n for m u l t i c o p y t a n d e m insertions. 1987a. In addition. nidulans will also b e m e n t i o n e d . o l i g o m y c i n . Skatrud et al. G e n e s h a v e b e e n c l o n e d primarily by " r e v e r s e g e n e t i c s " (that is. albeit at a l o w e r frequency. c e p h a l o s p o r i n . D r a m a t i c i m p r o v e m e n t in the transformation frequency w a s later o b t a i n e d by u s i n g the h o m o l o g o u s I P N S p r o m o t e r to drive expression of the h y g r o m y c i n g e n e (Skatrud et al. either singly or as t a n d e m . w h e r e instability d u e to h o m o l o g o u s r e c o m b i n a t i o n m i g h t h a v e b e e n predicted. 9. e x o g e n o u s D N A u p t a k e is induced b y C a C l 2 and p o l y e t h y l e n e g l y c o l . 1979. p h l e o m y c i n . primarily b e c a u s e of the high d e g r e e of relatedness of the p a t h w a y s and g e n e s .1 Transformation Systems D e v e l o p i n g a transformation s y s t e m is key to m o l e c u l a r biological m a n i p u l a t i o n s of an o r g a n i s m .4. and c e p h a m y c i n h a v e b e e n c l o n e d . T r a n s f o r m a t i o n s y s t e m s d e v e l o p e d for P. acremonium ( Q u e e n e r et al. Selectable m a r k e r s used for P . chrysogenum has b e e n reported by a n u m b e r of different g r o u p s (Beri and T u r n e r 1987.4 Molecular Biology of Penicillin and Cephalosporin Producing Fungi 229 industrially for antibiotic p r o d u c t i o n . acremonium are quite similar to those for yeast ( H i n n e n et al. 1984). the s a m e rules that h a v e b e e n established for other fungal transform a t i o n s y s t e m s apply to both P. 1984) used h y g r o m y c i n as the selectable m a r k e r . T r a n s f o r m a t i o n of P.s p e c i e s h y b r i d i z a t i o n . Bull et al. T h e s e q u e n c e c o n s e r v a t i o n . w h i c h w a s the first g e n e cloned and p r o v i d e d the strategies for c l o n i n g the other g e n e s . chrysogenum and C. 9. O n c e c l o n e d .4. cell walls are r e m o v e d e n z y m a t i c a l l y in the p r e s e n c e of o s m o t i c s u p p o r t . chrysogenum and C. W o r k d o n e with Streptomyces and A. In all c a s e s . K i n s e y and R a m b o s e k 1984. representatives of all the g e n e s in the biosynthetic p a t h w a y s of penicillin. In g e n e r a l . T h e first reported transformation s y s t e m for C. w e h a v e used the I P N S p r o m o t e r and the p h l e o m y c i n m a r k e r to transform C . T h e transform a n t s are mitotically quite stable. has led to the h y p o t h e s i s that they w e r e originally transferred . Yelton et al. e v e n in the a b s e n c e of selective p r e s s u r e . chrysogenum include a c e t a m i d a s e . as well as the g e n o m i c o r g a n i z a t i o n of the g e n e s . 1987b). acremonium (unpublished). At P a n l a b s . and multiple i n d e p e n d e n t insertions are also o b s e r v e d . W i t h the e x c e p t i o n of the g e n e e n c o d i n g C P C synthetase (acetyl transferase). T h e g e n e s are described in the order of the biosynthetic s t e p s . trpC and p y r 4 ) has b e e n used successfully. 1978) and other filamentous fungi ( C a s e et al. the g e n e s h a v e in m a n y cases b e e n used as c r o s s species hybridization p r o b e s to clone c o g n a t e g e n e s . Cantoral et al. with the e x c e p t i o n of I P N S .9. m u l t i c o p y insertions. c o m p l e m e n t a t i o n of a u x o t r o p h i c m a r k ers (for e x a m p l e . and b e n o m y l .2 Gene Cloning T h e c l o n i n g of g e n e s in the c e p h a l o s p o r i n and penicillin biosynthetic p a t h w a y s is d i s c u s s e d in this section. w h i c h a l l o w e d c r o s s . 1988). and selective p r e s s u r e applied to allow o u t g r o w t h of transformants. acremonium: transformation o c c u r s primarily as a c o n s e q u e n c e of n o n h o m o l o g o u s integration. using a m i n o acid s e q u e n c e to design o l i g o n u c l e otide p r o b e s ) .

T h a t the clones e n c o d e I P N S has been conclusively p r o v e n b y e x p r e s s i n g the c l o n e d g e n e in E. 1986). 4 . clavuligerus. 9. A. A l t h o u g h the A C V synthetase has n o w been cloned from Flavobacterium. T r a n s f o r m a n t s w e r e s h o w n to h a v e A C V synthetase activity and to p r o d u c e penicillin. chrysogenum. u n p u b l i s h e d ) .230 Molecular Biology and Biochemistry of the ^-Lactam Antibiotics from p r o k a r y o t e s to fungi. little is yet k n o w n of its s e q u e n c e or structure. 1989c) and A. 5 .t e r m i n a l a m i n o acid s e q u e n c e of the isolated protein. 1987). D . lipmanii ( W e i g e l et al. 1988). B a r r e d o et al. 1988). This high d e g r e e of s e q u e n c e similarity p r o v i d e d the first indication of a c o m m o n ancestral g e n e . chrysogenum c o s m i d clones c o n t a i n i n g I P N S and A A T plus the putative A C V synthetase. and at the a m i n o acid level the percent identity ranges from 5 4 % to 7 9 % (Miller and Ingolia 1989). P a n l a b s . S. cross-hybridization of b a n d s k n o w n to not contain I P N S w e r e inferred to contain A C V synthetase c o d i n g s e q u e n c e s . A. nidulans ( S m i t h . . T h e genes and the evolution of the p a t h w a y are discussed in the following p a r a g r a p h s .4. chrysogenum (Carr et al. 1988. are highly h o m o l o g o u s . T h e different I P N S g e n e s . At the D N A level. 1990a) and C. T h e g e n e e n c o d e s an a p p r o x i m a t e l y 9 . T h e C. nidulans ( M o n t e n e g r o et al. nidulans ( R a m o n et al. chrysogenum. and S. sharing 8 1 % identity at the a m i n o acid level. T h e g e n e from S. chrysogenum A A T m u t a n t . and the m i n i m u m size D N A fragment required for transfer of A C V synthetase by transformation is consistent with the transcript size. 4 . Flavobacterium produces c e p h a m y c i n .k b transcript. 2 .l a c t a m biosynthetic p a t h w a y to b e cloned ( S a m s o n et al. 1989. T h e cloned s e q u e n c e s w e r e s h o w n to e n c o d e A A T by c o m p l e m e n t i n g a P. the percent identity ranges from 6 2 % to 8 0 % . and therefore has only A C V synthetase and I P N S in c o m m o n with P. 1990. D . 9 . acremonium using o l i g o n u c l e o t i d e p r o b e s b a s e d on the a m i n o . T h e t w o g e n e s are very closely related. It w a s cloned from C. acremonium (Skatrud et al. acremonium I P N S g e n e has been used as a p r o b e to c l o n e I P N S from P. 1990b). et al. chrysogenum ( V e e n s t r a et al. P. 1990) h a v e been cloned and s e q u e n c e d . p r o v i n g the location of the A C V synthetase g e n e . chrysogenum ( B u r n h a m et al. et al. 9 . 2 . as well as the e n z y m e s they c o d e for. 1985). T h i s w a s s u b sequently p r o v e n by transforming Neurospora crassa and Aspergillus niger (organisms that d o not p r o d u c e /3-lactam antibiotics) with P. 2 A C V S y n t h e t a s e A C V synthetase w a s originally cloned as a c o n s e q u e n c e of cross-hybridization e x p e r i m e n t s b e t w e e n Flavobacterium and P. S m i t h .2. C o n s e q u e n t l y .1 I P N S ( p c b C ) I P N S w a s the first g e n e in the ß . coli. It also provided the initial insight that cross-species hybridization could be an effective strategy for cloning the s a m e g e n e s from different o r g a n i s m s . 3 A A T ( p e n D E ) T h e acyltransferase ( A A T ) g e n e s from P. clavuligerus has also b e e n c l o n e d using oligonucleotide p r o b e s based on a m i n o acid s e q u e n c e ( L e s k i w et al.

clavuligerus e p i m e r a s e g e n e as a hybridization p r o b e . chrysogenum transformants w o u l d indicate linkage of the t w o g e n e s . coli. chrysogenum e n z y m e are p r o c e s s e d at the identical position. T h e a m i n o t e r m i n u s of the small subunit (11 k D a ) corres p o n d s to the b e g i n n i n g of the g e n e . If the e p i m e r a s e and e x p a n d a s e / h y d r o x y l a s e are linked and e x p r e s s e d in P. 1990). the e p i m e r a s e from S.4. acremonium c l o n e s c o n t a i n i n g the e x p a n d a s e / h y d r o x y l a s e g e n e and flanking D N A .4 Molecular Biology of Penicillin and Cephalosporin Producing Fungi 231 D N A s e q u e n c i n g . chrysogenum e n c o d e s a protein that consists of t w o nonidentical s u b u n i t s . w h e r e a s the a m i n o t e r m i n u s of the large subunit (29 k D a ) c o r r e s p o n d s to a s e q u e n c e about 9 0 0 nucleotides into the g e n e . T h i s w o r k h a s s h o w n that the e p i m e r a s e is tightly linked to and i m m e d i a t e l y u p s t r e a m of the e x p a n d a s e g e n e . revealed that the A A T g e n e of P. T h e e n z y m e s m a y b e selfprocessing. T h u s p r o d u c t i o n of D A C b y P. O n e a p p r o a c h to cloning the fungal g e n e will u n d o u b t e d l y be to use the S. D A C should b e p r o d u c e d as a c o n s e q u e n c e of conversion of isopenicillin Ν to penicillin Ν b y e p i m e r a s e and s u b s e q u e n t ring e x p a n s i o n and h y d r o x y l a t i o n b y the e x p a n d a s e / h y d r o x y l a s e . w h i c h supports the idea that the e n z y m e is selfprocessing.a m i n o . T h i s w a s d e m o n s t r a t e d by s h o w i n g that extracts c o n v e r t e d penicillin Ν to D A O C . w a s conclusively p r o v e n b y e x p r e s s i n g the c l o n e d g e n e in E. . the e p i m e r a s e is linked to e x p a n d a s e / h y d r o x y l a s e in fungi. B e c a u s e there are n o stop c o d o n s b e t w e e n the s u b u n i t s . T h e bifunctional nature of the e n z y m e . chrysogenum and A. 1987b) by using an a m i n o acid s e q u e n c e from the purified e n z y m e (Dotzlaf a n d Y e h 1987) as a guide for synthesis of o l i g o n u c l e o t i d e p r o b e s . chrysogenum. by analogy with bacterial penicillin a m i d a s e s . D e t e r m i n a t i o n of the a m i n o t e r m i n u s of the large subunit of the A. C l o n e s expressing the g e n e contained both e x p a n d a s e a n d h y d r o x y l a s e activity.t e r m i n a l a m i n o acid s e q u e n c e . they m u s t initially b e translated as a proprotein that is subsequently p r o c e s s e d . chrysogenum with C. 9. b y a n a l o g y with the bacterial s y s t e m . A n o t h e r a p p r o a c h is to ask if. T h e g e n e e n c o d e s a 3 9 8 . acremonium ( S a m s o n et al.2.2.4 I P N E (cefD) T h e e p i m e r a s e g e n e h a s yet to b e c l o n e d from fungi. It has b e e n reported ( Q u e e n e r 1990) that a C D N A clone e n c o d i n g the e n z y m e is c a p a b l e of e x p r e s s i n g activity in E. w a s originally c l o n e d from C . W e h a v e transformed P.5 E x p a n d a s e / H y d r o x y l a s e (cefEF) T h e c e f E F g e n e . B o t h the P.9.4. e n c o d i n g the bifunctional e x p a n d a s e / h y d r o x y l a s e ( D A O C S / D A C S ) . A A T is thus the first fungal g e n e in the penicillin or c e p h a l o s p o r i n biosynthetic p a t h w a y s h o w n to contain introns. c o m b i n e d with N . w h i c h had b e e n a s s u m e d b e c a u s e of the inability to separate the t w o activities b i o c h e m i c a l l y . clavuligerus has recently b e e n c l o n e d ( K o v a c e v i c et al. and finally to D A C . coli. nidulans e n z y m e has revealed that it and the P.a c i d protein that is transcribed with e x p a n d a s e as a single transcript. 9. H o w e v e r . nidulans g e n e s contain three introns found at f identical positions g r o u p e d in the 5 half of the g e n e .

232
1)

Molecular Biology and Biochemistry of the /3-Lactam Antibiotics
QDIARISLFTLESGVILRDVPEAYKSWGRMNVSRDNCVIVCHTLTSSAHVTSWW

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3)

QRIVQVPELVLESGWINNFPIAYKTWGTLNEAGDNVLVICHALTGSADVADWW

1)

PTLFGQGRAFDTSRYFIICLNYLGSPFG

2)

GPLIGPGRAFDTSRYFIVCCNSMGSPYG

3)

GPLLGNDLAFDPSRFFIICLNSMGSPYG

a
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F I G U R E 9-2 Sequence similarities between (1) C. acremonium, (2) Ascobolus immersus,
and (3) Saccharomyces cerevisiae acetyltransferases. The A. immersus and S. cerevisiae
sequences are from homoserine acetyltransferases, and begin 34 and 31 amino acids from the
N-terminus, respectively. The C. acremonium sequence is from cephalosporin C synthetase
(acetyltransferase). The endpoints of the gene remain to be defined.
U n l i k e fungi, in Streptomyces

the e x p a n d a s e (cefE) and h y d r o x y l a s e (cefF)

activities represent different e n z y m e s that are c o d e d for by separate g e n e s (Jensen et
al. 1985). T h e e x p a n d a s e ( D A O C S ) g e n e from S. clavuligerus

has been c l o n e d and

characterized ( K o v a c e v i c et al. 1989). T h e proteins e n c o d e d by the fungal and
bacterial g e n e s are very similar ( 6 7 % identity), but the monofunctional bacterial
e n z y m e is about 2 0 a m i n o acids shorter at the c a r b o x y t e r m i n u s .

9.4.2.6

A c e t y l t r a n s f e r a s e (cefG)

W e h a v e recently cloned the g e n e c o d i n g for

the last step in C P C b i o s y n t h e s i s , the acetyltransferase (cefG). S e q u e n c i n g u p stream of the e x p a n d a s e / h y d r o x y l a s e (cefEF) revealed a long o p e n reading frame
transcribed in the opposite direction as c e f E F . T h e a m i n o acid s e q u e n c e c o d e d for
w a s searched for s e q u e n c e similarity to other p r o t e i n s , and strong identity w a s
found to regions of h o m o s e r i n e acetyltransferases of Saccharomyces
lus.

and

Ascobo-

T h e s e q u e n c e similarities are s h o w n in Figure 9 - 2 . Further u p s t r e a m

or

d o w n s t r e a m little s e q u e n c e similarity is found. T h e D N A fragment c o n t a i n i n g this
region has been s u b s e q u e n t l y s h o w n to c o m p l e m e n t cefG m u t a n t s , p r o v i n g the
identity and location on the cefG g e n e .

9.4.2.7
O t h e r G e n e s in /3-Lactam B i o s y n t h e s i s A clone c o n t a i n i n g the pathw a y for c e p h a m y c i n C synthesis has previously b e e n isolated ( C h e n et al. 1988),
but the g e n e s (that i s , cefF, c e f H , cefl, cefJ) r e m a i n to b e characterized. T h e cefl
g e n e of S. clavuligerus,
e n c o d i n g 0 - c a r b a m o y l - D A C h y d r o x y l a s e , is k n o w n to be
linked to e x p a n d a s e ( S m i t h , D . et al. 1990a).

9.4.3

Regulation of Gene Expression

A l t h o u g h m u c h has b e e n a c c o m p l i s h e d with respect to cloning and

physical

characterization of the g e n e s , less is k n o w n about their regulation. In g e n e r a l ,

9.4

Molecular Biology of Penicillin and Cephalosporin Producing Fungi

233

c o o r d i n a t i o n and regulation of g e n e expression during s e c o n d a r y m e t a b o l i s m is not
well u n d e r s t o o d . T h e c l o n e d g e n e s of /3-lactam p a t h w a y s are currently b e i n g used to
a d d r e s s these issues.
I P N S activity has been shown to be critical in regulation of C P C biosynthesis in C.
acremonium (Ramsden et al. 1989). A mutant, N - 2 , that accumulated A C V had been
previously described (Shirafuji et al. 1979). T h e mutant lacked a number of enzymatic
activities—IPNS, epimerase, and expandase/hydroxylase (Ramos et al. 1985)—yet
C P C production could be restored by introducing a functional I P N S gene (Chapman et
al. 1987). These data were consistent with IPNS activity regulating expression of
epimerase and expandase/hydroxylase, but could also be explained as a consequence of
a closely linked regulatory gene. Ramsden et al. showed that N - 2 produced an inactive
IPNS protein, and that the mutation inactivating the enzyme resulted from a single
amino acid change in a highly conserved region. The simplest interpretation of these
results is that isopenicillin N , or an active I P N S , is involved in the regulation of steps
further in the pathway. In this regard, it would be interesting to k n o w if N - 2 could
produce C P C if fed isopenicillin N .
T h e I P N S g e n e of C. acremonium
has also b e e n used in other g e n e regulation
studies ( S m i t h , A . et al. 1990). T h e s e investigators s h o w e d that I P N S specific
transcripts first a p p e a r e d with the onset of stationary p h a s e (day 2 of a 7-day
fermentation) and p e a k e d at a p p r o x i m a t e l y 4 d a y s . This correlated well with
p r o d u c t i o n of /3-lactams, and s u g g e s t e d that regulation of I P N S w a s at the transcriptional level. P r i m e r extension and S I m a p p i n g e x p e r i m e n t s indicated t w o major and
t w o m i n o r transcription start sites centered a r o u n d a p p r o x i m a t e l y - 7 0 b p with
respect to the start of the c o d i n g s e q u e n c e s . T h e relative ratios of the four transcripts
r e m a i n e d c o n s t a n t t h r o u g h o u t the fermentation, suggesting that the different transcripts did not reflect differential regulation of the g e n e . T h e transcription start site
for the P. chrysogenum
I P N S g e n e has also b e e n m a p p e d ( B a r r e d o et al. 1989a). In
contrast to the results with C. acremonium,
the start site w a s m a p p e d b y p r i m e r
e x t e n s i o n to position - 1 1 . O t h e r than a pyrimidine-rich stretch near the transcription
starts, n o other s e q u e n c e similarities are apparent.
T h e I P N S g e n e from P. chrysogenum
d o e s not s h o w c o m p a r a b l e regulation
( S m i t h et al. 1989). T h e level of I P N S transcripts in both high ( B W 1 8 9 0 ) and low
( N R R L 1 9 5 1 ) penicillin p r o d u c i n g strains a p p e a r e d to b e essentially constitutive. In
a d d i t i o n , I P N S activity w a s not strictly correlated with m e s s a g e levels: in B W 1 8 9 0 ,
I P N S activity at 14 h w a s only half that at 38 h , although the m e s s a g e levels w e r e
similar. T h i s o b s e r v a t i o n suggests the possibility of post transcriptional regulation
of I P N S in P.
chrysogenum.
In A . nidulans,
like C. acremonium,
the steady state level of I P N S specific
m e s s a g e , as well as e n z y m a t i c activity, increases as the cells enter stationary p h a s e
( P e n a l v a et al. 1989), suggesting transcriptional regulation. Translational lacZ
fusions h a v e b e e n used to define the s e q u e n c e s required for this regulation ( G o m e z P a r d o and P e n a l v a 1990). F u s i o n s w e r e created in w h i c h the first 35 nucleotides of
I P N S c o d i n g s e q u e n c e and a p p r o x i m a t e l y 2 kb of u p s t r e a m s e q u e n c e w e r e fused to
the a m i n o - t e r m i n u s of the reporter g e n e lacZ. T h e fusions w e r e introduced into A.
nidulans and t w o types of transformants w e r e e x a m i n e d for /3-galactosidase activ-

234

Molecular Biology and Biochemistry of the /3-Lactam Antibiotics

ity: o n e w h e r e the fusion had integrated at the n o r m a l I P N S locus and o n e w h e r e the
fusion had integrated at the argB l o c u s , w h i c h is on a different c h r o m o s o m e . In both
i n s t a n c e s , the s a m e t e m p o r a l expression of lacZ

from the I P N S p r o m o t e r w a s

o b s e r v e d . Activity p e a k e d at the s a m e time in both t r a n s f o r m a n t s , after the cultures
w e r e well into stationary p h a s e . T h e level of expression for the integrant at the argB
locus w a s , h o w e v e r , only about 5 0 % of that o b s e r v e d for the transformant at the
I P N S l o c u s . T h u s the 2 kb of 5 ' I P N S s e q u e n c e s contain sufficient information to
control the t e m p o r a l regulation of the I P N S p r o m o t e r , but not to p r o v i d e o p t i m a l
e x p r e s s i o n . T h e c h r o m o s o m a l context of the g e n e m a y p r o v e to b e i m p o r t a n t , or
regulatory s e q u e n c e s outside of the 2 kb of flanking D N A s e q u e n c e m a y

be

involved.

9.4.4

Evolution of the Biosynthetic Pathway

T h e o b s e r v a t i o n that the I P N S g e n e s of C. acremonium

and P. chrysogenum

share

e x t e n s i v e s e q u e n c e h o m o l o g y first led to the proposal that they e v o l v e d from a
c o m m o n ancestral g e n e (Carr et al. 1986). T h e current v i e w is that the p a t h w a y
e v o l v e d in bacteria and w a s transferred to a primordial fungus about 3 7 0 million
years a g o . In the original transfer, the p a t h w a y w a s split b e t w e e n t w o different
c h r o m o s o m e s . S o m e 7 0 million years after the initial transfer, the primordial fungus
split off the penicillin p r o d u c i n g fungi, with loss of o n e of the c h r o m o s o m e s and
r e c r u i t m e n t of acyltransferase ( A A T ) activity.
A n u m b e r of lines of e v i d e n c e support the horizontal transfer m o d e l . (1) T h e
s e q u e n c e d g e n e s c o m m o n to both bacteria and fungi (that is, I P N S , e x p a n d a s e ,
e x p a n d a s e / h y d r o x y l a s e ) are highly h o m o l o g o u s and h a v e G + C contents w h i c h are
typical of Streptomyces.

(2) T h e s e s a m e fungal g e n e s d o not contain introns. (3)

T h e g e n e s are clustered in both the fungi and bacteria. (4) F u n g i

synthesize

" t r u n c a t e d " versions of c e p h a m y c i n C , consistent with transfer of part of the
original cluster from bacteria.
T h e A A T g e n e s from both P. chrysogenum

and A. nidulans

h a v e b e e n s h o w n to

contain introns. It is therefore likely that the A A T g e n e w a s not part of the originally
transferred g e n e cluster a n d , instead, is of fungal origin. O n the other h a n d , the fact
that A A T is linked to I P N S and A C V synthetase in fungi ( S m i t h , D . et al. 1990a)
suggests that it, or s o m e part of it, w a s represented in the initial cluster. It will be
interesting to see w h e t h e r in an a n a l o g o u s position in bacteria (that is, d o w n s t r e a m
of the I P N S g e n e ) s e q u e n c e s related to fungal A A T will b e o b s e r v e d . W e h a v e
o b s e r v e d that the P. chrysogenum
clavuligerus

A A T g e n e cross-hybridizes with

Streptomyces

g e n o m i c D N A , but d o not yet k n o w if the cross hybridizing s e q u e n c e

forms part of the g e n e cluster.

9.5

APPLICATION OF MOLECULAR BIOLOGY TO
/3-LACTAM PRODUCTION

W h y are those involved in b i o t e c h n o l o g y and /3-lactam p r o d u c t i o n interested in
c l o n i n g and studying g e n e s for penicillin and cephalosporin b i o s y n t h e s i s ? R e a s o n s

9.5

Application of Molecular Biology to /3-Lactam Production

235

vary from strain i m p r o v e m e n t to novel antibiotics to n e w p r o d u c t i o n o r g a n i s m s .
Potential benefits from the application of m o l e c u l a r b i o l o g y are d i s c u s s e d b e l o w .

9.5.1

Use of Molecular Biology for Strain Improvement

It is p r o b a b l y fair to say that the potential for increasing antibiotic titers is a major
r e a s o n for interest in the application of m o l e c u l a r biology to /3-lactam p r o d u c i n g
microorganisms.

Classic

strain

improvement

techniques,

involving

random

m u t a g e n e s i s and s c r e e n i n g , in conjunction with m e d i a o p t i m i z a t i o n , h a v e p r o d u c e d
strains that can vastly o v e r p r o d u c e these antibiotics. T h e original strain of
lium

notatum

Pénicil-

p r o d u c e d penicillin titers of about 2 units p e r milliliter, w h e r e a s

current p r o d u c t i o n strains can p r o d u c e 7 0 , 0 0 0 - 1 0 0 , 0 0 0 units per milliliter. T h u s
e m p i r i c a l a p p r o a c h e s h a v e resulted in strains that are fifty-thousand-fold better with
respect to penicillin p r o d u c t i o n . H o w e v e r , as the strains h a v e b e c o m e o p t i m i z e d ,
further increases in titers h a v e b e c o m e difficult. T h u s the t i m e is right for looking at
alternative a p p r o a c h e s .
In t h e o r y , titer increases can b e a c c o m p l i s h e d by identifying the rate-limiting
e n z y m e and increasing its e x p r e s s i o n using the c l o n e d g e n e . F o r increased p r o d u c tion of C P C , this a p p r o a c h has already p r o v e n to be successful (Skatrud et al.
1989). T h e C. acremonium

p r o d u c t i o n strain 3 9 4 - 4 a c c u m u l a t e s penicillin N , thus

implicating e x p a n d a s e / h y d r o x y l a s e (cefEF) as rate limiting, since penicillin Ν is the
e n z y m e ' s substrate. A d d i n g o n e extra c o p y of c e f E F by transformation d o u b l e d the
e x p a n d a s e / h y d r o x y l a s e activity and increased the production of C P C b y a p p r o x imately 1 5 % in pilot scale fermenters. A n o t h e r a p p r o a c h to strain i m p r o v e m e n t is to
c o m p l e m e n t rate-limiting steps without k n o w i n g w h a t the rate-limiting steps a r e .
F o r e x a m p l e , altering the e x p r e s s i o n of a n u m b e r of u n k n o w n g e n e s c o u l d affect the
p r o d u c t i o n of s e c o n d a r y metabolites like antibiotics. C o s m i d libraries can be prep a r e d from a p r o d u c t i o n o r g a n i s m , and the D N A reintroduced into the p r o d u c t i o n
o r g a n i s m b y transformation. T h e transformants can then be screened for increased
antibiotic titers. T h e a s s u m p t i o n is that w h a t e v e r the rate-limiting step m a y b e , it
can b e o v e r c o m e by increased g e n e d o s a g e . A s it m a y be difficult to distinguish
relatively small increases in antibiotic titers against the b a c k g r o u n d of a p r o d u c t i o n
strain, it m a y b e desirable to introduce the library into a wild-type host and identify
transformants with increased titers. T h e clo n es identified in this m a n n e r can then be
i n t r o d u c e d into the p r o d u c t i o n strain and analyzed for yield i m p r o v e m e n t . A n o t h e r
a p p r o a c h is to introduce w i l d - t y p e D N A into production o r g a n i s m s on the a s s u m p tion that s o m e of the m u t a t i o n s the strains h a v e a c c u m u l a t e d d u r i n g the years of
m u t a g e n e s i s are adversely affecting antibiotic p r o d u c t i o n .
Strain i m p r o v e m e n t n e e d not be limited to titer increases. P r o d u c t i o n strains
m a y h a v e u n d e s i r a b l e qualities (for e x a m p l e , p o o r c o n i d i a t i o n , g l u c o s e repression)
that can also be a d d r e s s e d by this t y p e of m o l e c u l a r a p p r o a c h . In any e v e n t , only a
few t h o u s a n d c o s m i d transformants need to be analyzed to e n s u r e that the g e n o m e
has b e e n a d e q u a t e l y r e p r e s e n t e d . T h e s e types of n u m b e r s are h a n d l e d routinely in
" c l a s s i c " strain i m p r o v e m e n t p r o g r a m s . T h e strategy of introduction of r a n d o m
c o s m i d c l o n e s , c o m b i n e d with s c r e e n i n g , m a y best be thought of as a c o m b i n a t i o n
of m o l e c u l a r and classic strain i m p r o v e m e n t p r o c e s s e s .

236

Molecular Biology and Biochemistry of the /3-Lactam Antibiotics

9.5.2

Combining and/or Modifying /3-Lactam Pathway
Genes

H a v i n g the c l o n e d g e n e s p r o v i d e s the opportunity to c o m b i n e p a t h w a y s and to
modify g e n e s to m a k e either antibiotics novel to the p r o d u c i n g o r g a n i s m or novel
antibiotics. F o r e x a m p l e , can Pénicillium

b e e n g i n e e r e d to p r o d u c e c e p h a l o s p o r i n

C ? In p r i n c i p l e , all that w o u l d b e required w o u l d be to delete p e n D E and add c e f D ,
c e f E F , and c e f G . T h i s is certainly within the scope of current t e c h n o l o g y , and could
be quite v a l u a b l e b e c a u s e penicillin is currently m a d e at h i g h e r titers than c e p h a l o s p o r i n . F o r similar r e a s o n s , and using similar strategies, it m a y be feasible to m a k e
c e p h a m y c i n C in either Pénicillium

or

Cephalosporium.

C o m b i n i n g /3-lactam g e n e s from different p a t h w a y s has been p r o p o s e d as a
m e a n s of p r o d u c i n g 7 - a m i n o - d e a c e t o x y c e p h a l o s p o r o n i c acid ( 7 - D A C A ) ( C a n t w e l l
et al. 1990). T h i s c o m p o u n d is used as the starting material for the p r o d u c t i o n of
s e m i s y n t h e t i c oral c e p h a l o s p o r i n s , and is currently p r o d u c e d by c h e m i c a l ring
e x p a n s i o n of penicillin. C a n t w e l l et al. h a v e p r o p o s e d e x p r e s s i n g a modified
clavuligerus

penicillin Ν e x p a n d a s e (cefE) g e n e in P. chrysogenum.

S.

The gene

w o u l d be modified (that is, protein e n g i n e e r e d ) to ring e x p a n d penicillin V ( p e n V ) ,
a

substrate

it

does

not

currently

recognize.

The

resulting

compound,

de-

a c e t o x y c e p h a l o s p o r i n V , could be c o n v e r t e d to 7 - D A C A by bacterial penicillin V
a m i d a s e s . T h e y h a v e so far d e m o n s t r a t e d that an unmodified cefE g e n e can b e
e x p r e s s e d in P. chrysogenum

without affecting penicillin p r o d u c t i o n . T h e real trick

will b e altering the substrate specificity of the e x p a n d a s e so that it will ring e x p a n d
p e n V . T h i s s e e m s a large o r d e r , given the stringent substrate specificity that the
e n z y m e exhibits: it currently e x p a n d s only p e n N , and not i s o p e n N , w h i c h differ
only in the stereochemistry of the a m i n o a d i p y l side c h a i n . In contrast, p e n V has
p h e n o x y a c e t y l m o i e t y in place of an a m i n o a d i p y l g r o u p .
A n o t h e r a p p r o a c h to the s a m e goal w o u l d be to utilize the I P N a m i d o l y a s e
(A A T ) of P. chrysogenum

to p r o d u c e 7 - D A C A from D A O C or 7 - A C A from C P C .

7 - A C A is also used as a starting material for semisynthetic c e p h a l o s p o r i n p r o d u c tion. In this a p p r o a c h , A A T w o u l d be used in conjunction with I P N e p i m e r a s e
a c c o r d i n g to the following s c h e m e :
C P C or D A O C

i s o C P C or i s o D A O C

epimerase

7 - A C A or 7 - D A O C

amidolyase

It is p r o b a b l e that both A A T and e p i m e r a s e w o u l d also h a v e to b e e n g i n e e r e d to
accept alternative substrates. T h e native e p i m e r a s e is unlikely to accept C P C , as
i s o C P C is not p r o d u c e d by C. acremonium,

and the specificity of the a m i d o l y a s e is

p r o b a b l y not directed solely at the a m i n o a d i p y l side c h a i n .

9.5.3

Understanding Overproduction in Industrial Strains

H a v i n g c l o n e d g e n e s has b e e n useful in u n d e r s t a n d i n g the nature of o v e r p r o d u c t i o n
in industrial strains. In the case of penicillin b i o s y n t h e s i s , s o m e p r o d u c t i o n strains
h a v e amplified the biosynthetic g e n e s during strain i m p r o v e m e n t ( B a r r e d o et al.
1989a; S m i t h et al. 1989). In these strains, increased g e n e e x p r e s s i o n i s , at least

References

237

partly, the c o n s e q u e n c e of increased g e n e c o p y n u m b e r . P r e s u m a b l y p r o m o t e r or
other regulatory m u t a t i o n s m a y also be involved in increased expression of these
g e n e s . Interestingly, in the B - 1 0 strain, the structural g e n e s for I P N S and A A T are
identical to those from the p r o g e n i t o r strain N R R L 1 9 5 1 . T h u s the specific activities
of the e n z y m e s h a v e not b e e n altered during the years of m u t a g e n e s i s .
In C. acremonium,
there are n o reports of applification of any biosynthetic
g e n e s in p r o d u c t i o n strains. H e r e t o o , p r o m o t e r and regulatory m u t a n t s h a v e
p r o b a b l y b e e n instrumental in increased g e n e e x p r e s s i o n .

9.6

FUTURE PROSPECTS

All of the g e n e s for biosynthesis of penicillin, c e p h a l o s p o r i n , and c e p h a m y c i n h a v e
b e e n , or shortly will b e , cloned and characterized. T h e cloned g e n e s will be
essential in u n d e r s t a n d i n g the biosynthesis of these antibiotics at a m o l e c u l a r level.
This will afford the opportunity to address the issue of strain i m p r o v e m e n t in n e w ,
rational w a y s that w e r e not previously p o s s i b l e . This is particularly timely as
" c l a s s i c " m e t h o d s of strain i m p r o v e m e n t h a v e b e c o m e m o r e difficult. In addition,
m a n i p u l a t i o n s of the g e n e s will u n d o u b t e d l y lead to novel antibiotics and strains.

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CHAPTER

10
Therapeutic Metabolites
Prakash S. Masurekar

F u n g i are n a t u r e ' s m o s t a c c o m p l i s h e d c h e m i s t s . T h e c o m p o u n d s they synthesize
r a n g e from simple u b i q u i t o u s m o l e c u l e s such as gluconic acid and citric acid, often
referred to as p r i m a r y m e t a b o l i t e s , to c o m p l e x unusual m o l e c u l e s such as aflatoxin
and ergot a l k a l o i d s , w h i c h are called s e c o n d a r y metabolites. F r o m a m i n o acids
fungi p r o d u c e a w i d e s p e c t r u m of p r o d u c t s , w h i c h include antibiotics such as
penicillins and c e p h a l o s p o r i n s , p e p t i d e s such as c y c l o s p o r i n s , and e n z y m e s such as
α - a m y l a s e and lipase. M a n y fungal metabolites h a v e very interesting biological
activities. F o r e x a m p l e , penicillin, the first ß - l a c t a m antibiotic, d i s c o v e r e d b y
F l e m i n g , is a true miracle d r u g . It is c o m m e r c i a l l y p r o d u c e d b y
Pénicillium
chrysogenum
and is an inhibitor of cell wall synthesis in g r a m - p o s i t i v e bacteria. It
w a s s h o w n to act by inhibiting the transpeptidase that catalyzed the closure of
p e p t i d e bridges b e t w e e n the adjacent m u r e i n strands (Tipper and S t r o m i n g e r 1965;
G h u y s e n 1977). T h i s antibiotic is discussed in C h a p t e r 6.
O n the other h a n d , c y c l o s p o r i n s (Table 1 0 - 4 ) , a class of cyclic u n d e c a p e p t i d e s
p r o d u c e d by Beuveria
nivea, w e r e reported by Borrel et al. (1976) to h a v e imm u n o s u p p r e s s i v e activity. S u b s e q u e n t studies h a v e s h o w n that these block p r o d u c tion of interleukin 2 (Elliot et al. 1984; K r o n k e et al. 1984). U s e of c y c l o s p o r i n s has
dramatically r e d u c e d m o r b i d i t y and increased survival rates in h u m a n transplant
patients ( S h o w s t a c k et al. 1989; Starzl et al. 1989).
Ergot a l k a l o i d s , p r o d u c e d by Claviceps purpurea from t r y p t o p h a n , h a v e m a n y
I thank Drs. R. Monaghan, J. Nielsen, L. Kaplan, M. Masurekar, and Mrs. M. Sosa for their critical
review of the manuscript and suggestions. I am grateful to Dr. J. Tkacz for making preprint of his article
available.

241

242

Therapeutic Metabolites

p h a r m a c o l o g i c a l activities, w h i c h include vasoconstriction, induction of uterine
c o n t r a c t i o n s , stimulation of d o p a m i n e p r o d u c t i o n , and inhibition of prolactin synthesis ( B e r d e and S t u r m e r 1978). Ergot alkaloid derivatives h a v e found use in the
t r e a t m e n t of m i g r a i n e , in control of uterine m o t o r activity, therapy of P a r k i n s o n ' s
d i s e a s e , and of senile d e m e n t i a and disorders c a u s e d by h y p e r p r o l a c t i n e m i a
( S a a m e l i 1978; V e n n 1978; T h o r n e r et al. 1980; C a l n e et al. 1980; F r i e d m a n et al.
1989).
Asperlicin is a b e n z o d i a z e p i n , w h i c h is synthesized from t r y p t o p h a n and
leucine by Aspergillus
alliaceus.
This is a first n o n p e p t i d e , p o t e n t , specific antagonist of c h o l e c y s t o k i n i n , a neurotransmitter involved in the control of gut
motility and pancreatic secretion ( C h a n g et al. 1985). H e n c e asperlicin m a y h a v e a
use in the treatment of the disorders of the gastrointestinal system that i n v o l v e
cholecystokinin.
Lovastatin p r o d u c e d by Aspergillus
terreus via the p o l y k e t i d e p a t h w a y is a very
potent inhibitor of cholesterol b i o s y n t h e s i s , and b e c a u s e h y p e r c h o l e s t e r o l e m i a has
b e e n implicated in coronary heart d i s e a s e , this c o m p o u n d has b e e n used for
l o w e r i n g b l o o d cholesterol levels (Alberts et al. 1980; T o b e r t 1988).
Aflatoxins derive their n a m e from the fungus that w a s implicated in its p r o d u c tion, Aspergillus flavus. T h e s e , t o o , are p r o d u c e d by the p o l y k e t i d e p a t h w a y and
w e r e found to be the agents responsible for the outbreak of liver disease that killed
d u c k s , t u r k e y s , and p h e a s a n t s in 1960 in G r e a t Britain. D u e to their e c o n o m i c
i m p o r t a n c e a c o n c e r t e d research effort w a s m o u n t e d and four aflatoxins w e r e
isolated ( A s a o et al. 1965). T h e s e are designated as B 1 ? B 2 , G j , and G 2 . In addition
to their ability to c a u s e liver d i s e a s e , they possess c a r c i n o g e n i c properties w h i c h
w e r e first noted by L a n c a s t e r et al. ( 1 9 6 1 ) . S u b s e q u e n t studies established that a
dietary level of 0 . 0 1 5 p p m of aflatoxin w a s sufficient to c a u s e hepatic c a r c i n o m a in
rats and that this is o n e of the m o s t potent c a r c i n o g e n s ( W o g a n and N e w b e r n e
1967).
T h e discovery of aflatoxins sparked an interest in the study of m y c o t o x i n s , and
a large n u m b e r of fungal metabolites with toxic properties w e r e found. O n e such
c o m p o u n d , called p a r a h e r q u a m i d e , w a s isolated from Pénicillium
paraherquei
( Y a m a z a k i et al. 1981). Interestingly, later it w a s found to h a v e a n t h e l m e n t i c
activity (Ostlind et al. 1990).
T h e s e few e x a m p l e s are sufficient to illustrate the d i l e m m a o n e faces w h e n
selecting the c o m p o u n d s to be discussed. I m a d e m y c h o i c e based on the structural
variety and i m p o r t a n c e of their therapeutic activities. T h e c o m p o u n d s that will be
described in this c h a p t e r are lovastatin, c y c l o s p o r i n s , ergot alkaloids, and asperlicin. After a brief introduction, production p r o c e s s , m o d e of action, b i o c h e m i s t r y ,
and clinical and animal data for each will be discussed.

10.1

LOVASTATIN

H y p e r c h o l e s t e r o l e m i a has been implicated as a major risk factor in the d e v e l o p m e n t
of c o r o n a r y heart d i s e a s e . B e c a u s e as m u c h as t w o thirds of total b o d y cholesterol in

Synvinolin. It differs from lovastatin in that it lacks the 6 . F o o d and D r u g A d m i n i s t r a t i o n (Tobert 1987). a /3-hydroxy l a c t o n e . also called synvinolin and Z O C O R ® .m e t h y l g r o u p . Pravastatin. is m a d e by microbial transformation of m e v a s t a t i n ( S e r i z a w a et al.a . SQ-31000. 0 0 0 strains of m i c r o o r g a n i s m s for their ability to p r o d u c e an inhibitor of in vitro sterol synthesis b y rat liver e n z y m e s y s t e m resulted in the isolation of mevastatin ( E n d o 1985). Structures of lovastatin and other similar c o m p o u n d s are s h o w n in F i g u r e 1 0 . T h i s transformation involves Lovastatin (MK-803. Lovastatin contains a n a p h t h a l e n e ring s y s t e m . MEVACOR) Simvastatin (MK-733. w a s isolated from Pénicillium citrinum by E n d o et al. 1980). is m a d e c h e m i c a l l y from lovastatin (Hoffman et al. A screen of 8 . inhibition of d e n o v o synthesis w o u l d b e e x p e c t e d to l o w e r p l a s m a c h o l e s t e r o l . w h i c h is also k n o w n as eptastatin. Mevinolin. Eptastatin) FIGURE 10-1 Structures of lovastatin and related compounds. and methylbutyric acid. Compactin) Pravastatin (CS-514. Lovastatin is p r o d u c e d b y Aspergillus terreus (Alberts et al. M e v a s t a t i n . 1983a). It is the first c o m p o u n d of its kind to b e a p p r o v e d by the U .p o s i t i o n in the four-carbon side c h a i n . 1986). It h a s an additional m e t h y l g r o u p at the 2 ' .10. It is also k n o w n as m e v i n o l i n and is c o m m e r c i a l l y available as M E V A C O R ® for the treatm e n t of h y p e r c h o l e s t e r o l e m i a . A search for other c o m p o u n d s w h i c h also inhibited cholesterol synthesis resulted in the isolation of lovastatin. also called c o m p a c t i n . ZOCOR) Mevastatin (ML-236B.1 Lovastatin 243 h u m a n s is of e n d o g e n o u s origin.1 . . ( 1 9 7 6 ) . S i m v a s t a t i n . S . CS-500.

161-169.h y r o x y l a t i o n at the 6-position. terreus w h i c h p r o d u c e s lovastatin w a s isolated at C I B E Laboratories in M a d r i d . It is also p r o d u c e d b y Monoascus ruber ( E n d o 1979). T o test this hypothesis a n u m b e r of re-isolates w e r e tested for the p r o d u c t i o n .1.1 ) . effects of various carbon s o u r c e s . H o w e v e r . it w a s not h o m o g e n e o u s as far as the lovastatin synthesis potential w a s c o n c e r n e d .3 . O n e possible reason w a s that although the strain used w a s microbiologically p u r e . .7 . nitrog e n . A fermentation process with A. trace metals such as m a g n e s i u m . Society for Industrial Microbiology. W h e n these are in a b u n d a n t supply the g r o w t h is restricted only by the inherent properties of the fungus. Arlington. the conditions w h i c h are ideal for g o o d g r o w t h are not necessarily o p t i m u m for the production of s e c o n d a r y m e t a b o l i t e s . p h o s p h o r u s .244 Therapeutic Metabolites ß .h y d r o x y .1. M a r t i n and D e m a i n 1980). VA. terreus w a s d e v e l o p e d to m a n u f a c t u r e it on large scale ( B u c k l a n d et al. A h a r o n o w i t z 1980. T h e types of m e d i a used for isolation of the TABLE 10-1 Effect of Reisolation Lovastatin Culture ATCC 20542 Reisolate Mean Uli %CV 91 54 207 12 Reprinted by permission of the publisher from Buckland et al. Spain from soil. p H of the m e d i u m . This culture w a s h o m o g e n e o u s as far as the yields of lovastatin w e r e c o n c e r n e d (see T a b l e 1 0 . F o r this p u r p o s e culture h o m o g e n e i t y .1. sulfur. All of these c o m p o u n d s are very potent inhibitors of 3 . that is. terreus ( A T C C 2 0 5 4 2 ) .p r o d u c i n g re-isolates w a s selected and the p r o c e s s w a s repeated to obtain the strain designated as 4 6 . 1989). It w a s reisolated and " p u r i f i e d . T h e r e h a v e been n u m e r o u s studies on this subject of effects of various nutrients on the p r o d u c t i o n of secondary metabolites (Martin 1977. with very low production by 5 0 % of the re-isolates ( T a b l e 1 0 . and the effect of agitators w e r e studied. . (1989) in Novel Microbial Products for Medicine and Agriculture.1 Production 10. Interestingly.1 P r o d u c t i o n o f L o v a s t a t i n . 10. h o w e v e r . A s m e n t i o n e d in the p r e c e d i n g section lovastatin is p r o d u c e d by A. Nutrients essential for the g r o w t h include sources of assimilable carbon. iron. p o t a s s i u m e t c . pp. it did not contain any other c o n t a m i n a t i n g m i c r o o r g a n i s m .m e t h y l g l u t a r y l c o e n z y m e A ( H M G C o A ) reductase and thus l o w e r p l a s m a cholesterol levels. O n e of the h i g h .1 ) . w h e n it w a s used for fermentation p r o c e s s d e v e l o p m e n t it w a s found that yields fluctuated. Yields of lovastatin by these re-isolates varied o v e r a w i d e r a n g e . A. and o x y g e n . " w h i c h is a standard practice for all culture isolation p r o g r a m s .

and m e t h y l oleate w a s tried. g l y c e r o l . and p H m a y be important variables (Figures 1 0 . time of exhaustion of carbon source.10. Initial m e d i u m w a s d e s i g n e d with g l u c o s e as a carbon source and best titers w e r e o b s e r v e d with 100 g of glucose/1. P r o d u c tion w a s better than control with glycerol and methyl oleate. 1989). With glucose in the m e d i u m the p H dropped to 4 . modified starch. L a c t o s e and modified starch could replace g l u c o s e to a significant extent and yields u p to 9 0 % of control w e r e seen (Table 1 0 . U / l . studies w e r e initiated to o p t i m i z e the p r o d u c t i o n m e d i u m . respectively. 1989) suggested that the rate of carbon source utilization. of Cerelose (gll) Name and Cone.2 ) . 5 on the third day and recovered when glucose was exhausted. or 3-(Λ/^οφηοΗηο)-ρΓορ3ηε8υ1π3ηκ. T h e r e f o r e . Often the synthesis of a s e c o n d a r y m e t a b o l i t e can be e x t e n d e d by addition of c a r b o n source during the fermentation. 1989). A n attempt w a s m a d e to i m p r o v e yield by addition of either g l u c o s e or glycerol to the fermentation on the sixth d a y . 1989). Kinetics of fermentation with glucose or with glycerol as carbon source (Buckland et al. O n the other hand.2 and 10-3). with glycerol it did not go below 5 . 8 . TABLE 10-2 Effect of Other Carbon Sources as Partial Replacement of Glucose Cone. T h e p H profiles obtained with the two carbon sources showed some differences. VA. T h e rates of synthesis of lovastatin with glucose and glycerol as carbon source were 0. proflo oil. In both cases the rate declined after the sixth day which coincided with the time most of the carbon source was used u p . T h e titer w a s increased by 2 0 % and 5 0 % with glycerol and g l u c o s e a d d i t i o n s . respectively ( B u c k l a n d et al.h . pp. Total r e p l a c e m e n t of g l u c o s e with glycerol i m p r o v e d the p r o d u c t i o n b y 3 0 % . T h e importance of p H control was confirmed by the addition of 2-(7V-morpholino)ethanesulfonic acid ( M E S ) . W i t h this m e d i u m as a c o n t r o l . Arlington. partial replacem e n t of g l u c o s e with lactose. 161-169.8 U/l-h and l . This explains the improvement in the yields seen with the addition of a "shot" of glucose or glycerol. of Other Carbon Source 100 (control) None Lovastatin (Uli) 104 50 None 57 50 50 g lactose/1 94 50 50 g modified 88 50 45 g Proflo oil/1 69 50 45 g soybean oil/1 62 50 50 g glycerol/1 125 50 45 g methyl oleate/1 111 Reprinted by permission of the publisher from Buckland et al.1 Lovastatin 245 culture s u p p o r t e d g o o d g r o w t h but the yields of lovastatin w e r e l o w . s o y b e a n oil. phosphate. (1989) in Novel Microbial Products for Medicine and Agriculture. Society for Industrial Microbiology. In the first p h a s e of these studies the effect of various carbon sources w a s investigated ( B u c k l a n d et al. . acid ( M O P S ) as buffer in shake flasks (Buckland et al. l .

1 . T h e h y d r o x y l g r o u p at C .Δ . h o w e v e r .246 Therapeutic Metabolites ι Ο • ι 2 ι ι 4 ι ι 6 Days ι ι 8 1 1 10 1 ΐ12 F I G U R E 10-2 Kinetics of lovastatin production. 1 0 .a) profiles with these t w o impellers w e r e similar. T h e information obtained with the shake flask studies w a s c o m b i n e d with that from scaleup e x p e r i m e n t s to d e v e l o p a successful production p r o c e s s . 1989). A c o m p a r i s o n of the standard R u s h t o n flat blade turbine with the P r o c h e m agitator at 800-1 scale s h o w e d that although the o x y g e n transfer coefficient (AT L. 1 . 2 P r o d u c t i o n of S i m v a s t a t i n .O . the p e a k p o w e r d r a w with the P r o c h e m agitator w a s approximately 6 6 % of that with the R u s h t o n impeller ( B u c k l a n d et al. . Acylation of alcohol w a s carried out . . This observation is of great value b e c a u s e the fermentors used for manufacture are often u n d e r p o w e r e d and the cost of e n e r g y is h i g h . O n t a r i o . A novel design of impellers w a s used to meet the high o x y g e n r e q u i r e m e n t s of the lovastatin p r o c e s s . This hydrofoil axial flow impeller is designed by P r o c h e m M i x i n g E q u i p m e n t Ltd. ( B r a m p t o n . Details of the large-scale m a n u f a c t u r i n g p r o c e s s h a v e not b e e n published. a m e t h o d for laboratory scale synthesis w a s reported by Hoffman et al.1 3 of lovastatin w a s protected by preparation of silyl ether.in glycerol medium. C a n a d a ) . ( 1 9 8 6 ) .in cerelose medium. O n e of the difficulties e n c o u n t e r e d in the scaleup of mycelial fermentation p r o c e s s e s is h o w to m e e t the o x y g e n d e m a n d s of the culture efficiently.

· . T h e following p r o d u c t i o n m e t h o d s for mevastatin and pravastatin are from the reports that described their d i s c o v e r y . and Hypomyces chrysospermus ( B r o w n et al. citrinum S a n k 18767 w a s g r o w n aerobically in a m e d i u m c o n t a i n i n g 3 % malt extract. 1 % p e p t o n e for 9 6 h ( E n d o et al. Paecilmyces.1 Lovastatin 247 Days F I G U R E 10-3 Kinetics of fermentation.10. with 2 .Q .cerelose. Trichoderma longibraiatum. O n c e again the details of m a n u f a c t u r i n g p r o c e s s e s h a v e not b e e n p u b l i s h e d . 1 0 .pH in glycerol medium. T h e solution w a s stirred at 100°C u n d e r nitrogen a t m o s p h e r e for 4 h.O . F u n g i other than Pénicillium citrinum that p r o d u c e mevastatin are P. 1976. .pH in cerelose medium. T. In the a b s e n c e of acetic acid e x t e n s i v e d e g r a d a t i o n of the lactone ring w a s o b s e r v e d . . T h e d e p r o t e c tion of silyl ethers w a s d o n e in tetrahydrofuran ( T H F ) with t e t r a b u t y l a m m o n i u m fluoride. T h e . E n d o et al.d i e t h y l b u t y r y l chloride in dry pyridine containing 4 . . 1986). F o r p r o d u c t i o n of m e v a s t a t i n P. 1 . A c e t i c acid w a s a d d e d to attenuate basicity of fluoride. brevicompactum. 2 % g l u c o s e . and 0 . pseudokoningii. 2 .V . 1 .glycerol. .p y r r o l i d i n o p y r i d i n e . 1976). 3 P r o d u c t i o n o f M e v a s t a t i n a n d P r a v a s t a t i n . N o r is there any information available o n the effects of various e n v i r o n m e n t a l variables on the synthesis of either of the c o m p o u n d s .

p e p t o n e . F u r t h e r m o r e .2 d o n a t e d by m e t h i o n i n e ( C h a n et al. 1983b). 10. Culture filtrate ( 2 .9 0 % ( S e r i z a w a et al.. a c t i n o m y c e t e s . based on the o b s e r v a t i o n that P. (1985) 13 13 w h e n they studied the incorporation of [ C ] a c e t a t e . 1988). the s o d i u m salt of mevastatin w a s a d d e d and the fermentation w a s continued for an additional 5 . M.1. 0 to yield 327 g of crude active c o m p o u n d . O n further screening Streptomyces carbophillus w a s discovered as an efficient c o n v e r t e r with little b y . hiemalis w a s sensitive to the concentration of m e v a s t a t i n in the m e d i u m and the Nocardia s p . as m e n t i o n e d before n o details about the process used w e r e g i v e n . After the exploration of c h e m i c a l m e t h o d s to h y d r o x y l a t e mevastatin it w a s felt that the microbial transformation m a y b e a m o r e facile and e c o n o m i c a l a p p r o a c h . citrinum d o e s not p r o d u c e any c o m p o u n d s w h i c h contain the methyl g r o u p at . It w a s also m e n t i o n e d that a large a m o u n t of mevastatin w a s p r o d u c e d . and 1 8 0 .4 ) consists of a polyketide chain of nine intact acetate units with a m e t h i o n i n e . and S a n k 6 2 9 8 1 w e r e found to h y d r o x y l a t e the 6/3-position with c o n v e r s i o n yields of 3 0 . 1988). [ C ] p r o p i o n a t e . and 13 [ C ] m e t h i o n i n e by P. T h i s culture w a s g r o w n in a m e d i u m containing g l u c o s e .c o n t r o l l e d c o n t i n u o u s feeding of mevastatin w e r e reported to h a v e resulted in high c o n v e r s i o n rates and shortened the duration of the p r o c e s s (Arai et al. respectively. H . Sank 6 2 7 8 1 .d e r i v e d methyl g r o u p at C . H o w e v e r . and yeast extract. and bacteria w e r e screened ( S e r i z a w a et al. T h i s i n v o l v e d both strain d e v e l o p m e n t and optimization of cultural c o n d i t i o n s .5 g of mevastatin ( E n d o et al.248 Therapeutic Metabolites v o l u m e of culture broth from a 6. Pravastatin w a s originally discovered in the urine of d o g s treated with m e v a s t a tin.000-1 fermentor was 3 . 1988). T h e s e results w e r e confirmed by E n d o et al. 1983).. It w a s also reported that a large-scale process w a s d e v e l o p e d . Strain i m p r o v e m e n t and c o m p u t e r .000 strains of fungi. F r o m these Mucor hiemalis and Nocardia s p . 9 0 0 1) w a s c o n c e n t r a t e d to 4 5 0 1 in v a c u o and extracted with ethyl acetate at p H 4 . citrinum and Monoascus ruber into mevastatin and lovasta13 tin. T h e y found that w h e r e a s the label from [ C ] p r o p r i o n a t e w a s not incorporated into either mevastatin or lovastatin the results with the other t w o p r e c u r s o r s w e r e the s a m e as those of C h a n et al. especially in a fed-batch s y s t e m . terreus A T C C 2 0 5 4 2 of C .7 d a y s . A n industrial-scale fermentation p r o c e s s has reported to h a v e been d e v e l o p e d (Arai et al. w e r e found to p r o d u c e a dih y d r o x y l a t e d p r o d u c t along with pravastatin (Arai et al. T h e /3-methylbutyryl side chain is c o n f structed from t w o intact acetate units with a methyl g r o u p at C . ( 1 9 8 3 ) . h o w e v e r . 1983b).2 Biosynthesis I 3 2 Results of studies on incorporation by A.6 . 1983a). After 2 d a y s .p r o d u c t . Pravastatin w a s isolated by solvent extraction and c o l u m n c h r o m a t o g r a p h y (Arai et al.l a b e l e d precursors into lovastatin s h o w e d that the m a i n portion of the m o l e c u l e (Figure 1 0 . 1976). A b o u t 1. A simple and e c o n o m i c a l isolation s c h e m e w a s also d e v i s e d . Further c h r o m a t o g r a p h i c separations finally g a v e 10. T h e i r studies with 14 [ C ] a c e t a t e resulted in the suggestion that the hydroxylation and the m e t h y l b u t y r a tion at C-8 take place after cyclization. 0 0 0 1. 1988). m e a t extract. It w a s found to be m o r e potent than the parent c o m p o u n d in inhibition of cholesterol synthesis in vitro (Serizawa et al. S a n k 6 2 8 8 1 .

6 4 n M and 1. 10. T h i s is o n e m e c h a n i s m by w h i c h these c o m p o u n d s c a u s e reduction of p l a s m a cholesterol levels in d o g s (Tsujita et al.6 . a n u m b e r of m e c h a n i s m s h a v e b e e n p r o p o s e d and substantiated. T h e s e c o m p o u n d s also selectively r e d u c e low-density lipoprotein ( L D L ) in h u m a n s . Kx values for the s o d i u m salt of lovastatin and for the s o d i u m salt of m e v a s t a t i n w e r e found to be 0 . T h e reduction of H M G C o A to m e v a l o n a t e b y H M G C o A reductase ( E C 1. 1979. m e v a s t a t i n is not a p r e c u r s o r of lovastatin. 1980.10.34) is a rate-limiting step in this p a t h w a y ( R o d w e l l et al. and in h u m a n s ( Y a m a m o t o et al. B i o s y n t h e s i s of cholesterol involves m o r e than 25 e n z y m e s . Illingworth and Sexton 1984).4 n M . 1976).3 Mode of Action T h e information o b t a i n e d so far indicates that the m e c h a n i s m of action of lovastatin a n d related c o m p o u n d s is c o m p l e x and is not yet fully u n d e r s t o o d . Alberts et al. C . It w a s found that lovastatin and related c o m p o u n d s w e r e very efficient c o m p e t i t i v e inhibitors of this e n z y m e (Alberts et al.1. in m o n k e y s ( K u r o d a et al. At this time n o n e of the e n z y m e s i n v o l v e d in the biosynthesis has been isolated or studied.1 Lovastatin 249 Lovastatin (Mevinolin.1. H o w e v e r . respectively. 1980.1. that is. Mevacor®) F I G U R E 10-4 Biosynthesis of lovastatin. 1979). 1980). E n d o 1985). they suggested that the m e t h y l g r o u p is a d d e d before cyclization.

although it w a s increased to 10 m g b . O n e m e c h a n i s m for this reduction in L D L involves an increase in the hepatic L D L receptors ( K o v a n e n et al. T h e y consisted of 157 with h e t e r o z y g o u s familial h y p e r c h o l e s t e r o l e m i a ( F H ) and 192 n o n . i .2 5 % .4 Clinical Data A substantial a m o u n t of clinical data is n o w available on M E V A C O R ® (lovastatin). in 57 F H and 32 n o n . 1980.F H patients. and therefore its reduction reduces p l a s m a cholesterol. Slater and M a c D o n a l d 1988). H (high) D L cholesterol + 1 0 % . 1982). Similarly the neurotoxicity observed in some dogs treated with high doses of lovastatin has not been noted in clinical studies (Slater and MacDonald 1988).3 5 % . d . G r u n d y and B i l h e i m e r 1984).4 0 % . B i l h e i m e r et al. T o b e r t et al. Clinical studies h a v e been c o n d u c t e d on simvastatin ( Z O C O R ® ) and pravastatin (Arai et al. d . specifically L D L cholesterol. T h e changes in the liver are believed to be mechanism based rather than idiosyncratic. 0 0 0 patients (Slater and M a c D o n a l d 1988). W a l k e r 1989). A n o t h e r m e c h a n i s m involves reduction in L D L a p o l i p o p r o tein Β ( a p o B ) synthesis. It w a s c l a i m e d by Tsujita . as it has b e e n available for treatment of h y p e r c h o l e s t e r o l e m i a since A u g u s t 1987.F H patients. They occurred with all of the H M G C o A inhibitors tested and were reversed or prevented by feeding mevalonate.1. Pravastatin w a s tested in 3 4 9 patients for 12 w e e k s . L D L cholesterol . 1983.000 patients for u p to 4 years following c h a n g e s h a v e been observed: at 4 0 m g b . and a p o B . N o adverse ophthalmological effects of lovastatin or simvastatin in humans have been seen (Slater and MacDonald 1988). Huff et al. Total cholesterol w a s r e d u c e d b y 1 8 % in F H and by 1 7 % in n o n . T h e y s h o w e d that lovastatin induced m R N A for L D L receptors in livers of h a m s t e r s and rabbits. respectively. T h e dose w a s 5 m g b . It has b e e n s h o w n that lovastatin r e d u c e s a p o B in h u m a n s (Tobert 1987). T h e r e has b e e n s o m e d e b a t e on the tissue specificity of the three d r u g s (Tsujita et al. p l a s m a triglycerides . T h e formation of cataracts observed in dogs when given high doses of lovastatin appears to have resulted from a high drug plasma concentration. In the s a m e g r o u p s .250 Therapeutic Metabolites ( Y a m a m o t o et al. 1988. L D L cholesterol w a s reduced by 2 4 % and 2 3 % . d . respectively. Results of studies with o v e r 1. s e r u m triglycerides w e r e reduced by 9 % and 1 1 % (Arai 1988). i . Alberts 1988. It has b e e n prescribed for about 3 0 0 . total p l a s m a cholesterol .800 patients for u p to 2 years with a d o s e r a n g e of 1 0 . Tobert 1988). i . Results of the simvastatin study with 1. Lovastatin and simvastatin are well tolerated. (1985) s h o w e d that lovastatin selectively inhibited direct synthesis of L D L a p o B in miniature p i g s . all three drugs s e e m to be quite efficacious in the treatment of h y p e r c h o l e s t e r o l e m i a . with discontinuation of the treatment required only for 2 % receiving lovastatin and 0 .2 0 % (Tobert 1987). and H D L cholesterol w a s increased by 1 0 % ( W a l k e r 1989). T h e total and L D L cholesterol w e r e r e d u c e d b y 3 0 % and 4 0 % . T w o areas of potential safety concern were the variety of changes in the liver in some of the species of animal studied and the systemic effect at high doses in dogs. 5 % receiving simvastatin (Slater and MacDonald 1988. H D L cholesterol w a s increased by 1 3 % and 9 % . 10. lovastatin.F H patients. 1986.4 0 m g o n c e daily w e r e similar. T h u s . V (very) L D L cholesterol . L D L is involved in the translocation of c h o l e s t e r o l .3 3 % . E v i d e n c e in support of this m e c h a n i s m w a s obtained by M a et al. 1 9 8 1 . ( 1 9 8 6 ) .

(1976) as antifungal antibiotics p r o d u c e d b y a fungus then classified as Trichoderma polysporum.2 Cyclosporins 251 and his c o . XH. MeLeu H .5 and T a b l e 1 0 . T h e s p e c t r u m of antifungal activity w a s rather n a r r o w (Dreyfuss et al. C l | -CH-N—CO-CH XH3 MeLeu l 3 Ο I 66 CH3 D-Ala I CH—C—N—CH. w h i c h is the target o r g a n for these d r u g s .2 l II H 3 I.3 C H . Y CH. CH—CO-N—CH-C—Ν CO CH XH CH.CH CH. CH. In k i d n e y . CH-CO—Ν I 5 Ο 1 l Ο Ο I D l I l II OC-CH—N—CO—CH—N—CO-CH—Ν—ΟΙ I I I I CH2 CH. All c y c l o sporins are cyclic p e p t i d e s that contain 11 a m i n o acids. T h i s appears to b e an artifact of the e x p e r i m e n t a l protocol used (Alberts 1988. In these studies lovastatin and simvastatin w e r e used in the p r o d r u g lactone form w h i c h is used for treatment rather than the o p e n h y d r o x y a c i d form used b y Tsujita et al. 1987) and their structures are s h o w n in F i g u r e 1 0 . s p l e e n .I '3 ^CH2 5 j 2 HO. 2 10 C H .10. MeBmt MeVal MeLeu XH. tCH ΌΗ" XH.3 . S o m e of these a m i n o a c i d s . CH3 Η 11 I •CI I 8 77 Η Η I . H o w e v e r . and n o n g l a n d u l a r and g l a n d u l a r s t o m a c h the levels of pravastatin w e r e many-fold h i g h e r than those of lovastatin or simvastatin ( G e r m e r s h a u s e n et al. CH. 1989). I XH CH. 10.CH Ala II l I Η I I ^ 0 I 4 Ί N I . t e s t e s . Slater and M a c D o n a l d 1988). it w a s found to h a v e i m m u n o s u p p r e s s i v e activity (Borel et al.3 I 12 l Sar Abu Η I / .w o r k e r s that pravastatin w a s m o r e selective than lovastatin for the liver. T h e r e are 25 k n o w n naturally o c c u r r i n g cyclosporins (Traber et al. Of these c y c l o s p o r i n A ( C s A ) is the major p r o d u c t and other c y c l o s p o r i n s are p r o d u c e d in m i n o r a m o u n t s . XH.—Ν I l . a d r e n a l s . CH. 1976) w h i c h led to its use in organ transplant surgery. H I MeLeu CH. CH. T h e s u b s e q u e n t d r a m a t i c increase in the success of this p r o c e d u r e m a d e c y c l o s p o r i n A o n e of the very i m p o r t a n t therapeutic agents of the last d e c a d e (Jones and C a t t o 1989). 1976) and the interest in it as an antibiotic w a n e d . CH. Ç 3 C H . Val F I G U R E 10-5 Structure of cyclosporin A. ( 1 9 8 6 ) . C s A is used to p r e v e n t graft rejection in o r g a n transplantation surgery.2 Cyclosporins C y c l o s p o r i n s w e r e d i s c o v e r e d by Dreyfuss et al. Studies with rats given e a c h of the three drugs at 25 m g / k g s h o w e d that lovastatin and simvastatin levels in liver w e r e almost twice that of pravastatin.

m e t h y l . N o n e has b e e n found to b e superior to C s A (Borel 1989).o c t 6-enoic acid are not usually found in p r o t e i n s .a l a n i n e . only a few w e r e tested in v i v o . Borel 1989). . methylamino-3-hydroxy-4-methyl-oct-6-enoic acid. V a r i o u s c y c l o s p o r i n s differ in a m i n o acid c o m p o s i t i o n often by only o n e residue (Traber et al. T h e a m i n o acids in all positions but 3 and 8 can b e c h a n g e d . Nva. DOMBt. 1987. viz.252 Therapeutic Metabolites T A B L E 10-3 Structures of Cyclosporins Amino Acid in Position Cyclosporin 1 A MeBmt Β MeBmt C MeBmt 2 3 4 5 6 7 8 9 10 11 Abu Sar MeLeu Val MeLeu Ala D-Ala MeLeu MeLeu Ala Sar MeLeu Val MeLeu Ala D-Ala MeLeu MeLeu MeVal Thr Sar MeLeu Val MeLeu Ala D-Ala MeLeu MeLeu MeVal MeVal D MeBmt Val Sar MeLeu Val MeLeu Ala D-Ala MeLeu MeLeu MeVal Ε MeBmt Abu Sar MeLeu Val MeLeu Ala D-Ala MeLeu MeLeu Val F DOMBt Abu Sar MeLeu Val MeLeu Ala D-Ala MeLeu MeLeu MeVal G MeBmt Nva Sar MeLeu Val MeLeu Ala D-Ala MeLeu MeLeu MeVal H MeBmt Abu Sar MeLeu Val MeLeu Ala D-Ala MeLeu MeLeu D-MeVal I MeBmt Val Sar MeLeu Val MeLeu Ala D-Ala MeLeu Val Leu MeVal Κ DOMBt Sar MeLeu Val MeLeu Ala D-Ala MeLeu MeLeu MeVal L Bmt Abu Sar MeLeu Val MeLeu Ala D-Ala MeLeu MeLeu MeVal M MeBmt Nva Sar MeLeu Nva MeLeu Ala D-Ala MeLeu MeLeu MeVal Ν MeBmt Nva Sar MeLeu Val MeLeu Ala D-Ala MeLeu MeVAl 0 MeLeu Nva Sar MeLeu Val MeLeu Ala D-Ala MeLeu MeLeu MeVal Ρ Bmt Thr Ala D-Ala MeLeu MeLeu MeVal Q R MeBmt Abu Sar Ala D-Ala MeLeu MeLeu MeVal MeBmt Abu Sar MeLeu Val Leu(?) Ala D-Ala MeLeu Leu(?) MeVal S MeBmt Thr Val MeLeu Ala D-Ala MeLeu MeLeu MeVal T MeBmt Abu Sar MeLeu Val MeLeu Ala D-Ala MeLeu Leu(?) MeVal u MeBmt Abu Sar MeLeu Val Ala D-Ala MeLeu MeLeu MeVal V MeBmt Abu Sar MeLeu Val MeLeu Abu D-Ala MeLeu MeLeu MeVal w MeBmt Thr Sar MeLeu Val MeLeu Ala D-Ala MeLeu MeLeu Val X MeBmt Nva Sar MeLeu Val MeLeu Ala D-Ala MeLeu MeVal Y MeBmt Nva Sar MeLeu Val Ala D-Ala MeLeu MeLeu MeVal ζ MeOa Ala D-Ala MeLeu MeLeu MeVal Sar MeLeu Val MeLeu Sar Val Val Val MeLeu Leu Leu Abu Sar MeLeu Val MeLeu Leu Leu MeBmt. Adapted by permission of the publisher from Traber et al. Schweizerische Chemische Gesellschaft. α .3 . Chim. In addition to the naturally o c c u r r i n g c y c l o s p o r i n s about 7 5 0 semisynthetic or synthetic a n a l o g u e s h a v e b e e n p r e p a r e d and e v a l u a t e d in vitro. methylaminooctanoic acid.m e t h y l a m i n o . (1987) Helv.h y d r o x y . MeOa. Basel. 13-26. All amino acids are in L-configuration unless specified otherwise. Abu. Acta 70. D . desoxy-MeBmt. α-aminobutyric acid.4 .a m i n o b u t y r i c acid. norvaline. H o w e v e r . and 2 .

1976). 1989).500-1 fermentor.1 F e r m e n t a t i o n P r o c e s s .2 after sterilization. polysporum (Link ex P e r s . A n u m b e r of carbon sources supported as g o o d or better p r o d u c t i o n than g l u c o s e . Fifty liters of seed m e d i u m in a 75-1 fermentor w a s inoculated 9 with 5 x 1 0 conidia and aerated and agitated at o n e v o l u m e of air p e r v o l u m e of m e d i u m ( V V M ) and 2 0 0 r p m . T h e seed w a s g r o w n in t w o stages as in the s h a k e flask p r o c e s s . 1989). the last o n e being the m o s t efficient b a s e d on the specific p r o d u c t i o n rate. m a l t o s e . higher volumetric productivities w e r e o b s e r v e d with cellobiose a n d g a l a c t o s e . and F e S 0 4 .500-1 fermentor. A g a t h o s et al. E r l e n m e y e r flasks containing 100 ml of production m e d i u m w e r e inoculated with 10 ml of the i n o c u l u m d e v e l o p e d as described earlier.7 H 2 0 10 m g . M g S 0 4 . P r o d u c t i o n v o l u m e w a s 3 . and s o r b o s e . T h e fermentation w a s carried out for 12 d a y s . 0 0 0 1 in a 4. respectively. S u b s e q u e n t l y . g a l a c t o s e . Neocosmospora vasinfecta (Nakajima et al. 5 g. T h e duration of the second stage w a s 6 d a y s . After 7 2 h it w a s used to inoculate 5 0 0 1 of seed m e d i u m in a 750-1 fermentor. T h e flasks w e r e incubated at the s a m e c o n d i t i o n s as the seed flasks for 12 d a y s . ( 1 9 7 6 ) . N a N 0 3 3 g. A production p r o c e s s w a s d e scribed by Dreyfuss et al. N o explanation w a s offered for this difference n o r w a s there any discussion of the effects of e n v i r o n m e n t a l variables o n the p r o c e s s . H o w e v e r . K C l 0 . T h e rate of agitation w a s 150 r p m . fructose. T h e ratio b e t w e e n C s A and C s C s e e m e d to be l o w e r in the fermentor than in the shake flasks. ) Rifai (Dreyfuss et al. T h e production m e d i u m w a s the s a m e as in the s h a k e flask p r o c e s s .1. In s h a k e flasks the p r o d u c t i o n leveled off after 8 d a y s but in the fermentor it c o n t i n u e d albeit at a l o w e r rate to 12 d a y s . and Fusarium solani M C I . T h e p H of the m e d i u m w a s adjusted to 5. T h e aeration rate and the t e m p e r a t u r e w e r e the s a m e as described a b o v e . 5 g. T h e s e w e r e rayö-inositol. T h e p r o c e s s w a s also scaled u p to 4. T h e p r o d u c t i o n b e g a n as the g r o w t h s l o w e d d o w n and reached a titer of 1 7 0 . c e l l o b i o s e . T h e durations of the first and the second stages w e r e 7 2 and 4 8 h . w h i c h w e r e shaken at 27°C and at 2 0 0 r p m on a rotary shaker with 2-inch t h r o w . A s m e n t i o n e d in the p r e c e d i n g section c y c l o sporins w e r e d i s c o v e r e d in the fermentation broth of T. K H 2 P 0 4 2 g. (1986) studied the optimization of the p r o c e s s for the p r o d u c t i o n of C s A .m l E r l e n m e y e r flasks. In the initial 4 8 h the culture g r e w rapidly. O t h e r fungi that w e r e reported to p r o d u c e cyclosporins are Tolypocladium geodes (Kobel and T r a b e r 1982).2 0 0 mg/1. F o r s h a k e flask production the seed w a s d e v e l o p e d in t w o stages in 5 0 0 . T h i s culture w a s later reclassified as Tolypocladium inflation and a g a i n . m o r e recently. S e e d culture w a s g r o w n in a m e d i u m c o n t a i n i n g p e r liter: g l u c o s e 4 0 g.1 5 4 9 and M C I .2. T h e production m e d i u m w a s the s a m e as the seed m e d i u m e x c e p t the concentration of c a s e i n p e p t o n e w a s increased to 10 g/1. T h e kinetics of g r o w t h and production w e r e similar in the shake flasks and in the fermentor. Kinetics of c a r b o n source utilization and C s A production s h o w e d that the . respectively. c a s e i n p e p t o n e 5 g. s u c r o s e .1 Cyclosporins 253 Production 10. A e r a t i o n rate and the t e m p e r a t u r e w e r e the s a m e as before but the stirring speed w a s r e d u c e d to 100 r p m .1 5 5 0 ( S h o w a 1982). as Beauveria nivea ( L a w e n et al. T h e t e m p e r a t u r e w a s m a i n t a i n e d at 2 7 ° C .10.2 10.7 H 2 0 0 .2.

V a l . L-valine ( L V a l ) . r e s p e c t i v e l y . 1986). C s C . biotin. B y feeding D L . s o m e C s A w a s p r o d u c e d in all c a s e s . a n d p y r i d o x i n . 10. a n d v i t a m i n solution. A d d i t i o n of inorganic nitrogen sources d i d not g i v e g o o d p r o d u c t i o n . trace e l e m e n t solution.S e r a n d D L . T h e yields w e r e increased with a m i n o acid s u p p l e m e n t a t i o n in all cases e x c e p t with L .N v a ( W e n g e r et a l . C s D . S i m i l a r l y .2. T h e reason for this is that t h e broth in such fermentations is very v i s c o u s a n d p s e u d o p l a s t i c . T h e y used a defined m e d i u m c o n t a i n i n g s u c r o s e . m a l i c acid.A D U ) it w a s possible t o p r o d u c e only C s A . V i t a m i n s supplied w e r e t h i a m i n . L .p e p t o n e w a s t h e best. M o r e w o r k is n e e d e d to u n d e r s t a n d t h e r e a s o n s w h y these particular c a r b o n a n d nitrogen s o u r c e s s t i m u late t h e p r o d u c t i o n of c y c l o s p o r i n s ( A g a t h o s et a l . 1985).A b u than for the o t h e r four a m i n o a c i d s . inflatum in fermentation c a n b e affected b y feeding a p p r o p r i a t e a m i n o a c i d s . K H 2 P 0 4 . C a C l 2 . L .T h r .N v a ) resulted in p r o d u c t i o n of C s B . 10. O f t h e c o m p l e x nitrogen sources tested B a c t o . m o l y b d e n u m .254 Therapeutic Metabolites synthesis of C s A s t o p p e d as t h e c a r b o n source w a s e x h a u s t e d . p r o d u c t i o n b y a superior m u t a n t M 6 increased a l m o s t fourfold when m a l t o s e w a s t h e initial c a r b o n source ( A g a t h o s et a l .3 Production by Immobilized Cells. B o l l i n g e r et a l . T h e s e results s u g g e s t e d that either the d e n o v o synthesis of L .S e r . 1987). m a n g a n e s e . and iron.A l a . W i t h n o a m i n o acid s u p p l e m e n t a t i o n 7 7 % of t h e c y c l o s p o r i n p r o d u c e d w a s C s A a n d t h e rest w a s C s C .a . ( 1 9 8 3 ) reported synthesis of c y c l o s p o r i n s with an -allylgly. P r o d u c t i o n of C s A in c o n t i n u o u s culture w a s reported a l t h o u g h n o e x p e r i m e n t a l details w e r e given ( A g a t h o s et a l . they d e m o n s t r a t e feasibility of d e s i g n i n g n e w c y c l o s p o r i n s that will h a v e altered p h a r m a c o l o g i c a l properties such as increased activity o r r e d u c e d toxicity. o n e of t h e difficulties e n c o u n t e r e d in t h e scaleup of mycelial fermentations is a d e q u a t e transfer of o x y g e n .1. M g S 0 4 . a n d L-norvaline ( L . 1 9 8 6 ) . c y c l o s p o r i n s c o n t a i n i n g D-Ser at t h e 8-position a n d n o r v a l i n e at the 2-position w e r e p r e p a r e d b y s u p p l e m e n t i n g the m e d i u m with D . I m m o b i l i z e d cell s y s t e m s h a v e t h e potential t o e l i m i n a t e this .2 Directed Biosynthesis. A s m e n t i o n e d earlier. 1986). L .s o u r c e d e p l e t i o n .1.a l l y l g l y c i n e or D . T r a c e e l e m e n t solution p r o v i d e d sources of z i n c .t h r e o n i n e ( L . S u c h s u p p l e m e n t a t i o n with 2 % m a l t o s e i m p r o v e d t h e p r o d u c t i o n b y t h e parent threefold w h e n g l u c o s e w a s t h e initial c a r b o n s o u r c e a n d twofold with s o r b o s e as t h e initial c a r b o n source ( A g a t h o s et a l . additions of L-alanine ( L . H o w e v e r . L .a m i n o b u t y r i c acid ( D L . a n d L .2.T h r ) .A l a ) .A l a . S i m i l a r l y .N v a is rate-limiting w h e r e a s that of D L . c o p p e r . T h e yields of C s A w e r e increased b y these w o r k e r s from 5 0 m g / m l t o 5 3 7 m g / m l b y strain i m p r o v e m e n t a n d p r o c e s s d e v e l o p m e n t ( A g a t h o s et a l . S i m i l a r l y . F u r t h e r m o r e .A b u is p r o b a b l y not o r t h e affinity of t h e " s y n t h e t a s e " is h i g h e r for D L . K o b e l a n d T r a b e r ( 1 9 8 2 ) d e m o n s t r a t e d that the t y p e of c y c l o s p o r i n p r o d u c e d b y T. 1986). T h i s s u g g e s t e d t h e possibility of e x t e n d i n g t h e p r o d u c t i o n p h a s e b y t h e addition of a c a r b o n s o u r c e at the t i m e of c a r b o n .S e r ) residue in the 8-position b y feeding D .residue in t h e 2-position o r a D-serine ( D . a n d C s G .

(1989) J.1 m g C s A / g of cells in the large b e a d s y s t e m and that of 7. T h e former g r o u p used c a r r a g e e n a n to i m m o b i l i z e T. T h i s high initial loading suggested that specific activity in the i m m o b i l i z e d cell s y s t e m m a y be l o w e r than that in the free s y s t e m . large beads. C s A production in the i m m o b i l i z e d cell s y s t e m with the small b e a d s w a s higher than that with the large b e a d s w h i c h in turn w a s better than that with the free cells (Figure 1 0 .10. 7 m g of C s A / g of cells as c o m p a r e d to that of 13.2 0 7 μτη. A n o t h e r reason p r o p o s e d for the superior p e r f o r m a n c e of the i m m o b i l i z e d cell system w a s that they use a h i g h e r proportion of 300 Time (h) F I G U R E 10-6 Production of CsA by immobilized cells. T h e y studied the p r o d u c t i o n of C s A by the i m m o b i l i z e d cells in 2 5 0 . Celite g r a d e 5 6 0 b e a d s in t w o size r a n g e s w e r e u s e d . (1983) and b y C h u n and A g a t h o s ( 1 9 8 9 ) .-T. O n the other h a n d . T h e specific p r o d u c t i o n in the small b e a d s y s t e m w a s 2 7 .immobilized cells. N o data w e r e given o n the c o m p a r a t i v e cell concentration in the free and i m m o b i l i z e d s y s t e m s but the loading of the b e a d s in the i m m o b i l i z e d cell s y s t e m w a s 3 5 ^ 0 % . . found e n h a n c e m e n t of v o l u m e t r i c productivity as well as that of specific activity on i m m o b i l i z a t i o n .O .7 m g C s A / g of cells in the free cell s y s t e m ( C h u n and A g a t h o s 1989). inflatum. 9. . P r o d u c t i o n of C s A by i m m o b i l i z e d cells w a s reported by Foster et al. Synthetic m e d i u m used by K o b e l and T r a b e r ( 1 9 8 2 ) w a s modified and used as production m e d i u m . Elsevier Science Publishers. although n o c o n v i n c i n g e v i d e n c e w a s p r e s e n t e d in support of this s u g g e s t i o n .• . small beads. Biotech.Δ .m l s h a k e flasks in a batch fermentation. New York. and Agathos. C h u n and A g a t h o s ( 1 9 8 9 ) . inflatum.N. G. B e r k et al.free cells. . 237-254.immobilized cells. A n u m b e r of such s y s t e m s h a v e b e e n reported for other s e c o n d a r y m e t a b o l i t e s ( G b e w o n y o and W a n g 1983a and b . 1984).5 0 0 / i m and that of small b e a d s w a s in the r a n g e of 1 5 0 .. It w a s suggested by the authors that the e n z y m e s i n v o l v e d in the synthesis of c y c l o s p o r i n s are m o r e stable in i m m o b i l i z e d cells as c o m p a r e d to those in free c e l l s . T h e size of the large b e a d s w a s in the r a n g e of 2 9 5 .2 Cyclosporins 255 p r o b l e m . w h o used celite b e a d s to i m m o b i l i z e T. T h e y found that i m m o b i l i z e d cells h a d about the s a m e v o l u m e t r i c productivity as the free cells. S. . Cell m a s s p e r unit v o l u m e of the m e d i u m w a s h i g h e r in the large bead s y s t e m c o m p a r e d to that in the small b e a d s y s t e m or the free cell s y s t e m . Reprinted with permission of the publisher from Chun.6 ) .

6 . either with free cells or with i m m o b i l i z e d cells and h e n c e the cell-free s y s t e m c a n n o t be used for the production of C s A .m e t h y l a m i n o .H]methionine indicated that it w a s the m e t h y l d o n o r for the N . 3 / ? .m e t h y l .256 Therapeutic Metabolites c a r b o n source for the secondary metabolite production than d o the free cells.D . Precursors w e r e a d d e d 2 h after the inoculation. 5 .1 1 .m e t h y l a t i o n and methylation at position 4 of the C-9 acid as well as for the methylation of the other seven a m i n o a c i d s .N v a at C . C s A with L N v a at C-2 and C-5 and N . C s A with L-allo-Ile at C-5 and with N .l 1. 4 .e n o i c acid ( C .a l l o . w h o o p t i m i z e d the s y s t e m . It should be noted that the p r o d u c t i o n in the best o p t i m i z e d cell-free s y s t e m reported is still substantially l o w e r than that in the fermentation. T h e s e results suggested that the c a r b o n skeleton of unusual C-9 acid w a s synthesized by head to tail c o n d e n s a t i o n of 3 3 four acetate units. N o incorporation l 3 of C into any of the other a m i n o acids w a s seen.l 1. and that of H from [methyl. 3 13 l3 H from [l.o c t . T h u s . T h e s e included C s A with ^ V . T. 10. C s A with L . and 7 of C-9 13 acid w e r e enriched w h e n [ 1 . although the rate of synthesis is m a x i m u m at 2 4 ° C . C s A with D .d i m e t h y l o c t o n o i c acid at C .h y d r o x y 1 3 4 . 4 .C] methionine. T h e y found that incubation at low t e m p e r a t u r e s (6°C) for 1 w e e k resulted in h i g h e r yields of c y c l o s p o r i n s (50 /xg/ml) as c o m p a r e d to those seen at 24°C (30 / i g / m l ) .2 Biosynthesis Initial studies on the biosynthesis of cyclosporin w e r e directed t o w a r d the d e termination of the origin of the unusual ( 2 S . All w e r e found to h a v e i m m u n o s u p p r e s s a n t activity although n o n e w a s better than C s A .a m i n o b u t y r i c acid at C . C s A with L-allo-Ile at C-5 and C .3 . T h e results s h o w e d that carbon a t o m s 1.m e t h y l . 4 / c .N v a at C-5 and with yV-methyl-L-Nva at C . T h i s w o r k w a s e x t e n d e d further by L a w e n et al.( + ) .h y d r o x y .4 .8 . although the reasons for the better production by the i m m o b i l i z e d cell system are unclear at this t i m e . 1983). T h e authors exploited this cell-free system to synthesize novel c y c l o s p o r i n s w h i c h could not b e p r e p a r e d by directed b i o s y n t h e s i s . [methyl2 3 3 H 3 ] m e t h i o n i n e .I l e at C .2 .F r e e S y s t e m . 6 £ ) .a m i n o .C]acetate.l 1.3 6 0 s p e c t r o m e t e r .C ] a c e t a t e w a s used (Kobel et al.m e t h y l .C]acetate.2.L . and C s A with ß .H]methionine into C s A w a s investigated. Incorporation of H from [raei/ry/. inflatum w a s g r o w n in a c o m p l e x m e d i u m c o n t a i n i n g m a l t o s e a n d c a s e i n p e p t o n e and K H 2 P 0 4 .m e t h y l .A l a at C .C ] a c e t a t e w a s used and c a r b o n a t o m s 2 . it is a very powerful research tool to prepare n e w cyclosporins and to study their b i o s y n t h e s i s . the results did not support this suggestion u n e q u i v o c a l l y .2 .2. Cell-free e n z y m a t i c synthesis of C s A w a s first reported by Billich and Z o c h e r ( 1 9 8 7 ) . Fo r this p u r p o s e incorporation of C and 2 .2 . ( 1 9 8 9 ) . 3 . 6. it d o e s look attractive for scaleup studies as it offers the a d v a n t a g e s of increased production and r e d u c e d broth viscosity.1. T h i s w a s . [2.c h l o r o .L . 13 1 3 C .l . O n c e a g a i n . H o w e v e r .8 .3 . [methyl. and 8 13 w e r e enriched w h e n [ 2 . O n e possible explanation for these o b s e r v a tions is that the biosynthetic e n z y m e s are less stable at 24°C than at l o w e r t e m p e r a t u r e s .N u c l e a r m a g n e t i c r e s o n a n c e ( C .N M R ) spectroscopy w a s d o n e with a B r u k e r W H . 10.9 acid).4 P r o d u c t i o n in a C e l l .

G l y .d i m e n s i o n a l thin-layer c h r o m a t o g r a p h y and the c o r r e s p o n d i n g spots located by a u t o r a d i o g r a p h y w e r e scraped off for the d e t e r m i n a t i o n of radioactivity. In light of the similarities b e t w e e n these t w o c o m p o u n d s and c y c l o s p o r i n s . C s C .( D .Cyclosporins 10. it w a s suggested by Z o c h e r et al.C]methionine.L e u . L . and L . (2) formation of c y c l o .C H 3 ] m e t h i o n i n e suggested that m e t h y l transfer to C-9 acid o c c u r s via intact C H 3 u n i t s . efforts w e r e m a d e to d e t e r m i n e the p r e c u r s o r s of other a m i n o acids in C s A . After the e s t a b l i s h m e n t of the origin of the C-9 acid. m o l a s s e s m e d i u m and the m y c e l i u m w a s r e s u s p e n d e d in tap w a t e r with 14 C .p y r o p h o s p h a t e e x c h a n g e . ammonium sulfate precipitation (30-50% cut). r e m o v a l of nucleic acids with P o l y a m i n e (BASF).( D . Results 257 with 2 [ m e f / i v / .M e L e u ) c o i n c i d e d in the fraction n u m b e r s 12 to 18 from Ultrogel c h r o m a t o g r a p h y . T h e purification s c h e m e involved disruption of freeze-dried m y c e l i u m with sand in a m o r t a r .l a b e l e d a m i n o acid for 10 m i n . T h e activities c a p a b l e of catalyzing D . T h e c a u s e of this d i s c r e p a n c y m a y be the differences in the e x p e r i m e n t a l conditions used by these t w o g r o u p s . Z o c h e r et al. T h e s e results are consistent with those of K o b e l and T r a b e r ( 1 9 8 2 ) .A l a . ( 1 9 8 6 ) that c y c l o s p o r i n s are synthesized b y a m u l t i . c o u l d be selectively labeled with L . (1983) neither the m e t h y l a t i o n at 14 position 4 of the C-9 acid with L-[raei/ry/. inflatum was g r o w n in c o r n s t e e p liquor. T h e a m i n o acids tested w e r e l4 C y c l o s p o r i n s synthesized in the L .M e t is the m e t h y l d o n o r for the TV-methylation of a m i n o a c i d s .A l a . possibly b y nucleophilic attack of an appropriate e n o l a t e . 0 . 3 by 13 C incorporation from 13 [mei/iv/. 14 C . and p r e c l u d e s a p o s s i b l e m e c h a n i s m via c y c l o p r o p a n e or m e t h y l e n e intermediate ( K o b e l et al. and L-[methyl.M e L e u ) .A l a .C]Met. In contrast to the results reported by K o b e l et al. 1983). and size Ρ exclusion c h r o m a t o g r a p h y on an Ultrogel A c A 2 2 c o l u m n . C y c l o s p o r i n s contain unusual a m i n o acids and N . 1983) that L .P P j e x c h a n g e and formation of c y c l o . 1984). T h e exl4 p e r i m e n t s with L-[methyl. T o d o this T.C]Met confirmed the earlier observation ( K o b e l et al. T h e a m i n o acids in the h y d r o l y s a t e w e r e separated with t w o . C-9-acid d e p e n d e n t .d e p e n d e n t A T P .2 confirmed .A l a .a m i n o b u t y r i c acid.l a b e l from each p r e c u r s o r a m i n o acid w a s found exclusively in that constituent a m i n o acid. D L .m e t h y l a t e d p e p t i d e b o n d s w h i c h are also present in the fungal d e p s i p e p t i d e s enniatin and b e a u v e r i c i n .L e u .7 .V a l .d e p e n d e n t . and (3) TV-methylation of c o n stituent a m i n o a c i d s . s u s p e n s i o n s y s t e m w e r e isolated and h y d r o l y z e d with 6 Ν H C l . T h e o p t i m u m p H for the activation of a m i n o acids and for the formation of cyclic dipeptide w e r e respectively in the r a n g e of 7 . T h i s fraction w a s c a p a b l e of forming covalent enzyme-substrate c o m p l e x e s and c a t a l y z e d ( 1 ) constituent a m i n o acid d e p e n d e n t A T P .[ C ] T h r ( Z o c h e r et al.e n z y m e c o m p l e x from p r e c u r s o r a m i n o acids as are these t w o (Kleinkauf and v o n D o h r e n 1987). nor g o o d incorporation of 14 [ C ] a c e t a t e into the C-9 acid w a s o b s e r v e d . (1986) reported five-hundred-and-forty-fold purification of an e n z y m e fraction from T. 3 .T h r in place of 1 4 α . T h e next step in the elucidation of the biosynthetic p a t h w a y w a s the isolation and the characterization of the e n z y m e s involved.C]Met. w h o d e m o n s t r a t e d incorporation of u n l a b e l e d a m i n o acids into c y c l o s p o r i n s . w h i c h contains L .m e t h y l a t i o n and peptide b o n d formation. inflatum. B a s e d o n this h y p o t h e s i s it can b e postulated that the biosynthetic s c h e m e involves activation of constituent a m i n o acids followed by N .

1986) to that with m a l t o s e and casein p e p t o n e . T h e a u t h o r s . and constituent a m i n o acids (Billich and Z o c h e r 1987). L . TV-Methylation of constituent a m i n o acids b y this e n z y m e fraction required S-adenosyl m e t h i o n i n e ( S A M ) . Further characterization of the c o m p l e x s h o w e d its m o l e c u l a r m a s s to b e 7 0 0 k D a . F u r t h e r m o r e . L . nivea w h i c h w a s a superior p r o d u c e r of C s A in s u b m e r g e d cultures than that used by Billich and Z o c h e r ( 1 9 8 7 ) . different from the o n e used in the studies described in the p r e v i o u s p a r a g r a p h . 1986). 4 .m e t h y l a t e d a m i n o acids or the a m i n o acids that are not present in c y c l o s p o r i n s . Dissociation studies with performic acid s h o w e d that the N . Isolation of the active fraction involved a m m o n i u m sulfate precipitation and gel filtration on Ultrogel A c A 3 4 . iodoacetic acid. di- thioerythritol ( D E T ) . T h e y p r e p a r e d their e n z y m e fraction from a strain of B. w a s d e m o n s t r a t e d by Billich and Z o c h e r ( 1 9 8 7 ) . T h e formation of an e n z y m e a m i n o acid c o m p l e x w a s d e m o n s t r a t e d with 14 C-labeled amino acids. 1986) w a s replaced with Tris buffer with K C l . T h e y c h a n g e d the m e d i u m used to g r o w the seed from that c o n t a i n ing c o r n steep liquor and m o l a s s e s ( Z o c h e r et al. T h e results described so far strongly suggested that this e n z y m e fraction w a s involved in the b i o s y n t h e s i s of c y c l o s p o r i n s .C]SAM.A l a . 1986). D E T w a s found to stabilize the C s A synthesizing activity. 1 .2 . or i o d o a c e t a m i d e p r e v e n t e d the c o m p l e x formation b e t w e e n the e n z y m e and the a m i n o a c i d s . T h e e n z y m e fraction failed to activate N . h o w e v e r . T h e l o w e r m o l e c u l a r m a s s e n z y m e fraction catalyzed A T P . M g 2 + . 0 . 2 . T h e s e results suggested i n v o l v e m e n t of different e n z y m e s / s u b u n i t s in catalyzing these t w o reactions ( Z o c h e r et al.N v a w e r e substituted for L . with this fraction synthesis of C s A w a s not observed ( Z o c h e r et al. and glycerol. did not indicate the extent to w h i c h this e n z y m e activity w a s purified (Billich and Z o c h e r 1987). w e t m y c e l i u m w a s used the e n z y m e fraction isolated had m o l e c u l a r m a s s of 2 0 0 k D a . ( 1 9 8 9 ) . N-ethylmaleimide. . This complex c o u l d be cleaved with performic acid but not with formic acid. or L . 5 .c h l o r o m e r c u r y benzoate. e t h y l e n e d i a m i n e tetraacetic acid ( E D T A ) . inflatum.m e t h y l a t i o n o c c u r s w h i l e the a m i n o acid is b o u n d to the e n z y m e by thioester l i n k a g e . p h e n y l m e t h a n e s u l f o n y l fluoride ( P M S F ) .P P j e x c h a n g e d e p e n d e n t on constituent a m i n o acids but w a s not c a p a b l e of formation of cyclic d i p e p t i d e . novel c y c l o sporins w e r e synthesized w h e n L . with this e n z y m e fraction it w a s p o s s i b l e to d e m o n strate the formation of the naturally occurring cyclosporins such as C s A . T h i s w a s supported b y the observation that thiol g r o u p blocking compounds such as / 7 .a m i n o b u t y r i c acid in the reaction m i x t u r e . w h i c h s u g g e s t e d the p r e s e n c e of thioester l i n k a g e . Interestingly. T h e p h o s p h a t e buffer used previously for the extraction of m y c e lium ( Z o c h e r et al. In vitro synthesis of C s A and other h o m o l o g o u s c y c l o s p o r i n s by an e n z y m e fraction isolated from a strain of T. C y s B . if instead of freeze-dried m y c e l i u m . T h i s e n z y m e catalyzed formation of C s A w h e n incubated with A T P . h o w e v e r .T h r .9 . M g C l 2 . T h e potential of a cell-free system for the p r o d u c t i o n of novel c y c l o s p o r i n s w a s e x p l o r e d further b y L a w e n et al. A s m e n t i o n e d in section 1 0 .258 Therapeutic Metabolites and 9 . T h e p r o c e d u r e s used b y these w o r k e r s w e r e similar to those of Billich and Z o c h e r ( 1 9 8 7 ) .V a l . fraction l4 [methyl. w h i c h led the authors to suggest the possibility of using an e n z y m e reactor for the production of c y c l o s p o r i n s (Billich and Z o c h e r 1987). F o r the isolation of the e n z y m e the a b o v e m e n t i o n e d p h o s p h a t e buffer w a s replaced with Tris buffer c o n t a i n i n g D E T and g l y c e r o l .

b o u n d a m i n o acids and catalyzed formation of the p o l y p e p t i d e c h a i n .5 0 % (saturation) ( N H 4 ) 2 S 0 4 involved precipitation. It is clear that the c o n s i d e r a b l e p r o g r e s s m a d e in u n d e r s t a n d i n g the biosynthesis of c y c l o s p o r i n s is d u e to the excellent w o r k d o n e by Z o c h e r and his c o l l e a g u e s . S t r u c t u r e . F o r . 3 0 . Cyclosporins 259 C y s D . Initial studies established that C s A inhibited the activation of Τ l y m p h o c y t e s by various m i t o g e n s and allo-antigens ( H e s s et al. It is a p o l y p e p t i d e chain with a m o l e c u l a r m a s s of 8 0 0 k D a . I L . (2) inhibition of I L .A l a ] C s A .a l l y l g l y c i n e ] C s A . antifungal.2 CsC.5 5 c o l u m n c h r o m a t o g r a p h y . for e x a m p l e . it m a y be possible n o w to apply m o d e r n m o l e c u l a r biological t e c h n i q u e s to the study of the genetics of cyclosporin synthesis. h e n c e . those p r e p a r e d by directed b i o s y n t h e s i s . 10. 1 Effect o n Τ a n d Β L y m p h o c y t e s . is d esi g n at ed as " m u l t i e n z y m e p o l y p e p t i d e " ( L a w e n and Z o c h e r 1990). 2 . interferon 7 . Fractogel H W . ( 4 ) inhibition of activation of Β l y m p h o c y t e by a n t i . T h e purification s c h e m e p o l y e t h y l e n e i m i n e precipitation.d e p e n d e n t clonal amplification.m e d i a t e d and h u m o r a l i m m u n i t y . Investigations in tissue culture s y s t e m s h a v e c o n f i r m e d this c o n c l u s i o n and h a v e e n h a n c e d o u r u n d e r s t a n d i n g of the action of c y c l o s p o r i n s . 1 0 . T h e s e effects are briefly described in s u b s e q u e n t s e c t i o n s .D . L a w e n a n d Z o c h e r ( 1 9 9 0 ) w e r e finally able to purify c y c l o s p o r i n synthetase to n e a r .2 synthesis.i m m u n o g l o b u l i n M ( I g M ) antibodies. for e x a m p l e .5 0 % ) centrifugation and resulted in 72-fold purification. and antiparasitic activities.m e t h y l a t e d appropriate t h i o e s t e r . 1982). Studies in w h o l e a n i m a l s s h o w e d that C s A s u p p r e s s e d b o t h c e l l .10. 2 [ L . It is n o w k n o w n that C s A has the following effects: (1) inhibition of proliferation of cy to to x ic Τ l y m p h o c y t e s ( C T L ) via m o d u l a t i o n of interleukin 2 (IL-2) r e c e p t o r o n p r e c u r s o r C T L and inhibition of I L . m a c r o p h a g e c h e m o t a c t i c factor. and C y s G . N .3 Mode of Action Cyclosporins have immunosuppressive. (5) n o inhibition of s u p p r e s s o r Τ l y m p h o c y t e activation.c 7 z / < ? A O . Purification and characterization of this e n z y m e has e n h a n c e d o u r k n o w l e d g e of n o n r i b o s o m a l p ep tid e synthesis in filamentous fungi. and glycerol gradient ( 2 5 . I L . It is i m p o r t a n t to note that while it carried out multiple catalytic functions it is a single p o l y p e p t i d e a n d . and m i g r a t i o n inhibition factor. 3 . I L .4 . T h e extent of inhibition w a s d e p e n d e n t o n the m i t o g e n u s e d .5 .2 . It activated all constituent a m i n o acids of c y c l o s p o r i n . (3) inhibition of synthesis of o t h e r l y m p h o g n e s . (6) alteration of p h y s i o l o g y and function of m o n o c y t e s and m a c r o p h a g e s resulting in i m p a i r m e n t in their ability to p r o c e s s and present antigens. i m m u n o s u p p r e s s i v e action of c y c l o s p o r i n s has b e e n m o s t extensively investigated and yet its m e c h a n i s m is not fully u n d e r s t o o d . respectively. R e a d e r s interested in m o r e information on these topics are referred to t w o excellent b o o k s edited by W h i t e (1982) and by T h o m s o n ( 1 9 8 9 ) .a c t i v i t y relationship studies h a v e suggested that the m e c h a n i s m of e a c h of these activities m a y be different (Borel 1989).2.3 .h o m o g e n e i t y from the s a m e o r g a n i s m . and (7) stimulation of p r o s t a g l a n d i n synthesis. a n d those that w e r e not m a d e with directed biosynthesis such 8 as [ ß . In addition. Of these t h r e e .

Similar results w e r e obtained by Ryffel et al. . 1983). 1981). IL-5 (Granelli-Piperno et al. m o r e information is n e e d e d to u n d e r s t a n d fully the effect of C s A on IL-2 r e s p o n s i v e n e s s of p r e c u r s o r C T L (Hess et al. H e s s et al. T h e significance of these and other similar observations is that they indicate that C s A m a y be useful in suppressing the r e s p o n s e of sensitized individuals (Hess et al.5 .260 Therapeutic Metabolites e x a m p l e . Activation and proliferation of C T L w e r e inhibited by C s A (Borel 1976). 1986. migration inhibition factor. T h e r e f o r e . 1982).i n d u c e d Τ l y m p h o c y t e d e v e l o p m e n t and of I L . 1986).s t i m u l a t e d h u m a n l y m p h o c y t e s although it did r e d u c e the e x p r e s sion of other activation antigens such as class II major histocompatibility a n t i g e n . IL-2 production is quite sensitive to C s A . H o w e v e r . It is important to note that C s A did not inhibit the activation of s u p p r e s s o r Τ l y m p h o c y t e s . C s A w a s found to prevent IL-2 production by previously sensitized cells w h e n restimulated with antigen or m i t o g e n ( A n d r u s and Lafferty 1981).1 . IL-2 is also called Τ cell g r o w t h factor b e c a u s e it is involved in Τ cell g r o w t h regulation. T h e latter w o r k e r s d e m o n s t r a t e d in h u m a n p r i m a r y m i x e d l y m p h o c y t e reaction that C s A did not inhibit the ability of the cells from these cultures to suppress in either antigen-nonspecific or specific m a n n e r d e p e n d ing on the d o s e of C s A used. 1981). ( 1 9 8 5 ) . ( 1 9 8 3 ) . In contrast. F o r C T L r e s p o n s e it is necessary to activate precursor C T L w h i c h m u s t acquire I L . T h e c a u s e of this difference is believed to be the nature of the signal delivered to the cell and not a characteristic of the cell ( K a y 1989). 1986). T h e inhibition of I L . T h u s .2 receptors ( L a r s s o n 1980. in both these studies proliferation of C T L w a s inhibited by C s A . It is o n e of the l y m p h o k i n e s p r o d u c e d by Τ helper l y m p h o c y t e s ( T h l ) ( H o d e s 1989). S u b s e q u e n t studies on the effect of C s A d e m o n s t r a t e d that C s A also inhibited production of other l y m p h o k i n e s such as IL-1 (Bunjes et al. M i y a w a k i et al. Interestingly e n o u g h . IL-4 (Granelli-Piperno et al. It w a s s h o w n that C s A inhibited the production of IL-2 (Bunjes et al. activation by a n t i . h o w e v e r .3 ( O r o s z et al.2 0 ng/ml can c a u s e 5 0 % inhibition and 5 0 ng/ml can c o m p l e t e l y suppress it (Hess 1989). 1984). F u r t h e r m o r e .2 r e c e p tors and u n d e r g o clonal amplification. w h o used a m o n o c l o n a l antibody ( T A C ) . for w h i c h a specific antigen w a s required.C D 3 m o n o c l o n a l antibody O K T 3 w a s m o r e sensitive to C s A inhibition than that by lectin c o n c a n a v a l i n A (Con A) and that in turn w a s m o r e sensitive than that by p h y t o h e m a g g l u t i n i n A ( P H A ) ( K a y and B e n z i e 1983 and 1986). and m a c r o p h a g e c h e m o t a c t i c factor ( T h o m s o n 1983a and b ) .or C o n Α . 1988). M o h a g h e g h p o u r et al. it does not act as a m i t o g e n (Hess et al. 1983). T h e s e observations w e r e followed by similar results from other laboratories w h i c h studied this p h e n o m e n o n in different s y s t e m s ( H e s s et al.d e p e n d e n t Β l y m p h o c y t e proliferation are s o m e of the effects of inhibition of l y m p h o k i n e p r o d u c t i o n . 1 0 . Further studies established that C s A had different effects on the various subsets of Τ l y m p h o c y t e s . C s A did not suppress either the activation of a regulatory subset of Τ h e l p e r cells w h i c h are responsible for amplification of suppressor Τ l y m p h o c y t e s or the activation and proliferation of suppressor Τ cells (Hess and T u t s c h k a 1980. 1986). it did not induce their function. found that C s A did not inhibit T A C e x p r e s s i o n o n P H A . interferon y ( R e e m et al. H e s s 1989). I L . 1983). C s A w a s found to block the acquisition of r e s p o n s i v e n e s s to IL-2 and it w a s believed to b e d u e to the a b s e n c e of I L .

A l t h o u g h these reports s h o w e d that C s A inhibited activation or proliferation of Β cells. 1983). stimulation of proliferation of preactivated Β cells b y eosinophil differentiation factor/B cell g r o w t h factor II ( E D F / B C G F II) w a s sensitive to C s A . t h y m u s . Chemotaxis. the action of C s A w a s d e p e n d e n t on the m i t o g e n u s e d . T h u s . 1986. O n the other h a n d .d e p e n d e n t ( T D ) r e s p o n s e to k e y h o l e limpet h e m o c y a n i n ( K L H ) w a s inhibited in b o n e m a r r o w transplant patients treated with C s A w h e r e a s n o inhibition of t h y m u s . there w e r e other studies with other m i t o g e n s w h i c h s h o w e d that in those s y s t e m s C s A did not h a v e any effect ( M o t t a and Truffa-Bachi 1989). it w a s found that m a c r o p h a g e p h a g o c y t o s i s . w h o d e m o n s t r a t e d susceptibility of t h y m u s i n d e p e n d e n t antigen 2 (TI-2) responses to C s A in m o u s e . 1 0 . V a r e y et al. Studies on irradiated spleen cells pulsed with antigen in the p r e s e n c e of C s A indicated that these cells had lost their ability to present the antigen to Τ cells ( M a n c a et al. E x t e n s i o n of these studies to h u m a n Β l y m p h o c y t e s s h o w e d that their activation b y a n t i . stimulation of Β cells w a s found to b e b l o c k e d by C s A ( T o s a t o et al. A n t i g e n presenting cells ( A P C ) or a c c e s sory cells are required for the stimulation of l y m p h o c y t e s by a n t i g e n s . In the c a s e of C s A action o n m a c r o p h a g e s .μ antibody w a s sensitive to C s A only in the first 2 4 h of the c u l t u r e . K n i g h t and Bedford 1987). after w h i c h the cells b e c a m e refractory to the effect of C s A ( M a r a g u c h i et al. 1 0 .F i c o l l ) w a s seen ( A m l o t et al. 3 . 2 . 3 Effect o n H u m o r a l I m m u n i t y . 1986). T h e s e w o r k e r s further s h o w e d that a c c u m u l a t i o n of antigen by these cells w a s r e d u c e d on in v i v o administration of C s A to m i c e ( K n i g h t et al. Similarly. 1986). w h i c h b e l o n g to another class of accessory cells. 2 Effect o n A c c e s s o r y C e l l s . A l t h o u g h it can be safely said that u n d e r certain c o n d i t i o n s C s A m o d u l a t e s the h u m o r a l r e s p o n s e the literature is full of conflicting r e p o r t s . H o w e v e r . In a n o t h e r s y s t e m . T h e dendritic cells from rabbits and m o u s e w e r e u n a b l e to function as accessory cells w h e n incubated with C s A ( K n i g h t et al. with p o k e w e e d m i t o g e n ( P W M ) . T h e s e cells p r o c e s s antigen m o l e c u l e s and present their i m m u n o g e n i c epitopes to appropriate l y m p h o c y t e s . 1982). antibodies w a s sensitive to C s A ( D o n g w o r t h and K l a u s 1982). and the synthesis as well as the excretion of e n z y m e s such as p l a s m i n o g e n activator and l y s o z y m e w e r e not affected ( W e i s i n g e r and Borel 1979).10. 1988). w h e n patients with c h r o n i c uveitis w h o w e r e being treated with C s A w e r e chal- . their accessory functions w e r e found to be impaired by the addition of C s A as d e t e r m i n e d b y m i t o g e n . 1985. T h e Β l y m p h o c y t e proliferation i n d u c e d b y anti-μ. 2 . O t h e r w o r k e r s h a v e s h o w n that C s A b l o c k e d antigen oresentation by m a c r o p h a g e s ( M a n c a et al. 1983). 1985). 3 . T h i s w a s s h o w n not to b e c o m p l e t e l y true by K u n k l and K l a u s ( 1 9 8 0 ) . F o r e x a m p l e .i n d u c e d proliferation ( U y e m u r a et al.2 Cyclosporins 261 It w a s m i s t a k e n l y c o n c l u d e d from the early studies in w h i c h L P S w a s used to i n d u c e Β cell proliferation that C s A did not h a v e any effect on the activation and proliferation of Β l y m p h o c y t e s (Borel 1976).i n d e p e n d e n t (TI) r e s p o n s e to dinitrophenyl Ficoll ( D N P .

A s m e n t i o n e d earlier in this p a r a g r a p h . in h u m a n v o l u n t e e r s C s A h a d n o effect on the response to the T I class 2 antigen D N P . 1989) provided e v i d e n c e that c y c l o p h i lin is a p e p t i d y l . erythrocyte (Foxwell et al. A l t h o u g h these studies strongly suggested that cyclophilin w a s inv o l v e d in the action of C s A . forms of this protein in t h y m o c y t e s . 1989. 1 0 . C o n v e r s e l y . W i t h particulate antigens such as vesicular stomatitis virus it w a s found that C s A did not h a v e any effect on the early I g M production but it eliminated the later I g G r e s p o n s e ( C h a r a n et al. 3 . T h e y h a v e similar C s A . T h e apparent m o l e c u l a r m a s s of the C s A . major and m i n o r . I g M antibody production w a s normal (Palestine et al. S u b s e q u e n t l y . 1984). 1989. T w o later reports (Fischer et al. the t y p e of antigen. T h i s m a c r o m o l e c u l e w a s identified as a protein and w a s purified first from b o v i n e t h y m o c y t e s and n a m e d cyclophilin ( H a n d s c h u m a c h e r et al. 1987). 2 . h u m a n peripheral blood l y m p h o c y t e s from volunteers p r e v i o u s l y i m m u n i z e d with K L H or tetanus toxoid (TT) and subsequently treated with C s A w e r e not able to p r o d u c e I g M or I g G antibodies w h e n c h a l l e n g e d by either of the t w o antigens (Harley and Fauci 1983).F i c o l l . 1988). T h e dissociation constant 8 7 for C s A is in the r a n g e 1 0 " . T h e y h a v e nearly identical a m i n o acid c o m p o s i t i o n and the exact difference b e t w e e n t h e m is not k n o w n . the exact m e c h a n i s m of its action r e m a i n s to be elucidated. C s A w a s concentrated in the cytosol and it a p p e a r e d to be associated with a m a c r o m o l e c u l e . H a n d s c h u m a c h e r et al. T h e latter w o r k e r s d e m o n s t r a t e d that cyclophilin binds to the region of cyclosporin involved in its i m m u n o s u p p r e s sive activity.000 to 2 0 .b i n d i n g activity of about a m o l e of C s A per m o l e of the protein. it w a s also isolated from calf spleen ( Q u e s n i a u x et al. 1984.262 Therapeutic Metabolites lenged with K L H .m a c r o m o l e c u l e c o m p l e x w a s 15. T a k a h a s h i et al. 1986). 1985). It w a s o b s e r v e d by M e r k e r and H a n d s c h u m a c h e r (1984) that in a m u r i n e t h y m o m a cell line. T h u s . the exact nature of its role w a s not clear. 4 R o l e of C y c l o p h i l i n a n d M e c h a n i s m of A c t i o n . Q u e s n i a u x et al. 1988). T h e picture of the effect of C s A on the TI h u m o r a l r e s p o n s e is not clear. 1989). pig k i d n e y (Fischer et al. (1984) s h o w e d that there w e r e t w o . T h e y found the a m i n o acid . F u r t h e r m o r e . A l t h o u g h the targets of C s A are well k n o w n . only those c y c l o s p o r i n s that inhibited the m i x e d l y m p h o c y t e reactions w e r e able to bind to cyclophilin ( H a n d s c h u m a c h e r et al. and Saccharomyces cerevisiae ( T r o p s c h u g et al. T h e r e are a n u m b e r of h y p o t h e s e s to explain the effects of C s A and I will briefly describe t h e m . this sensitivity w a s d e p e n d e n t on the time of C s A administration. it s e e m s that the effect of C s A on the h u m o r a l r e s p o n s e is d e p e n d e n t on the species of the animal tested. F u r t h e r m o r e . B W 5 1 4 7 . 1987). Neurospora crassa ( T r o p s c h u g et al. T a k a h a s h i et al. and the time of its use and the type of r e s p o n s e . 0 0 0 D a as d e t e r m i n e d by gel filtration c h r o m a t o g r a p h y . S e c o n d a r y r e s p o n s e to T D antigens in m i c e w a s insensitive to C s A ( K u n k l and K l a u s 1980). A s far as the s e c o n d a r y r e s p o n s e to the T I antigens is c o n c e r n e d it w a s found to be unaffected by C s A (Motta and Truffa-Bachi 1989).1 0 ~ .p r o l y l cis-trans i s o m e r a s e ( P P I a s e ) . w h i c h indicated its ubiquitous n a t u r e . 1989). In m i c e C s A w a s able to suppress the p r i m a r y r e s p o n s e to T I class 2 antigen but not to TI class 1 antigen ( M o t t a and Truffa-Bachi 1989).

T h i s is followed b y sustained elevation of 2+ the c y t o p l a s m i c C a c o n c e n t r a t i o n . T h i s s e c o n d p h a s e is essential for successful activation of Τ and Β cells. 5 . O n e p o s s i b l e explanation for these o b s e r v a t i o n s is that N F . O n e of the p r o p o s e d m e c h a n i s m s of 2+ c y c l o s p o r i n action involves C a as a m e d i a t o r . T h e other g r o u p w a s m o r e cautious in assigning a role to cyclophilin in the i m m u n o s u p p r e s s i o n by C s A (Fischer et al. or b o t h . It has b e e n p r o p o s e d that C s A inhibits IL-2 and γ-interferon p r o d u c t i o n d u r i n g T . activation p r o t e i n . T h e inhibition of the P P I a s e activity by C s A prevents these e v e n t s from t a k i n g p l a c e . C a l m o d u 2 + lin is a m e d i a t o r of m a n y effects of C a . h o w e v e r . 4 . 1989). Diacylglycerol activates protein k i n a s e C 2+ and i n o s i t o l . it is not possible to d e t e r m i n e 2 + w h e t h e r it affects the u p t a k e of C a . n u c l e a r factor of activated Τ cells ( N F . T h e concentration r a n g e of C s A required for this inhibition correlated with that n e e d e d for the inhibition of Τ cell activation.3 ) . and that P P I a s e (cyclophilin) activity is n e e d e d for this refolding. This indicated that C s A interfered with s o m e p h y s i o l o g i c process essential for the a p p e a r a n c e of the activity. ( 1 9 8 9 ) noted the s a m e d e p e n d e n c e of both catalytic and b i n d i n g activities on sulfhydryl g r o u p s as well as the inhibition of P P I a s e activity and that of acceleration of protein folding.t r i p h o s p h a t e ( K a y 1989). 1989). T h e y s u g g e s t e d that P P I a s e m a y not only facilitate protein folding but also m a y b e involved in a signal transduction p r o c e s s through cis-trans isomerization of the partner m o l e c u l e s . C s A 2 + p r e v e n t s the transient elevation of C a . to Τ cell activation g e n e s ( E m m e l et al.1 . c a l m o d u l i n .10. 1989). 1989).t r i p h o s p h a t e increases the intracellular C a pool transiently b y 2+ releasing C a from intracellular stores. M o r e studies o n t h e s e reactions will h e l p to establish the exact m e c h a n i s m of C s A action. T h e y did not think that there w a s e n o u g h e v i d e n c e to c o n c l u d e that C s A b i n d i n g to cyclophilin w a s involved in i m m u n o s u p p r e s s i o n .m e d i a t e d h y d r o l y s i s of p h o s p h a t i d y l i n o s i t o l b i s p h o s p h a t e to g e n e r a t e diacylglycerol and i n o s i t o l .3 (or a protein n e c e s s a r y for their activity) require refolding for D N A b i n d i n g or transcriptional a c t i v a t i o n .l y m p h o c y t e activation via m o d u l a t i o n of P P I a s e activity ( T a k a h a s h i et al.3 ( A P . It had b e e n o b s e r v e d that activation of both Τ and Β cells b y antigens or m i t o g e n s is initiated by r e c e p t o r .t e r m i n a l s e q u e n c e of 38 a m i n o acids of pig P P I a s e w a s identical to cyclophilin from h u m a n spleen and b o v i n e t h y m u s .1 . 1989). T h e y also o b s e r v e d that the N .A T ) . w h i c h in turn p r e v e n t s the activation of Τ cells ( E m m e l et al. w h i c h is a result of influx of extracellular 2 + C a . F u r t h e r studies indicated that C s A did not act primarily by inhibiting the u p t a k e but rather by b l o c k i n g the cells from r e s p o n d i n g .2 Cyclosporins 263 s e q u e n c e of p o r c i n e P P I a s e to be identical to b o v i n e cyclophilin and P P I a s e w a s inhibited by C s A ( T a k a h a s h i et al.k B ) .1 0 ~ ( Q u e s n i a u x et al.A T and A P . 4 . A n o t h e r u b i q u i t o u s p r o t e i n . 5 . A m o r e recent article s h o w e d that C s A specifically inhibited b i n d i n g of n u c l e a r p r o t e i n s . and nuclear factor k of Β cells ( N F . F o r C s A to inhibit the b i n d i n g it w a s essential that it b e a d d e d to the J u r k a t cells d u r i n g activation and prior to the preparation of the extracts for the a s s a y . 1987). T h e P P I a s e activity is affected by sulfhydryl b l o c k i n g r e a g e n t s as is the b i n d i n g of C s A to cyclophilin. also binds with c y c l o s p o r i n s with 7 6 dissociation constants in the r a n g e of 1 0 ~ . C s A w a s ineffective if a d d e d to the b i n d i n g reaction of the cell extract p r e p a r e d from the cells stimulated in the a b s e n c e of C s A . F i s c h e r et al.

r h e u m a t o i d arthritis.4 Clinical Use C s A is accepted today as the first-line treatment in transplantation (Borel 1989). w h e t h e r h a p l o t y p e w a s m a t c h e d or m i s m a t c h e d .2. the survival w a s i m p r o v e d to 8 9 . C s A w a s also effective in preventing graft-versus-host d i s e a s e . A s m e n t i o n e d a b o v e c a l m o d u l i n is involved in the mediation of C a signal and it w a s suggested that C s A binding of c a l m o d u l i n interfered with this role ( C o l o m b a n i et al. H o w e v e r . called posterior uveitis (Forrester et al. at this time the exact m e c h a n i s m of C s A activity is not fully elucidated. r e s p e c t i v e l y . there is rapid patching and c a p p i n g of C o n A receptors on the Τ l y m p h o c y t e surface w h i c h is c a l m o d u l i n d e p e n d e n t and is affected by p h e n a t h i a z i n e inhibitors of c a l m o d u l i n but not by C s A ( M i z u s h i m a et al. T h e rate. at the end of 1 year (Jones and C a t t o 1989). H o w e v e r .c y c l i c nucleotide p h o s p h o d i e s t e r a s e by c a l m o d u l i n in b o v i n e brain. T h e success of heart transplants d u e to the use of C s A has e n c o u r a g e d attempts to multiple organ transplant such as heart/liver transplants in patients suffering from atherosclerosis c a u s e d by hyperc h o l e s t e r o l e m i a and heart/lung transplants (Jones and Catto 1989).i m m u n e diseases such as intra-ocular inflammatory d i s e a s e . insulin- . S e c o n d . This suggestion w a s based on their results. 1982). In the transplants with k i d n e y s from living d o n o r s .3 5 % with c o n v e n t i o n a l i m m u n o s u p p r e s s i o n with a z a t h i o p r i n e . 1987).d o s e p r e d n i s o l o n e (Starzl et al. psoriasis. Clinical uses of C s A in areas other than organ transplantation include t r e a t m e n t of various a u t o .7 0 % with C s A and l o w . w a s increased 6 0 . 1985). c a l m o d u l i n plays in i m m u n o s u p p r e s s i o n by C s A . 1983). Lise of C s A in h u m a n b o n e m a r r o w transplantation in adults from H L A identical sibling has r e d u c e d the incidence of early graft failure to about 1 0 % ( A t k i n s o n 1989). T h e first use of C s A in c a d a v e r kidney transplant w a s reported by C a l n e and his c o . p r e d n i s o l o n e . 5 ' . c a l m o d u l i n binds all cyclosporins equally well regardless of their i m m u n o s u p p r e s s i v e activity ( Q u e s n i a u x et al. it is not clear w h a t r o l e . 2 0 % o v e r the control at the end of 1 and 2 y e a r s . w h i c h s h o w e d ? a m o n g other effects that C s A directly inhibited activation of 3 .264 Therapeutic Metabolites 2+ to the signal. these studies h a v e helped in analyzing the p r o c e s s of l y m p h o c y t e activation and h a v e e n h a n c e d our understanding of it. it could not be c o n firmed by other w o r k e r s . 1979a and b) and since then it has been used extensively. in the cardiac transplants treated with C s A ( O y e r et al. A d r a m a t i c i m p r o v e m e n t in graft survival w a s obtained with C s A in liver transplants (Jones and C a t t o 1989). C s A also m a d e it possible to use c a d a v e r i c d o n o r s with ages b e l o w 6 y e a r s . the survival of the retransplant recipients w a s greatly i m p r o v e d by C s A (Jones and Catto 1989). L a r g e multicenter trials in E u r o p e and C a n a d a with C s A s h o w e d i m p r o v e m e n t in the renal graft survival from 5 2 % to 7 2 % and 6 4 % to 8 0 % . 1989). 10.9 0 % . 1987). T h e r e f o r e . if a n y . which w a s 3 0 . In s o m e cases w h e r e retransplantation w a s n e e d e d d u e to the technical or p r i m a r y graft failure. and a n t i l y m p h o c y t e g l o b u l i n . T h u s . A followup study c o n d u c t e d b y S h u m w a y s h o w e d a substantial i m p r o v e m e n t in the graft survival. L a s t l y .w o r k e r s in 1979 (Calne et al.

p u l m o n a r y e d e m a . ) and contain the characteristic tetracyclic ergoline ring s y s t e m . Borel 1989). these side effects can b e m i n i m i z e d to s o m e extent b y l o w e r i n g the d o s e of C s A . It is quite e v i d e n t that C s A is a very effective d r u g available to m e d i c a l science t o d a y . F u r t h e r m o r e . C s A has fulfilled the p r o m i s e s h o w n in the initial studies and has contributed significantly to the reduction in h u m a n morbidity and mortality. p e r m a n e n t d a m a g e is o b s e r v e d . Both a c u t e . T h i s list is b y n o m e a n s c o m p l e t e and m o r e c o n d i t i o n s are a d d e d to it e v e r y d a y . nephrotoxicity b e i n g the m o s t n o t a b l e . T h e i r n a m e suggests their basic nature. it m a y b e possible to d e v e l o p a cyclosporin a n a l o g u e that has the s a m e i m m u n o s u p p r e s s i v e profile as C s A yet r e d u c e d toxicity. is an increase in b l o o d p r e s s u r e (Thiru 1989). Hepatotoxicity w a s noticed in the pilot studies w h e r e the d o s e of C s A w a s high ( C a l n e et al. M o r e than 4 0 alkaloids h a v e b e e n isolated from ergot and h u n d r e d s of derivatives h a v e b e e n c h e m i c a l l y p r e p a r e d from t h e m . T o distinguish t h e m from simple a m i n e s they are defined as c o m p o u n d s w h i c h contain nitrogen in a heterocyclic ring. O n the w h o l e . 1981). and systemic lupus e r y t h e m a t o s u s (Graffenried et al. and gastrointestinal s y m p t o m s and gingival h y p e r p l a s i a (Thiru 1989. It w a s s u g g e s t e d b y C a l n e et al. H y p e r t r i c h o s i s on the face and u p p e r trunk affected 8 0 % of the patients treated with C s A (Thiru 1989). C o a r s e n i n g and thickening facial features w a s seen in recipients of m a r r o w allografts w h o received C s A ( A t k i n s o n 1989). d o s e . 1989). J o n e s and C a t t o 1989). 1989). Initial c o n c e r n c a u s e d by the o b s e r v a t i o n s that patients i m m u n o s u p p r e s s e d with C s A d e v e l o p e d l y m p h o m a s w a s r e d u c e d w h e n it w a s found that it w a s d u e to the strong i m m u n o s u p p r e s s i v e action of C s A and that C s A itself is not an o n c o g e n e (Thiru 1989.d e p e n d e n t reversible a n d c h r o n i c . C r o h n ' s disease. multiple sclerosis. T h e effect o n liver functions w a s r e d u c e d w h e n a l o w e r d o s e of C s A w a s used. H o w e v e r . e v e n with the r e d u c e d d o s e the hepatotoxicity w a s not c o m p l e t e l y eliminated ( K l i n t m a l m et al.10. It can c a u s e fluid retention and e d e m a ( A t k i n s o n 1989). T h e s e d r u g s h a v e b e e n used for the treatment of m i g r a i n e h e a d a c h e s and to . especially in the recipients of heart and b o n e m a r r o w t r a n s p l a n t s . Fortun a t e l y .3 ERGOT ALKALOIDS T h e alkaloids are a g r o u p of naturally occurring organic c o m p o u n d s c o n t a i n i n g nitrogen. 1978). it also h a s undesirable side effects. h o w e v e r . H o w e v e r . In the United States alone a p p r o x i m a t e l y 5 0 proprietary and generic drug products are available ( R o b b e r s 1984). b a s e d on the historical u s a g e there are e x c e p t i o n s to this definition such as c o l c h i c i n e and m e s c a l i n e ( R o b i n s o n 1968). and substitution (Borel 1989. O t h e r m i n o r p r o b l e m s associated with C s A are t r e m o r s .3 Ergot Alkaloids 265 d e p e n d e n t d i a b e t e s . 10. It m a y b e d u e either to the nephrotoxicity or to s o m e effect o n the s y s t e m i c v a s c u l a t u r e . c o m b i n a t i o n of d r u g s . Ergot alkaloids are p r o d u c e d b y ergot fungus (Claviceps s p . A second a d v e r s e side effect of C s A . Graffenried et al. In spite of c o n s i d e r a b l e effort the exact m e c h a n i s m of the d a m a g e is not k n o w n (Thiru 1989). (1978) in their first report o n use of C s A in renal transplant that the drug has a direct toxic effect on the renal tubules o r on the b l o o d supply of the t u b u l e s .

1 0 d o u b l e b o n d and c a r b o x y l g r o u p at C .1 0 . Structures of c o m m o n classes of ergot alkaloids are s h o w n in Figure 1 0 .266 Therapeutic Metabolites m o d u l a t e uterine c o n t r a c t i o n s . 3 . Suffice to say that these g r o u p s of c o m p o u n d s are p h a r m a c o l o g i c a l l y very potent and of substantial e c o n o m i c i m p o r t a n c e . T h e third class is c o m p r i s e d of d-lysergic acid a m i d e s and the last contains the d-lysergic acid p e p t i d e s . O n l y the c o m p o u n d s with an 8/3-configuration are biologically active. In clavine alkaloids (8-ergolenes) there are t w o a s y m m e t r i c centers at C-5 and C . T h e 5-H a t o m a l w a y s has a /^-configuration and the 10-H a t o m is trans. If the d o u b l e b o n d of 9-ergolenes is r e d u c e d another a s y m m e t r i c center is created at C .8 . In lysergic acid and its d e r i v a t i v e s . or a.7 and T a b l e 1 0 .9 d o u b l e b o n d . This topic is discussed in m o r e depth in Section 1 0 .4 .1 0 . 4 . w h i c h are called 9-ergolenes. .8 . T h e d-lysergic acid g r o u p of c o m p o u n d s contains a 9 . T h e clavine alkaloids h a v e a basic tetracyclic ring system with an 8. the t w o a s y m m e t r i c centers are at C-5 and C . H Ergot peptide alkaloids FIGURE 10-7 Structure of ergot alkaloids. to 5-H.

. Inc. After k a r y o g a m y and m e i o s i s cylindrical asci. R o b b e r s 1984). giving rise to fruiting bodies ( s t r o m a t a ) . 3. the g r o w t h of t h e sporulating tissue stops a n d m o r e c o m p a c t type of g r o w t h b e g i n s . e a c h with eight threadlike a s c o s p o r e s .10-trans c o m p o u n d s h a v e so far b e e n of i m p o r t a n c e ( B e r d e a n d S t ü r m e r 1978. M a s s a c h u s e t t s w e r e affected b y ergot alkaloids ingested u n k n o w i n g l y (Caporael 1976). Reprinted by permission of Wiley-Liss. T h e m a l e nuclei of t h e antheridia m i g r a t e into a s c o g o n i u m during p l a s m y . T h e fungi in this class form a spore-bearing structure called a s c u s a n d their spores are called a s c o s p o r e s . T h e life cycle of Claviceps b e g i n s w h e n the a s c o s p o r e s carried b y the w i n d infect the ovary of the floret. O n l y 5. (1984) in Advances in Biotechnological Processes.( C H 3) 2 H /3-Ergokryptine CH3 CH3 C H ( C H 3) C H 2( C H ) 3 H Ergocornine CH3 CH3 C H ( C H 3) 2 H Bromocriptine CH3 CH3 C H 2C H ( C H 3) 2 Br Reprinted by permission of publisher from Robbers. 197-239.10. It contains several alkaloids. R u t s c h m a n n a n d Stadler 1978). E r g o t toxicity c a u s e d b y eating c o n t a m i n a t e d grain p r o d u c t s h a s b e e n k n o w n since the m i d d l e ages ( R o b b e r s 1984) a n d it h a s b e e n suggested that the w o m e n tried in 1692 for witchcraft in S a l e m . sclerotial.C H . R2 Rs R4 Ergotamine H H CH2-C6H5 H Ergocristine CH3 CH3 CH2-C6H5 H a-Ergokryptine CH3 CH3 C H 2. ) w h o s e preferred host is t h e r y e plant. A t t h e b a s e of this are o n e o r m o r e multinucleate antheridia. n o n s p o r u l a t i n g structure. T h e Sclerotium dries a n d b e c o m e s as hard as stone a n d falls to t h e g r o u n d w h e r e it r e m a i n s d o r m a n t till spring. J. A s m e n t i o n e d in t h e p r e c e d i n g p a r a g r a p h s these c o m p o u n d s are p r o d u c e d b y the ergot fungus (Claviceps s p . the characteristic ergot Sclerotium. w h i c h in turn h a s ramifications in d e v e l o p i n g processes for production of these c o m p o u n d s as d i s c u s s e d in the next section. a division of John Wiley and Sons. U p to this point little o r n o alkaloids are p r o d u c e d . Vol. are formed w h i c h b e g i n t h e cycle a n e w ( M a n t l e 1 9 7 5 . U n d e r favorable c o n d i t i o n s it g e r m i n a t e s . w h i c h leads to a h a r d . a l t h o u g h it can infect m a n y grasses a n d cereal plants such as w h e a t a n d barley. e a c h with o n e m u l t i n u c l e a t e a s c o g o n i u m . Claviceps is a g e n u s that b e l o n g s to t h e class Ascomycetes. s w e e t e x u d a t e called " h o n e y d e w . T h e h e a d s of the stromata contain several m i n u t e cavities. T h e o v a r y is p e r m e a t e d by t h e m y c e l i u m w h i c h after about a w e e k bears t h e asexual s p o r e s .3 Ergot Alkaloids 267 TABLE 10-4 Structures of Ergot Peptide Alkaloids Compound R. w h i c h spread the fungus to other plants. A t this t i m e the host p r o d u c e s a thick. " It not only p r o v i d e s t h e nutrients for t h e fungal g r o w t h but also acts as a vehicle for t h e d i s s e m i n a t i o n of c o n i d i a .E. It c a n b e seen that t h e p r o d u c t i o n of ergot alkaloids is associated with the sclerotial stage in the c y c l e . Copyright © Wiley-Liss. A b o u t 2 w e e k s after the initiation of t h e infection.

1967). it has b e e n used as a s o u r c e . 2 S t r a i n S e l e c t i o n for F e r m e n t a t i o n . 1964). in the early d a y s they w e r e not very successful. R o b b e r s 1984). T o increase the y i e l d s . the whole crop can be mechanically harvested and the sclerotia separated from the grain. 1 . although c a p a b l e of p r o d u c i n g lysergic acid derivatives on r y e . T h e strains that p r o d u c e d alkaloids s e e m to lose their biosynthetic ability u p o n a few serial transfers. w e r e p r o d u c e d . fusiformis. there w e r e a n u m b e r of p r o b l e m s e n c o u n t e r e d in the selection of strains for the p r o c e s s d e v e l o p m e n t . These include use of land for one crop of rye per year. the strains w e r e recycled on the natural host. and Spacelia sorghi (Mantle 1973 and 1975. 3 . T h e third p r o b l e m w a s the preservation of the active c u l t u r e . T h e sclerotia can b e h a n d p i c k e d . or alternatively. T h e first difficulty w a s the availability of strains. rather than e c o n o m i c a l l y m o r e i m p o r t a n t lysergic acid or lysergic acid p e p t i d e s . These factors led to intensive efforts to develop a fermentation process. r a n d o m screening of cultures isolated from sclerotia to screening of those b a s e d on the m o r p h o l o g i c a l or physiological characteristics w e r e used ( A m i c i et al. T h e s e latter t w o p r o b l e m s w e r e solved b y careful selection of culture and preservation of stock cultures at . Yields w e r e low and very often only clavine alkaloids.268 10. A l t h o u g h the n u m b e r of strains reported in the literature is high very few h a v e b e e n deposited in public culture collection ( R o b b e r s 1984). T h e reason for this genetic instability w a s found to be that the p r o d u c i n g strains are h e t e r o k a r y o n s w h i c h segregate into n o n p r o d u c i n g p r o g e n y and thus give rise to sectored c o l o n i e s . R o b b e r s 1984). C. F u r t h e r m o r e . As in the fermentation p r o c e s s . their c o n i d i a are u n i n u c l e a t e ( A m i c i et al. 1 . T h e s e c o n d p r o b l e m w a s that a n u m b e r of strains isolated from sclerotia. 1967.3. R e h a c e k and K o z o v a 1975. C. E v e r since it w a s r e c o g n i z e d that the sclerotia of rye ergot contain the alkaloids. At the h o n e y d e w stage the spread of conidia w a s facilitated by m e c h a n i c a l m e a n s . T o isolate cultures c a p a b l e of p r o d u c i n g in s u b m e r g e d fermentation a n u m b e r of m e t h o d s r a n g i n g from largescale. R o b b e r s 1984). cost-inefficient development of inoculum in the laboratory and its dissemination. rye flowers w e r e inoculated with a selected strain of Claviceps. C. 3 . T h e agricultural production of ergot alkaloids has a number of drawbacks. M u t a g e n e s i s has also . In spite of the relatively large n u m b e r of strains c a p a b l e of alkaloid p r o d u c t i o n . T h e r e f o r e . It is o b v i o u s h o w this can lead to the loss of alkaloid production on serial transfers. papsali isolates w h i c h w e r e i m p r o v e d p r o d u c e r s (Bianchi et al. 1 Field P r o d u c t i o n of E r g o t A l k a l o i d s .7 0 ° C to m i n i m i z e the need for subculturing ( M a n t l e 1975. T h i s led to selection of C . vulnerability to climatic conditions. and overall labor intensive nature of the process (Mantle 1975). C. as well as Pénicillium sizove (Kozlovskii and V e p r i t s k a y a 1987) and Aspergillus fumigatus ( N a r a y a n and R a o 1982). r y e . 1 0 . A l t h o u g h the fungus w a s isolated and attempts w e r e m a d e to p r o d u c e the alkaloids in f e r m e n t a t i o n . field p r o d u c t i o n of alkaloids w a s practiced. papsali. T h e ergot fungi that p r o d u c e ergot alkaloids include Claviceps purpurea. sulcata. gigantea. p r o d u c e d only clavine alkaloids in s u b m e r g e d fermentations.1 Therapeutic Metabolites Production of Ergot Alkaloids 1 0 .

m o s t of the alkaloids p r o d u c e d w e r e associated with the m y c e l i u m . 1 % . paspali Steven and Hall in production m e d i u m containing m a n n i t o l 3 % .3. .) T u l . 5 m g / m l in 1 1 . and isolysergic acid m e t h y l c a r b i n o l a m i d e . Z n S o 4 . T h i s fermentation w a s scaled u p to an 800-1 fermentor ( A r c a m o n e et al. 2 ) . labeled 2 7 5 F . 0 5 % . paspali in s u b m e r g e d fermenta- tion ( A r c a m o n e et al. (1960) w e r e the first to report p r o d u c t i o n of lysergic acid derivatives by C. I . and trace e l e m e n t m i x t u r e of F e S 0 4 . K H 2 P 0 4 0 .7 H 2 0 0 .e r g o l - 8-ene-carboxylic acid and it w a s c o n v e r t e d into D-lysergic acid by r e a r r a n g e m e n t of the d o u b l e b o n d from the 8 . isolysergic acid a m i d e .m l s h a k e flasks. 2 ) . paspali. w h i c h used a different strain of C . lysergic acid m e t h y l carbinola m i d e .N . L a t e r the Farmitalia g r o u p described p r o d u c t i o n of lysergic acid derivatives by C. C a ( N 0 3 ) 2 · 4 H 2 0 0 .7 H 2 0 . A b o u t 8 0 % of the alkaloids p r o d u c e d consisted of e r g o t a m i n e . A n o t h e r p r o c e s s to obtain D-lysergic acid.7 H 2 0 0 .N ' . K H 2 P 0 4 0 . Superior strains thus isolated w e r e used to d e v e l o p s u b m e r g e d fermentation p r o c e s s e s to p r o d u c e various ergot a l k a l o i d s . 1 % . 1 .m e t h y l . 1960). T h e agitation w a s adjusted b a s e d on the dissolved o x y g e n c o n c e n t r a t i o n . ( A m i c i et al. I n o c u l u m v o l u m e w a s 1 0 % .9 position to the 9 . 1970). and K C l in tap w a t e r ( p H 5 . T h e yield r e a c h e d 1 . yeast extract 0 . 1962. 0 3 % in tap w a t e r (pH 5 .n i t r o .5%. 10. T h e i n o c u l u m w a s g r o w n in m e d i u m c o n t a i n i n g sucrose 1 0 % .10. T h i s p r o c e s s w a s o p t i m i z e d further to yield 5 m g / m l ( M a n t l e 1975). T h e p r o d u c t i o n m e d i u m w a s similar to the i n o c u l u m m e d i u m e x c e p t the c o n centrations of sucrose and citric acid w e r e increased to 3 0 % and 1.n i t r o s o g u a n i d i n e N T G ) to isolate i m p r o v e d m u t a n t s . a m m o n i u m succinate 3 % . w h i c h w e r e s h a k e n at 2 2 0 r p m at 2 4 ° C . Srikrai and R o b b e r s 1983). It w a s started at 100 r p m at 25 h and increased to 150 r p m at a b o u t 7 5 h. Interestingly. T h e fermentation p r o c e s s inv o l v e d g r o w i n g C .1. T h i s culture p r o d u c e d 6 .3 Ergot Alkaloids 269 b e e n used for the isolation of superior m u t a n t s ( K o b e l et al. T h e p r o d u c t i o n b e g a n at 5 0 h a n d r e a c h e d the m a x i m u m of 1.3 Fermentation Development. M g S 0 4 . A r c a m o n e et al.m l s h a k e flasks agitated on a rotary shaker as well as on the large scale in fermentors of u p to 5 0 0 1 c a p a c i t y . w a s d e v e l o p e d ( K o b e l et al. 1964). T h e former g r o u p used ultraviolet (UV)-irradiation and the latter w o r k e r s used t w o cycles of mutation with A ^ m e t h y l . 0 1 % . 1966 and 1967). T h e alkaloids w e r e almost c o m p l e t e l y released into the m e d i u m . T h e y isolated the strain from the grass Paspalum L and r e g r e w it o n rye e m b r y o g r o w n on agar. F e r m e n t a t i o n s w e r e d o n e in 5 0 ml of the p r o d u c t i o n m e d i u m in 3 0 0 . citric acid 1%. T h e r e g r o w n culture distichum p r o d u c e d lysergic acid a m i d e . T h e fermentations w e r e d o n e in 100 ml of m e d i u m in 5 0 0 .1 m g / m l at 2 9 0 h. A six-nozzle sparger w a s used for aeration. The o p e r a t i n g v o l u m e w a s 5 0 0 1. purpurea (Fr. T h e alkaloid p r o d u c t i o n b e g a n on the s e c o n d d a y and c o n t i n u e d until it reached 2 m g / m l on the ninth d a y . T h e m e d i a u s e d for the i n o c u l u m d e v e l o p m e n t and the p r o d u c t i o n of e r g o t a m i n e w e r e the s a m e as those used b y A m i c i et al. respectively. 0 2 5 % . T h e fermentor w a s agitated with t w o four-blade turbine i m p e l l e r s . ( 1 9 6 6 ) .1 2 d a y s . M g S 0 4 .1 0 chemical position ( M a n t l e 1975).1 .7 H 2 0 . F e r m e n t a t i o n t e m p e r a t u r e w a s 2 4 ° C .

F e S 0 4 · 7 H 2 0 0 . T h e presterilization p H w a s adjusted to 5. 2 5 g. the p H w a s adjusted to 5. M g S 0 4 · 7 H 2 0 0 . 2 5 g.7 H 2 0 0 . K H 2 P 0 4 0 . A variant strain. Z n S 0 4 . T h e p H w a s adjusted to 5. F e r m e n t a t i o n s w e r e d o n e in 100 ml of m e d i u m in 5 0 0 .a s p a r a g i n e 10 g. T h e alkaloid production b e g a n on the second day and r e a c h e d the m a x i m u m of 4 m g / m l on the sixth d a y . 0 3 3 . K C l 0 . T h e p H w a s controlled at 5 . 2 5 g.7 H 2 0 0 . w a s found to p r o d u c e d i h y d r o e r g o s i n e ( M a n t l e 1973). and n o F e S 0 4 · 7 H 2 0 . 1 0 8 g. K H 2 P 0 4 0 . 0 2 7 g. Agitation and aeration w e r e p r o v i d e d by single disc turbine and ring sparger. m o s t of the alkaloids w e r e released in the m e d i u m but the proportion of the m y c e l i u m . T h e c o m p o s i t i o n of the seed m e d i u m w a s as follows: sucrose 100 g. C a ( N 0 3 ) 2 4 H 2 0 1 g. M g S 0 4 · 7 H 2 0 0 . T h e r e w a s n o increase in the concentration of the alkaloids from the sixth to the tenth d a y . festuclavine. fusiformis w a s reported by B a n k s et al. per liter: K H 2 P 0 4 0 . K H 2 P 0 4 0 . T h e p r o d u c t i o n fermentors w e r e 400-1 size with operating v o l u m e of 3 6 0 1.8 g. T h e alkaloid production b e g a n at about 12 d a y s and r e a c h e d 0 . purpurea had b e e n successful to s o m e extent (Kobel and Sanglier 1978). T h e alkaloids p r o d u c e d c o n t a i n e d m a i n l y A g r o c l a v i n e ( > 9 0 % ) and traces of e l y m o c l a v i n e . and p e n n i c l a v i n e . Z n S 0 4 · 7 H 2 0 0 . L a r g e . respectively. paspali. T h e first a t t e m p t s of directed synthesis with prototrophic cultures of C . 7 m g / m l after 22 d a y s . L .b o u n d alkaloids increased with time of incubation.s c a l e production of clavine alkaloids by C. A s in the case of the seed m e d i u m . 5 g.2 with N a O H . 2 5 g. setoclav i n e . distilled water 1 1.7 H 2 0 0 .2 0 0 0 ( D o w C h e m i c a l ) w a s used as an antifoaming agent. ( 1 9 7 4 ) . 0 3 3 g. 0 2 7 g. T h e flasks w e r e incubated w i t h o u t s h a k i n g at 27°C in the dark. 10.m l E r l e n m e y e r flasks. and Z n S 0 4 · 7 H 2 0 0 . Z n S 0 4 · 7 Η 2 0 0 . T h e agitation rate w a s varied from the initial 153 r p m to 3 0 6 r p m w h e n the dissolved o x y g e n concentration d r o p p e d to 3 0 % of the 2 saturation v a l u e . w a s used for the d e v e l o p m e n t of s u b m e r g e d fermentation.4 Directed Biosynthesis and Biotransformation.a s p a r a g i n e 15 g. F e S 0 4 · 7 H 2 0 0 . 0 3 3 g. 0 with N a O H . M g S 0 4 . T h e p r o d u c t i o n m e d i u m c o n t a i n e d . T h e seed w a s d e v e l o p e d in four stages. and d i h y d r o e l y m o c l a v i n e w e r e also p r o d u c e d . T h e m a i n c o m p o n e n t of the alkaloids p r o d u c e d w a s d i h y d r o e r g o s i n e but small a m o u n t s of c h a n o c l a v i n e . w h i c h autolysed the g l u c a n . P o l y g l y c o l P .5 with N a O H . 1974). w h i c h led to highly viscous broths and p r o b l e m s in supplying a d e q u a t e o x y g e n ( B a n k s et al. p e r liter of distilled water: sucrose 150 g.2 with N a O H . 0 2 7 g. yeast extract 0.270 Therapeutic Metabolites Sphacelia sorghi M c R a e .3. T h e yields w e r e i m p r o v e d to 6 m g / m l by the use of a m e d i u m r e p l a c e m e n t / m u l t i s t a g e process with m e d i u m c o n t a i n i n g . 0 1 g. 1 2 5 g. T h e aeration rate w a s 4 0 0 1/min. 2 5 g. c h a n o c l a v i n e . F e S 0 4 . O n s u p p l e m e n t i n g the production m e d i u m . A s in the case of C . T h e incubation t e m p e r a t u r e w a s 2 7 ° C . 5 g.1. T h e o p t i m i z e d production m e d i u m constituents w e r e : sucrose 2 0 0 g. A m m o n i u m sulfate w a s sterilized separately. a fungus parasitic on Sorghum vulgare P c r s . L . B a c k p r e s s u r e of 100 k N / m w a s m a i n t a i n e d . isolated in N i g e r i a . distilled water 1 1. M g S 0 4 · 7 H 2 0 0 .1 g. ( N H 4 ) 2 S 0 4 11. 2 5 g. L .c y s t e i n e h y d r o c h l o r i d e 0 . T h e original strain of this culture isolated from bulrush millet g r o w n in Senegal w a s not suitable for s u b m e r g e d fermentations as it p r o d u c e d extracellular glucan during the g r o w t h p h a s e .

A p r o l i n e a n a l o g u e .1 K i n e t i c s of F e r m e n t a t i o n . and 6 5 % 5 ' . T h e bulk of the alkaloids w a s p r o d u c e d d u r i n g the linear g r o w t h p h a s e . T h e culture w a s found to a c c u m u late an o l i g o s a c c h a r i d e w h e n sucrose w a s used as a c a r b o n s o u r c e .10. w e r e tested with different a n a l o g u e s of p h e n y l a l a n i n e such as /?-fluorophenylalanine and those of l e u c i n e .I l e . fusiformis W l w h e n fed c h a n o c l a v i n e c o n v e r t e d it to c h a n o c l a v i n e I 0-/3-fructofuranoside and c h a n o c l a v i n e I O-jß-D-fructofuranosyl-(2 —» l ) . succinic acid. C.c h l o r o p h e n y l a l a n i n e w a s a d d e d and the fermentation was c o n t i n u e d for 10 m o r e d a y s . 5 . L a t e r it w a s found that a n a l o g u e s of ergot alkaloids can b e p r o d u c e d by feeding a n a l o g u e s of the a m i n o acids present in the p ep t i d e sidechain ( B e a c c o et al. 1974). h o w e v e r . In all cases directed b i o s y n t h e s i s of alkaloids c o n t a i n i n g these a n a l o g u e s w a s o b s e r v e d ( B e a c c o et al. 4 g of D L . S u b s e q u e n t l y other strains. B a n k s et al. w h i c h p r o v i d e d the e n e r g y a n d the p r e c u r s o r s for the alkaloid sy n t h esi s. w h i c h were either p h e n y l a l a n i n e or leucine a u x o t r o p h s . 1978). and /3-hydroxyleucine./ ? . 1967 a n d 1969. L . M g S 0 4 . they will g i v e the r e a d e r a g o o d idea of the different types used. U s e of a u x o t r o p h s greatly i m p r o v e d the efficiency of the p r o c e s s b y r e d u c i n g the c o n c e n t r a t i o n of the alkaloid n o r m a l l y p r o d u c e d by this fungus. 5 .2 Physiology and Regulation of the Alkaloid Production 10.L e u .7 H 2 0 . thiazolidine-4-carboxylic acid. 10. s h o w e d that inorganic p h o s p h a t e w a s utilized rapidly and the alkaloid synthesis did not begin until it w a s e x h a u s t e d ( A m i c i et al.0 . A r c a m o n e et al./ 3 .d i b e n z y l . After 4 d a y s of g r o w t h . Initial m e d i u m p H w a s adjusted with N H 4 O H . 1970. purpurea ( B a u m e r t et al.3 Ergot Alkaloids 271 with either L . T h e alkaloids p r o d u c e d c o n t a i n e d 3 5 % e r g o c r i s t i n e . K H 2 P 0 4 . these w o r k e r s did not see any increase in the g r o w t h or the total alkaloid p r o d u c t i o n . w a s incorporated into e r g o s i n e b y C. 5 . B i o t r a n s f o r m a t i o n of c h a n o c l a v i n e to m o n o . Another e x a m p l e of incorporation of a m i n o acid a n a l o g u e s w a s reported b y B a u m e r t et al.f r u c t o f u r a n o s i d e . iron. Kinetics of fermentation in s h a k e flasks and in f e r m e n t o r s . D u r i n g the initial rapid g r o w t h m a r k e d b y the utilization of inorganic p h o s p h a t e . for e x a m p l e . ( 1 9 8 2 ) . and z i n c . w h i c h w e r e c o n s u m e d at a faster rate.and d i g l y c o s i d e w a s recently d e s c r i b e d (Flieger et al. purpurea w a s g r o w n in a m e d i u m c o n t a i n i n g s u c r o s e . T h e p r o d u c t i o n of sterols and lipids paralleled that of a l k a l o i d s . L ./ 7 . 1982).1 4 d a y s as c o m p a r e d to the c a r b o x y l i c acids in the m e d i u m . w h e n a m m o n i a . or L . 1978).n o r l e u c i n e . w h i c h is the major p r o d u c t of this culture in the a b s e n c e of the a n a l o g u e . and salts of p o t a s s i u m . T h i s o l i g o s a c c h a r i d e w a s h y d r o l y z e d in the later p h a s e of the fermentation.c h l o r o b e n z y l e r g o c r i s t i n e .2. S u g a r s w e r e used m o r e slowly o v e r the 1 0 . 1990). there w a s n o detectable alkaloid sy n t h esi s. the distribution of individual alkaloid w a s m a r k e d l y affected. yeast extract.t r i f l u o r o l e u c i n e .D .3. .3.V a l . regardless of the strain u s e d . A p h e n y l a l a n i n e a u x o t r o p h of C. A l t h o u g h these e x a m p l e s by n o m e a n s c o v e r all the k n o w n p r o c e s s e s to p r o d u c e ergot a l k a l o i d s .

d e t e r m i n a t i o n of e n z y m e levels w a s not attempted. T h e s e w o r k e r s found that the highest yields w e r e obtained at 0 . the g r o w t h w a s i m p r o v e d at the higher concentration of the p h o s p h a t e ( W a a r t and T a b e r 1960). purpurea w a s d o n e by K y b a l et al. 1 0 . w h i c h are tricarboxylic acid ( T C A ) cycle i n t e r m e d i a t e s . A l t h o u g h n o explanation w a s offered by the authors for this difference. ( 1 9 8 1 ) . A r c a m o n e et al. fumigatus and S. and with the latter fungus these w e r e 1. In the cases of A.C o A c a r b o x y l a s e by citrate. 7 5 g/1. A similar effect of p h o s p h a t e on the production of lysergic acid derivatives in 5-1 fermentors w a s reported (Arcam o n e et al. A s m e n t i o n e d in the p r e c e d i n g section. It w a s found that citric. it a p p e a r s that p h o s p h a t e limitation is needed for o p t i m u m alkaloid p r o d u c t i o n . T h e y noted that rye leaves contain citric and malic acid and this led t h e m to testing of carboxylic acids in the fermentation m e d i u m . especially during the production p h a s e . 9 1 g/1) n e e d e d for the o p t i m u m g r o w t h of C.272 Therapeutic Metabolites nitrogen w a s exhausted ( A m i c i et al. A detailed analysis of organic acid m e t a b o lism in the biosynthesis of ergot alkaloids by C. T h e y postulated that the T C A cycle acids feedback control citrate synthase and aketoglutarate d e h y d r o g e n a s e . 2 . 1 0 .C o A into m e v a lin and fatty acid synthesis. sorghi t o o . A n o t h e r effect of citric acid is the inhibition of p h o s p h o f r u c t o k i n a s e which in turn leads to the preferential use of the p e n t o s e . W i t h the former o r g a n i s m the o p t i m u m concentrations of K H 2 P 0 4 for the synthesis and the g r o w t h w e r e 0 . 1970. 3 Effects of O r g a n i c A c i d s . respectively (Mantle 1973. U n f o r t u n a t e l y . 2 5 g/1 to 0 . paspali in defined m e d i u m w a s l o w e r than that (18 g/1) for the highest alkaloid yields ( R o s a z z a et al. T h e latter is stimulated further by activation of a c e t y l . respectively. c a r b o h y d r a t e s .0 g/1 and 10. for the m a x i m u m g r o w t h 1 g/1 w a s n e e d e d .0 g/1. R a o et al. it w a s found that the K H 2 P 0 4 concentration ( 0 . 1974). T h e s e observations provided a basis for the optimization of the production medium. Further studies on the c o m p o s i t i o n of the m y c e l i u m w h e n g r o w n at these t w o concentrations of p h o s p h a t e s h o w e d that at the higher c o n c e n t r a tion the m y c e l i u m contained m o r e p o l y o l s . 1977). it is possible that the specific e x p e r i m e n t a l design used by t h e m w a s the c a u s e . reducing s u g a r s . 3 . 1977). w h i c h results in c h a n n e l i n g of a c e t y l . 2 . In contrast to these reports. 5 g K H 2 P 0 4 / 1 . B a n k s et al. In g e n e r a l . 1967). 3 . alkaloid synthesis b e g a n after the exhaustion of p h o s p h a t e . H o w e v e r . W a a r t and T a b e r (1960) found that alkaloid p r o d u c t i o n by C. the concentrations of p h o s p h a t e n e e d e d for the best p r o d u c t i o n w e r e l o w e r than those needed for the m a x i m u m g r o w t h ( M a n t l e 1 9 7 3 . purpurea strain P R L 1578 w a s almost c o m p l e t e l y e l i m i n a t e d w h e n the concentration of K H 2 P 0 4 w a s increased from 0 . 2 5 g/1 and 1. indicated that high p h o s p h a t e repressed the e n z y m e s involved in the biosynthesis of alkaloids. It w a s suggested that the r e d u c e d R N A levels. R a o et al. 2 Effect of I n o r g a n i c P h o s p h a t e . w h i c h s u g g e s t e d that it is inhibited by p h o s p h a t e . h o w e v e r . s u c c i n i c . 1967 and 1969. and malic a c i d s .0 g/1. and lipids and less R N A (Taber and Vining 1963). 1970). supported the p r o d u c t i o n of alkaloids but tartaric and lactic acid did not.

m a n n i t o l . At least 0 .K G acid w a s present. P o t a s s i u m is involved in t r y p t o p h a n biosynthesis in Bacillus subtilis ( S c h w a r t z and B o n n e r 1964). 5 Effect of T r a c e E l e m e n t s . T h u s . and mineral salts m e d i u m ( S a m b a m u r t h y and R a o 1971). regardless of the carboxylic acid used (Kozlovskii and V e p r i t s k a y a 1987). shizovae s h o w e d that m a n n i t o l with either of the three acids g a v e the highest yields. A l m o s t n o synthesis w a s o b s e r v e d with sorbitol. 2 . a n d g l u c o s e with succinic. T h e s e o p t i m u m c o m b i n a t i o n s a p p e a r to lead to the preferential use of the p e n t o s e monophosphate pathway. A systematic study of various c o m b i n a t i o n s either of m a n n i t o l . K o z l o v s k i i and V e p r i t s k a y a 1987). lactose. or fumaric acid o n the alkaloid synthesis by P. and t r i a m m o n i u m citrate g a v e the highest yields. 1970. (1965) and by R o s a z z a et al. ( 1 9 6 7 ) . or starch ( B a n k s et al. 3 . and sorbitol. O n the other h a n d . P o o r g r o w t h w a s o b s e r v e d in the m e d i a containing fructose. a . 1974). 4 Effect o f C a r b o n S o u r c e . 2 g of potassium/1 w a s n e e d e d to obtain the m a x i m u m g r o w t h and alkaloid p r o d u c t i o n . 1 0 . sorbitol. a m m o n i u m s u c c i n a t e . S a m b a m u r t h y and R a o 1 9 7 1 . T h e studies on the optimization of m e d i u m for p r o d u c t i o n of alkaloids by A . w a s substituted for m a n n i t o l in m a n n i t o l . fumigatus (Fresenius) indicated that a c o m b i n a t i o n of g l u c o s e . sorbitol. the best c a r b o n source for the production b y Sphacelia sorghi w a s either sucrose or g l u c o s e ( M a n t l e 1973). 1974. the production of alkaloids i m p r o v e d w h e n p o t a t o starch. fusiformis in defined m e d i u m in shake flasks. w h e r e a s m a n n i t o l and m a n n o s e w e r e not as effective. H i g h yields w e r e o b t a i n e d in c o m b i n a t i o n with fumaric acid. T h e latter w o r k e r s found that p o t a s s i u m exerted a strong effect on the g r o w t h and the p r o d u c t i o n of lysergic acid derivatives b y C . purpurea. B a n k s et al.3 Ergot Alkaloids 273 p h o s p h a t e p a t h w a y o v e r the glycolytic p a t h w a y and so the critical cofactor N A D P H and p r e c u r s o r t r y p t o p h a n are p r o v i d e d for the alkaloid synthesis. M a n t l e 1 9 7 3 . W i t h C. 3 . In light of the role of t r y p t o p h a n as a p r e c u r s o r of the ergoline . T h e s e results with different fungi d e m o n s t r a t e that n o o n e carbon source is o p t i m u m for alkaloid p r o d u c t i o n by all cultures and that the best production is o b t a i n e d with a c o m b i n a t i o n of o n e or m o r e sugars and organic acids d e p e n d i n g o n the culture u s e d . g l y c e r o l . m a l t o s e . T o u n d e r s t a n d the effect of s u c r o s e . T h e r e q u i r e m e n t s of various " t r a c e " elem e n t s w e r e studied e x h a u s t i v e l y by M a r y et al.10. 1 0 . N a r a y a n and R a o 1982. paspali.k e t o g l u t a r i c ( α . individually or as a m i x t u r e . 2 . it w a s replaced with e q u i m o l a r a m o u n t s of g l u c o s e and fructose. G l u c o s e supported a l o w level of p r o d u c t i o n w h e n either succinic or α . g a l a c t o s e . carbon sources that can b e used for g o o d p r o d u c t i o n of the alkaloids are s u c r o s e . m a n n i t o l . or a c o m b i n a t i o n of soluble starch or w h e a t starch and g l u c o s e at a ratio of 1 1 : 2 . S u c r o s e and g l u c o s e w e r e found to support m a x i m u m g r o w t h and alkaloid p r o d u c tion by C . the stimulation of alkaloid p r o d u c t i o n b y T C A cycle intermediates m a y be the result of their action on m a n y different targets.K G ) . 1970). In all cases the yields w e r e r e d u c e d ( A r c a m o n e et al. S i m i l a r l y . Effects of various carbon sources o n the alkaloid p r o d u c t i o n h a v e been described ( A r c a m o n e et al.

paspali that of the total 1 4 DL-[3. that is. T h u s . S i m i l a r l y . F u r t h e r m o r e . sulfur.5 g/1 to 2 . Neither U L . H o w e v e r . ( 1 9 6 5 ) did not find a n y r e q u i r e m e n t for either c o p p e r or m a n g a n e s e . the results described so far led to the belief that the t r y p t o p h a n effect w a s m e d i a t e d t h r o u g h its role as precursor of the ergoline m o i e t y . iron. M a g n e s i u m w a s also n e e d e d for the g r o w t h and the production of alkaloids ( M a r y et al. In contrast.C ] T r p w a s .6 R o l e of T r y p t o p h a n . B e r t r a n d and D e W o l f 1959). stimulation of alkaloid p r o d u c t i o n b y tryptophan w a s seen. A n u m b e r of other w o r k e r s also reported a similar effect of t r y p t o p h a n ( B r a d y and T y l e r 1959 and 1960. they noted that the cells g r o w n with t r y p t o p h a n c o n t i n u e d to synthesize alkaloids w h e n transferred to a m e d i u m without t r y p t o p h a n .[ C ] L . w h e n u s e d at a concentration of 0 . purpurea culture P R L 1578 w h e n used as a sole nitrogen source at 11 g/1.2. 1961). is reported to be involved in tryptophan biosynthesis ( N a s o n et al. w h o s h o w e d that tryptophan stimulated the p r o d u c t i o n only if added early. 1965. 1 9 5 1 .T y r w a s in1 4 c o r p o r a t e d into the ergoline moiety of the alkaloids. 1967). and m a n g a n e s e w e r e n eed ed for the o p t i m u m g r o w t h and alkaloid synthesis ( R o s a z z a et al. T h e y s u p p l e m e n t e d the m e d i u m containing a m m o n i u m succinate with 1 g of tryptophan/1 and found that the yield w a s increased from 1. paspali Steven and Hall ( A r c a m o n e et al. Early w o r k e r s suspected from the structure of ergot alkaloids that t r y p t o p h a n might be a precursor and therefore they studied its effect on p r o d u c t i o n . the beneficial effect of p o t a s s i u m can be readily u n d e r s t o o d . A r c a m o n e et al. 10. z i n c . l o w e r levels of c a l c i u m .[ C ] L . purpurea ( V i n i n g and T a b e r 1 4 Re- was examined 1 4 1963). O n the other h a n d . Increase concentration of a m m o n i u m succinate had n o effect o n the yield. succinic acid. w h e r e a s D L .[ 2 . T r y p t o p h a n w a s not able to support g r o w t h of C .C]Trp i n c o r p o r a t e d .274 Therapeutic Metabolites n u c l e u s . Interestingly. T h i s h y p o t h e s i s w a s c h a l l e n g e d by Floss and M o t h e s ( 1 9 6 4 ) . A r c o m o n e et al. Z i n c . In these e x p e r i m e n t s t o o . c o p p e r . 1962). M a r y et al. In another study incorporation of labeled t r y p t o p h a n . R o s a z z a et al. (1962) found with C. nitrate had a negative effect on the alkaloid p r o d u c t i o n . 0 g/1. A s c o m p a r e d to these t w o . 5 g/1 with a m m o n i u m succinate as a nitrogen source in a defined m e d i u m c o n t a i n i n g g a l a c t o s e . 1967). it d o u b l e d the p r o d u c t i o n of alkaloids as c o m p a r e d to that with a m m o n i u m succinate alone ( T a b e r and V i n i n g 1958). 5 9 % w a s in the alkaloid. 1965).h y d r o x y e t h y l a m i d e by C.P h e nor U L . T h e s e studies s h o w e d the effect of the trace m e t a l s on the yields of alkaloids quite clearly. tryptophan w a s found to stimulate the p r o d u c t i o n of d-lysergic acid α . and p h e n y l a l a n i n e by C . S u b s e q u e n t studies on b i o s y n t h e s i s with isotope-labeled tryptophan s h o w e d it to b e incorporated into the alkaloid. A s o m e w h a t a m b i g u o u s effect of trace m e t a l s on the distribution of the alkaloids p r o d u c e d has also b e e n reported ( M a r y et al. in the g r o w t h p h a s e and that the tryptophan a n a l o g u e s w h i c h are not precursors could also c a u s e this stimulation. t y r o s i n e . T h i s is consistent with the p r e v i o u s observation that tryptophan c a n n o t be used as a sole nitrogen source ( T a b e r and V i n i n g 1958). there w a s n o extensive degradation of these aromatic a m i n o acids and they w e r e incorporated into proteins intact.3. and salts. If the a b o v e m e n t i o n e d hypothesis w a s correct then these o b s e r v a t i o n s c o u l d not b e . as in the c a s e of p o t a s s i u m .

T h i s led the authors to suggest that tryptophan inhibited the alkaloid p r o d u c t i o n in the last p h a s e . B e c a u s e this s e c o n d derivative w o u l d b e a m e a s u r e of the synthesis of the e n z y m e . did not stimulate the s y n t h e s i s .T r p and 5 . this observation w o u l d support the n o tion of a regulatory role of t r y p t o p h a n . F u r t h e r e v i d e n c e from the laboratories of Floss c o n c l u s i v e l y s h o w e d that t r y p t o p h a n is an i n d u c e r of the alklaoid biosynthesis ( R o b b e r s and Floss 1970.L e u . w h i c h s u g g e s t e d that t r y p t o p h a n did not act as a preferred nitrogen source ( R o b b e r s et al.to twenty-five-fold increase in tryptophan synthetase activity d u r i n g the transition b e t w e e n the g r o w t h and alkaloid p r o d u c t i o n w a s o b s e r v e d . an- . the target e n z y m e for induction by t r y p t o p h a n w a s identified to b e d i m e t h y l a l l y l t r y p t o p h a n ( D M A T ) s y n t h e t a s e .h e p t u l o s o n i c acid ( D A H P ) s y n t h e t a s e . T h e a p p r o a c h used b y these w o r k e r s i n v o l v e d the u s e of D L . V i n i n g ( 1 9 7 0 ) found that in a Claviceps species addition of t r y p t o p h a n at the b e g i n n i n g of the fermentation resulted in the stimulation of alkaloid synthesis and this stimulation d e c r e a s e d as t r y p t o p h a n w a s a d d e d later and later in the fermentation. T h e y noted that the a n a l o g u e w a s not i n c o r p o r a t e d into either alkaloids or p r o t e i n s . paspali (Steven et Hall. In a d d i t i o n . S u b s e q u e n t studies s h o w e d that a n o t h e r a m i n o acid. t h e s e results indicate a relationship b e t w e e n that and the intracellular c o n c e n t r a t i o n of t r y p t o p h a n . F u r t h e r m o r e .3 Ergot Alkaloids 275 e x p l a i n e d . h o w e v e r .D L .m e t h y l t r y p t o p h a n others w e r e not able to d o so ( A r c a m o n e et al. It is indeed possible that the former w a s the effect of the latter. V i n i n g 1970). B u ' L o c k and Barr ( 1 9 6 8 ) d e m o n s t r a t e d that the s e c o n d derivative of the alkaloid p r o d u c t i o n c u r v e correlated with the internal (pool) c o n c e n t r a t i o n of t r y p t o p h a n . that of t r y p t o p h a n did not. 1972. the first e n z y m e of the alkaloid b i o s y n t h e s i s (Krupinski et al. In the m i d d l e p h a s e the e n d o g e n o u s c o n c e n t r a t i o n of t r y p t o p h a n increased in parallel with that of the alkaloid. w h i c h s u g g e s t e d that p h o s p h a t e inhibition m a y be m e d i a t e d t h r o u g h limiting the s u p p l y of t r y p t o p h a n . K r u p i n s k i et al.m e t h y l . w h i c h w a s a c c o m p a n i e d by the reduction in d e n o v o protein sy n th esis. 1962. R o b b e r s et al.5 8 and found a sevenfold increase in the alkaloid p r o d u c t i o n .to threefold in the internal t r y p t o p h a n level and t w e n t y . A l t h o u g h Floss and M o t h e s ( 1 9 6 4 ) h a d r e p o r t e d the stimulation b y 5 . A n alternative e x p l a n a t i o n offered by these authors w a s that p r o b a b l y t r y p t o p h a n acts as an i n d u c e r of the alkaloid biosynthetic p a t h w a y .a r a b i n o . to stimulate the p r o d u c t i o n of alkaloids by Claviceps strain S D . In the last p h a s e the alkaloid synthesis stopped.2 . w h i c h are a n a l o g u e s of t r y p t o p h a n .d e o x y .5 8 and the determination of c o n c e n t r a t i o n s of 3 .( l . L . T o test if the reason for this m i g h t h a v e been the p r o d u c t i o n m e d i u m used by these w o r k e r s . t r y p t o p h a n w a s found to reverse the inhibition of synthesis b y high p h o s p h a t e c o n c e n t r a t i o n .D . In these e x p e r i m e n t s a transient increase of t w o . 1976). Finally.b en zo thien-3-yl)-alanine ( t h i o t r y p t o p h a n ) .T r p and ) 3 . E v i d e n c e in support of this s u g g e s t i o n w a s obtained by o t h e r s . 1976). 1972). rate-limiting for alkaloid s y n t h e s i s . concentration of t r y p t o p h a n T h e s e o b s e r v a t i o n s suggested that a high d u r i n g alkaloid p r o d u c t i o n w a s not required for stimulation. Intracellular tryptophan c o n c e n t r a t i o n s w e r e also d e t e r m i n e d in C . R o b b e r s and Floss ( 1 9 7 0 ) r e p e a t e d the e x p e r i m e n t in a defined m e d i u m with Claviceps strain S D .10. T h e y found that in the a b s e n c e of a d d e d t r y p t o p h a n the intracellular level fell d u r i n g the early p h a s e of alkaloid s y n t h e s i s . R e h a c e k and M a l i k 1971). w h i l e the protein synthesis c o n t i n u e d to slow d o w n .

3.7 E n d P r o d u c t Inhibition inhibit D M A T synthetase of Claviceps and Floss E l y m o c l a v i n e and agroclavine w e r e found to strain S D . T h e t w o t r y p t o p h a n a n a l o g u e s differ in their effectiveness in stimulating the alkaloid p r o d u c t i o n . 1980). T h i s suggested that the in v i v o inhibition of alkaloid formation w a s d u e to the inhibition of D M A T synthetase and not its repression. efforts w e r e initiated to elucidate the biosynthetic p a t h w a y . 3 .m e t h y l t r y p t o p h a n w a s less effective. M a n n and Floss 1977). Heinstein 1976). 10. and after t h o r o u g h w a s h i n g to r e m o v e the alkaloid w e r e tested for the incorporation in the a b s e n c e of the alkaloid. It w a s difficult to d e m o n s t r a t e in vivo inhibition of alkaloid synthesis b y e l y m o c l a v i n e in intact m y c e l i u m .I c y c l a s e and an- thranilate synthetase (Erge et al. 1962. H o w e v e r .3. A h i g h e r level of alkaloid synthesis is obtained with thiotryptophan than with 5 . 1973. h o w e v e r .3 Biosynthesis of Ergot Alkaloids 1 0 . n o or slight repression w a s o b s e r v e d with thiotryptophan.[ . O n the other h a n d .m e t h y l t r y p t o p h a n . there w a s n o a g r e e m e n t on the p r e c u r s o r s ) of the rest of the m o l e c u l e . F u r t h e r m o r e . 3 . h o w e v e r it c o u l d be clearly s h o w n in protoplasts ( C h e n g et al. t r y p t o p h a n s y n t h e t a s e .m e t h y l t r y p t o p h a n . a n u m b e r of g r o u p s h a v e s h o w n the incorporation of L . Interestingly. w h e r e a s 5 . the first three are involved in the biosynthesis of t r y p t o p h a n and w e r e strongly repressed by b o t h D L .276 Therapeutic Metabolites thranilate s y n t h e t a s e . T h e observation that the tryptophan p o o l w a s high in the p r e s e n c e of either t r y p t o p h a n or thiotryptophan as c o m p a r e d to that with 5m e t h y l t r y p t o p h a n supported this h y p o t h e s i s . 1 F e e d i n g S t u d i e s . t r y p t o p h a n and t h i o t r y p t o p h a n d e r e p r e s s e d D M A T synthetase equally w e l l . A n e x a m i n a t i o n of the structure indicated that the rings A and Β (Figure 1 0 . M o t h e s and his colleagues (1958) w e r e first to 14 d e m o n s t r a t e incorporation of ß .C ] t r y p t o p h a n into ergot alkaloids with ergotinfected rye plants ( M o t h e s et al. Later. Vining and T a b e r 1963). H o w e v e r . 3 .5 8 (Floss et al. 10.l a b e l e d t r y p t o p h a n into alkaloids b y the protoplasts w a s inhibited. It w a s found that in the p r e s e n c e of e l y m o c l a v i n e succinate the incorporation of 14 C . to o b s e r v e the m a x i m u m effect of the derepression the intracellular t r y p t o p h a n level m u s t not b e limiting.t r y p t o p h a n into ergot alkaloids in s a p r o p h y t i c culture ( A r c a m o n e et al. and D M A T synthetase. until it w a s p r o p o s e d that it is an isoprenoid C-5 unit ( M o t h e s et al. 1974. 1972). if the cells w e r e g r o w n in the p r e s e n c e of e l y m o c l a v i n e s u c c i n a t e . [ C O O H ] t r y p t o p h a n and [alanine-3- . E l y m o c l a v i n e also inhibited c h a n o c l a v i n e . thiotryptophan is able to reverse p h o s p h a t e inhibition but 5 .2. 1958). O n c e the structure of lysergic acid w a s d e t e r m i n e d .7 ) m a y h a v e their origin in the indole ring s y s t e m of t r y p t o p h a n .T r p and 5 . 2 . Of the four e n z y m e s studied.m e t h y l t r y p t o p h a n is not ( R o b b e r s et al. n o inhibition w a s o b s e r v e d . T h e s e results indicate that the derepression of D M A T s y n t h e t a s e is responsible for the stimulation of alkaloid synthesis. 1958). as m e n t i o n e d in Section 1 0 . T o d e 14 t e r m i n e if t r y p t o p h a n w a s incorporated intact. 6 .

H ] m e v a l o n a t e ( G r o e g e r et al. [ 2 . the location of the incorporation w a s not k n o w n as they did not d o d e g r a d a t i o n studies ( G r o e g e r et al. isolation of s t e r e o i s o m e r s of c h a n o c l a v i n e suggested the n e e d to repeat these e x p e r i m e n t s carefully with the individual i s o m e r s (Stauffacher and T s c h e r t e r 1964). 1964). F l o s s et al. 1 9 6 1 . 1959 and 1960. A g u r el l and R a m s t a d 1962).I I and i s o c h a n o c l a v i n e .H ] t r y p t o p h a n w e r e tested ( G r o e g e r et al. B a x t e r et al. H o w e v e r . A very efficient incorporation of the label 1 4 3 3 w a s found with D L . T h e retention of a m i n o nitrogen w a s found in the s u b s e q u e n t studies (Floss et al. B a r r o w and Q u i g l e y 1975). F e e d i n g e x p e r i m e n t s with m e v a l o n i c acid d e m o n s t r a t e d that the s e c o n d postulated p r e c u r s o r . w h i c h is an intermediate in sterol b i o s y n t h e s i s .[ C H 3 ] M e t h i o n i n e w a s used to establish that it w a s the p r e c u r s o r of Nm e t h y l g r o u p ( B a x t e r et al. a C-5 isoprenoid unit. T h e s e results are consistent with the earlier finding of acetate incorporation. T a y l o r and R a m s t a d 1960). w h i c h w a s not. 1961).w o r k e r s 14 ( 1 9 5 9 ) h a d earlier s h o w n that [ C ] a c e t a t e is incorporated into ergot alkaloids.. 1 4 L . w h e r e a s [ C ] e l y m o c l a vine w a s incorporated into l y s e r g o l .C ] . T h e s a m e g r o u p . [ C ] A g r o c l a v i n e w a s c o n v e r t e d to 14 e l y m o c l a v i n e . 3 . S u b s e q u e n t l y it w a s found that c h a n o c l a v i n e . D e g r a d a t i o n of labeled alkaloids p r e p a r e d from 14 [2. T h e e s t a b l i s h m e n t of 3 .I 14 w e r e not ( F e h r et al.d i m e t h y l a l l y l p y r o p h o s p h a t e . 1960.[ 2 . found that C . w a s incorporated into alkaloid ( G r o e g e r et al. 1960. 1966. h o w e v e r . p y r o c l a v i n e . indicating that the d e c a r b o x y l a t i o n takes p l a c e after incorporation of t r y p t o p h a n into the clavine n u cl eu s ( B a x t e r et al. 1 4 1961). Birch et al. 1967b). p e n n i c l a v i n e . 1 9 7 3 . 1960).[ C H 3 ] m e t h i o n i n e s h o w e d that the m e t h y l g r o u p w a s incorporated as a unit b e c a u s e the ratio of the t w o isotopes did not c h a n g e (cf. and setoclavine. G r o e g e r and his c o . 1962. as a p r e c u r s o r . Initial e x p e r i m e n t s to d e m o n s t r a t e the c o n v e r s i o n of c h a n o c l a v i n e into tetracyclic ergolines w e r e unsuccessful (Baxter et al. Floss et al. 3 .10.C ] m e v a l o n a t e . Bassett et al.d i m e t h y l a l l y l p y r o p h o s p h a t e r e d u c e d the incorporation of m e v a l o n a t e s e e m s to b e the s a m e as that for the synthesis of t e r p e n e s ( B a x t e r et al. T h e s e results suggested that e l y m o c l a v i n e is formed by the p a t h w a y ..I w a s c o n v e r t e d into tetracyclic a g r o c l a v i n e and e l y m o c l a v i n e but c h a n o c l a v i n e . T h e results s h o w e d that t r y p t o p h a n w a s incorporated intact with the e x c e p t i o n of the c a r b o x y l g r o u p . T h e next step w a s to elucidate the relationship b e t w e e n various clavine alkaloids. also e x p l a i n s the o b s e r v a t i o n that high alkaloid p r o d u c t i o n is a c c o m p a n i e d by high sterol accumulation. and [ 4 . neither t r y p t a m i n e nor TV-methyltryptamine w a s incorporated.3 14 Ergot Alkaloids 277 3 C .I to a g r o c l a v i n e to e l y m o c l a vine. and i s o p e n n i c l a v i n e (Agurell and R a m s t a d 1962). F l o s s 1976).H ] . 1964. 1964). 1 4 3 S t u d i e s with L . h o w e v e r . 1959). 1960.C]mevalonate s h o w e d that > 9 0 % of the label w a s in C . It is important to note that e l y m o c l a v i n e w a s not c o n v e r t e d to a g r o c l a v i n e (Agurell and R a m s t a d 1962). T h e structure of e r g o p e p t i n e s i n v o l v e s a clavine n u c l e u s and a peptide sidec h a i n . c h a n o c l a v i n e . It w a s p r o p o s e d that e l y m o c l a v i n e is c o n v e r t e d to lysergic acid and its . isolysergol. o n feeding [ l . T h e m e c h a n i s m of m e v a l o n a t e incorporation suggested by the observation that 3-isopent e n y l p y r o p h o s p h a t e or 3 . 3 . . Floss and Groeger 1963).l w a s not i n c o r p o r a t e d . Interestingly.l 7 and the rest in C-7 of the alkaloids (Birch et al. festulaclavine.

1 9 7 1 . O t h e r p r e c u r s o r a m i n o a c i d s . 1962). [ 2 . M a i e r et al.278 Therapeutic Metabolites p e p t i d e derivatives ( M o t h e s et al. 1968.( y .[ U . 4 1 4 C ] P r o . ( 1 9 8 1 ) followed their . 1971 and 1981).C ] A l a into the appropriate p e p t i d e alkaloid ( M a i e r et al.C ] L e u with w h o l e cells with t h o s e in a cell-free s y s t e m ( M a i e r et al.[ U . T h e s e results indicated that alanine m a y not b e a direct p r e c u r s o r .[ U . A n u m b e r of w o r k e r s h a v e s h o w n the labeling of the p e p t i d e m o i e t y with constituent a m i n o a c i d s . 1970).[ 8 . 10. B a s m a d j i a n a n d Floss 1 9 7 1 .C . alaninol w a s not incorporated ( N e l s o n I4 3 and A g u r e l l 1969). the feeding studies established the precursors of all moieties of the ergot alkaloids.t r y p t o - .H ] l y s e r g i c acid w e r e incorporated into e r g o m e t r i n e . Interestingly. L .C ] A l a into the p e p t i d e m o i e t y (Bassett et 1 4 al. 1971).C ] P h e served as a p r e c u r s o r of lysergic acid a n d isolysergic acid. C a s t a g n o l i et al. T h e y found that leucine w a s efficiently incorporated into e r g o s i n e by the cell-free s y s t e m p r e p a r e d from either p r o t o p l a s t s or m y c e l i u m .d i m e t h y l a l l y l ) t r y p t o p h a n ( D M A T ) . Incorporation of L .[ l .C ] P r o and L . 1970).C ] A l a and [ l . T h i s w a s later confirmed by other w o r k e r s w h o also d e m o n s t r a t e d 1 4 1 4 incorporation of L . N ] a l a n i n e w a s used ( C a s t a g n o l i et al. H o w e v e r . 1968.C ] P r o into the proline part of the p e p t i d e chain of e r g o t o x i n e w a s also o b s e r v e d ( G r o e g e r and E r g e 1970).C ] P h e . A s d e s c r i b e d in Section 1 0 . C a s t a g n o l i et al.C ] p y r u v a t e and [ l . 1970).[ U . the s a m e s y s t e m w a s c a p a b l e of incorporating L . alanine w a s found to label the alaninol side c h a i n ( N e l s o n and Agurell 1969).[ U . In their studies on the incorporation of a m i n o acids V i n i n g 1 4 and T a b e r ( 1 9 6 3 ) found that L . F u r t h e r m o r e . T h e substrates of this e n z y m e w e r e identified as D M A . y .P P into D M A T (Heinstein et al. This reaction is catalyzed by d i m e t h y l a l l y l p y r o p h o s p h a t e : t r y p t o p h a n dimethylallyl transferase. T h u s .C ] p y r u vate w e r e also incorporated a n d the ratio 1 5 1 4 N / C increased substantially m o r e than 1 4 1 5 e x p e c t e d from the loss of o n e c a r b o n a t o m .3. t h i o g l y c o l a t e . 1981). 1 the first step in the ergot alkaloid b i o s y n t h e s i s is addition of C-5 isoprenoid unit to t r y p t o p h a n to form 4 . L . 1966). T h e s a m e g r o u p s also 1 5 d e m o n s t r a t e d that the label from [ N ] A l a w a s found in a m i d e nitrogen ( G r o e g e r et 14 1 4 al.t r y p t o p h a n and dimethylallylpyrophosphate 1 4 [ 1 .h y d r o x y v a l i n e and 3 7 % in valine ( B a s m a d j i a n and Floss 1971). T h e m y c e l i u m of Claviceps s p e c .h y d r o x y e t h y l a m i d e 14 (Agurell 1 4 1966).[ U .[ U 1 4 .C ] V a l 5 8 % of the label w a s found in the α . L .C ] A l a s h o w e d that the f o r m e r but not the latter w a s incorporated and that m o s t of the label w a s in the c a r b i n o l a m i d e m o i e t y ( G r o e g e r et al.3.C ] D M A . S u b s e q u e n t studies with various labeled 1 4 c l a v i n e alkaloids c o n f i r m e d this h y p o t h e s i s (Floss et al.P P a n d L . 1973). 3 . In the c a s e of e r g o n o v i n e .C ] A l a n i n e w a s s h o w n to b e incorporated into lysergic acid α . w h e n [ U . In feeding 1 4 studies with D L . 4 earlier studies on the incorporation of L .2 Enzymology.[ U . strain S D 5 8 w a s g r o u n d in Tris buffer c o n t a i n i n g d i e t h y l d i t h i o c a r b a m a t e . T h e feeding studies with [ 2 . M a i e r et al. 1971). T h e y also found that d . valine a n d l e u c i n e . 3 . w e r e also incorporated into the p e p t i d e m o i e t y (Floss et al. and m e r c a p t o e t h a n o l to obtain cell-free preparation w h i c h efficiently c o n v e r t e d L . w h i c h is also called D M A T s y n t h a s e (Heinstein et al.[ U . 1981).[ C ] l y s e r g i c acid and i / .

a p r e c u r s o r of e l y m o c l a v i n e . M g . Floss 1976).3 Ergot Alkaloids 279 p h a n . T h e c o n v e r s i o n 2 + required a d e n o s i n e t r i p h o s p h a t e . M g . T h e activity r e a c h e d a m a x i m u m a n d sharply d e c l i n e d after 10 d a y s . T h e activity could be restored b y the addition of F A D to the fractions.10. M n .w o r k e r s (cf. 1973). 1976). It is a single p o l y p e p t i d e chain with a m o l e c u l a r m a s s of 7 3 . In their system A T P . T h i s inhibition is n o n c o m p e t i t i v e with either substrate. it p e a k s m u c h earlier ( O t s u k a et al.I to agroclavine and not to e l y m o c l a v i n e .I c y c l a s e activity w a s d e m o n s t r a t e d in the cell-free extracts of alkaloid p r o d u c i n g 2 + strains of C. A l t h o u g h in this s y s t e m as m u c h as 2 0 % c o n v e r s i o n of c h a n o c l a v i n e . 1 9 7 3 . and N H j had n o effect. this e n z y m e preparation c o n v e r t e d c h a n o c l a v i n e . O n the other h a n d . Floss 1976) found cyclase activity in the extracts of Claviceps s p . Floss and c o . and C a . D M A T s y n t h a s e w a s purified to h o m o g e n e i t y (Lee et al. 1971). It c o n t a i n s t w o half-cystine r e s i d u e s . 1 9 7 3 . C a . In this case A T P w a s not required and both agroclavine and e l y m o c l a v i n e w e r e p r o d u c e d . p r e c e d ing the alkaloid p r o d u c t i o n by 1 d a y .I into e l y m o c l a v i n e w a s seen. 1970). A cell-free s y s t e m c a p a b l e of c o n v e r t i n g c h a n o c l a v i n e . T h e fact that the c o n c e n t r a t i o n s of the e n d p r o d u c t s required for the inhibition are similar to the final yields o b t a i n e d in the fermentation suggests its physiological significance (Floss 1976). T h e s e c o n d pathway-specific reaction that w a s d e m o n s t r a t e d in the cell-free s y s t e m p r e p a r e d from the s a m e fungus discussed in the p r e c e d i n g p a r a g r a p h w a s the transfer of the m e t h y l g r o u p of S A M to the a m i n o g r o u p of D M A T ( O t s u k a et al. Later this c h a n o c l a v i n e . T h e kinetics of p r o d u c t i o n of this e n z y m e w a s similar to that of D M A T synthase ( E r g e et al. w h i c h suggested i n v o l v e m e n t of flavine c o e n z y m e in the cyclization reaction (Floss 1976). and N A D P H w e r e n e e d e d for the c o n v e r s i o n but not F A D or free o x y g e n . Interestingly. 2 mM and 0 . 1971). T h e p H o p t i m u m is b e t w e e n 7 and 8 (Heinstein et al. 2+ 2 + and to lesser extent b y F e . purpurea (Erge et al. strain S D 5 8 . and free o x y g e n as cofactors. Further . A s e x p e c t e d this decline resulted in the cessation of the alkaloid s y n t h e s i s . and C o . T h e activity w a s strongly inhibited b y Z n 2 + 2 + 2 + 2 + 2 + C u . T h e Km values for these as d e t e r m i n e d with the purified e n z y m e are 0 . T h e cyclase accepted either c h a n o c l a v i n e . 1980). M g . 0 0 0 D a . it is inhibited b y a g r o c l a v i n e and e l y m o c l a v i n e . It 2 + 2 + 2 + is activated b y F e . but not isochanoclavine-I or d i h y d r o c h a n o c l a v i n e . r e d u c e d n i c o t i n a m i d e a d e n i n e dinucleotide p h o s p h a t e . in spite of the stabilizer the c h r o m a t o g r a p h y o n S e p h a d e x or D E A E cellulose resulted in the loss of the e n z y m e (Erge et al. 1976). 0 6 7 m M . Interestingly. T h e efforts to purify this e n z y m e h a v e not b e e n successful d u e to its instability.I or c h a n o clavine-I a l d e h y d e as a substrate. w a s found. n o a c c u m u l a t i o n of a g r o c l a v i n e . Floss 1976). h o w e v e r . respectively (Lee et al. T h e e n z y m e activity w a s very low d u r i n g the g r o w t h p h a s e and increased rapidly d u r i n g the p r o d u c t i o n p h a s e .I into e l y m o c l a v i n e w a s d e v e l o p e d from Claviceps strain 231 ( O g u n l a n a et al. A d d i t i o n of glycerol stabilized the activity to s o m e extent. 1980). w h i c h suggests feedback control of this e n z y m e (Heinstein et al. M g . H o w e v e r . T h i s e n z y m e is p r o d u c e d after the cessation of g r o w t h as w a s o b s e r v e d for D M A T s y n t h a s e . w h i c h s u g g e s t e d that either the e n z y m e involved in the c o n v e r s i o n of agroclav i n e to e l y m o c l a v i n e w a s lost during its preparation or the conditions used w e r e not o p t i m u m for the c o n v e r s i o n .

and m o l e c u l a r o x y g e n as substrates + and e l y m o c l a v i n e . This m u t a n t a c c u m u l a t e d chanoclavine-I and c h a n o c l a v i n e . g a v e the best extracts.I . T h e o p t i m u m p H of the e n z y m e is about 7. purpurea P R L 1980. (1981) described a cell-free s y s t e m prepared from C. T h e reaction required N A D P H and w a s inhibited by ethylenediaminetetraacetic acid ( E D T A ) and c y a n i d e (Hsu and A n d e r s o n 1971). and agroclavine into e r g o p e p t i n e s . T h e e n z y m e preparation required all the p r e c u r s o r s and liver concentrate for activity. A d d i t i o n s of g l y c e r o l . cells at the b e g i n n i n g of production p h a s e .280 Therapeutic Metabolites e v i d e n c e for the i n v o l v e m e n t of this e n z y m e in the alkaloid biosynthesis w a s o b t a i n e d by M a i e r et al. T h e observation that the dialyzed e n z y m e is active supports this s u g g e s 1 8 0 incorporation studies it w a s s h o w n earlier that the o x y g e n of tion. c h a n o c l a v i n e . e l y m o c l a v i n e . T h e . T h e 6 0 . e l y m o c l a v i n e .I a l d e h y d e and contained substantially l o w e r cyclase activity but n o r m a l levels of D M A T s y n t h a s e . and p h e n y l m e t h y l s u l f o n y l fluoride ( P M S F ) w e r e necessary to stabilize the activity. T h i s apparent contradiction is p r o b a b l y b e c a u s e the metal ion required is b o u n d to the e n z y m e . 3 . T h e m o s t preferred substrate w a s agroclavine and the least preferred w a s d-lysergic acid. T h r e e . T h e cells w e r e b r o k e n in a Virtis h o m o g e n i z e r and the cell debris w a s r e m o v e d by l o w speed centrifugation. and m e t h i o n i n e b y a partially purified e n z y m e preparation from C. 1 M a i e r et al. T h e supernate w a s further centrifuged at 1 0 5 . w h o isolated a m u t a n t b l o c k e d in the p r o d u c t i o n of a l k a l o i d s . 1970). T h i s reaction w a s not catalysed by the extracts prepared by O g u n l a n a et al. and chanoclavine-II from t r y p t o p h a n . B a s e d on e l y m o c l a v i n e is derived from m o l e c u l a r o x y g e n (Floss et al. w h i c h catalyzed the c o n v e r s i o n of a g r o c lavine to e l y m o c l a v i n e (Hsu and A n d e r s o n 1970). 0 0 0 g for 1 h. T h e cell-free s y s t e m w a s p r e p a r e d either by h o m o g e n i z i n g the protoplasts or by grinding lyophilized m y c e lium in a mortar.I to e l y m o c l a v i n e ( O g u n l a n a et al.d a y . A b o u t the s a m e time as this w o r k w a s reported C a v e n d e r and A n d e r s o n (1970) described synthesis of a g r o c l a v i n e . N A D . that i s . 1981). N o n e of the divalent metal ions tested stimulated the activity. N A D P H . S u b s e q u e n t refinement of the p r o c e d u r e s resulted in a m o r e active preparation (Maier et al. 1981). isopentenyl p y r o p h o s p h a t e . ( 1 9 7 0 ) w h i c h c o n v e r t e d c h a n o c l a v i n e .o l d m y c e l i u m . and water as products (Hsu and A n d e r s o n 1971). A s m e n t i o n e d in Section 1 0 . A similar p r o c e d u r e w a s used to partially purify agroclavine h y d r o x y l a s e . 3 . T h e c o n v e r s i o n of agroclavine to e l y m o c l a v i n e involves h y d r o x y l a t i o n . T h e c r u d e extract c o n verted d-lysergic acid. T h e a g e of the m y c e l i u m used w a s critical in obtaining g o o d activity.8 0 % (saturation) ( N H 4 ) 2 S 0 4 fraction had the activity. purpurea that converted either clavine alkaloids or dlysergic acid into e r g o p e p t i n e s ( M a i e r et al. dithioerythrol. 1981). T h e s e o b s e r v a tions c o u p l e d with the fact that the addition of agroclavine increased the rate of N A D P H oxidation suggested that the h y d r o x y l a s e is a m i x e d function o x y g e n a s e ( m o n o o x y g e n a s e ) with a g r o c l a v i n e . T h e failure to obtain this reaction with the crude cell-free preparation suggested a p r e s e n c e of an inhibitor. ( 1 9 8 0 ) . 1967a). the specific o n e p r o d u c e d w a s d e t e r m i n e d by the a m i n o acids a d d e d . T h e r e f o r e . they c o n c l u d e d that agroclavine w a s not an intermediate in the synthesis of e l y m o c l a v i n e . T h e c o n v e r sion w a s d e p e n d e n t on A T P (Maier et al. T h e e n z y m e solution w a s fractionated with ( N H 4 ) 2 S 0 4 .

10. ( 1 9 6 2 ) i n v o l v e s nucleophilic attack by C-5 of D M A . T h e formation of the ring C is a c o m p l e x reaction and not yet fully u n d e r s t o o d . 2 m M . 1984). 1984).l y s e r g i c acid w a s 0 .d o u b l e b o n d is involved . n a m e l y C-4 of the indole ring or the α . 6 . agroclav i n e .A l a .3. A n e n z y m e w a s found in the cell-free extracts of C. T h e e n z y m e activated d-lysergic acid and dihydrolysergic acid but not J . w h e r e a s L . a n d L .P r o resulted in their incorporation into e r g o s i n e . It a p p e a r s that cis-trans isomerization in the isoprenoid unit with respect to 8 . c o n s i d e r a b l e w o r k is yet to b e d o n e to isolate all of the e n z y m e s involved in the c o n v e r s i o n of clavine alkaloids to e r g o p e p t i n e s . 9 . T h e direct addition of D M A .3 M e c h a n i s m of F o r m a t i o n o f R i n g s C a n d D a n d P e p t i d e S i d e C h a i n . 1962). 1 . T h e e n z y m e did not catalyze p e p t i d e b o n d formation nor did it activate any other constituent a m i n o a c i d s . It is i m p o r t a n t to note that these extracts contained not only the e n z y m e s i n v o l v e d in the formation of p ep tid e b o n d s but also those that catalyzed a- h y d r o x y l a t i o n of the appropriate a m i n o acid and cyclol formation.a g a r o s e .a n d forty-fivefold purification. as seen from the rates of the reaction. d i h y d r o l y s e r g i c acid a m i d e . 1981). a n d L . resulting in o n e . Floss 1976).l y s e r g i c acid m e t h y l ester.0 . 8 . T o d e c i d e b e t w e e n these t w o possibilities.h u n d r e d . T h e analysis of the e l y m o c l a v i n e p r o d u c e d s h o w e d that the addition o c c u r s at C-4 of the indole ring. for e x a m p l e . T h e m e c h a n i s m as p r o p o s e d by B a x t e r et al.L e u .5 and 9 .P P to t r y p t o p h a n as suggested b y the feeding studies can o c c u r at t w o sites. Characterization of these will help d e t e r m i n e the b i o s y n thetic p a t h w a y of lysergil p e p t i d e s . 0 0 0 D a . F u r t h e r m o r e . Earlier it w a s postulated that the p e p t i d e synthesis o c c u r s on a m u l t i e n z y m e c o m p l e x similar to that i n v o l v e d in g r a m i c i d i n biosynthesis (Floss et al. purpurea. p r o p y l . and e r g o t a m i n e . w h i c h activated d-lysergic acid (Keller et al.P h e . If this is the case then the constituent a m i n o acids will h a v e to b e activated via a d e n y l a t e formation (Keller et al. e l y m o c l a v i n e .3. 0 0 0 and 1 4 0 . d-lysergic acid w a s preferred. S o it appears that although M a i e r et al. the reaction w a s c o m p l e t e in 2 . L . ( 1 9 8 1 ) w e r e able t o d e m o n s t r a t e the synthesis of e r g o p e p t i n e s in the cell-free s y s t e m . the predicted p r o d u c t s of this reaction w e r e synthesized and w e r e fed to the fungus ( W e y g a n d et al.c a r b o n of the side c h a i n of t r y p t o p h a n .3 Ergot Alkaloids 281 addition of L . c h a n o c l a v i n e .A l a . T h e purification s c h e m e involved c h r o m a t o g r a p h y o n D E A E . T h e m o l e c u l a r m a s s of the e n z y m e as d e t e r m i n e d b y gel-filtration w a s b e t w e e n 1 3 5 .P P on the C-4 of t r y p t o p h a n b y the loss of a p y r o p h o s p h a t e ion ( B ax ter et al. T h e Km for J .c e l l u l o s e . Plieninger et al. T h e e n z y m e w a s active o v e r the p H r a n g e b e t w e e n 6. L . 1963). and D E A E . 10.P r o w e r e incorporated into e r g o t a m i n e ( M a i e r et al. w h i c h w a s surprising as this is a w e a k l y reactive position. that is. 5 m i n with d-lysergic acid as c o m p a r e d to 25 min n e e d e d with d i h y d r o l y s e r g i c acid. Of the t w o substrates. 0 with a slight o p t i m u m at p H 8 a l t h o u g h it w a s m o r e stable at l o w e r p H . 1962 and 1964.c e l l u l o s e . 1976). It w a s suggested by Floss (1976) that the e n z y m e D M A T s y n t h a s e binds the t w o substrates in such w a y as to o p t i m i z e the direct alkylation at C-4 (cf. 1974. Ultrogel A c A 3 4 . a l m o s t three t i m e s m o r e product w a s formed with the former than the latter. F l o s s .

P P ( S h i b u y a et al.P P and c h a n o c l a v i n e . T h i s indicated that s d u r i n g the cyclization of r i n g .4 . T o explain these results the authors p r o p o s e d that the h y d r o g e n at C-9 is transferred from o n e substrate m o l e cule to s o m e acceptor on the e n z y m e and then back to the second substrate m o l e c u l e (Floss et al. 1974). and trideuterated m e v a l o n a t e into clavine alkaloids w a s d e t e r m i n e d . oxidation of C .C ] m e v a l o n i c and [ I . It w a s found that c h a n o c l a v i n e . 1974). c h a n o c l a v i n e . T h e c o n v e r s i o n of chanoclavine-I to agroclavine involves cyclization to form 14 14 r i n g .I (Floss 1976). as calculated from the 3 1 4 ratio of H / C .I and at C-17 of e l y m o c l a v i n e obtained from both precursors (Floss et al.I w a s trideuterated but e l y m o c l a v i n e and p e n n i c l a v i n e w e r e not. s h o w e d that 9 0 % of the label from m e v a l o n a t e w a s at C-7 of c h a n o c l a v i n e .c h a n o c l a v i n e . T h e e x p e r i m e n t s with [ 2 . T h e s e results confirmed the loss of o n e h y d r o g e n from C-17 as well as cis-trans isomerization during cyclization (Floss et al. T h e incorporation studies with [ 2 . 1968). T h e d e g r a d a t i o n of e l y m o c l a v i n e s h o w e d that the incorporation of the a l d e h y d e w a s specific and that the h y d r o g e n at C-17 w a s completely retained d u r i n g the cyclization. T h e m e c h a n i s m for the ring formation inv o l v e d a potential carbocation at the benzylic carbon and a potential c a r b a n i o n at C-a. T o test this hypothesis r e p l a c e m e n t cultures of Claviceps w e r e in 1 8 c u b a t e d in an a t m o s p h e r e of 0 2 / N 2 .I and C .. All these observations establish c h a n o c l a v i n e . 1974).D cis-trans isomerization a r o u n d A d o u b l e b o n d 14 3 14 3 o c c u r s .I . S o m e s c r a m b l i n g of the label from the C-2 of m e v a l o n a t e b e t w e e n C-7 and C-17 of e l y m o c l a v i n e .I . 5 3 % of the tritium label w a s retained in e l y m o c l a v i n e and all of that w a s in C-7 position.a l d e h y d e w a s tested as a precursor of e l y m o c l a v i n and w a s found to b e incorporated with fourfold m o r e efficiency than chanoclavine-I (Floss et al. respectively. d i . Further feeding studies with [ 1 7 .I w h i c h s h o w e d that.I . F u r t h e r studies indicated that the h y d r o g e n at C-9 u n d e r g o e s i n t e r m o l e c u lar transfer to the s a m e position (Floss et al.C . 1968). T h e s e q u e n c e of reactions p r o p o s e d for the formation of the ring C includes methylation of the a m i n o nitrogen.C .282 Therapeutic Metabolites b e t w e e n D M A .H ] m e v a l o n i c acid and [ 7 .1 0 of D M A T . 1974).C ] c h a n o c l a v i n e into c h a n o c l a v i n e . O n e 3 of the d e u t e r i u m labels from C-17 w a s lost. T h i s can b e explained by postulating that the h y d r o x y m e t h y l g r o u p of c h a n o c l a v i n e . w a s s h o w n to o c c u r in the formation of D M A . This suggestion w a s tested with m e v a l o n a t e labeled at a p p r o 1 3 2 priate positions with C and H and results consistent with the h y p o t h e s i s w e r e o b tained.I g o e s through the oxidation stage of an a l d e h y d e during this p r o c e s s .a l d e h y d e to be a n o r m a l i n t e r m e d i a t e in ergot b i o s y n t h e s i s .H ] 14 c h a n o c l a v i n e .9 .D .I and e l y m o c l a 1 8 vine p r o d u c e d s h o w e d 7 2 a t o m % 0 e n r i c h m e n t of both ( K o b a y a s h i and Floss 1987). 1990). e l y m o c l a v i n e . w h i c h w a s o b s e r v e d p r e v i o u s l y . In the s a m e set of e x p e r i m e n t s incorporation of d e u t e r i u m from m o n o . and d e c a r b o x y l a t i o n at C-5 with simultaneous cyclization resulting in c h a n o c l a vine-I ( K o b a y a s h i and Floss 1987). T h e analysis of c h a n o c l a v i n e . 1967a). and e l y m o c l a v i n e .. . T h i s suggested that the o x y g e n of chanoclavine-I and e l y m o c l a v i n e is introd u c e d by an o x y g e n a s e and therefore m u s t c o m e from a t m o s p h e r i c o x y g e n (Floss et al.H ] c h a n o clavine s h o w e d that 7 0 % of the label is retained in C-9 of e l y m o c l a v i n e (Floss et al. T h e r e f o r e .

V a l to L . 1974).h y d r o x y t r y p t a m i n e . T h e intermediates in the biosynthesis are transferred o n t h i o t e m p l a t e p r o b a b l y via p h o s p h o p a n t e t h e i n e . b l o c k e r s and stimulants of α .b o u n d pep tid e w h i c h is followed b y head-to-tail cyclization to give g r a m i c i din S ( K l e i n k a u f a n d v o n D o h r e n 1987).I .I to c h a n o c l a v i n e . T h e s e c o n d acceptor g r o u p will transfer this h y d r o g e n from C-9 to the C-9 of a n o t h e r substrate m o l e c u l e (Floss et al.I to e l y m o c l a v i n e w a s proposed: o x i d a t i o n of c h a n o c l a v i n e . T w o multifunctional e n z y m e s catalyze the formation of enz y m e .a l d e h y d e . After addition of L ./3-unsaturated c a r b o n y l s y s t e m of the a l d e h y d e into the "active s i t e .3 Ergot Alkaloids 283 F r o m the results of the studies described in the p r e v i o u s p a r a g r a p h the following set of reactions to c o n v e r t c h a n o c l a v i n e . d o p a m i n e a n t a g o n i s t s . T h i s w a s followed by h y d r o x y l a tion and cyclol ring formation to give e r g o p e p t i n e (Floss et al.8 .4 Pharmacological Effects and Clinical Studies E r g o t alkaloids h a v e so m a n y p h a r m a c o l o g i c a l effects that they h a v e b e e n referred to as " t r e a s u r e chest for p h a r m a c o l o g i s t s " and " t r e a s u r e .P r o to form the p e p t i d e . isomerization a r o u n d the Δ 8 d o u b l e b o n d resulting in the synthesis of i s o c h a n o c l a v i n e . 10.w o r k e r s is responsible for a significant portion of o u r k n o w l e d g e on this t o p i c . 1974.9 d o u b l e b o n d to bring N . A m e c h a n i s m w a s postulated for the i s o m e r i z a t i o n that involved the i n t e r m o l e c u l a r e x c h a n g e of h y d r o g e n . F l o s s 1976). It should be noted that so far neither c h a n o c l a v i n e .H T ( 5 . T h e isolation of d-lysergic acid activating e n z y m e p r o v i d e s support to this h y p o t h e s i s (Keller et al.a l d e h y d e has been isolated.a l d e h y d e . 1984). and reduction leading to agroclavine and its h y d r o x y l a t i o n giving rise to e l y m o c l a v i n e (Floss et al. and e n h a n c e r s of re- . formation of Schiff b a s e . It m u s t h a v e b e c o m e clear to the r e a d e r that a vast a m o u n t of the w o r k has been d o n e o n the biosynthesis of ergot alkaloids and it is not possible to d o j u s t i c e to that in the present chapter. Floss and A n d e r s o n 1980). A c c o r d i n g to this the substrate b i n d s the e n z y m e in such a w a y as to insert the α. the p e p t i d e w a s transferred to J .6 in p r o x i m i t y to the a l d e h y d e c a r b o n and an acceptor g r o u p on the e n z y m e . K l e i n k a u f and v o n D o h r e n 1987). t w o excellent r e v i e w s by F l o s s . are r e c o m m e n d e d (Floss 1976. A s indicated by the results of feeding studies the p ep t i d e side chain of the e r g o p e p t i n e s is p r o b a b l y synthesized by a s e q u e n c e of reactions similar to that d e s c r i b e d for the p ep tid e antibiotics like g r a m i c i d i n S (Floss 1976.I a l d e h y d e n o r i s o c h a n o c l a v i n e . T h e y are both stimulants and b l o c k e r s of 5 . T h e nitrogen a t o m transfers its p r o t o n to the acceptor g r o u p .l y s e r g i c acid and released from the e n z y m e . F o r those w h o w o u l d like to read m o r e about it.L e n and L .h o u s e for d r u g s " ( B e r d e and S t u r m e r 1978). T h e s e t w o e n z y m e s also activate all the a m i n o acids i n v o l v e d . T h i s is followed b y transfer of h y d r o g e n from C-9 to a n o t h e r a c c e p t o r g r o u p and either c a r b i n o l a m i n e or isochanoclavine-I-aldehyde is released from the e n z y m e . 1974).a d r e n o r e c e p t o r s .8 / C . " T h i s is followed by p r o t o n a tion of o x y g e n and C-9 and partial rotation a r o u n d the C . A biosynthetic p a t h w a y b a s e d on the present state of o u r u n d e r s t a n d i n g is s h o w n in F i g u r e 1 0 .I . serotonin) r e c e p t o r s . w h o a l o n g with his c o .10.I .3. It w a s suggested that the p e p t i d e side chain w a s synthesized on a thiotemplate b e g i n n i n g with p r o l i n e .V a l or L . e n h a n c e r s of prostaglandin s y n t h e s i s .

T h e activities are found useful include control of p r e s s u r e and h e m o d y n a m i c s . 297-304. (1990) J. Reprinted with permission from Shibuya et al. Soc. and control of and t h o s e of the e n d o c r i n e s y s t e m .284 Therapeutic Metabolites 1 COOH Elymoclavine Lysergic Acid (R =OH) Ergopeptines (R = Peptide side chain) F I G U R E 10-8 Biosynthesis of ergot alkaloids. Am. Chem. control of b l o o d diseases of the central n e r v o u s s y s t e m . n o n v a s c u l a r s m o o t h m u s c l e s (Mullermajor therapeutic areas w h e r e t h e s e uterine m o t o r activity. Copyright 1990 American Chemical Society. s p o n s e s to biogenic a m i n e s in vascular and S c h w e i n i t z e r and W e i d m a n n 1978). 112.

a ergotriptine ( b r o m o c r i p t i n e ) c a u s e d a long-lasting reduction in b l o o d p r e s s u r e in anesthetized d o g s with d o s e of 6 m g / k g given intravenously. O t h e r uses of o x y t o c i c effect of these c o m p o u n d s are for the induction of labor and for therapeutic a b o r t i o n s .3. D i h y d r o e r g o t o x i n e and its c o m p o n e n t s h a v e a long-lasting ( 3 .0 . 0 0 5 . E q u a l l y intriguing o b s e r v a t i o n s are those on the species difference.4. h o w e v e r . d o s e s e v e n l o w e r than those used in g u i n e a pig and d o g w e r e effective in h u m a n (Garrett 1959). Clark et al. 0 0 2 . rabbit.0 . 1977). 3 . A l k a l o i d s of the lysergic acid a m i d e class and e r g o p e p t i n e class are effective.2 Control of Blood Pressure and H e m o d y n a m i c s .5 h) d e p r e s s o r effect in n o r m o t e n s i v e and h y p e r t e n s i v e subjects (Clark et al. 0 0 8 m g / k g had an o x y t o c i c effect for i n d u c i n g or e n h a n c i n g labor near t e r m . on the other h a n d . 5 m g / k g w a s required in d o g (cf.0 . Ergopeptines have universally pressor effect on the blood pressure. d i h y d r o e r g o p e p t i n e s .a d r e n e r g i c receptors as c o m p a r e d to that for those of peripheral v a s c u l a t u r e . T h e m e c h a n i s m of the . and cat (Saameli 1978). T h e h o r m o n a l condition of w o m a n has a significant influence on the effect of the alkaloids. and d i h y d r o e r g o t a m i n e (cf. T h e effect of o x y t o c i n is not b l o c k e d by the b l o c k e r s of α .B r o m o . A n u m b e r of factors influence the effect of alkaloids. S t u m p e et al. O v e r a l l . T h i s d r u g has b e e n reported to c a u s e reduction in b l o o d pressure in patients with P a r k i n s o n ' s disease or a c r o m e g a ly. T h e s e d r u g s are o x y t o c i c . S a a m e l i . Interestingly. T h e y affect frequency. T h e preferred route of administration a p p e a r s to b e i n t r a v e n o u s . 4 . G u i n e a pig and d o g are m o r e sensitive than rat. w h o are b e i n g treated with high d o s e s of it. e r g o m e t r i n e (also k n o w n as e r g o n o v i n e ) and m e t h y l e r g o m e t r i n e are used to treat uterine b l e e d i n g . Initially it w a s believed that c o m p o u n d s with 9. and basal t o n e of uterine contractility positively ( B e r d e and S t u r m e r 1978). E r g o m e t r i n e is only slightly active in the first half of the menstrual cycle but very active during m e n s t r u a t i o n . 1 C o n t r o l of U t e r i n e M o t o r A c t i v i t y . t h o u g h . w h e r e the 9 . a m p l i t u d e . F o r e x a m p l e . 1978). d i h y d r o e r g o t o x i n e . and in patients with essential h y p e r t e n s i o n ( G r e e n a c r e et al.3 Ergot Alkaloids 285 1 0 . and d o s e . p h e n t o l a m i n e . T h e s e include c h e m i c a l structure. 0 1 m g / k g w e r e effective in w o m e n d u r i n g the third p h a s e of labor. It is important to note that o x y t o c i n and these alkaloids d o not act on the s a m e target. species affected.10-unsaturation w e r e o x y t o c i c .a d r e n e r g i c r e c e p t o r s . w h e r e a s 0 . 1978). although at h i g h e r d o s e s this effect d i m i n i s h e d (cf. T h e duration of the effect in w o m e n w a s found to be 2 0 m i n . O n e reason for their selection is that they d o not elevate the b l o o d p r e s s u r e . 2 . h o r m o n a l condition of the subject. e r g o m e t r i n e is less effective o n the n o n p r e g n a n t uterus than on the puerperal u t e r u s . w h i c h results in rapid r e s p o n s e usually within 1-2 m i n . T h e lack of effect of e r g o m e t r i n e and m e t h y l e r g o m e t r i n e on blood pressure is p r o b a b l y d u e to their higher affinity for uterine α . B e r d e 1978). doses of 0 . 10. 1978). 1976. 0 0 1 . in contrast 0 . 1 0 d o u b l e b o n d is r e d u c e d . s e e m e d to h a v e a d e p r e s s o r effect. T h e oldest k n o w n use of ergot alkaloids is for the control of the uterine m o t o r activity. In w o m e n the effect is d o s e d e p e n d e n t (Saameli 1978). h o w e v e r a n u m b e r of c o m p o u n d s are e x c e p t i o n to this notion. T h e effect on the s m o o t h m u s c l e of the uterus w a s t h o u g h t to be d u e to their action on α-adrenergic receptors and it can b e b l o c k e d b y α-adrenergic b l o c k i n g agents such as p h e n o x y b e n z a m i n e .10.

reduction (Clark et al. A n o t h e r factor thought to trigger these h e a d a c h e s is a sudden reduction in serotonin concentration (Clark et al. t r e m o r . Clark et al. 4 . Interestingly. the effect on the arteries is u n p r e d i c t a b l e . 1978). U n d e r certain c o n d i t i o n s they c a u s e increase in the flow rate in the arteries of the extremities and u n d e r other. Clark et al. 1978).286 Therapeutic Metabolites h y p o t e n s i v e action of these c o m p o u n d s is suggested to b e o n e involving stimulation of peripheral d o p a m i n e receptors resulting in vasodilation. T h e r e is also cessation of . and similar inhibition of s y m p a t h e t i c n e r v e terminal in vascular s m o o t h m u s c l e (cf. 3 . A v e r s i o n to light and painful sensation in the skin of face and scalp are c o m m o n (Clark et al. loss of nigrostriatal 3 . T h e i r effect on the veins is better defined. P a r k i n s o n i s m is indicated by four major signs: rigidity. a multic e n t e r . and s h o u l d e r s . although Cafergot P-B w a s m o r e effective than Cafergot in r e d u c i n g the n a u s e a . 1973). O n e of the causes of these h e a d a c h e s is thought to be stretch and dilation of the arteries to the scalp and dura w h i c h leads to increased blood flow.a d r e n o r e c e p t o r s or d o p a m i n e receptors in n o r a d r e n e r g i c nerves resulting in l o w e r i n g the release of this transmitter.b l i n d . T h e s e signs h a v e been linked to d e g e n e r a t i o n of the large p i g m e n t e d n e u r o n s in the substantia nigra. Ergot alkaloids also significantly affect the blood flow in both the arteries and the v e i n s . 3 P a r k i n s o n i s m . H o w e v e r . r a n d o m i z e d study to c o m p a r e Cafergot ( e r g o t a m i n e tartarate and caffeine). d i m i n u t i o n in the increase in the heart rate c a u s e d by the stimulation of cardiac sympathetic n e r v e . w h i c h is another type of severe h e a d a c h e . 1978). 1 0 . O n e of the m o s t important uses of alkaloids is for the treatment of m i g r a i n e h e a d a c h e s . T h e alkaloids h a v e been s h o w n to be effective in the treatment and p r e v e n tion of m i g r a i n e attack ever since the observations of T z a n c k (1928) and T r a u t m a n n ( 1 9 2 8 ) (cf. 1978). It w a s suggested that a prostaglandin-like c o m p o u n d secreted upon the administration of the alkaloids is r e s p o n s i b l e for these differences (Muller-Schweinitzer and B r u n d e l 1975). 1989). D u r i n g the attack the pain can e n c o m p a s s the w h o l e h e a d . T h u s . D y s k i n e s i a a n d Senile C e r e b r a l Insufficiency. prophylactic therapy with e r g o n o v i n e m a l e a t e w a s found to h a v e s o m e value (Gallagher 1989). T h e results indicated that either Cafergot or Cafergot P-B w a s significantly m o r e effective than the p l a c e b o for the treatment of m i g r a i n e . T h e m o s t effective c o m p o u n d is semisynthetic l-methyl-d-lysergic acid-L-2-butanolamide hydrogen maleinate (methylsergide). b r a d y k i n e s i a . Cafergot P-B (Cafergot with pentobarbital and bellafoline). R e c e n t l y . T h e y i m p r o v e the v e n o u s tone and are venoconstrictors. 1978). this c o m p o u n d w a s s h o w n to h a v e a less p r o m i n e n t antiserotonin role in the carotid bed of d o g although it had a vasoconstrictive activity ( S a x e n a 1972). and gait disorder ( L i e b e r m a n 1974). T h e differences in their effect on the arteries and the veins h a v e b e e n p u z z l i n g . n e c k . and p l a c e b o w a s reported ( F r i e d m a n et al. and is a c c o m p a n i e d by nausea and v o m i t i n g . In the treatment of menstrual m i g r a i n e . stimulation of p r e j u n c t i o n a l α . d o u b l e . it appears that it acts by vasoconstriction and by reversing the effects of the reduction in serotonin concentration ( B e r d e and S t ü r m e r 1978). 4 d i h y d r o x y p h e n y l a l a n i n e ( D O P A ) d e c a r b o x y l a s e activity and d e c r e a s e in striatal d o p a m i n e contents and turnover ( B i r n h e i m e r et al.

T w o other u n d e r investigation are lisuride and p e r g o l i d e (Calne et al. Serby et al. after w h i c h the efficacy of a m a n t a d i n e declined and after a n o t h e r 2 w e e k s . chronic a l c o h o l i c s . c h r o n i c o r g a n i c brain s y n d r o m e . 1978). In o n e study with 3 0 subjects. T h e c o n c l u s i o n s s e e m to b e that b r o m o c r i p t i n e is effective. b r o m o c r i p tine w a s significantly m o r e effective e v e n in this later p h a s e (Giannini et al. the disease c o n t i n u e s to progress and in 2 . T h e s e involve cognitive and behavioral c h a n g e s . h o w e v e r . 1980). and e p i n e p h r i n e w e r e e v e n lower. it w a s n o m o r e effective than the p l a c e b o . 1989. H o w e v e r . A n u m b e r of studies with b r o m o c r i p t i n e h a v e b e e n reported ( L i e b e r m a n et al. w h i c h t o o . T h e c o m p o u n d s w h i c h h a v e been d e m o n s t r a t e d to b e effective are b r o m o c r i p t i n e and lergotrile. 1989). In the o t h e r e x p e r i m e n t . 1980). M o n t a s t r u c et al. T o c o u n t e r this reduction in d o p a m i n e l e v e l s . residual type ( C a v a n a g h et al. 1980. 1989). 1989). w e r e also tested for the therapy of dyskinesia.10.D O P A therapy w a s tried and found to b e useful in the initial p h a s e s . 1989). and n o r m a l controls w e r e e x a m i n e d p o s t m o r t e m (Carlsson et al. 5 H T . very little or n o success w a s obtained ( T a m m i n g a 1980. b e c a u s e of their d o p a m i n e agonist p r o p e r t i e s . c a u s i n g d o p a m i n e depletion could be c o u n t e r a c t e d with the ergot a l k a l o i d s . b r o m o c r i p t i n e w a s c o m p a r e d with a m a n t a d i n e and p l a c e b o for its ability to d i m i n i s h the w i t h d r a w a l s y m p t o m s (Giannini et al. In an extensive study several transmitters or transmitter m a r k e r s in h u m a n brain from senile d e m e n t i a patients. 5 . w a s found to b e hepatotoxic in m a n and to c a u s e psychiatric effects ( C a l n e et al. L i e b e r m a n et al. w h e r e a s those of m o n o a m i n e o x i d a s e Β increased and m o n o a m i n e o x i d a s e A and c h o l i n e acetyltransferase r e m a i n e d u n c h a n g e d . although effective. are d o p a m i n e a g o n i s t s . choline acetyltransferase w a s r e d u c e d and m o n o a m i n e o x i d a s e Β w a s increased. L e r g o t r i l e . a d o p a m i n e agonist. It w a s found that levels of n o r e p i n e p h r i n e . E r g o t a l k a l o i d s .H T . its usefulness is limited in s o m e cases by lack of efficacy and a d v e r s e effects such as mental c h a n g e s and h y p o t e n s i o n . b r o m o c r i p t i n e w a s tried for the reduction of p a t i e n t ' s c r a v i n g for c o c a i n e and at the s a m e t i m e to address p a t i e n t ' s attention deficit d i s o r d e r . 1980. and d o p a m i n e r e d u c e d significantly with a g e . In recent years c o n s i d e r a b l e effort has b e e n directed at u n d e r s t a n d i n g the c h a n g e s taking p l a c e in the brain. and senile cerebral insufficiency are used to describe t h e s e .5 years L . T h e r e h a v e b e e n recent reports on trials of alkaloids in the treatment of c h e m i c a l d e p e n d e n c y . T h e rationale for the use of ergot alkaloid w a s that the w i t h d r a w a l of c o c a i n e . A s a result of the spectacular progress of medical science in the last four d e c a d e s p e o p l e are living longer and h e n c e h a v e to face the p r o b l e m s c a u s e d by the a g i n g p r o c e s s of the b r a i n . the levels of d o p a m i n e . h o w e v e r . N a k a n i s h i et al. A n u m b e r of t e r m s such as senile d e m e n t i a ( A l z h e i m e r ' s d i s e a s e ) . L . 1989).3 Ergot Alkaloids 287 function of d o p a m i n e receptors ( L o e w et al.D O P A t h e r a p y b e c o m e s less satisfactory. B r o m o criptine a n d a m a n t a d i n e w e r e both effective in reducing the s y m p t o m s in the first 2 w e e k s post w i t h d r a w a l . T h e results indicated an e q u i v o c a l r e s p o n s e to b r o m o c r i p t i n e . H o w e v e r . T h e c o n c e n t r a t i o n s of these in the brains of the c h r o n i c alcoholics w e r e similar to those seen in the brains of senile . 1980). In senile d e m e n t i a . In such cases treatment with d o p a m i n e agonists such as ergot alkaloids s e e m s to b e beneficial.

4 . c a b e r g o l i n e . O t h e r ergot alkaloids that h a v e been found to be effective are lisuride. 1989). H y p e r p r o l a c t i n a e m i a . m o r e c o m m o n in w o m e n than in m e n . T h u s . In a n u m b e r of studies w h e r e E E G w a s used as o n e of the m e a s u r e s of evaluating the response to the d r u g . it w a s thought that the senile d e m e n t i a w a s c a u s e d b y the reduction in the cerebral blood flow and d i h y d r o e r g o t o x i n e w a s used in its therapy b e c a u s e it w a s believed to be a cerebral vasodilator.or m a c r o a d e n o m a ) . interference with d o p a m i n e delivery to the pituitary ( m i c r o . T h e failure rate of this drug (about 2 6 % ) w a s high but that for all other d r u g s available is higher. Originally. 1989. loss of libido. a m e n o r r h e a . 1989).or m a c r o a d e n o m a ) . V e n n 1980. and d i h y d r o e r g o t o x i n e m e s y l a t e (Ciccarelli et al. efficacy of d i h y d r o e r g o t o x i n e w a s established with a large series of double-blind controlled studies ( R a o and Norris 1972. h e a d a c h e s . has s o m e s h o r t c o m i n g s and search for a better drug c o n t i n u e s . H o w e v e r . and/or galactorrhea. w h e r e a s those in m e n are i m p o t e n c e . T h o r n e r and Besser (1978) d e m o n s t r a t e d that b r o m o c r i p t i n e therapy restored the menstrual cycle in 8 3 % of the patients within 2 m o n t h s . high correlation w a s found b e t w e e n the s y m p t o m a t i c i m p r o v e m e n t and E E G c h a n g e s ( V e n n 1978). A l t h o u g h these observations offer a rationale for the use of ergot alkaloids in the therapy of senile d e m e n t i a . is caused by h y p o t h a l m i c d o p a m i n e deficiency (diseases of the h y p o t h a l a m u s such as t u m o r ) . the actual use c a m e about in an unrelated fashion ( V e n n 1980). the effectiveness of the drug is p r o b a b l y d u e to its effect on the transmitters. T a m u r a et al. menstrual irregularities. I n d e e d . lactotroph insensitivity to d o p a m i n e ( m i c r o . T h e a b o v e r e v i e w of the p h a r m a c o l o g i c a l effects and clinical application of ergot alkaloids is far from c o m p l e t e as the n u m b e r of studies in this field is . A c o m p r e h e n s i v e analysis of 12 different trials s h o w e d i m p r o v e m e n t in m o r e than 5 0 % of the patients treated and 2 3 % had a m a r k e d s y m p t o m i m p r o v e m e n t ( V e n n 1980). B r o m o c r i p t i n e w a s d e v e l o p e d specifically as an inhibitor of prolactin secretion. b r o m o c r i p t i n e therapy resulted in p r e g n a n c y in 7 1 % of the w o m e n within 3 . and infertility. an i m p r o v e m e n t in cognitive function and dysphoric m o o d states and b e h a v i o r of the patients w a s o b s e r v e d . S u b s e q u e n t l y .8 m o n t h s (al-Sulieman et al. h o w e v e r . although effective. 3 . 4 H y p e r p r o l a c t i n e m i a a n d R e l a t e d D i s o r d e r s . R o u y et al. T h e r e f o r e . A n u m b e r of studies d o c u m e n t the success of the treatment with this alkaloid. and visual d i s t u r b a n c e s . Prolactin is a h o r m o n e secreted by the anterior pituitary and d o p a m i n e is probably physiologically the m o s t important prolactin-release inhibitory factor.288 Therapeutic Metabolites d e m e n t i a patients. T h e early reports of the success of this alkaloid w e r e received with s k e p t i c i s m as the e x p e r i m e n t a l design used in these investigations w a s flawed. menstrual d i s t u r b a n c e s . it a p p e a r s that the therapy with d i h y d r o e r g o t o x i n e . It m a d e its efficacy difficult to u n d e r s t a n d until it w a s found that senile d e m e n t i a w a s not caused by c e r e b r o v a s c u l a r insufficiency. or stimulation of lactotroph cells ( T h o r n e r et al. 1980). In another study with 136 h y p e r p r o l a c t i n e m i c w o m e n suffering from infertility. 1989). 1 0 . B r o m o c r i p t i n e w a s also found to be effective in the treatment of pituitary a d e n o m a s . T h e clinical manifestations in w o m e n include galactorrhea. later this c o m p o u n d w a s s h o w n to h a v e n o relevant vasodilator action ( V e n n 1980).

A l s o it should be kept in m i n d that not all c o m p o u n d s are equally effective and that the structure-activity relationships are not fully understood. Its structure is s h o w n in Figure 10-9. V a r i o u s m o l e c u l a r forms of C C K h a v e been identified in gut and brain.d e o x y a s p e r l i c i n s . and t w o anthranilate m o i e t i e s . L i e s c h et al. D . c o m p e t i t i v e . . is p r o d u c e d by Aspergillus alliaceus.a c i d peptide found in the gastrointestinal tract and in the central n e r v o u s s y s t e m ( M o r l e y 1982. T h e p r e d o m i n a n t and the biologically m o s t active form is C C K . h o w e v e r . and therefore m a y be involved in physiological regulation of appetite (cf. 1988. C . A n e x a m i n a t i o n of the structure of asperlicin s h o w s that it is c o m p o s e d of t r y p t o p h a n . leucine. A n attempt w a s m a d e to ex- F I G U R E 10-9 Structure of asperlicin. 1985).a m i n o . in D carbonyl g r o u p is associated with t r y p t o p h a n residue rather than with the anthranilate moiety as in the c a s e of other asperlicins. asperlicin has the potential to be used for therapy in gastrointestinal disorders w h e r e C C K is involved. C C K is a major stimulator of gall bladder contraction and pancreatic e x o c r i n e secretion. A s p e r l i c i n . Asperlicin Β is N 5 . it should give the reader a g o o d idea of the usefulness of this class of c o m p o u n d s . Rehfeld 1985). w h i c h is a 3 3 . 1988). alliaceus A T C C 2 0 6 5 5 and 2 0 6 5 6 ( G o e t z et al. 10. Asperlicin Ε is also a deleucyl-asperlicin with the α-nitrogen of t r y p t o p h a n residue c o n d e n s e d with indole m o i e t y . 1986). Asperlicin C a n d D are both d e l e u c y l . It is also k n o w n to p r o d u c e satiety.10. H o w e v e r . B o c k et al. In light of this role of C C K . and Ε — w e r e isolated from the fermentation broths of A. and a regulator of gut motility ( W i l l i a m s 1982).h y d r o x y a s p e r l i c i n . a natural b e n z o d i a z e p i n . F o u r m o r e C C K a n t a g o n i s t s — a s p e r l i c i n B .8 w h i c h m a y be derived from the larger C C K . n o n p e p t i d e antagonist of neurotransmitter c h o l e c y s t o k i n i n ( C C K ) .4 Asperlicin 289 e n o r m o u s .3 3 ( C h a n g et al.4 ASPERLICIN Asperlicin is a p o t e n t .

g l y c e r o l . A n o t h e r m e d i u m formulation called J K 1 which contained t r y p t o p h a n and p h a r m a m e d i a in place of anthranilic acid and soybean meal w a s used in the fermentors and g a v e titers of 2 2 0 mg/1 ( M o n a g h a n et al. T h e p r o d u c t i o n p r o c e s s w a s d e v e l o p e d with A T C C 2 0 6 5 6 . alliaceus—namely ATCC 1 6 8 9 1 . 0 .290 Therapeutic Metabolites ploit these observations for the d e v e l o p m e n t of the m e d i u m for the production of asperlicin as described in the next section. 2 6 3 ) . D u r i n g the d e v e l o p m e n t of this m e d i u m s o m e interesting o b s e r v a t i o n s o n the o p t i m u m carbon source w e r e m a d e ( M a s u r e k a r and I h n e n . P r o c . d e x t r o s e .6 H 2 0 ( M o n a g h a n et al. as e x p e c t e d . T h e original soil isolate A T C C 2 0 6 5 5 . 2 0 6 5 5 . sixfold higher yields . 1985). In an effort to assist the s c a l e u p . N e x t . T h i s m e d i u m w a s s u p p l e m e n t e d with anthranilic acid and tryptophan to i m p r o v e the yields to 180 mg/1. s h o w e d a population distribution capable of p r o d u c i n g asperlicin from < 8 to 2 6 mg/1 ( M o n a g h a n et al. P r o c . 1 0 .1 Production Asperlicin is p r o d u c e d by four different strains of A. s o y b e a n m e a l . 1989). T h e following ingredients w e r e tested using this protocol: a r d a m i n e P H . corn steep liquor. corn m e a l . 1989). K H 2 P 0 4 . 1987. a n u m b e r of formulations containing the four constituents m e n t i o n e d a b o v e along with four others that had a small positive or negative effect. It is standard practice to begin m e d i u m d e v e l o p m e n t by screening a large n u m b e r of carbon and nitrogen sources and the Plackett and B u r m a n m e t h o d allows o n e to d o so in a m i n i m u m n u m b e r of e x p e r i m e n t s . and poly glycol P 2 0 0 0 . s o d i u m citrate. Bact. 4 . 2 6 3 ) . p e p t o n i z e d milk 3 7 . 1989). a r d a m i n e P H . 5 g. In this m e d i u m the production of asperlicin w a s increased to 9 0 0 mg/1. 1 9 8 7 . T h e m e d i u m used in the early studies consisted of t o m a t o p a s t e . p . 1989). T h e results of this screen s h o w e d that a r d a m i n e P H . Bact. T h i s m e d i u m w a s found to be limiting and s i m p l e d o u b l i n g of the concentrations of the ingredients resulted in an a l m o s t twofold increase in the y i e l d s . 10. A reisolate w h i c h consistently g a v e superior yields w a s used for the d e v e l o p m e n t of the fermentation p r o c e s s . and K H 2 P 0 4 . w e r e tried. and s o d i u m citrate had a strong positive effect on the production of asperlicin w h e r e a s the rest had a small positive or n e g a t i v e effect. and p h e n y l a l a n i n e 10 g. g l y c i n e . T h e duration of the fermentation w a s 7 d a y s . ( N H 4 ) 2 S 0 4 . 1989). ( N H 4 ) 2 S 0 4 . tryptophan 4 g. A statistical t e c h n i q u e k n o w n as Plackett and B u r m a n protocol w a s used in the early w o r k ( M o n a g h a n et al. cod liver oil. 1 F e r m e n t a t i o n D e v e l o p m e n t . lard w a t e r . corn m e a l . corn m e a l . This m e d i u m c o n t a i n e d . T h i s isolate is designated as A T C C 2 0 6 5 6 ( M o n a g h a n et al. F r o m these o n e m e d i u m w a s found to support the production of 79 mg/1. s o y b e a n m e a l .4. 1989). c o d liver oil. per liter: glycerol 2 0 g. p . and C o C l 2 . indicating that careful m e d i u m d e v e l o p m e n t w o u l d result in substantial yield i m p r o v e m e n t s ( M o n a g h a n et al. poly glycol P 2 0 0 0 . T h e presterilization p H w a s adjusted to 7 . lard w a t e r . t o m a t o p a s t e . p e c t i n . 1 . a simple m e d i u m w a s d e s i g n e d ( M a s u r e k a r and I h n e n . n a m e l y . a r d a m i n e P H 1 g. G l u c o s e w a s not as g o o d a carbon source as glycerol. 2 0 6 5 6 . and N R R L 315 (Goetz et al.

N H 4 C 1 2 g. but w a s sensitive to m. 1 . M g S 0 4 . Z n S 0 4 . 1988). pH 6 . T h e c o m p o s i t i o n of this m e d i u m w a s as follows. W e w e r e also surprised by the stimulation of s y n t h e sis b y a r o m a t i c a m i n o acids in this c o m p l e x m e d i u m ( M a s u r e k a r and I h n e n . increasing the concentration of glycerol a b o v e 25 g/1 did not i m p r o v e the p r o d u c t i o n w h i c h indicated that the concentration of the nitrogen source w a s p r o b a b l y limiting. T h e p r o d u c t s w e r e isolated and identified. A defined p r o d u c t i o n m e d i u m w a s d e v e l o p e d to study the biosynthesis of asperlicin. 1988). et al. 5 . 1 9 8 7 . S i m i l a r l y . increasing L . 1988). 10.3 Directed Biosynthesis. 5 g. 2 6 3 . increasing L . c o n t a i n i n g appropriate a m i n o acid or its a n a l o g u e and w e r e incubated for 36 h.5 ( H o u c k et al. p . T h i s suggested that it m a y be p o s s i b l e to synthesize a n a l o g u e s of asperlicin b y feeding the a n a l o g u e s of t r y p t o p h a n . Bact. A 3 0 % stimulation of the synthesis w a s o b s e r v e d u p o n the addition of anthranilic acid ( H o u c k et al.f l u o r o p h e n y l a l a n i n e w a s isolated and it w a s found to be a 2 0 % superior p r o d u c e r as c o m p a r e d to the parent ( M a s u r e k a r and I h n e n . In a m e d i u m c o n t a i n i n g 4 g of L . T h e culture w a s resistant to all of the 2 0 t r y p t o p h a n a n a l o g u e s and 8 out of 10 p h e n y l a l a n i n e a n a l o g u e s tested. 0 1 g. 10. glycerol 15 g. A d d i t i o n of L . p. A n efficient incorporation of t r y p t o p h a n and leucine a n a l o g u e s into asperlicin to give 25 n e w asperlicin a n a l o g u e s w a s o b s e r v e d ( H o u c k et al. 1987. B a c t . p e r liter: g l u t a m a t e 5 g. 1 for 4 8 h and w e r e harvested and w a s h e d . S e c o n d . Bact.4 Asperlicin 291 w e r e o b t a i n e d in the m e d i u m containing the latter as c o m p a r e d to those with the former. W i t h this m e d i u m the yields of asperlicin u p to 4 0 0 mg/1 w e r e o b t a i n e d . Proc.P h e w e r e a d d e d ( H o u c k et al. attempts w e r e m a d e to isolate m u t a n t s d e r e p r e s s e d for the p r o d u c t i o n of aromatic a m i n o acids ( M a s u r e k a r and Ihnen. in the m e d i u m c o n t a i n i n g 2 g L . Κ Η 2 Ρ 0 4 2 g.2 Mutagenesis. T h e presterile p H w a s adjusted to 6.4. T h e w a s h e d cells w e r e s u s p e n d e d in 5 0 mM [3-{N-movpho\mo)tihd^\^u\iomc acid].P h e from 0 to 10 g/1 g a v e a fourfold i m p r o v e m e n t in the yield. 1 9 8 7 . A d d i t i o n of aromatic a m i n o acids is not an e c o n o m i c a l - ly attractive option at the p r o d u c t i o n scale. T h e r e f o r e . 263). M o n a g h a n . suggested that these a m i n o acids w e r e rate limiting for the biosynthesis of asperlicin. 5 g . 2 6 3 ) .1. P r o c . lecithin 1 g.7 H 2 0 0 .L e u did not h a v e a n y effect on the synthesis of asperlicin. K C l 0 . T h e p r o c e d u r e used w a s as follows: the cells w e r e g r o w n in the synthetic m e d i u m d e s c r i b e d in section 1 0 . p . 0 1 g and F e S 0 4 . 1989). F o r this p u r p o s e m u t a g e n i z e d spores w e r e subjected to the toxic a n a l o g u e s of either tryptophan or p h e n y l a l a n i n e .7 H 2 0 0 . O n e m u t a n t resistant to m .P h e / 1 .1. . T h e length of fermentation w a s 6 d a y s . e v e n in the c o m p l e x m e d i u m .10.7 H 2 0 0 .f l u o r o p h e n y l a l a n i n e .T r p and L .and / 7 . 4 . 1988). P r o c .T r p / 1 . In a modified version of this m e d i u m lecithin w a s replaced with 1 g of algin/1 and 2 g/1 of e a c h of L .4. T h e stimulation of p r o d u c t i o n by t r y p t o p h a n and p h e n y l a l a n i n e .T r p from 0 to 4 g/1 resulted in a fivefold increase in the titer.

w a s c o n v e r t e d into asperlicin and asperlicin Ε ( H o u c k et al. designated as L .) yV-(2. Asperlicin E . In another study with isolated islets from rats. It has high affinity for the p a n c r e a t i c . 1989). w h i c h limits its usefulness. It d e m o n s t r a t e d specificity for a n t a g o n i s m of c o n tractions of gallbladder and ileum. and anthranilic acid s h o w e d t h e m to b e incorporated efficiently into asperlicin. 10.4. N i e d e r a u et al. asperlicin C . it w a s s h o w n to antagonize stimulation by C C K (Zawalich and D i a z 1987).l a b e l e d leucine. 1988). Similarly.p h e n y l . 7 1 8 w a s three-thousand-fold m o r e active in in vitro b i n d i n g assay as well as in vitro secretion assay with rat p a n c r e a s and five-hundred-fold m o r e active in in vivo secretion assay ( E v a n s et al. T h e r e f o r e .4.l 8 is converted into asperlicin. Incorporation studies with 1 4 3 C . 1989). which on the c o n d e n s a t i o n with leucine and h y d r o x y l a t i o n at C . T h e b i n d i n g to pancreatic receptors w a s competitive with C C K . N o w o r k on the incorporation of putative precursors labeled with the stable isotopes has been r e p o r t e d .l .3 Pharmacological Studies Asperlicin is a C C K antagonist. 3 5 ( . there are n o reports of any e n z y m o l o g i c a l studies.3-dihydro-l-methyl-2-oxo-5-phenyl-l//-l.2 Biosynthesis V e r y little is k n o w n about the biosynthesis of asperlicin. In a n o t h e r series of e x p e r i m e n t s a n u m b e r of 3-substituted 5 . 1985). w a s m o r e soluble ( B o c k et al. 4 b e n z o d i a z e p i n e a n a l o g u e s w e r e synthetically prepared and o n e of t h e s e . This supported the intuitive h y p o t h e s i s that these w e r e building blocks of asperlicin ( H o u c k et al. T h r e e a n a l o g u e s w h i c h w e r e modified at N . It inhibited cerulenin-stimulated pancreatic secretion in rats (Niederau et al.4-benzodiazepine-3-yl)-l//-indolec a r b o x a m i d e . t r y p t o p h a n . 1987. 4 . h o w e v e r . 1986).292 Therapeutic Metabolites 1 0 . although equally potent. w h i c h is formed by an intramolecular c o n d e n s a t i o n . it w a s modified c h e m i c a l l y to obtain 17 n e w a n a l o g u e s with i m p r o v e d solubility and p o t e n c y . 6 ± 0 . 10.and H . T h e s a m e w o r k e r s also s h o w e d that the naturally p r o d u c e d asperlicin a n a l o g u e . ileal and gallbladder C C K receptors but not to the brain C C K receptors ( C h a n g et al. with a Kx of 0 . At this point. 10. 2 μΜ ( C h a n g et al. T h e s e results indicated that asperlicin C is p r o b a b l y an intermediate. Asperlicin has low solubility in water.5 w e r e m o r e active than asperlicin in the pancreatic C C K receptor assay and o n e . a target disorder to be treated with this drug has not b e e n identified. 1988). 4 S e m i s y n t h e t i c a n d S y n t h e t i c A n a l o g u e s of A s p e r l i c i n .3 6 4 . 1 .5 FUTURE PRODUCTS In light of the large n u m b e r of fungal products with a w i d e variety of p h a r m a c o l o g i cal activities it is logical to predict that a n u m b e r of t h e m will be a d d e d to o u r . 1985). is probably a shunt p r o d u c t .

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M i l s o m 1987).M e y e r h o f f p a t h w a y . S m i t h et al. M a n y excellent r e v i e w s h a v e b e e n written c o v e r i n g the p r o d u c t i o n of o r g a n i c acids by fermentation ( P e r l m a n and Sih 1960.CHAPTER 11 Organic Acids Mary Jo Zidwick T h e biological production of o r g a n i c acids b y fermentation p r o c e s s e s h a s b e e n studied for m a n y y e a r s . M e y r a t h 1967. w h e r e a s the citric acid fermentation industry b e g a n at the start of the 20th c e n t u r y . L o c k w o o d 1975 and 1979. and the K r e b s tricarb o x y l i c acid ( T C A ) c y c l e . 1974. rather. K n o w l e d g e in basic b i o c h e m i c a l m e t a b o l i s m a d v a n c e d with elucidation of the E m b d e n . A l t h o u g h a great deal of research has b e e n d o n e . I n d e e d . D e p a r t m e n t of Agriculture in Peoria. A l s o . 303 . a great deal of practical fermentation research w a s d o n e in the 1940s and 1950s b y the U . S . It is b e y o n d the s c o p e of this c h a p t e r to c o v e r the historical evolution of the k n o w l e d g e of various p a t h w a y s . T h i s research still forms the basis of m u c h of o u r k n o w l e d g e on the p r o c e s s e s involved in several o r g a n i c acid fermentations. in m a n y cases the m e c h a n i s m for o v e r p r o d u c tion of these p r i m a r y metabolites is still not k n o w n . this report will c o v e r the basic outline of the p r o c e s s e s and recent literature on n e w insights into the control of m e t a b o l i s m . only the fungal p r o c e s s e s will b e described here. Miall 1978. T h e s e p r o c e s s e s w e r e originally run w i t h o u t a true u n d e r s t a n d i n g of the b i o c h e m i c a l p a t h w a y s of the o r g a n i s m or the i m p o r t a n c e of p r o c e s s p a r a m e t e r s such as sterility or aeration. the formation of lactic acid by Lactobacillus was put into c o m m e r c i a l practice in the late 1800s. A l t h o u g h certain o r g a n i c acids can be p r o d u c e d b y bacteria as well as fungi. Bigelis 1985. the p e n t o s e p h o s p h a t e shunt.

Citric acid is mainly sold as a white free-flowing p o w d e r . P r o b l e m s of stability and yield kept this p r o c e s s from being c o m m e r c i a l l y v i a b l e . w h i c h b e g a n in 1 9 2 3 . and has G R A S (generally regarded as safe) status from the U . Often. S . H e s h o w e d the i m p o r t a n c e of using p u r e r e a g e n t s .8-3. Citric acid also has the ability to c o m p l e x h e a v y m e t a l s . Currie j o i n e d Pfizer and C o . 11.0 .4-3. Various salts of citric acid are used in certain applications. citric acid is regarded as the standard against w h i c h other food acidulants are m e a s u r e d . and w i n e . Citric acid is also regarded as a very safe food additive. until C u r r i e ' s report in 1917 of the production of citric acid by Aspergillus niger (Currie 1917). and w a s partly responsible for the d e v e l o p m e n t of their c o m m e r c i a l fermentation p r o c e s s . soft d r i n k s . In food formulation. S .304 11. D e c r e a s i n g use of p h o s p h a t e in detergent has boosted the use of citric acid in cleaning p o w d e r s .5 0.1 Organic Acids CITRIC ACID Of the various organic acids p r o d u c e d by fungi. that p r o d u c e d significant quantities of citric acid. he used a surface fermentation p r o c e s s using shallow p a n s . citric acid sequesters h e a v y metals from catalyzing oxidative deterioration in flavor. M o s t of this production is believed to b e by s u b m e r g e d fermentation p r o c e s s e s . Citric acid is u s e d for tartness and p H control in j a m s and jellies. Citric acid is also used in cosmetics and p h a r m a c e u t i c a l s . W e h m e r (1903) described certain m o l d s . with an estimated U . M e d i c i n a l p o w d e r s contain citric acid for its effervescence.6-2. T h e p r i m a r y use of citric acid is in the food industry.1 Fermentation Process It w o u l d be remiss not to describe the early history of our k n o w l e d g e of citric acid a c c u m u l a t i o n by fungi. c o n s u m p t i o n of 3 0 0 million p o u n d s in 1987. T h e basic m e d i u m w h i c h w a s d e v e l o p e d by Currie and is in general use today can be described as follows: Sugar source N H 4 N O 3 or other ammonium salt K H 2P 0 4 M g S 0 4 · 7 H 20 HCl or H 2 S 0 4 to pH 3. w h i c h later turned out to be Pénicillium. Its chelating ability also m a k e s it useful for metal c l e a n i n g . It serves as an antioxidant and for p H adjustment in c o s m e t i c s . It is also used to adjust the acid flavor in b e v e r a g e s such as fruit j u i c e s .5 Grams per liter 125-200 1. a less-pure liquid grade of citric acid is sold for a variety of industrial applications. F o o d and D r u g A d m i n i s t r a t i o n . Currie d e v e l o p e d a m e d i u m that has served as a basis for s u b s e q u e n t w o r k on the citric acid fermentation.1. F r o z e n foods are often treated with citric acid to retain vitamin C and reduce enzymatic browning.2-1. In foodstuffs. Certain c a n d i e s contain citric acid for a sharp taste. Like m o s t other w o r k e r s in his t i m e . citric acid is by far the m o s t important in e c o n o m i c t e r m s .0 0.

1 .8 .0 . A variety of substrates h a v e b e e n described for c o m m e r c i a l p r o d u c t i o n of citric acid b y A. T h e y further c l a i m that addition of 1. T h e y also r e c o m m e n d that a concentration of 0 . O v e r the last 5 0 y e a r s . the literature has b e e n full of various attempts to control and e n h a n c e citric acid p r o d u c t i o n . and that the r e m a i n i n g ferrocyanide ions are a d d e d before inoculation. as heat is n e e d e d to effect the actual ferrocyanide r e m o v a l of m e t a l s . W h i l e the use of p o t a s s i u m ferrocyanide has b e e n well d o c u m e n t e d . ferrocyanide can be a d d e d at certain specific times during the fermentation. in the form of m o l a s s e s . P e r q u i n ( 1 9 3 8 ) . 5 g/1 p h o s p h a t e and 0 . h o w e v e r . and that a separate sterile solution of s o d i u m or p o t a s s i u m ferrocyanide be a d d e d to c o o l e d m e d i u m that is at a p H of 6 . a c o m p l e t e u n d e r s t a n d i n g of the m e c h a n i s m s of o v e r p r o d u c tion of citrate has not b e e n attained. T o m a x i m i z e the inhibitory effect on cell g r o w t h and stimulatory effect o n citric acid p r o d u c t i o n . it is u n c l e a r w h e n first industrial practice b e g a n . 5 . t o d a y it is b e l i e v e d that m o s t industrial fermentations are b y the s u b m e r g e d p r o c e s s . on the other h a n d . in his thesis w o r k . M a r t i n and W a t e r s (1952) describe a s u b m e r g e d fermentation p r o c e s s with beet m o l a s s e s at about 1 2 % sugar containing 0 . C o n t r o l of c o n t a m i n a t i n g metal ions w a s difficult. E v e n t o d a y .11. c l a i m s that heating of the ferrocyanide salts is to b e a v o i d e d . G o l d ( 1 9 6 7 ) .1 .2 0 0 x 1 0 g / m l .1 Citric Acid 305 W h i l e early w o r k d e s c r i b e s the production of citric acid using surface fermentat i o n s . d e v e l o p e d the shaking t e c h n i q u e for studying citric acid p r o d u c t i o n . Besides chelation of u n w a n t e d m e t a l s . Surface fermentation w a s d o n e in shallow p a n s and required acres of surface areas a n d m a n u a l labor for m i x i n g and aeration of the fungus and substrate. H u s t e d e and R u d y ( 1 9 7 6 a ) specify that r o u g h l y half of the ferrocyanide ions n e e d e d to c o m p l e x h e a v y metals be added prior to sterilization. T h e citric acid fermentation h a s historically b e e n k n o w n as o n e that is difficult to c o n t r o l . T h e r e are conflicting reports on the treatment of ferrocyanide in the m e d i u m .e x c h a n g e materials or ferrocyanide r e m o v e s s o m e of the inhibitory materials. niger. Clark (1964) c l a i m s that ferrocyanide ion should b e in e x c e s s at a 6 c o n c e n t r a t i o n of 1 0 . T h e t w o p r i m a r y substrates in use today are s u c r o s e . T h e y state that p r e t r e a t m e n t of m o l a s s e s with c a t i o n . if not i m p o s s i b l e . 0 g/1 p o t a s s i u m ferrocyanide trihydrate. P e r l m a n et al. ( 1 9 4 6 b ) c l a i m e d that inorganic constituents of c a n e m o l a s s e s are at least partially responsible for low yields of citric acid. O n e of the m o s t c o m m o n m e t h o d s w a s the addition of various c h e m i c a l s to stimulate the fermentation yield. they will not b e discussed in this chapter. ferrocyanide ion is c l a i m e d to p r o v i d e a stimulatory effect to the m i c r o o r g a n i s m to increase citric acid y i e l d s . T h e resulting precipitate need not b e r e m o v e d . 4 p p m u n c o m p l e x e d excess ferrocyanide b e m a i n tained d u r i n g the fermentation.5-2 g of p o t a s s i u m ferrocyanide p e r liter . M a r t i n (1956) c l a i m s that ferrocyanide should b e added to the fermentation m a s h and sterilized. Certain species of Candida yeast h a v e also b e e n e x a m i n e d that p r o d u c e citric acid at high yields on h y d r o c a r b o n and paraffin substrates as well as d e x t r o s e . M e z z a d r o l i (1938) is usually cited as o n e of the first to publish its u s e . and d e x t r o s e . T h i s heralded the b e g i n n i n g of a shift in interest from the surface culture to s u b m e r g e d fermentation.

r e d u c e d cationic impurities and increased citric acid yield ( N a s i m et al. Addition of s o d i u m dithionite. and acid s o d i u m bisulfite w e r e reported to h a v e stimulatory effects on m o l a s s e s m e d i a treated with ferrocyanide. N o n i o n i c surface active agents such as sucrose m o n o s t e a r a t e and S p a n . This addition stimulates citric acid production in the p r e s e n c e of m a n g a n e s e . T h e s e agents are s u p p o s e d to be especially useful with an i m p u r e sugar s o u r c e . R e c e n t l y .5 0 h after inoculation. T h i s effect w a s b e lieved to be related to the metal c o m p l e x i n g ability of the d i t h i o c a r b a m a t e s . m o l a s s e s has also been treated by ion e x c h a n g e to r e m o v e undesirable c o n t a m i n a n t s from the citric acid p r o c e s s . on the other h a n d . A n early M i l e s patent c l a i m s that addition of m o r p h o l i n e b e t w e e n 100 and 1. and n a p h t h o l s .k n o w n additives to the citric acid fermentation is m e t h a nol. T h e y found that m a n g a n e s e s u p p r e s s e d citric acid a c c u m u l a t i o n . A n o t h e r patent describes the addition of quaternary a m m o n i u m c o m p o u n d s or a m i n e oxides to counteract an inhibitory effect of iron on the fermentation (Miles 1969). used at an o p t i m a l level of 2 % . niger cells to g r o w as c o m p a c t aggregates with a b n o r m a l l y short and b u l b o u s m y c e l i u m ( S c h w e i g e r and Snell 1949). 1949). T h e actual metal content of the Aspergillus m y c e l i u m w a s unc h a n g e d ( C h o u d h a r y and Pirt 1966). and p r o p a n o l s added at the 1 . Sublethal d o s e s of these c o m p o u n d s w e r e added at 4 0 .3 % r a n g e in the fermentation. and can also be used to increase yields in certain other o r g a n i c acid . 1981). O t h e r chelating agents such as ethylenediaminetetraacetic acid ( E D T A ) and related c o m p o u n d s w e r e also s h o w n to boost citric acid production u p to about a tenfold increase. m e t h a n o l .306 Organic Acids of fermentation m e d i a to the citric acid m i c r o o r g a n i s m during the physiological stage of spore-swelling to spore-germinating will c a u s e high acid p r o d u c t i o n ( H u s tede and R u d y 1976b) w h e r e a s Kabil ( 1 9 7 3 ) . A n early M i l e s patent d i s c u s s e s the use of a t w o .1 0 p p m increments o v e r the c o u r s e of the fermentation to m a i n t a i n a desirable m o r p h o l o g y and high productivity. O n e of the m o s t w e l l .s t e p decationization treatment that r e d u c e s the level of iron in the m o l a s s e s ( W o o d w a r d et al. A l t h o u g h the m e c h a n i s m of action is unclear. results indicate that its p r i m a r y effect o c c u r s with m o r e i m p u r e substrates. 1965). T h e r e h a v e b e e n reports of m a n y other additives besides ferr o c y a n i d e that h a v e been used to e n h a n c e citric acid yield. B e s i d e s ferrocyanide. w e r e s h o w n to stimulate citric acid yields in solidstate fermentations with b a g a s s e ( K h a n n a and G u p t a 1986). A n o t h e r patent cites the usefulness of toxic organic agents such as cresylic acid and various x y l e n o l s . r e c o m m e n d s a d d i n g ferrocyanide in 0 . This addition w a s most beneficial for surface fermentations and m o l a s s e s of p o o r quality (Ilczuk 1983). sulfur-containing c o m p o u n d s . or d i t h i o c a r b a m a t e s . N o g u c h i and J o h n s o n ( 1 9 6 1 ) purified c o m m e r c i a l d e x t r o s e with a chelating resin and found the best yields in the p r e s e n c e of limited a m o u n t s of iron and z i n c . M o y e r (1952) d o c u m e n t e d the use of m e t h y l a c e t a t e . 0 5 . G r o w t h w a s altered from small pellets to a filamentous form and overall yield w a s increased from r o u g h l y 3 8 % to 5 0 % ( T a k a h a s h i et al. e t h a n o l .2 0 w e r e also s h o w n to increase citric acid yields. T r e a t m e n t of m o l a s s e s by b e n t o n i t e .000 p p m i m p r o v e d yield and c a u s e d the A. and are reputed to interfere with the respiratory e n z y m e s of the m i c r o o r g a n i s m ( L o c k w o o d and Batti 1965). c r e s o l s . p o t a s s i u m pyrosulfite.

z i n c . and 1950). possibly d u e to s o m e disturbance of cell m e t a b o l i s m . is a p o o r substrate for citric acid p r o d u c t i o n .1. It has taken m a n y years of investigation to begin to u n d e r s t a n d the significance of the effects of certain metal ions. all four e l e m e n t s w e r e detrimental to citric acid a c c u m u l a t i o n ( T o m l i n s o n et al. Z i n c . niger m o r p h o l o g y in a s u b m e r g e d fermentation. Bertrand and d e W o l f 1961a and b ) .2 Mechanism of Citric Acid Overproduction M u c h of the literature in the citric acid field has dealt with the i m p o r t a n c e of metal ions to the fermentation. T h e decationization treatment itself increased the c o n v e r s i o n of sugar to citric acid. S c r u p u l o u s care w a s used in glass w a s h i n g and reagent g r a d e c h e m i c a l s w e r e u s e d . 1939a. 1986). c o p p e r and m a n g a n e s e a p p e a r e d to interact in s o m e w a y .11. and although utilized for g r o w t h . T h i s sugar is a constituent of w h e y . A systematic study w a s d o n e w h e r e nutrients were e x a m i n e d for their interactions and effects on fermentation yields. 1 mg/1 also increased sugar c o n v e r s i o n . M a n g a n e s e w a s found to r e d u c e citric acid yield. T r a c e e l e m e n t nutrition in relation to a m i n o acid synthesis and nucleic acid synthesis w a s explored (Steinberg 1939b. niger w e r e d o n e o v e r a span of m a n y years (Steinberg 1919. but the m e c h a n i s m w a s not certain ( T o m l i n s o n et al. 1 9 4 6 . Several very detailed studies of the i m p o r t a n c e of various m i n e r a l s and other nutrients for g r o w t h of A. h o w e v e r . m a n g a n e s e . M e t h a n o l m a y increase the p e r m e a b i l i t y of the cell to citrate. iron. 1951). iron. and c o p p e r in citric acid. By using a m m o n i u m c a r b o n a t e as a . and the cell r e s p o n d s b y increasing citrate p r o d u c t i o n through repression of 2-oxoglutarate d e h y d r o g e n a s e ( M a d d o x et al. F u r t h e r w o r k confirmed the i m p o r t a n c e of z i n c . A m m o n i u m nitrate and m a g n e s i u m sulfate had n o specific effect on citrate p r o d u c t i o n . and a l u m i n u m a d d e d at 0 . In e x c e s s i v e a m o u n t s . w a s found to be inhibitory at all concentrations tested ( P e r l m a n et al. and m a n g a n e s e . especially iron. S m a l l a m o u n t s of m a n g a n e s e and c o p p e r permitted higher yield. Severely limiting key minerals m a y increase citric aid yield but will also h a v e effects on n o r m a l cell g r o w t h . M e t h a n o l w a s also found to b e stimulatory to citric acid yields on g a l a c t o s e . U n d e r s t a n d i n g the essentiality of certain e l e m e n t s for g r o w t h aids in the control of the citric acid p r o c e s s . a l l o w e d a c c u m u l a tion of citric acid. 11. m o r e detailed w o r k looking at the effect of different a m o u n t s of the metals w a s d o n e . O n e study u s e d d e c a t i o n i z e d sugar to w h i c h minerals w e r e added b a c k . T h e s e studies provided b a c k g r o u n d k n o w l e d g e on g r o w t h r e q u i r e m e n t s that formed the basis of further w o r k d o n e on the interaction of nutrients in the citric acid p r o c e s s . on the other h a n d . and p h o s p h a t e — w h e n present in g r o w t h limiting a m o u n t s . A n early patent from M i l e s describes the effects that metal ions can h a v e on A. and 1942. 1950). T o clarify further the conflicting reports on certain metal i o n s . 1 9 4 1 . the effect w a s less p r o n o u n c e d in a phosphate-free m e d i u m (Shu and J o h n s o n 1948).1 Citric Acid 307 f e r m e n t a t i o n s . Early reports conflicted on the effects of metal ions on citric acid p r o d u c t i o n . m a n g a n e s e . T h r e e other c o m p o n e n t s — z i n c . Iron. 1946a). Z i n c and iron w e r e found to b e essential c o m p o n e n t s of the m e d i a .

A. F u r t h e r insights into the i m p o r t a n c e of m a n g a n e s e to the citric acid fermentation w e r e reported by Clark et al. the citric acid production w a s retarded. M u c h later w o r k reiterated the i m p o r t a n c e of ion control by u s i n g deionized sugar solutions and c o p p e r addition as growth-inhibiting agents (Jernejc et al. while taking care to not maintain the p H at this high level for too long (Batti 1967a). Besides the i m p o r t a n c e of inhibiting isocitrate d e h y d r o g e n a s e or a c o n i tase for citrate overflow (Kubicek and R o h r 1986). 1979). A s little as 2 p p b of m a n g a n e s e r e d u c e d the yield and caused o r g a n i s m m o r p h o l o g y to switch from pellet form to filamentous. M y c e l i u m structure s h o w e d granulation and m a n y vacuoles and n o n o r m a l r e p r o d u c t i v e b o d i e s (Snell and S c h w e i g e r 1949).c y c l e e n z y m e s are repressed u n d e r m a n g a n e s e d e ficiency with the exception of citrate s y n t h a s e . Iron w a s s h o w n to adversely affect this cellular m o r p h o l o g y and thus productivity. If too m u c h c o p p e r w a s a d d e d . D e s p i t e the e c o n o m i c i m p o r t a n c e of citric acid. Addition of c o p p e r to the c a r b o h y d r a t e m e d i a o v e r c a m e the iron effect and restored p r o p e r m o r p h o l o g y (Miles patent 1956). w o r k d o n e by Christian K u b i c e k and M a x R o h r in Austria has greatly increased our k n o w l e d g e of s o m e of the possible m e c h a n i s m s involved in control of this fermentation. stubby b u l b o u s m y c e l i u m . a reduced 2-oxoglutarate d e h y d r o g e n a s e activity w a s also s h o w n to increase citric acid ( K u b i c e k and R o h r 1978). . 1982). the o r g a n i s m g r e w as a c o m p a c t aggregate with short. 1 9 8 3 . T h e authors s h o w e d that the N H j ions m a y ant a g o n i z e the inhibition of phosphofructokinase by citrate and thus p r o v i d e a m e c h a n i s m for o v e r p r o d u c t i o n (Habison et al. citric acid production is retarded but cell g r o w t h increases. an u n d e r s t a n d i n g of the fund a m e n t a l m e c h a n i s m of the metal ion effects on production has not b e e n attained. P h o s p h o f r u c t o k i n a s e is a key regulatory point for m o s t o r g a n i s m s .308 Organic Acids nitrogen source and k e e p i n g the ion concentration b e l o w 1 p p m . ( 1 9 6 6 ) . L e v e l s of a l m o s t all the o x o a c i d s c h a n g e d in the transition b e t w e e n g r o w t h and p r o d u c t i o n . N u m e r o u s other metals th