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Biot echnology

Second Edition

Volume 7

Products of Secondary Metabolism

VCH4b

A Wiley company

Biot echnology

Second Edition

Fundamentals

Special Topics

Volume 1 Biological Fundamentals

Volume 9 Enzymes, Biomass, Food and Feed

Volume 2

Volume 10

Genetic Fundamentals and Genetic Engineering

Special Processes

Volume 3

Volumes lla and b Environmental Processes

Bioprocessing

Volume 4 Measuring, Modelling, and Control

Volume 12 Legal, Economic and Ethical Dimensions

Products

Volume 5 Recombinant Proteins, Monoclonal Antibodies, and Therapeutic Genes

Volume 6 Products of Primary Metabolism

Volume 7 Products of Secondary Metabolism

Volume 8 Biotransformations

A Multi-Volume Comprehensive Treatise

Biotechnology

Second,

Completely Revised Edition

Edited by H.-J. Rehm and G. Reed in cooperation with A. Piihler and P. Stadler

Volume 7

Products of

Secondary Metabolism

Edited by

H. Kleinkauf and H. von Dohren

VCH4b

A Wiley company

Series Editors:

Volume Editors:

Prof. Dr. H.-J. Rehm

Dr. G. Reed

Prof. Dr. H. Kleinkauf

Institut fur Mikrobiologie

1914 N. Prospect Ave. #61

Dr. H. von Dohren

Universitat Munster CorrensstraBe 3

Milwaukee, WI 53202-1401 USA

Institut fur Biochemie Technische Universitat

D-48149 Munster

Franklin-StraBe 29

FRG

A-10587 Berlin

 

Prof. Dr. P. J. W. Stadler

Germany

Prof. Dr. A. Piihler

P.O. Box 100131

Bayer AG

Biologie VI (Genetik) Universitat Bielefeld

Verfahrensentwicklung Biochemie Leitung Friedrich-Ebert-StraBe 217

D-33501 Bielefeld

D-42096 Wuppertal

FRG

FRG

This book was carefully produced. Nevertheless, authors, editors and publisher do not warrant the information contained therein to be free of errors. Readers are advised to keep in mind that statements, data, illustrations, procedural details or other items may inadvertently be inaccurate.

Executive Editor: Dr. Hans-Joachim Kraus Editorial Director: Karin Dembowsky Production Manager: Hans-Jochen Schmitt

Library of Congress Card No.: applied for

British Library Cataloguing-in-Publication Data:

A catalogue record for this book is available from the British Library

Die Deutsche Bibliothek - CIP-Einheitsaufnahme

Biotechnology : a multi volume comprehensive treatise I ed. by H.-J. Rehm and G. Reed. In cooperation with A. Piihler and P. Stadler. - 2., completely rev. ed. -VCH.

ISBN 3-527-28310-2 (Weinheim

.

..

)

NE: Rehm, Hans J. [Hrsg.]

Vol. 7. Products of secondary metabolism I ed. by H. Kleinkauf and H. von Dohren - 1997 ISBN 3-S27-28317-X

OVCH Verlagsgesellschaft mbH, D-69451 Weinheim

(Federal Republic of Germany), 1997

Printed on acid-free and chlorine-free paper.

All rights reserved (including those of translation into other languages). No part of this book may be reproduced in any form -by photoprinting, microfilm, or any other means-nor transmitted or translated into a machine language without written permission from the publishers. Registered names, trademarks, etc. used in this book, even when not specifi- cally marked as such, are not to be considered unprotected by law. Composition and Printing: Zechnersche Buchdruckerei, D-67330 Speyer. Bookbinding: J. SchSiffer, D-67269 Griinstadt. Printed in the Federal Republic of Germany

Preface

In recognition of the enormous advances in biotechnology in recent years, we are pleased to present this Second Edition of “Biotech- nology” relatively soon after the introduction of the First Edition of this multi-volume com- prehensive treatise. Since this series was ex- tremely well accepted by the scientific com- munity, we have maintained the overall goal of creating a number of volumes, each de- voted to a certain topic, which provide scien- tists in academia, industry, and public institu- tions with a well-balanced and comprehensive overview of this growing field. We have fully revised the Second Edition and expanded it from ten to twelve volumes in order to take all recent developments into account. These twelve volumes are organized into three sections. The first four volumes consid- er the fundamentals of biotechnology from biological, biochemical, molecular biological, and chemical engineering perspectives. The

series. Its members come from key institu- tions representing scientific input from about twenty countries. The volume editors and the authors of the individual chapters have been chosen for their recognized expertise and their contribu- tions to the various fields of biotechnology. Their willingness to impart this knowledge to their colleagues forms the basis of “Biotech- nology” and is gratefully acknowledged. Moreover, this work could not have been brought to fruition without the foresight and the constant and diligent support of the pub- lisher. We are grateful to VCH for publishing “Biotechnology” with their customary excel- lence. Special thanks are due to Dr. Hans- Joachim Kraus and Karin Dembowsky, with- out whose constant efforts the series could not be published. Finally, the editors wish to thank the members of the Scientific Advisory Board for their encouragement, their helpful

H.-J. Rehm

next four volumes are devoted to products of industrial relevance. Special attention is given here to products derived from genetically en-

suggestions, and their constructive criticism.

gineered microorganisms and mammalian

  • G. Reed

cells. The last four volumes are dedicated to

A.

Puhler

the description of special topics. The new “Biotechnology” is a reference work, a comprehensive description of the state-of-the-art, and a guide to the original literature. It is specifically directed to micro- biologists, biochemists, molecular biologists, bioengineers, chemical engineers, and food and pharmaceutical chemists working in indus- try, at universities or at public institutions. A carefully selected and distinguished Scientific Advisory Board stands behind the

  • P. Stadler

Scientific Advisory Board

Pro$ Dr. M. J. Beker

August Kirchenstein Institute of Microbiology Latvian Academy of Sciences Riga, Latvia

Pro$ Dr. T. K. Ghose

Biochemical Engineering Research Centre Indian Institute of Technology New Delhi, India

Pro$ Dr. J. D. Bu’Lock

Weizmann Microbial Chemistry Laboratory Department of Chemistry

Pro$ Dr. I. Goldberg

Department of Applied Microbiology The Hebrew University

University of Manchester Jerusalem, Israel

Manchester, UK

Pro$ Dr. C. L. Cooney

Department of Chemical Engineering Massachusetts Institute of Technology Cambridge, MA, USA

Pro$ Dr. H. W. Doelle

Department of Microbiology University of Queensland St. Lucia, Australia

Prof Dr. J. Drews

F. Hoffmann-La Roche AG Basel, Switzerland

Pro$ Dr. A. Fiechter

Institut fur Biotechnologie Eidgenossische Technische Hochschule Zurich, Switzerland

Pro$ Dr. G. Goma

Departement de GCnie Biochimique et Alimentaire Institut National des Sciences AppliquCes Toulouse, France

Sir D. A. Hopwood

Department of Genetics John Innes Institute Norwich, UK

Pro$ Dr. E. H. Houwink

Organon International bv Scientific Development Group Oss. The Netherlands

Pro$ Dr. A. E. Humphrey

Center for Molecular Bioscience and Biotechnology Lehigh University Bethlehem, PA, USA

VIII

Scientific Advisory Board

Prof. Dr. I. Karube

Research Center for Advanced Science and Technology University of Tokyo Tokyo, Japan

Prof. Dr. M. A. Lachance

Department of Plant Sciences University of Western Ontario London, Ontario, Canada

Prof. Dr. Y. Liu

China National Center for Biotechnology Development Beijing, China

Prof. Dr. J. F. Martin

Department of Microbiology University of Leon Leon, Spain

Prof. Dr. B. Mattiasson

Department of Biotechnology Chemical Center University of Lund Lund, Sweden

Prof. Dr. M. Roehr

Institut fur Biochemische Technologie und Mikrobiologie Technische Universitat Wien Wien, Austria

Prof. Dr. H. Sahm

Institut fur Biotechnologie Forschungszentrum Julich Julich, Germany

Prof. Dr. K. Schiigerl

Institut fur Technische Chemie Universitat Hannover Hannover, Germany

Prof. Dr. P. Sensi

Chair of Fermentation Chemistry and Industrial Microbiology Lepetit Research Center Gerenzano, Italy

Prof. Dr. Y. H. Tan

Institute of Molecular and Cell Biology National University of Singapore Singapore

Prof. Dr. D. Thomas

Laboratoire de Technologie Enzymatique UniversitC de Compibgne Compibgne, France

Prof. Dr. W. Verstraete

Laboratory of Microbial Ecology Rijksuniversiteit Gent Gent, Belgium

Prof. Dr. E.-L. Winnacker

Institut fur Biochemie Universitat Munchen Munchen, Germany

Contents

Introduction

H. von Dohren, H. Kleinkauf

General Aspects of Secondary Metabolism 1

H.

von Dohren, U. Grafe

 

Regulation of Bacterial Antibiotic

 

Production

57

K.

Chater, M. Bibb

 

Screening of Novel Receptor-Active

Compounds of Microbial Origin

107

H.

Tanaka, S. Omura

 

Microbial Lipids

133

C.

Ratledge

Microbial Siderophores

199

G.

Winkelmann, H. Drechsel

 

Advances in the Molecular Genetics of

PLactam Antibiotic Biosynthesis

247

P.

15. Skacrud, T. Schwecke,

H.

v. Liempt, M. B. Tobin

 

Peptide Antibiotics

277

H.

Kleinkauj H. von Dohren

 

Lantibiotics

323

R.

Jack, F. Gotz, G. Jung

 
  • 9 Glycopeptide Antibiotics

(Dalbaheptides)

369

G.

Lancini, B. Cavalleri

 
  • 10 Aminoglycosides and Sugar

Components in Other Secondary

Metabolites 397

 

W.

Piepersberg, J. Distler

 
  • 11 Products from Basidiomycetes

489

G.

Erkel, T. Anke

  • 12 Cyclosporins: Recent Developments in

Biosynthesis, Pharmacology and

 

Biology, and Clinical Applications 535

J.

Kallen, V. Mikol, V. F. J. Quesniaux,

M.

D. Walkinshaw,E. Schneider-Scherzer,

K.

Schorgendorfer, G. Weber, H. Fliri

  • 13 Secondary Products from Plant Cell Cultures 593

J.

Berlin

  • 14 Biotechnical Drugs as Antitumor Agents 641

    • U. Grafe, K. Dornberger, H.-P. Saluz

Index

707

Contributors

Prof. Dr. Timm Anke

Lehrbereich Biotechnologie Universitat Kaiserslautern Postfach 3049 D-67618 Kaiserslautern Germany

Chapter I1

Dr. Jochen Berlin

Gesellschaft fur Biotechnologische Forschung Mascheroder Weg 1 D-38124 Braunschweig Germany

Chapter 13

Dr. Mervin Bibb

John Innes Centre Norwich Research Park Colney Lane Colney, Norwich NR4 7UH UK

Chapter 2

Dr. Bruno Cavalleri

MMDRI - Lepetit Research Center Via R. Lepetit, 34 1-21040 Gerenzano (Varese) Italy

Chapter 9

Prof. Keith Chater

John Innes Centre Norwich Research Park Colney Lane Colney, Norwich NR4 7UH UK

Chapter 2

Jurgen Distler

Bergische Universitat GH Mikrobiologie - FB 19 Gauss-StraSe 20 D-42097 Wuppertal Germany

Chapter 10

Dr. Hans von Dohren

Institut fur Biochemie Technische Universitat Franklin-Str. 29 D-10587 Berlin Germany

Chapters I, 7

Dr. Klausjiirgen Dornberger

Hans-Knoll-Institut fur Naturstoff-Forschung Bereich Naturstoffchemie BeutenbergstraBe 11 D-07745 Jena Germany

Chapter 14

XI1

Contributors

Dr. Hartmut Drechsel

Prof. Dr. Gunter Jung

Mikrobiologie und Biotechnologie

Universitat Tubingen

Universitat Tubingen Auf der Morgenstelle 1 D-72076 Tubingen Germany

Institut fur Organische Chemie Auf der Morgenstelle 18 D-72076 Tubingen Germany

Chapter 5

Chapter 8

Dr. Gerhard Erkel

Dr. Jorg Kallen

Lehrbereich Biotechnologie Universitat Kaiserslautern Postfach 3049 D-67618 Kaiserslautern Germany Chapter I1

Sandoz Pharma Ltd. Preclinical Research CH-4002 Basel Switzerland Chapter I2

Dr. Hans Fliri

Prof. Dr. Horst Kleinkauf

RhBne Poulonc Rorer S.A. Centre de Recherche de Vitry-Alfortville 13, quai Jules Guesde F-94403 Vitry-sur-Seine Cedex France Chapter 12

Institut fur Biochemie Technische Universitat Franklin-Str. 29 D-10587 Berlin Germany Chapter 7

Dr. Friedrich Gotz

Dr. Giancarlo Lancini

Lehrstuhl fur Mikrobielle Genetik Universitat Tubingen Auf der Morgenstelle 18 D-72076 Tubingen Germany Chapter 8

MMDRI - Lepetit Research Center Via R. Lepetit, 34 1-21040 Gerenzano (Varese) Italy Chapter 9

Prof. Dr. Udo Grafe

Dr. Henk van Liempt

Hans-Knoll-Institut fur Naturstoff-Forschung

DRL, BT-FDG

Bereich Naturstoffchemie

SudstraSe 125

BeutenbergstraSe 11

D-53175 Bonn

D-07745 Jena

Germany

Germany Chapters I, 14

Chapter 6

Dr. Ralph Jack

Dr. Vincent Mikol

Universitat Tubingen Institut fur Organische Chemie Auf der Morgenstelle 18 D-72076 Tubingen Germany Chapter 8

Sandoz Pharma Ltd. Preclinical Research CH-4002 Basel Switzerland Chapter 12

Prof. Dr. Satoshi Omura

School of Pharmaceutical Sciences Kitasato University The Kitasato Institute 9-1, Shirokane 5-chome Minato-ku, Tokyo 108 Japan Chapter 3

Prof. Dr. Wolfgang Piepersberg

Bergische Universitat GH

Mikrobiologie - FB 19 Gauss-Strafie 20

D-42097 Wuppertal Germany Chapter 10

Dr. Valkrie F.J. Quesniaux

Sandoz Pharma Ltd. Preclinical Research CH-4002 Basel Switzerland Chapter 12

Prof. Colin Ratledge

The University of Hull Department of Applied Biology Hull HU6 7RX UK Chapter 4

Contributors

XI11

Dr. Kurt Schorgendorfer

Biochemie GmbH A-6330 Kufstein-Schaftenau Austria Chapter 12

Dr. Torsten Schwecke

Institute of Biochemistry University of Cambridge Tennis Court Road Cambridge, CB2 1QW UK Chapter 6

Dr. Paul L. Skatrud

Infectious Diseases Research Eli Lilly and Company Lilly Corporate Center Indianapolis, IN 46285 USA Chapter 6

Dr. Haruo Tanaka

School of Pharmaceutical Sciences Kitasato University The Kitasato Institute Minato-ku, Tokyo 108 Japan Chapter 3

Prof. Dr. habil. Hans-Peter Saluz

Hans-Knoll-Institut fur Naturstoff-Forschung Bereich Naturstoffchemie BeutenbergstraSe 11 D-07745 Jena Germany Chapter 14

Dr. Matthew B. Tobin

Infectious Diseases Research Eli Lilly and Company

Lilly Corporate Center Indianapolis, IN 46285 USA Chapter 6

Dr. Elisabeth Schneider-Scherzer

Biochemie GmbH A-6330 Kufstein-Schaftenau Austria

Chapter 12

Dr. Malcolm D. Walkinshaw

Sandoz Pharma Ltd. Preclinical Research CH-4002 Basel Switzerland Chapter 12

XIV

Contributors

Dr. Gerhard Weber

Biochemie GmbH A4330 Kufstein-Schaftenau Austria Chapter 12

Prof. Dr. Giinther Winkelmann

Mikrobiologie und Biotechnologie Universitat Tubingen Auf der Morgenstelle 1 D-72076 Tubingen Germany

Chapter 5

This volumes provides an overview of sec- ondary metabolites illustrating most aspects of their discovery, formation, exploitation, and production. Compared to the first edition the focus when has clearly shifted towards the molecular genetic background of the produc- ing organisms. These efforts serve not only our understanding of the production proc- esses to permit improvements by genetic ma- nipulations, but also promote our apprecia- tion of the environmental significance of sec- ondary metabolites. The term “secondary metabolite” has been discussed widely, and a shift in perception took place in the last years. From a play- ground of nature leading to mostly disparable products ideas focus now on special purpose products promoting evolutionary advantages. This shift is connected to the impressive eluci- dation of the genetics of multistep synthetic processes of secondary metabolite formation. Genes encoding biosynthetic reaction se- quences have been found clustered together with resistance or export genes and are under the control of specific signals. Biosynthetic functions or unit operations reside on mod- ules, and these modules in their functional protein state interact to assure the fidelity of the multistep processes. The genetic burden for many of these processes seems remarka- ble, and genes assembled from modules often display sizes of 10 to more than 45 kilobases. Since some of the now established microbial genomes are devoid of such multistep path- ways, their unique placement in other ge- nomes indicates important functions for their producers.

Biotechnology Edited by H.-J. Rehm and G. Reed OVCH Verlagsgesellschaft mbH, 1997
Biotechnology
Edited by H.-J. Rehm and G. Reed
OVCH Verlagsgesellschaft mbH, 1997

Still largely unconnected to the back- ground of their producers secondary metabol- ites generally are high-value compounds es- tablished mainly in pharmacology, veterinary medicine, agriculture, and biochemical and medical research. The introductory chapter points to product fields and to the genetic in- vestigation of biosynthetic unit operations. Regulatory mechanisms are then considered in the most advanced fields of the proka- ryotes. As the central field of present drug discovery approaches target-based screenings are discussed. Compound groups considered are lipids siderophores, aminoglycosides, and peptides (p-lactams, dalbaheptides, cyclospo- rins, lantibiotics). Producer groups presented are basidiomycetes and plant cells. As a tar- get group antitumor drugs are evaluated. An updated chapter on macrolides as sec- ondary metabolites including reprogramming strategies will be included in Volume 10 of the Second Edition of Biofechnofogy(see also Volume 4 of the First Edition). Further chapters to be consulted are espe- cially on biopolymers and surfactants (Vol- ume 6), on the overproduction of metabolites and the treatment of producer organisms like bacilli, streptomycetes and filamentous fungi (Volume 1) as well as on reactor modeling (Volume 3). We thank our colleagues for their valuable contributions, the publisher for their patience and cooperativity, and the se- ries editors for many helpful suggestions.

Berlin, March 1997

Hans von Dohren Horst Kleinkauf

Biotechnology Edited by H.-J. Rehm and G. Reed OVCH Verlagsgesellschaft mbH, 1997
Biotechnology
Edited by H.-J. Rehm and G. Reed
OVCH Verlagsgesellschaft mbH, 1997

1 General Aspects of Secondary Metabolism

HANSVON DOHREN

Berlin, Germany

UDO GRAFE

Jena, Germany

1 Introduction: The Importance of Secondary Metabolites as Drugs

2

  • 2 Secondary Metabolism, an Expression of Cellular and Organismic Individuality

11

  • 2.1 Roles of Secondary Metabolites in Producing Organisms 11

  • 2.2 Regulation of Microbial Secondary Metabolism 17

    • 2.2.1 Genetic Organization of Product Formation 17

    • 2.2.2 Regulatory Mechanisms 23

    • 2.2.3 Genetic Instability 26

    • 2.2.4 Developmental Processes 27

    • 2.2.5 The A-Factor and the Signal Cascade of Cytodifferentiation in Streptomyces 27

    • 2.2.6 Overproduction of Microbial Secondary Metabolites and Precursor Pools 29

    • 2.2.7 Biotechnical Production of Secondary Metabolites 31

  • 3 The Biosynthetic Pathways

31

  • 3.1 Precursors and the Main Biosynthetic Pathways 31

  • 3.2 Secondary Metabolites Formed through Biosynthetic Modifications of a Single Precursor 31

  • 3.3 Polyketides 32

  • 3.4 Terpenes 35

  • 3.5 Sugar-Derived Oligomeric Structures 35

  • 3.6 Oligo- and Polypeptides 36

  • 3.7 Biosynthetic Modifications of Structures and Precursor-Directed Biosyntheses 37

  • 4 Variability of Structures of Secondary Metabolites

38

  • 4.1 Secondary Metabolites as Products of Biological “Unit Operations” 38

  • 4.2 Structural Classifications of Secondary Metabolites 38

  • 5 Future Perspectives: New Products of the Secondary Metabolism

40

2

1 General Aspects of Secondary Metabolism

1 Introduction: The Importance of Secondary Metabolites as Drugs

Today, bioactive secondary metabolites of microorganisms and of plants, and their syn- thetic derivatives as well, are among the most frequently used therapeutics in human and veterinary medicine (Scrip, 1993). The inven- tion of antibiotic therapy contributed greatly to the successful control of most of the epi- demic infectious diseases and even promoted their disappearance. Moreover, it contributed to the general increase in the lifespan of man, not only in industrialized countries. New ap- plications for bioactive biotechnical products in medical care like their use as immunosup- pressants or antiatherosclerotics, and as ani- mal growth promoters and pesticides in agri- culture rendered research on new secondary metabolites an apparently endless story (SAN- GLIER and LARPENT,1989; ComitC Editorial, 1992; LANCINIand LORENZE-ITI,1993; VIN- ING and STUTTARD,1995). In the past, natural products supplied 54% of the annual increase in the world’s total pharmaceutical market. The list of the 25 worldwide best-selling drugs for application in humans in 1992 includes a series of drugs of microbial origin which are used either in their native structures or as chemical deriva- tives (see, e.g., Mevacor, Cefaclor or other cephalosporins, Augmentin, Sandimmun) (Scrip, 1993). Many plant products, from digitalis glyco- sides and neuroactive alkaloids to the pyre- thrines, serve as therapeutics for human dis- eases and as agricultural agents (Comitk Edi- torial, 1992). Sometimes, the experiences made in folk medicine initiated the discovery of new plant-derived antitumor drugs, anti- neuralgic, antihypertonic, antidepressant, in- secticidal, nematicidal, and other bioactive compounds. Antiinfective chemotherapy once was the classical domain of biotechnical drug produc- tion due to the discovery of p-lactam antibiot- ics, such as penicillins, cephalosporins, clavu- lanates, and carbapenems. Even today, the in-

crease in resistant nosocomial and opportu- nistic pathogens (particularly dangerous to immunosuppressed AIDS and tumor pa- tients) requires both improvement of known drugs and search for new drugs (GRAFE, 1992; LANCINIand LORENZETTI,1993; HUT- CHINSON, 1994). Microbial products such as doxorubicin, bleomycin, and mitomycin C are indispensa- ble as cancerostatics (Fox, 1991). The same is true for plant metabolites such as the vinca al- kaloids, taxol, and their chemical derivatives which exert excellent antitumor activity by in- teraction with the cellular mitotic system (NOBLE, 1990 Fox, 1991; HEINSTEINand CHANG,1994 POTIERet al., 1994). However, even the non-therapeutic fields of application, such as in animal husbandry and plant protection, contributed to a high degree to the continuing interest in secondary metabolite production. Last but not least, nat- ural products of biotechnical and agricultural origin play an important role as “biochemical tools” in molecular biology and in the investi- gation of cellular functions. More than loo00 antibiotics and similar bioactive secondary metabolites have been isolated so far from microbes, and a compara- bly higher number of drugs was derived from plants and even from animals (see, e.g., ma- rine tunicates, molluscs, toxic insects, snakes, and toads) (BERDYet al., 1980; LAATSCH, 1994). Approximately 500 new representa- tives of low-molecular weight compounds are published every year. In addition to this huge and still growing number of bioactive molecules, more than 1OOOOO derivatives as representatives of some few basic structures (e. g., p-lactams, macro- lides, aminoglycosides, tetracyclines, anthra- cyclines) were obtained by means of synthetic derivatizations (LAATSCH,1994). Irrespective of this plethora of drug molecules a little more than a hundred basic structures gained practical importance. We owe much progress in the detection of new drug structures to modern physico- chemical approaches such as mass-spectrome- try, high-field nuclear magnetic resonance spectroscopy and X-ray diffractometry. Com- pilations of the numerous structural data (BERDY et al., 1980; BYCROFT, 1988;

I

Introduction: The Importance of Secondary Metabolites as Drugs

3

LAATSCH,1994) provide indispensable

assist-

ance in the identification of new drug mole- cules. Thus, the enormous number of already known metabolites from microbes and plants increased the detection and isolation of alrea- dy known structures dramatically. A compilation of about 200 recently de- scribed products illustrates the current trends in screening efforts (Tab. 1). These have been published during the last two years. It is evi- dent from these data that highly selective screens prevail and yet the majority of com- pounds originate from the classical Actino- mycete pool. Rare bacteria and fungi, marine microorganisms and plants now have a signifi- cant share. It is obvious that well-known or- ganisms again contribute with newly isolated substances to new, e. g., receptor targeted screens. Strategies of such screens are dis- cussed in this volume in Chapter 3 by TANA- KA and OMURA. The development of new drugs from natu- ral sources is common practice of the pharma- ceutical industry. 6000 to loo00 chemicals have to be tested in a given assay system to obtain one single compound suitable as a

therapeutical agent (OMURA,1992; KROHN et al., 1993). No wonder that research and de- velopment for a new approved drug may cost up to one billion US$. In most cases, a new natural “leading structure” is intensively modified by chemical means to improve its activity and to reduce side effects. Chemistry is also extremely helpful if rather rare natural products occurring in low amounts or in or- ganisms from sensitive ecological areas have been proposed as drugs. For example, 40000 yew trees, i. e., the whole population of Northern America, would be required to pro- duce 25 kg of taxol, a new promising cancero- static drug, and even this amount would not be sufficient to treat every cancer patient. Fortunately, taxol derivatives of similar activ- ity (taxotere) can be obtained by chemical derivatization of taxoid metabolites which are obtainable in large quantities from the dried leaves of European yews (HEINSTEINand CHANG,1994). Alternatively, cell cultures (ELLISet al., 1996) or endophytic fungi such as Pestalotiopsis microspora (STIERLEet al., 1994, 1995; STROBELet al., 1996) of Taxus species could be exploited for production.

From the recently completed chemical syn- thesis of taxol it is evident that, as in bicyclic plactams, classical approaches cannot com- pete with natural producers. Instead, increas- ing attention is given to the recruitment of biocatalysts for certain key reactions in metabolite production. In addition, directed biosynthesis in microbial cultures (THIER- ICKE and ROHR, 1993), production of plant products in cell cultures (BERLIN,Chapter 14, this volume), and cell free in vitro systems of enzymatic synthesis and peptide and protein producing translation systems are considered as complementary methods in structure-func- tion studies (ALAKHOVand VON DOHREN, unpublished data). Only 30% of the total developmental ef- forts have been spent to the search for a new drug. However, for the estimation of its effi- cacy and evaluation of safety often more than 50% are needed. Taking into account a quota of approximately 1: 15 000 for a hit structure, the challenges of modern pharmaceutical de- velopment become visible. In general, natural products seem to offer greater chances than synthetically derived agents. Hence, a great research potential is still dedicated to the dis- covery of new natural drugs and their bio- technical production. Classical strategies of drug development are being more and more supplemented by new biomedical approaches and ideas and by the use of genetically engi- neered microbes and cells as screening organ- isms (TOMODAand OMURA,1990; ELDERet al., 1993). These tools initiated a “renais- sance” in the search for new leading struc- tures. New sources of bioactive material, such as marine organisms, and new microbes from ecological “niches” promoted the recent ad- vances in the discovery of drugs (WILLIAMS and VICKERS1986; RINEHARTand SHIELD 1988; MONAGHANand TKACZ,1990; JACOB and ZASLOFF,1994; JENSENand FENICAL, 1994) (Tab. 1). Present research activities were also stimu- lated by the discovery of block busters (Scrip, 1993) such as cyclosporin A (KAHAN,1987), avermectins (CAMPBELL, 1989), acarbose (MULLER, 1989), and monacolin (ENDO, 1979) in microbial cultures. A series of very promising new screening drugs (zaragozic acid, squalestatins) (HASUMI,1993), erbstatin

4

I

General Aspects of Secondary Metabolism

Tab. 1. Selected Natural Products Detected by Screening Efforts Published in 1995196

Compound

Producing Organisms

Structural

Selected

Research Group

Reference

Type'

Properties

Involved

Antimicrobial Drugs:

Griseusin

Actinomycete

PK

antibacterial

Institute of Microbial

derivatives

(unidentified)

Chemistry

BE-24566B

Streptomyces

PK

antibacterial

Banyu Pharm. Co.

violaceus-niger

Amicenomycin

Streptomyces sp.

PK-GLYC

antibacterial

Institute of Microbial Chemistry

Kalimantacins

Alcaligenes sp.

PK, mod.

antibacterial,

Yamanouchi Pharm. Co.

 

MDR strains

and PT Kalbe Pharma

A21459

Actinoplanes sp.

PEP

antibacterial

Lepetit

Epoxyquino-

Amycolatopsis

acyl AA

antibacterial

Institute of Microbial

mycins

Chemistry

GE 37468

Streptomyces sp.

PEP

antibacterial

Lepetit

Phencomycin

Streptomyces sp.

PK

antibacterial

Hoechst

Chrysoapermin

Apiocrea chrysosperma

PEP

antibacterial

Hans Knoll Institute

 

antifungal

and Univ. Tiibingen

Bacillaene

Bacillus subtilis

PK

antibacterial

Bristol Myers Squibb

GE2270

Planobispora rosea

PEP

antibacterial

Lepetit

AL072

Streptomyces sp.

PK, mod.

antilegionella

Cheil Foods & Chem.

Ripostatin

Sorangium cellulosum

PK

antibacterial

Inc and NIH Korea GBF

Sorangiolid

Sorangium cellulosum

PK

antibacterial

GBF

Thiomarinol

Alteromonas rava

antibacterial

Sankyo

(marine)

Echinoserine

Streptomyces tendae

PEP + PK

antibacterial

Univ. Tiibingen and Hans Knoll Institute

07F275

unidentified fungus

PK

antibacterial

Lederle

Pyralomycins

Actinomadura spiralis

PK, mod.

antibacterial

Institute of Microbial Chemistry

RS-22

Streptomyces

PK

antibacterial

RIKEN

violaceusniger

Ochracenomy-

Amycolatopsis sp.

PK

antibacterial

Institute of Microbial

cins

Chemistry

Azicemycins

Amycolatopsis

PK

antibacterial

Institute of Microbial

sulphurea

Chemistry

Amythiamycin

Amycolaotopsis sp.

PEP

antibacterial

Institute of Microbial Chemistry

APHE 31~

Streptoverticillium

ALK

antibacterial

Univ. Alcala

griseocarnum

Aurantimycin

Streptomyces

PEP

antibacterial,

Hans Knoll Institute

aurantiacus

cytotoxic

Cineromycins

Streptomyces

antibacterial

Univ. Tlibingen, Univ.

griseoviridus

Gattingen, Hans Knoll Institute

Papyracon

Lachnum payraceum

TERP

antibacterial

Univ. Lund and Univ. Kaiserslautern

Cephem

Penicillium chryso-

PEP, mod.

antibacterial

Panlabs

derivatives

genum

Sorrentanone

Penicillium chryso-

PK

antibacterial

Bristol Myers Squibb

genum

I

Introduction: The Importance of Secondary Metabolites as Drugs

5

Tab. 1. (Continued)

Compound

Producing Organisms

Reference

Benzastatin

Streptomyces

nitrosporeus

Jerangolides

Sorangium cellulosum

BE29602

Fusarium sp.

Dibefurin

unidentified mushroom

Darlucins

Sphaerellopsis filu

Fusaricidin

Bacillus polymyxa

Helioferin

Mycogone rosea

Azalomycin

Actinomycete

Liposidolide

Streptomyces sp.

Chirosazol

Sorangium cellulosum

Ratjadon

Sorangium cellulosum

Chrysospermin

Apiocrea chrysosperma

Hydroxystro-

Pterula sp.

bilurin

Fusacandin

Fusarium sambucinum

Favolon

Favolaschia

Aureobasidins

A ureobasidium

pullulans

Phosmidosine

Streptomyces sp.

NP-1OlA

Streptomyces

aurantiogriseus

YM-47522

Bacillus sp.

Australifungin

Sporomiella australis

AKD-2

Streptomyces sp.

UK-2AIBICID

Streptomyces sp.

Prodimicin

Actinomadura spinosa

Pradimicin

Actinomadura spinosa

Ascosteroside

Ascotricha amphitricha

Epothilone

Mucor hiemalis

Fusarielin

Fusarium sp.

Saricandin

Fusarium sp.

Furanocandin

Tricothecium sp.

Siamycin

Streptomyces sp.

L-671,776,

Stachybotrys sp.

derivatives

Structural

Type '

ALK

PK

PK-GLYC

PK

PK-mod.

PEP-PK

PEP-PK

PK

PK

PK

PK

PEP

PK

PK-GLYC

TERP

PEP

NUC

A-mod.

PK

PK

PK

PEP

PK

PK

TERP-GLYC

PK-AA

PK

PK

PK-GLYC

PEP

PK-AA

Selected

Research Group

Properties

Involved

antifungal,

KRIBB

antiviral free

radical scaven-

ger

antifungal

GBF

antifungal

Banyu Pharm. Co.

antifungal

Abbott

antibacterial,

Univ. Kaiserslautern

antifungal

and Univ. Munich

antifungal,

Wakunaga Pharm. Co.

antibacterial

and PT Kalbe Pharma

antifungal,

Hans Knoll Institute

antibacterial

antifungal

Hoechst, AgrEvo

antifungal

RIKEN

antifungal

GBF

cytotoxic

antifungal

GBF

antifungal

Hans Knoll Institute

antifungal

Univ. Kaiserslauern and

antifungal

Univ. Munich Abbott

antifungal

Univ. Kaiserslautern

antifungal

and Univ. Munich Takara Shuzo Co.

antifungal

RIKEN and SynPhar

antifungal

Lab. Inc. Hokkaido Univ.

antifungal

Yamanouchi Pharm. Co

antifungal

Merck Sharp & Dohme

antibacterial

Univ. Osaka City

antifungal

antifungal

Osaka Univ. and

antifungal

Suntory Ltd. Meijo U, Toyama Pref.

antifungal

Univ. Toyama Pref. Univ. and

antifungal

Bristol Myers Squibb Bristol Myers Squibb

antifungal,

GBF

cytotoxic

antifungal

Univ. Tokyo

antifungal

Abbott

antifungal

Meiji Seika Kaishi Ltd

antiviral, HIV

and Mitsubishi Chem. Corp. Bristol Myers Squibb

HIV protease

Ciba-Geigy

inhib., endothe-

lin antag.

6

I

General Aspects of Secondary Metabolism

Tab. 1. (Continued)

Compound

Producing Organisms

Structural

Reference

Type'

Benzastatins

Streptomyces

ALK

nitrosporeus

Triterpene-

Fusarium compactum

TERP

sulfates

Quinoxa-

Betula papyrifera

PEP-PK

peptides

Karalicin

Pseudomonas

PK

fluorescens

AH-758

Streptomyces sp.

PK

Eulicin

Streptomyces sp.

PEP-mod.

Sattabacin

Bacillus sp.

A-mod.

Sattazolin

Bacillus sp.

AA-mod.

GE20372

Streptomyces sp.

PEP

Isochromo-

Penicillium sp.

PK

philones

Antitumor Drugs

Sch5290011 Gliocladiurn sp.

PEP

Rakicidins Micromonospora sp.

PEP-PK

Esperamicin Actinomadura

PK-GLYC

verrucosospora Ossamycin Streptomyces

PK

hygroscopicus Acetophthalidin Penicillium sp. (marine)

PK

Tryprostatins

Aspergillus fumigatus

'S

PEP-TERP

Sparoxomycin

Streptomyces sparsogenus

NUC-mod.

Cochleamycins

Streptomyces sp.

PK

Himastatin

Streptomyces

PEP

Chondramide

hygroscopicus Chondromyces crocatu

PEP

Anguinomycin

Streptomyces sp.

PK

Clovalicin

Sporothrix sp.

PK

Clecarmycin

Streptomyces sp.

PK

Piericidin

Streptomyces sp.

PK-GLYC

derivatives

Hydroxymyco-

Bacillus sp.

PK

trienine

FR901537 Bacillus sp.

PEP-PK

Medelamine

Streptomyces sp.

PK, mod.

Naphthablin

Streptomyces sp.

PK

Macquarimicin Micromonospora sp.

PK

Selected

Research Group

Properties

Involved

free radical

KRIBB

scavenger anti-

fungal, antiviral

rhinovirus pro-

Abbott

tease inhib.

antiviral:

Merck Sharp & Dohme

HIV1,2,RT

antiviral: HSV

Univ. Cagliari and Univ.

antiviral: HSV

Cattolin (Rome) Kumamoto Univ.

antiviral: HIVl

Jikei Univ., Institute of

antiviral: HSV

Microbial Chemistry Univ. Cagliari and Univ.

antiviral: HSV

Rome Univ. Cagliari and Univ.

antiviral: HIV

Rome Lepetit

antiviral: HIV

Kitasato

DGAT, AC-

TAT inhib.

antitumor

Schering-Plough

cytotoxic

Bristol Myers Squibb

antitumor

Bristol Myers Squibb

cytotoxic

Lilly

cell cycle

RIKEN

inhibitor cell cycle inhib. proliferation

RIKEN Toyama Pref. Univ.

mod. antitumor antitumor

Kirin Brewery Co. Bristol Myers Squibb

cytotoxic

GBF

cytotoxic

Univ. Tokyo

cytocidal

Kitasato

antitumor

Kyowa Hakko Kogyo

antitumor

Snow Brand Milk Co.

cancerostatic

and Kamagawa Univ. Institute of Microbial

aromatase

Chemistry and Showa College Fujisawa

inhib.

anticancer

Nippon Kayaku

oncogen

Keio Univ. and Institute

function inhib.

of Microbial Chemistry

cytotoxic

Abbott

I

Introduction: The Importance of Secondary Metabolites as Drugs

7

Tab. 1. (Continued)

Compound

Producing Organisms

Structural

Selected

Research Group

Reference

Type'

Properties

Involved

Thiazinotrieno-

Streptomyces sp.

PK-PEP

cytostatic

Institute of Microbial

mycin

(cancer)

Chemistry and Showa College

Cremeduycin

Streptomyces cremeus

A, mod.

cytotoxic

Univ. Illinois

Tryprostatin

Aspergillus fumigatus

PEP, mod.

cell cycle inhib.

RIKEN

Sch50673,6

Nattrassia mangiferae

PK

antitumor

Schering-Plough

Terpentecin

Streptomyces sp.

PK

antitumor:

Kyowa Hakko Kogyo

 

topoisomerase

inhib.

FD-211

Myceliophthora lutea

PK

cytotoxic: MDR

Taisho Pharm. Co.

Cytogenin

Streptoverticillium

PK

antitumor

Institute of Chemother-

eurocidium

apy (Shizuoka) and In- stitue of Microbial Chemistry

Enaminedonin

Streptomyces sp.

PEP-PK

detransforming

RIKEN

 

tumor cells

Dihydroepi-

Penicillium patulum

PK-mod.

IL-1 receptor

Upjohn

epoformin

antag.

EI-1507-1/2

Streptomyces sp.

PK

IL-1-converting

Kyowa Hakko Kogyo

 

enz. inhib.

TAN-15 11

Streptosporangium

PEP-PK

induces

Takeda

amethystogenes

cytokines

CJ-12,371,2

unidentified fungus

PK

DNA gyrase

Pfizer

 

inh.

Pharmacological Activities

 

FR901,483

Cladybotryum sp.

ALK-P-ester

immunosuppr.

Fujisawa Pharm. Co.

PA-48,153

Streptomyces prunicolor

PK

immunosuppr.

Shionogi

27-0-demethyl-

Steptomyces

PK-AA

immunosuppr.

Smith Kline Beecham

Rapamycin

hygroscopicus

NFAT 68,133

Streptomyces sp.

PK

immnuosuppr.

Abbott

Stevastatin

Penicillium sp.

PEP-PK

immunosuppr.

Nippon Kayaku

Trichstatin

Streptomyces sp.

PK

immunosuppr., histidine decar- boxylase inhib.

Kyowa Hakko Kogyo

Cytosporin

Cytospora sp.

PK

angiotensin bdg. inhib.

Merck Sharp & Dohme

Leustroducsin

Streptomyces platensis

PK-P-ester

t hrombocytosis inhib.

Sankyo Co.

Plactins

Agonomycetales

PEP

stimulates fibri- nolytic activity

Tokyo Noko Univ.

TAN1323CID

Streptomyces

PK

angiogenesis

Takeda

purpurescens

inhib.

Monamidocin

Streptomyces sp.

PEP

fibrinogen rec. antag.

Nippon Roche

A-72363

Streptomyces nobilis

GLYC

heparanase

Sankyo Co.

Trachyspic acid

Talaromyces trachyspermus

PK

inhib. hep a r a na se inhib.

Sankyo and Univ. Tokyo

Carbazo-

Streptomyces violaceus

AA-PK

antioxidant

Univ. Tokyo

quinocins

8

I

General Aspects of Secondary Metabolism

Tab. 1. (Continued)

Compound

Producing Organisms

Structural

Selected

Research Group

Reference

Type'

Properties

Involved

Phenopyrazin ,

Penicillium sp.

PK-AA

radical scavenger

Kitasato

Balmoralmycin

Streptomyces sp.

PK

protein kinase

Ciba-Geigy

Staurosporine

Streptomyces

ALK

inhib. protein kinase

Ciba Geigy

analogs

longisporoflavus

C inhib.

Paeciloquinones

Paecilomyces carneus

PK

protein tyrosine kinase inhib.

Ciba Geigy and Panlabs

Factor AIC

unidentified fungus

PK, mod.

myoinositol Pase inhib.

Lepetit

MS-444

Micromonospora sp.

PK

myosin light chain kinase inhib.

Kyowa Hakko Kogyo

WS79089B

Streptosporangium

PK

endothelin con-

Fujisawa

roseum

verting enzyme inhib.

Stachybocin

Stachybotrys sp.

PK-AA

endothelin rec. antag.

Asahi

RES-1149

Aspergillus sp.

PK

endothelin rec. antag.

Kyowa Hakko Kogyo

RES-701

Streptomyces sp.

PEP

endothelin rec. antag.

Kyowa Hakko Kogyo

L-671,776

Stachybotrys sp.

PK-AA

endothelin rec.

Ciba-Geigy

derivatives

antag.

Mer-A2026

Streptomyces pactum

PK

vasodilatory

Mercian Corp.

ET

Penicillium sclerotium

PK

endothelin rec. antag.

Xenova and Parke Davis

Drirnane-ses-

Aspergillus ustus

TERP-PK

entothelin rec.

Xenova

quiterpenes

bdg.

Bassiatin

Beauveria bassina

PEP

platelet aggr.

Taisho Pharm.

Herquline

Penicillium herquei

ALK

inhib. platelet aggr. inhib.

Kitasato

Schizostatin

Schizophyllum

TERP

squalene synth.

Sankyo

commune

inhib.

Macrosphelide

Microsphaeropsis sp.

PK

cell adhesion inhib.

Kitasato

Sulfobacins

Chryseobacter sp.

PK-S

Willebrand fac-

Nippon Roche

Lateritin

Gibberella lateritium

PEP

tor rec. antag. ACAT inhib.

Tokyo Noko Univ.

Isohalobacillin

Bacillus sp.

PEP-PK

ACAT inhib.

Tokyo Noko Univ.

GERI-BP002-A

Aspergillus fumigatus

PK

ACAT inhib.

KRIBB

Pyripyropenes

Aspergillus furnigatus

PK

ACAT inhib.

Kitasato and Pfizer

Amidepsine

Humicola sp.

DGAT inhib.

Kitasato

Terpendole

Albophoma yamanashiensis

TERP-mod.

ACAT inhib.

Kitasato

Epi-cochlio-

Stachybotrys bisbyi

TERP-PK

ACAT inhib.

Sankyo

quinone

F1839

Stachybotrys

TERP-PK

cholesterol

Tokyo Tanabe Co. and

 

esterase inhib.

Univ. Tokyo

CETPI

Cytospora (insect

PK

cholesteryl ester

Cornell Univ. and

associated)

transfer protein

Schering-Plough

 

inhib.

I

Introduction: The Importance of Secondary Metabolites as Drugs

9

Tab. 1. (Continued)

 

~~

Compound

Producing Organisms

Structural

Selected

Research Group

Reference

Type'

Properties

Involved

Fluvirucin

Streptomcyces sp.

PK-GLYC

phospholipase

Univ. Keio

 

inhib.

Thermorubin

Thermoactinomyces sp.

PK

aldose reduct-

UNITIKA Co. and

 

ase inhib.

Univ. Osaka

Salfredins

Crucibulum sp.

PK-mod.

aldose reduct-

Shionogi

 

ase inhib.

Panosialins

Streptomyces sp.

PK-mod.

glycosidase

Kitasato

 

inhib.

Xenovulene

Acremonium strictum

TERP

GABA-benzo-

Xenova

 

diazepine re-

ceptor binding

Arisugacin

Penicillium sp.

TERP

AChE-inhib.

Kitasato

Nerfilin I

Streptomyces halstedii

PEP-PK

neurite out-

Somtech and Univ.

 

growth ind.

Tokyo

Michigazones

Streptomyces halstedii

PEP

neuronal cell

Univ. Tokyo

 

protecting

Aestivophoerin

Streptomy ces

PK-mod.

neuronal cell

Univ. Tokyo

purpeofuscus

protecting

Lavandu-

Streptomyces

neuronal cell

Univ. Tokyo

quinocin

virdochromogenes

protecting

Epolactaene

Penicillium sp. (marine)

PK

neuritogenic

RIKEN and Kaken Pharm. Co.

MQ-387

Streptomyces

PEP

aPase N inhib.

KRIBB

nayaga waensis

YL-01869P

Actinomadura

PEP-mod.

matrix metallo-

Sankyo

ultramentaria

proteinase

 

inhib.

YM 4714112

Flexibacter sp.

PEP

elastase inhib.

Yamanouchi Pharm. Co.

Poststatin

Streptomyces

PEP

Pro-endopeptid-

Institute of Microbial

virdochromogenes

ase inhib.

Chemistry

Cathstatins

Microascus longirostris

PEP-mod.

proteinase

SynPhar Lab Inc. and

 

inhib.

Institute of Marine Bioscience (Halifax)

BE-40644

Actinoplanes sp.

PK

t hioredoxin

Tsukuba Res. and

 

inhib.

Banyo Pharm. Co.

RPR113228

Chrysosporium lobatum

TERP

farnesyl protein

Rhhe Poulenc Rorer

 

transferase

inhib.

Andrastin

Penicillium sp.

TERP-PK

farnesyl protein

Kitasato and Keio Univ.

 

transferase

inhib.

Saquayamycins

Actinomycetes

PK

farnesyl protein

Keio Univ. and Institute

 

transferase

of Microbial Chemistry

inhib.

Agricultural Uses

Rotihibin

Streptomy ces

PEP-PK

plant growth

Univ. Tokyo and

graminofaciens

regulator

Ajinimoto

Pironetin

Streptomyces sp.

PK

plant growth

Nippon Kayaku

 

regulator

Phthoxazolin

Streptomyces

PK

herbicidal

Univ. Paul Sabatier

griseoaurantiacus

(Toulouse)

10

1

General Aspects of Secondary Metabolism

Tab. 1. (Continued)

Compound

Producing Organisms

Structural

Selected

Research Group

Reference

Type'

Properties

Involved

Methylstrept-

Streptomyces sp.

PK-mod.

herbicidal

Hoechst India

imidon-deri-

vatives

Fudecalone

Penicillium sp.

PK

anticoccidial

Kitasato

Arohynapene

Penicillium sp.

PK

anticoccidial

Kitasato

Xanthoquinodin

Humicola sp.

PK

anticoccidial

Kitasato

Hydrantomycin

Streptomyces sp.

PK

herbicidal

Kitasato

 

antibiotic

Iturins

Bacillus subtilis

PEP-PK

phytopathogens

USDA, Univ. Texas and

Trichorzins

Trichoderma harzianum

PEP

antifungal

Univ. Purdue CNRS (Paris)

Azalom ycin

Actinomycete Streptomyces hygroscopicus

PK

antifungal

Hoechst and AgrEvo Merck Sharp and Dohme

Phthoxazolines

Streptomyces sp.

PK-mod.

antifungal

Kit asat o

Phenamide

Streptomyces albospinus

AA-mod.

antifungal

Monsanto

Patulodin

Penicillium urticae

PK

antifungal

Osaka Univ.

Gualamycin

Streptomyces sp.

GLYC

acaricidal

Nippon Kayaku Co.

NK-374200

Taralomyces sp.

NUC-PEP

insecticidal

Nippon Kayaku Co.

Melanoxadin

Trichoderma sp.

melanine bios.

Teikyo Univ. and

 

inhib.

Tokyo Univ.

Albocycline

Streptomyces sp.

PK

melanogenesis

Kitasato

 

inhib.

CI-4

Pseudomonas sp.

PEP

chitinase inhib.

Shimizu Labs.

(marine)

Oligosperons

Arthrobotyrys

TERP

nematocidal

Australian National

Isocoumarins

oligospora Lachnum sp. (Ascomy- cete)

PK

nematocidal

Univ. Univ. Kaiserslautern (FRG) and Univ. Lund (Sweden)

Milbemycins

Streptomyces sp.

PK

antihelminthic

Smith Kline Beecham

Sulfinemycin

Streptomyces albus

PK-mod.

antihelminthic

Lederle

Musacins

Streptomyces

antihelminthic

Univ. Gottingen, Univ.

griseoviridis

Tubingen, Hans Knoll Institute

Lachnum-

Lachnum papyraceum

PK

nematocidal,

Univ. Lund and Univ.

lactone

cytotoxic

Kaiserslautern

' Structural type: PEP - peptide, PK - polyketide, TERP - terpenoid, GLYC - glycoside, AA - amino acid, NUC - nucleoside, mod. - modified. * Property: antag. - antagonist; bios. - biosynthesis; ind. - inducer; inhib. - inhibitor; rec. - receptor. Group identification: Univ. - University of.

(AZUMA,1987), bestatin (OCHIAI, 1987), to- postins (SUZUKIet al., 1990), etc., are to be introduced into future therapy. The large-scale biotechnical production of bioactive compounds has been developed in a highly effective manner. Fermentations of high-producing microorganisms are carried

out up to a volume of more than 300 m3. The yield is sometimes more than 40 g L-' (VAN- DAMME, 1984), and up to 1OOgL-' in peni- cillin fermentations. This demonstrates the ef- ficiency of strain selection which supported knowledge of biosynthesis and strain genetics. Optimum bioprocess control and suitable fer-

2 Secondary Metabolism, an Expression of Cellular and Organismic Individuality

11

mentation equipment were developed as fur- ther prerequisites of a highly efficienct pro- duction of biotechnical drugs. As an introduction to this volume, this chapter summarizes some of the general as- pects of secondary metabolism in microorgan- isms such as:

  • - the biological role of bioactive compounds in the producer strains,

  • - the biosynthetic pathways and their organi- zation,

  • - natural and induced variations of second- ary metabolite structures and problems of their structural classification.

Finally, future perspectives of drug screen- ing from microbial sources are discussed.

2 Secondary Metabolism, an Expression of Cellular and Organismic Individuality

2.1 Roles of Secondary Metabolites in Producing Organisms

The majority of bioactive products of mi- croorganisms and plants is generated by sec- ondary metabolism. This part of the meta- bolic machinery of microbes, plants, and ani- mals may play no essential role in the vegeta- tive development of the producing organisms, but seems to convey advantages to the perti- nent species concerning its long-term survival in the biological community and environment (LUCKNERet al., 1977; KLEINKAUFand VON DOHREN, 1986; WILLIAMSet al., 1989; LUCKNER,1989 VINING,1992; WILLIAMSet al., 1992; CAVALIER-SMITH, 1992; OLESKIN, 1994; VININGand STUTTARD,1995) (Tab. 2). Further interpretations imply the formation of certain secondary metabolites by relatively

small, but systematically defined groups of or- ganisms (e.g., special species and genera of microbes, plants, animals) and point to the enormous variability of chemical structures (ComitC Editorial, 1992). In microbes, the ca- pacity to generate secondary metabolites is frequently lost by genomic mutations, but this feature misses any concomitant effect on the

vegetative development of the pertinent strains (SHAPIRO,1989; OLESKIN, 1994). An

inverse correlation is usually observed be- tween specific growth rate and the formation

of secondary metabolites such as antibiotics. Particular features of morphological differen- tiation in surface or submerged cultures, such as the formation of spores and conidia, seem to be related to the production capacity of secondary metabolism. Moreover, a maxi- mum production rate of antibiotics and other secondary metabolites (pigments, alkaloids, mycotoxins, enzyme inhibitors, etc.) has fre- quently been observed when growth-promot- ing substrates were depleted from the me- dium (DEMAIN,1992). This phenomenon was called “catabolite regulation” (DEMAIN, 1974). This may be one of the reasons for the phase-dependency of biosynthesis of many microbial drugs. Thus, during the microbial growth phase (trophophase) secondary metabolism is often suppressed, but increased later during the “idiophase” (VINING,1986). Sometimes this feature is not present and depends on the par- ticular strains and growth conditions. For in- stance, the formation of phytotoxins by some phytopathogenic microbes such as Alternaria and Fusarium strains is not a subject of catab- olite regulation and even occurs in a growth- associated manner (REUTER,1989). On the other hand, the production of antifungal ef- fectors including peptaibol trichorzianine may be induced, as shown in Trichoderma har- zianum by cell walls of the plant pathogen

Botrytis cinerea (SCHIRMBOCKet al., 1994).

Likewise, certain plant metabolites may in- duce the synthesis of peptide antibiotics in the respective pathogenic Pseudomonas strains (MAZZOLAand WHITE,1994; MO et al., 1995). In general, the phase-dependency or specific inducibility indicates that the sec- ondary metabolism is strictly governed by in- herent regulatory systems (see Sect. 2.2).

12

1

General Aspects of Secondary Metabolism

Tab. 2. Presumed “Roles” of Secondary Metabolites in Their Producer Organism

Exogenous “role” in the environment

Endogenous “role” in the producing organism

-

-

 

protection against competing organisms

endogenous regulatory signals triggering morpho-

 

-

genesis

-

regulation of commensalism and cohabitation

endogenous signals regulating mating processes such as

-

pheromones

 

protection against physicochemical noxes (UV light)

endogenous detoxification

 

of metabolites

-

-

acquisition of trace elements

supply of special building

detoxification of trace elements

material of the cell wall

endogenous reserve material not accessible to other micro- organisms

Most of the secondary metabolites are bio- synthesized in microbes and plants via com- plex multistep pathways involving many enzy- matic and even non-enzymatic events. These appear to be integrated in a coordinated man- ner into the global microbial processes of cy- todifferentiation such as formation of spores, conidia, and aerial mycelia (LUCKNER,1989), or in the processes of invasion or defense. The same is true for plants in which second- ary metabolite formation occurs in different tissues, e. g., roots, leaves, flowers, and seeds. Hence, it seems obvious that secondary metabolism does not reflect an occasional

feature but is the result of a very long evolu- tionary development. As was shown for the tetracycline antibiotics from Sfrepfomyces spp. more than 200 genes may affect the bio- synthetic pathway (VANEKand HOSTALEK, 1985). No wonder that speculation about the endogenous “function” and “roles” of sec- ondary metabolites in the producing organ- isms themselves never came to an end (VA-

NEK et al., 1981; VINING,

1992; OLESKIN,

1994; VININGand STUTTARD, 1995). To maintain such a great number of genes, generally linked into clusters, during evolu- tion should be of advantage to the pertinent organism. Obviously, in plants many second- ary metabolites are involved in the protection against microorganims and animals (CUND-

LIFFE, 1992; JOHNSON and ADAMS, 1992). Others act as chemoattractants or as repel- lents towards insects fructifying flowers or damaging plant tissues. A series of plant hor- mones (cytokinins, gibberelic acid, jasmonic acid, etc.) are similar in structure but per defi- nitionem are not secondary metabolites. An- other function of secondary metabolites in plants is the detoxification of poisonous metabolites via an endogenous compartmen- tized storage (LUCKNER,1989). The role of secondary metabolism in microbes is even more difficult to understand. Cellular efforts needed for secondary pathways are rather low in the wild-type strains (only a small amount of the overall substrate intake is con- verted to bioactive secondary metabolites). This part of metabolism would possibly have been eliminated during phylogenesis without any selective advantage of secondary metab- olite production. It appears to be a generally accepted view that microbial secondary

metabolites play an important but not gener- alizable rote, at least in special situations, e. g., in warranting the survival in particular environmental systems, during limitation of nutrient supply or even in the course of mor- phological development (LUCKNERet al., 1977; KLEINKAUFand VON DOHREN,1986;

VINING,1992; KELL et

al., 1995; VININGand

STUTTARD, 1995). From this point of view,

  • 2 Secondary Metabolism, an Expression of Cellular and Organismic Individuality

13

the formation of large amounts of antibiotics by high-producing strains (substrate conver- sion rates Yglucose,drug > 0.1) would be consid- ered as a “pathophysiological” problem (VA- NEK et al., 1981). In order to better under- stand the general roles of secondary metabol- ites in microbes one could refer to the color of hairs and feathers in animals, their odorous pheromones, and other metabolic products which do not contribute per se to the vegeta- tive life of the pertinent species. But they could have outstanding importance during the adaptation to changing media, in the pro- tection against competing organisms, and in the regulation of sexual and asexual processes of genetic exchange. General discussions of secondary metabolite formation in microbes consider four major fields of importance (LUCKNERet al., 1977; KLEINKAUFand VON DOHREN,1986; LUCKNER,1989; WILLIAMS et al., 1989, 1992; VINING,1992; CAVALIER- SMITH,1992; OLESKIN,1994; VININGand STU~TARD, 1995) (Tab. 2):

(1) The formation of secondary metabolites facilitates the adaptation to metabolic im- balances as a kind of a “metabolic valve”, which is needed to remove an excess of toxic, endogenous metabolites that other- wise are accumulated during a partial lim- itation of substrates. (2) Secondary metabolism could be a source of individual building blocks of cells or of metabolic reserves which warrant the in- dividuality and particular functionality of the given strain. (3) Secondary metabolites could be regarded as endogenous signals triggering particu- lar stages of morphogenesis and the ex- change of genetic material (see Fig. 1). This hypothesis was particularly sup- ported by the observation that the major- ity of the “good” producers (e.g., actino- mycetes, fungi, bacteria) display a life cy- cle involving several stages of morpholog- ical differentiation. (4) Secondary metabolite formation is partic- ularly important in biosystems as a signal of interspecific “communication” be- tween microbes and other microbes, plants, and animals. Symbiosis, commen- salism, and antagonism could be regu-

lated by secondary metabolites in hetero- logous populations.

The self-protecting mechanism in antibiot- ic-producing microbes should be mentioned as a further evidence of an ecological function of antibiotics, as a “weapon” against competi- tors (ZAHNERet al., 1983; BRUCKNERet al., 1990; CUNDLIFFE, 1989,1992; WILLIAMSand MAPLESTONE, 1992). By this means the mi- crobe prevents suicide due to its own second- ary metabolite either by enzymatic modifica- tions of the drug, by alteration of its biologi- cal target, or by an active transport-directed export (see, e. g., the tetracycline efflux) (JOHNSON and ADAMS, 1992; NIKAIDO, 1994). Usually, resistance mechanisms of the antibiotic-producing microorganisms are the same as in antibiotic-resistant bacteria. The analysis of the gene sequences encoding re- sistance determinants support the idea that the transfer of resistance occurs from the anti- biotic producers to the non-producing mi- crobes (JOHNSONand ADAMS, 1992; SA- LYERS et al., 1995; HIRAMATSU, 1995; DAV- IES, 1994). In addition, the emergence of new types of resistance factors by the formation of mosaic genes has been analyzed in P-lactam- resistant pneumococci (SPRATT, 1994; COF- FEY et al., 1995). The great variation of both active and inac- tive secondary metabolites, that were ob- served in microorganims and plants supplied the main arguments against their determined function. Obviously, the formation of a bioac- tive secondary metabolite, such as an anti- biotic, rather appears as an exception than as a rule. Frequently, many inactive “shunt-me- tabolites” and congenors are produced in ad- dition to the few active metabolites. It is not reasonable to believe that all these metabo- lites are needed in a single organism. It might be that a “function” of a secondary metabo- lite could become apparent only in a particu- lar, exceptional situation or in special stages of development. Hence, the selection pres- sure on structures and secondary pathways is necessarily low (ZAHNERet al., 1983). As a consequence, spontaneously evolving variants and mutants could survive with the same probability as their parents, and modifications of secondary pathways and structures would

14

1 General Aspects of Secondary Metabolism

 

A-factor

VB-factorS

 

J

0

autoinducer from

,

,

,

factorfrom Str. vlrldochromugenes

M0

Q

$?

Vibrio Rscheri

 

on

 

OH

0

 

Butalactin

Basidifferquinone

Gennicidin

 

no

v

v-0

 
 

antheridiol

oogonlol

trlsporlc acid C

differolide

ChOH

sirenin

Fig. 1. Structures of some representatives of signaling molecules from bacteria (streptomycetes) and fungi (for references, see text).

be preserved (secondary metabolism as a “playground of evolution”) (ZAHNER et al., 1983). This might explain the existence of the numerous similar structures. According to this hypothesis, the limited substrate specifici- ty of some enzymes of secondary metabolism has to be mentioned (LUCKNER,1989). How- ever, it should be noted that in many multi- step processes this limited specificity is re- stricted to certain steps and thus less re- stricted structural regions of the compounds (KLEINKAUFand VON DOHREN,in press). A few secondary metabolites, out the pool of the many non-functional metabolites, have apparently acquired an essential role in growth and differentiation. The siderophores, e.g., are microbial vehicles of iron transport formed in variable structures as constitutive parts of the iron uptake system (VON DER HELMand NEILANDS,1987; WINKELMANN, 1991; WINKELMANNand DRECHSEL, Chapter 5, this volume). Per definitionern, they should not be regarded as secondary metabolites. Highly specialized biomolecules such as cy-

tochromes, chlorophylls, sexual pheromones of fungi and bacteria, etc. might have been evolved similarly. Some of them may be at- tested to defined “functions” of microbial sec- ondary metabolites (Tab. 2, Fig. 1). A role of secondary metabolism in the ad- aptation to changing nutrient conditions is a realistic position since an excessive supply of metabolic intermediates (precursors) usually induces or stimulates drug production (DE- MAIN 1974,1984,1992). Growth may become imbalanced and precursors are accumulated during the limitation of a given substrate in the medium, while others are still available in excess. Excessive precursors could be re- leased into the medium or converted to hard- ly metabolizable products which would not support the growth of competitors. Moreover, colored secondary metabolites, such as pig- ments, could protect cells and spores from damage by ultraviolet radiation or also could promote the acquisition of rare elements via complex formation as, e. g., siderophores. Complex formation could also protect the

2

Secondary Metabolism, an Expression of Cellular and Organismic Individuality 15

cells from high concentrations of toxic heavy metals. The incorporation of secondary metabo- lites into cellular structures has been sug- gested to contribute to their individual char- acteristics. Thus, streptomycin and its build- ing moiety, streptidine, were established as a constitutent of the cell wall of the producing Sfrepfomyces griseus (DEMAIN,1984; DIST- LER et al., 1992). Otherwise, the production of secondary metabolites (so-called “idio- lites”) (DEMAIN,1992), could serve as a kind of a metabolic reserve which cannot be metabolized by other microbes. Some anti- biotics (anthracyclines, tetracyclines, cyclos- porins, etc.), e. g., are stored within the myce- lium and their complete degradation requires a series of specialized enzymatic steps. Other- wise, bioconversions of antibiotics are a con- stitutive part of the self-protecting mecha- nisms of the producer strain. Moreover, concentrations of several anti- biotics were shown to decrease in the course of prolonged cultivation, thus indicating the onset of degradative processes. Some fungi are well-known to degradate their own poly- ketides such as, e.g., citrinin (BARBERet al., 1988) and zearalenon and even to use them for additional syntheses. Active antibiotics were usually not detected in soil samples, al- though recently sensitive procedures have permitted the detection of phenazines (COOK et al., 1995). Their complete degradation un- der natural conditions seems very likely. Most likely, a series of signaling molecules is supplied by the secondary metabolism that possess interspecific (ecological) or species- dependent functions, e. g., as signals trigger- ing morphogenesis and the exchange of ge- netic material (Fig. l). By growth inhibition of competing microbes a producer strain could attain an advantage (c. f. the production of herbicidal antibiotics by phytopathogenic bacteria which damage plant tissues and facil- itate nutrient acquisition from the host) (KOHMOTOand YODER, 1994; MAZZOLA and WHITE,1994; Mo et al., 1995). Vice versa, secondary metabolism could confer a particu- lar advantage in symbiotic systems, such as Pseudomonaslplant roots, to both the produc- ing strain and the symbiont. An example is the control of phytopathogenic Fusarium or

Rhizocfonia fungi on plant roots by products of cohabiting streptomycetes and bacteria. In- terspecific effects have also been postulated for volatile compounds which are formed, e. g., by streptomycetes and cyanobacteria. Geosmin, isoborneol, and mucidon are the constituents of the typical earthy odor. It has been shown that sclerin and scleroid from the fungus Sclerofinia liberfiana stimulate the bio- synthesis of aminoglycosides by streptomy- cetes, but also the growth of some plants (KUBOTAet al., 1966; OXFORDet al., 1986). The formation of phytotoxins by phytopa- thogenic microbes is mentioned as another in- terspecific communication system (KOHMO- TO and YODER, 1994). Constituents of the microbial cell wall (elicitors such as p1,3-1,6- glucans from Phytophfora megasperma) are recognized by specific plant cell membrane receptors. Subsequently, a series of protective mechanisms is induced in the plant (e.g., hy- persensitivity reactions, de novo synthesis of tissues, secretion of enzymes lysing microor- ganisms, and formation of antimicrobial phy- toalexins). On the other hand, some of the phytoalexins are inactivated by enzymes of phytopathogenic microbes. In the natural habitat genetic information can be transferred from one microbe to an- other interspecifically. Both biosynthetic pro- cedures and resistance mechanisms thus can be spread among various heterologous spe- cies and genera. Apparently this is also true for genetic exchanges between plants and mi- crobes. A recent intriguing example is the dis- covery of a taxol producing fungus living in taxol producing yew trees (STIERLE et al., 1994). Typical plant hormones such as gibber- ellins and jasmonic acid are also produced by some microorganisms. Aflatoxins formed via complicated biosynthetic pathways in fungi, such as Aspergillus, have been established in actinomycetes. Sequence analyses of the genes encoding penicillin and cephalosporin biosynthetic clusters (ACV synthase, isopeni- cillin N-synthase, acyltransferase, deacetoxy- cephalosporin C-synthase, and deacetoxy- cephalosporin C-hydroxylase) in Penicillium chrysogenum, Acremonium chrysogenum, and Streptomyces spp. strongly suggested that fun- gi received the pertinent genes from the pro- karyotic actinomycetes during evolution

16

I

General Aspects of Secondary Metabolism

(LANDANet al. 1990 MILLERand INGOLIA, 1993; BUADESand MOYA, 1996). The pro- duction of cephabacins, chitinovorins, clavu- lanates, olivanic acids, carbapenems, and thiopeptides by unicellular bacteria and strep- tomycetes may indicate that an original bio- synthetic pathway was spread horizontally among different microbes, thus giving rise to evolutionary variations of structures and pathways. The evolution of secondary metabolism even appears to create hybrid structures by the combination of genetic material originat- ing from heterologous hosts. Recently, thio- marinol (SHIOZAWAet al., 1993) was isolated from the marine bacterium Alteromonas rava as a composite compound formed by the es- terification of pseudomonic acid (found in Pseudomonas fluorescens) and holomycin (a pyrrothine antibiotic, found in Streptomyces

Spa). The involvement of secondary metabolism in the regulation of microbial cytodifferentia- tion seems to be important, at least in some cases. The morphogenesis of antibiotic-pro- ducing microorganisms (streptomycetes, fun- gi, Mycobacteria, etc.) is obviously mediated by a plethora of biochemical steps, which dis- play a high specificity for the given organism. The pathways are regulated by individual sig- nals in a highly coordinated manner (Fig. 1) (LUCKNER,1989). During morphogenesis, si- lent genes are activated that have not been expressed during the growth phase. Accord- ingly, several endogenous non-antibiotic reg- ulators of the cell cycle were discovered in Streptomyces cultures, and their structure was elucidated (see below) (KHOKHLOV,1982; GRAFE, 1989 HORINOUCHIand BEPPU, l990,1992a,b, 1995; BEPPU,1992,1995). Cor- relations between the biogenesis of some pep- tidic antibiotics and morphogenesis were also described for synchronously growing Bacillus cultures (MARAHIELet al., 1979). Tyrocidin, gramicidin, and bacitracin are produced dur- ing the onset of sporulation, suggesting that their function concerns the control of tran- scription, spore permeability, dormancy of spores, and their temperature stability (MA- RAHIEL et al., 1979,1993). The y-butyrolactones represent a particu- larly important group of endogenous regula-

tors of Streptomyces differentiation (Fig. 2) (KHOKHLOV,1982; GRAFE 1989; HORINOU- CHI and BEPPU,l990,1992a, b, 1995; BEPPU, 1995). They are required as microbial “hor- mone-like” substances in few species such as streptomycin, virginiamycin or anthracycline producing strains. These effectors permit the formation of antibiotics and aerial mycelium by some blocked, asporogenous, antibiotic- negative mutants even in very low concentra- tions. Several other autoregulators of mor- phogenesis have been investigated (see, e. g., factor C) (SZESZAKet al., 1991). Otherwise, germicidin B (PETERSENet al., 1993) from Streptomyces violaceusniger inhibits germina- tion of its own spores by interference with en- dogenous ATPase. Antibiotics such as hor- maomycin (ROSSNERet al., 1990) and pama- mycin (KONDOet al., 1986) were shown to have autoregulatory functions. Moreover, streptomycetes can produce interspecific in- ducers such as anthranilic acid and basidiffer- quinone (Fig. 1) which affect basidiomycetes and the formation of fruiting bodies (AZUMA et al., 1980; MURAOet al., 1984). Moreover, regulatory molecules inducing cytodifferentiation were isolated from fungi and molds confirming that morphogenesis can be mediated by the aid of an agency of specialized endogeneous factors (HAYASHIet al., 1985). They can be regarded as secondary metabolites since they do not possess any function in vegetative development. In addition, sexual factors from fungi and yeasts can be considered as functionalized secondary metabolites. They trigger zygo- spore formation by haploid cells belonging to different mating types (GOODAY,1974). Dur- ing the evolution of signal systems, from the simple pro- and eukaryotes up to the hor- monal control in mammalians, some struc- tures and activities have been conserved. The alpha-factor of the yeast Saccharornyces cere- visiae as one of its sexual pheromones, e.g., appears to be partially homologous to the hu- man gonadotropin releasing hormone (Lou- MAYE et al., 1982). Moreover, inducers of dif- ferentiation of Friend leukemia cells were iso- lated from soil organism such as Chaetomium sp. These chlorine containing substituted di- phenols (Fig. 1) also induce morphogenesis (stalk cell differentiation) of Dictyostelium

2

Secondary Metabolism, an Expression of Cellular and Organismic Individuality

17

2 Secondary Metabolism, an Expression of Cellular and Organismic Individuality 17 Fig. 2. Regulatory events suggested

Fig. 2. Regulatory events suggested to be involved in morphogenesis and secondary metabolism of Strep-

tomyces griseus (P: promotor) (HORINOUCHI

and BEPPU,1992a).

discoideum, suggesting the similarity of mam- malian and fungal control of the cell cycle (KUBOHARAet al., 1993). Recently, the oc- currence of sexual pheromones was even es- tablished for the prokaryote Streptococcus faecalis. Its pheromones stimulate or inhibit the transfer of conjugative plasmids from do- nor to recipient strains (WIRTHet al., 1990). Peptides triggering competence in Bacillus subtilis have been characterized and were termed pheromones (D’SOUZAet al., 1994; SOLOMONet al., 1995; HAMOEN et al.,

1995).

2.2 Regulation of Microbial Secondary Metabolism

2.2.1 Genetic Organization of Product Formation

A large number of biosynthetic genes were isolated and characterized and, in general, they have been found assembled in clusters (Tab. 3). Such clusters may contain informa- tion for the biosynthesis of the basic structure of the metabolite, its modification, resistance determinants, e. g., promoting modification of products, targets, altered targets, or export systems, as well as regulatory elements; indi- vidual gene products which might as well ex- ert regulatory functions.

18

1

General Aspects of Secondary Metabolism

Tab. 3. Biosynthetic Clusters Identified

Compound Type

0 r g a n i s m

Selected References'

A54145

acylpeptidolactone

Streptomyces fradiae

BALTZet al., 1996'

Aflatoxins

polyketide

Aspergillus parasiticus,

BROWNet al. 1996

Actinomycin

chromopeptidolactone

Aspergillus fzavus Streptomyces chrysomallus

MAHANTIet al., 1996 KELLERet al., 19962

Anguibactin

modified peptide

Vibrio anguillarum