Biotechnology of Vitamins, Pigments and Growth Factors

BIOTECHNOLOGY OF
VITAMINS, PIGMENTS AND GROWTH FACTORS

ELSEVIER APPLIED BIOTECHNOLOGY SERIES
Other Titles in this Series:
Y. Chisti: Airlift Bioreactors
BIOTECHNOLOGY
OF
VITAMINS, PIGMENTS
AND
GROWTH FACTORS
Edited by
ERICK J. VANDAMME
Laboratory of General and Industrial Microbiology,
State University of Ghent, Belgium
ELSEVIER APPLIED SCIENCE

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Biotechnology of vitamins, pigments and

growth factors
1. Industrial chemicals: organic compounds
I. Vandamme, Erick J.
661'.8
ISBN·13:978-94-010-6991-5
Library of Congress Cataloging-in-Publication Data
Biotechnology of vitamins, pigments, and growth factors/edited by

Erick J. Vandamme.
p. cm.-(Elsevier applied biotechnology series)
Includes bibliographies and index.
ISBN-13: 978-94-01 0-6991-5 e-ISBN -13: 978-94-009-1111-6
001: 10.1007/978-94-009-1111-6
1. Vitamins-Biotechnology. 2. Growth promoting substances-

-Biotechnology. 3. Pigments (Biologyl--Biotechnology.
I. Vandamme, Erick J., 1943- II. Series.
[DNLM: 1. Biotechnology. 2. Growth Substances. 3. Pigments.
4. Vitamins. QT 34 B61545]
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To Mireille
PREFACE
Vitamins and related growth factors belong to the few chemicals with a
positive appeal to most people; the name evokes health, vitality, fitness,
strength .... each one of us indeed needs his daily intake of vitamins, which
should normally be provided via a balanced and varied diet. However, current
food habits or preferences, or food processing and preservation methods do
not always assure a sufficient natural daily vitamin supply, even for a healthy
human being; this is all the more true for stressed or sick individuals. Although
modern society is seldom confronted with the notorious avitaminoses of the
past, they do still occur frequently in overpopulated and poverty- and
famine-struck regions in many parts of the world. Apart from their in-vivo
nutritional-physiological roles as growth factors for man, animals, plants and
micro-organisms, vitamin compounds are now being introduced increasingly as
food/feed additives, as medical-therapeutical agents, as health-aids, and also
as technical aids. Indeed, today an impressive number of processed foods,
feeds, cosmetics, pharmaceuticals and chemicals contain extra added vitamins
or vitamin-related compounds, and single or multivitamin preparations are
commonly taken or prescribed.
These reflections do indicate that there is an extra need for vitamin supply,
other than that provided from plant and animal food resources. Most added
vitamins are indeed now prepared chemically and/or biotechnologically via
fermentation/bioconversion processes.
Similarly, other related growth factors, provitamins, vitamin-like com-
pounds, i.e. special unsaturated fatty acids, gibberellins and certain pigments-
some of which are increasingly used in agriculture, food/feed production or
processing and as health aids-are equally important biotechnological pro-
ducts, where production via microbial fermentation or micro-algal bioconver-
sion is now applied industrially. Indeed, biotechnology-based on bacteria,
yeasts, fungi and micro-algae-here again has been very instrumental in
procuring sufficient amounts of several of these valuable complex molecules via
vii
viii Preface
natural processes, although for certain products there is fierce competition with
chemical synthesis. Abundant and excellent literature is available on the
chemical properties, biochemistry, nutritional aspects and clinical aspects of
vitamins and related products, while that on microbial synthesis and produc-
tion methods is rather scarce or difficult to find.
In this respect, this book intends to assemble useful information on the
(potential) industrial synthesis of economically important vitamins, growth
factors and pigments, with emphasis on biotechnological aspects including
microbiology, genetics, biochemistry and bioprocess technology; so far, such
information is scattered widely in the scientific literature: for some products,
only secrecy and sparse data are available, other excellent volumes deal with
only one specific vitamin compound, while others then stress the chemical
synthesis processes. Therefore, I felt that there was a scientific need for a
biotechnological survey of the world of vitamins and related compounds
synthesis.
The help of several colleagues and friends in suggesting potential authors for
difficult-to-get chapters has been invaluable in constructing a rather com-
prehensive volume; so was the positive interaction with all my contributors. In
this respect, I am particularly indebted to:
Dr S. Anderson, Genentech, USA; Dr G. C. Barrere, Rhone-Poulenc,
France; Dr E. Cerda-Olmedo, University of Seville, Spain; Dr D. De Buyser,
N. V. Vandemoortele, Belgium; Dr. D. Defterdarovic, Pliva, Yugoslavia;
Professor Dr A. L. Demain, MIT, USA; Dr J. Florent, Rhone-Poulenc,
France; Dr A. Furuya, Kyowa Hakko Kogyo Co., Ltd, Japan; Dr H. Nelis,
University of Ghent, Belgium; Dr L. Segers, Orffa, Belgium; Dr S. Shimizu,
Kyoto University, Japan; Dr G. Smits, Tiense Suikerraffinaderij, Belgium;
Professor Dr Y. Tani, Kyoto University, Japan.
The editor also thanks the staff of Elsevier Science Publishers Ltd.
I suspect that my wife, Mireille, could only have withstood my 'mental
absence', strengthened with multivitamin preparations, although my sole
vitamin-shot was her encouraging and moral support during this biotechnologi-
cal enterprise.
E. J . VANDAMME
CONTENTS
Preface . . . . . . vii
List of Contributors . xi
1. Vitamins and Related Compounds via Micro-organisms: a

Biotechnological View (E. J. Vandamme) . . . . . . . 1
Fat-Soluble Vitamins and Pigments

2. p-Carotene (Provitamin A) Production with Algae (L. J.
Borowitzka & M. A. Borowitzka) . . . . . . . . . . 15
3. Production of Carotenoids with Fungi (E. Cerda-Olmedo 27
4. Microbial Production of Carotenoids other than p-Carotene (H.
J. Nelis & A. P. De Leenheer) . . . . . . . . . . . . 43
5. Vitamin D: The Biotechnology of Ergosterol (P. Margalith) . . 81
6. Algal and Microbial Production of Vitamin E (Y. Tani) . . . . 95
7. Microbial Production of Polyunsaturated Fatty Acids (Vitamin-
F Group) (S. Shimizu & H. Yamada) . . . . . . . . . 105
8. Microbial Production of Vitamin K2 (Menaquinone) and
Vitamin Kl (Phylloquinone) (Y. Tani) . . . . . . . . 123
Water-Soluble Vitamins
9. Microbial Synthesis of Vitamin Bl (Thiamine) (A. Iwashima) 137
10. Microbial Production of Vitamin B2 (Riboflavin) (T. Kutsal &
M. T. Ozbas) . . . . . . . . . . . . . . . . . . . . . 149
11. Microbial Production of D-Ribose (K. Sasajima & M. Yoneda) 167
12. Pantothenic Acid (Vitamin B s), Coenzyme A and Related
Compounds (S. Shimizu & H. Yamada). . . . . . . . . . 199
13. Microbial Production of Vitamin B6 and Derivatives (Y. Tani) 221
ix
x Contents
14. Microbial Production of Biotin (Y. Izumi & H. Yamada) 231

15. Microbial Production of Vitamin B12 (C. Spalla, A. Grein, L.
Garofano & G. Feroi) . . . . . . . . . . . . . . 257
16. Microbial Production of Orotic Acid (Vitamin B 13) (K.
Takayama & A. Furuya) . . . . . . . . . . . . . 285
17. Microbial Reactions for the Synthesis of Vitamin C (L-Ascorbic
Acid) (V. Delic, D. Sunic & D. Vlasic) . . . . . . . . . . . 299
Other Growth Factors

18. Microbial Production of ATP (Y. Tani) . . . . . . . . . 337
19. Adenosylmethionine, Adenosylhomocysteine and Related
Nucleosides (S. Shimizu & H. Yamada) . . . . . . . . . 351
20. Other Vitamin-Related Coenzymes (S. Shimizu & H. Yamada) 373
21. Fungal Gibberellin Production (B. Brueckner, D.
Blechschmidt, G. Sembdner & G. Schneider) 383
Index. 431
LIST OF CONTRIBUTORS
D. BLECHSCHMIDT,
Friedrich Schiller University Jena, Department of General Microbiology,
DDR-6900 Jena, Neugasse 24, GDR
L. J. BOROWITZKA,
Western Biotechnology Ltd, 2-6 Railway Parade, Bayswater, WA 6063,
Australia
M. A. BOROWITZKA,
Algal Biotechnology Laboratory, School of Biological and Environmental
Sciences, Murdoch University, Murdoch, WA 6150, Australia
B. BRUECKNER,
Friedrich Schiller University lena, Department of General Microbiology,
DDR-6900 lena, Neugasse 24, GDR
E. CERDA-OLMEDO,
Departmento de Genetica y Biotecnia, Universidad de Sevilla, Apartado
1095, Sevilla, Spain
A. P. DE LEENHEER,
Laboratoria voor Medische Biochemie en voor Klinische Analyse,
Rijksuniversiteit Ghent, Harelbekestraat 72, B-9000 Ghent, Belgium
V. DELle,
PLIVA Pharmaceutical, Chemical, Food and Cosmetic Industry, Research
Institute, Zagreb, Yugoslavia
G. FERNI,
Farmitalia Carlo Erba, Via dei Gracchi 35, 20146, Milano, Italy
A. FURUYA,
Kyowa Hakko Kogyo Co. Ltd, Tokyo Research Laboratories, 3-6-6
Asahi-machi, Machida-shi, Tokyo 194, Japan
L. GAROFANO,
A. GREIN,
A. IWASHIMA,
Department of Biochemistry, Kyoto Prefectural University of Medicine,
Kawaramachi-Kirokoji, Kamigyo-ku, Kyoto 602, Japan
Y. IZUMI,
Department of Agricultural Chemistry, Kyoto University, Kyoto 606, Japan
xi
xii List of Contributors
T. KUTSAL,
Hacettepe University, Chemical Engineering Department, 06532 Beytepe,
Ankara, Turkey
P. MARGALITH,
Department of Food Engineering & Biotechnology, Technion-Israel
Institute of Technology, Haifa, 32000, Israel
H. J. NELIS,
Laboratoria voor Medische Biochemie en voor Klinische Analyse,
Rijksuniversiteit Ghent, Harelbekestraat 72, B-9000 Ghent, Belgium
M. T. (hBAS,
Ankara, Turkey
K. SASAJIMA,
Central Research Division, Takeda Chemical Industries Ltd, 2-17-85
Jusohonmachi, Yodogawa-ku, Osaka 532, Japan
G. SCHNEIDER,
Institute of Plant Biochemistry, Academy of Sciences of the GDR, DDR-
4050 Halle, Weinberg 3, GDR
G. SEMBDNER,
Institute of Plant Biochemistry, Academy of Sciences of the GDR, DDR-
4050 Halle, Weinberg 3, GDR
S. SHIMIZU,
C. SPALLA,
D. SUNIC,
K. TAKAYAMA,
Kyowa Hakko Kogyo Co. Ltd, Tokyo Research Laboratories, 3-6-6
Asahi-machi, Machida-shi, Tokyo 194, Japan
Y. TANI,
Research Center for Cell and Tissue Culture, Faculty of Agriculture, Kyoto
University, Kyoto 606, Japan
E. J . VANDAMME,
Laboratory of General and Industrial Microbiology, Faculty of Agricultural
Services, State University of Ghent, Coupure Links 653, B-9000 Ghent,
Belgium
D. VLASIC,
H. YAMADA,
M. YONEDA,
Corporate Strategy, Takeda Chemical Industries Ltd, 2-17-85
Jusohonmachi, Yodogawa-ku, Osaka 532, Japan
Chapter 1
VITAMINS AND RELATED COMPOUNDS VIA

MICRO·ORGANISMS: A BIOTECHNOLOGICAL VIEW
E. J. VANDAMME
Laboratory of General and Industrial Microbiology, Faculty of Agricultural
Sciences, State University of Ghent, Coupure Links, 653, B-9000 Ghent,
Belgium
1 HISTORICAL
The start of the history of vitamins can be traced back to 400 Be, when
Hippocrates reported that eating liver could cure night-blindness. Much later,
in the 16th century, the therapeutical effects of lemon juice against scurvy or
scorbut became known; scorbut had inter alia caused the loss of 100 crew
members on Vasco da Gama's journey around Cape Hope. The English ship
doctor James Lind studied this disease further and described in 1757 in his
book A Treatise of Scurvy the beneficial effect of eating fresh vegetables and
fruits in preventing it. For another nutritional deficiency disease (already
mentioned in 1762 by Oviedo), the Italian doctor Francesco Frapoli used the
name pellagra (pella = skin; agra = rough). In the 19th century in Japan, the
Hikan child disease (keratomalacia and xerophthalmia) was successfully
treated by including ale-fat, cod liver oil or chicken liver in the diet. Trousseau
discovered that cod liver oil and also, direct sunlight, had a curing effect on
rickets, a disease already well described by Whistler in 1645. In the Far-East,
when hulled rice was replaced by de hulled or polished rice, a sharp increase in
the occurrence of beriberi was observed. In 1897 Eijkman observed that
poultry fed with polished rice developed polyneuritis, a disease very similar to
human beriberi. This disease could also be prevented and cured by feeding
rice and the silver fleece of the rice kernel. Grijns in 1901 hypothesised that
beriberi was caused by a protecting factor, which was obviously lacking in
dehulled rice. We now know that all these diseases are a result of nutritional
vitamin deficiencies (Machlin, 1984).
Around 1910, Hopkins in the UK and Osborne & Mendel in the USA
initiated modern vitamin research and developed a theory, stating that diseases
such as scurvy, pellagra, rickets, beriberi, etc., are the result of a lack of
certain essential food components. The Polish chemist Casimur Funk isolated
in 1912 from rice bran a beriberi-preventing compound, displaying chemical
1
2 E. J. Vandamme
properties of an amine; this led him in 1912 to coin the name vitamin for this
type of compounds.
In 1913, McCollum & Davis demonstrated a liposoluble factor A in butter
fat and egg yolk, and in 1915, a water-soluble factor B was found in
wheat-germ. It was Drummond in 1920 who named the fat-soluble factor,
vitamin A; the water-soluble anti-beriberi factor was named vitamin B; the
water-soluble anti-scorbut factor vitamin C. In 1925, the fat-soluble anti-rickets
factor was named vitamin D. After 1930, discovery and isolation of several
other vitamins followed quickly and their structure, nutritional and chemical
properties and synthesis were studied in great detail in the following decades.
These aspects of vitamins and growth factors are compiled in several
excellent standard references (Goodwin, 1963; Sebrell & Harris, 1971; De
Luca, 1978; Machlin, 1984; De Leenheer et al., 1985; Diplock, 1985; Chytil &
McCormick, 1986; Adrian, 1988; Friedrich, 1988).
2 VITAMINS: WHAT'S IN A NAME?
Vitamin nomenclature was initially based on the use of letter symbols,

alphabetically arranged to time of discovery. Soon, it appeared that one-letter
named vitamins were multiple complexes, and this led to the addition of an
index to the original letters (Bb B 2 , . • • ). Often, when the function of the
vitamin became known, an appropriate letter symbol was chosen, i.e. vitamin
K, with K as the first letter of the German word Koagulation; other names
reflected deficiencies, i.e. aneurin (B 1) for anti-polyneuritis vitamin; vitamin
PP = "pellagra-preventing" vitamin. Letter names or trivial names are gener-
ally more in use rather than the IUPAC names. The division into fat- and
water-soluble vitamins as introduced by McCollum & Davis, is still universally
applied.
A listing of well-recognised vitamin compounds is presented in Table 1.
From a chemical point of view, vitamins are a very heterogeneous mixture of
compounds, yet they can be considered as a single group, since they are all
organic compounds which are essential for a healthy development of humans
and animals and need to be present in their food, since their body is not able at
all--or not sufficiently able-to synthesize them. Indeed, certain vitamins can
be formed partially or indirectly in the body:
(a) compounds--often called provitamins--with no apparent vitamin ac-
tivity can be converted within the body into a vitamin, i.e.
Jj-carotene (in vegetables, fruits) - - vitamin A
tryptophan (in protein-rich food) - - nicotinic acid (niacin)
7-dehydrocholesterol (in skin) uv) vitamin D3 (cholecalcifenol)
(b) other vitamin compounds are formed by the intestinal bacterial flora i.e.
vitamin K, some B-vitamins, i.e. B 12 , etc.
Most vitamin compounds thus have to be provided via daily food/feed intake.
Vitamins and Related Compounds via Micro-organisms 3
Table 1
Survey of Vitamins and Growth Factors
Vitamin group Important examples Important natural International Recommended adult

sources Units (IU) daily intake
Provitamin A group ,B-carotene Vegetables, I IV = 0,6 pg 2·4 mg,B-carotene

y-carotene, fruits carotene
a-carotene, I pg = 3·34 IV
kryptoxanthine
Vitamin A group Retinol (vitamin A.), Fish oil, liver, I IV = 0·30 pg 0·75 mg retinol
3-dehydroretinol milk and daily retinol
(vitamin A 2 ), products, eggs
retinal (vitamin A.-
aldehyde)
Provitamin D 7-Dehydrocholesterol,
ergosterol Yeast
Vitamin D group Ergocalciferol (D2 ), Fish oil, liver, I IV = 0·025 pg
egg yolk, milk, vitamin D,
cholecalciferol (D,) dairy products Illg = 40 IV
Vitamin E group a- Tocoferol, Plant oils, I IV= I·Omg 2·5 pg vitamin D3
,B-tocoferol, vegetables dl-tocoferol- 30mg
y-tocoferol, liver, eggs, acetate
6-tocoferol dairy products
Vitamin F group Linolic acid, Fish oil, algae,
(polyunsaturated y-linolenic acid, fungi
fatty acids, PVFAs), eicosa-pentaenoic
w-3-fatty acids) acid (EPA),
DHA
Vitamin K group Phylloquinone (K.) Liver, vegetables O·lmg
or phytomenadion, bacterial flora,
phamoquinone (K 2 ) or plant oils
menaquinone,
menadion (K,),
menadiol (K.)
Vitamin B. Thiamine Cereals, liver, 1 IV = 31lg 0·8-1·6mg
meat, vegetables, thiamine
dairy products, lllg = 0·333 IV
yeast
Vitamin B2 Riboflavin Liver, eggs, 1·2-1·3mg
(vitamin G) dairy products,
vegetables, meat,
bacterial flora
Vitamin PP Nicotinamide, Liver, cereals, 10-15 mg
(vitamin B 3 ) nicotinic acid meat, potatoes,
(niacin) vegetables,
yeast, trypto-
phane-protein
Pantothenic acid Pantothenic acid, Liver, eggs, molasses 10-20mg
(vitamin B 5 ) coenzyme A vegetables,
cereals
Vitamin B6 group Pyridoxine Liver, potato, 2mg
(pyridoxol), vegetables,
pyridoxal meat, yeast
pyridoxamine
Biotin Biotin Liver, egg yolk, 0·3mg
(vitamin H, B 8 ) vegetables, cane
molasses
bacterial flora
(continued)
4 E. J. Vandamme
Table l---contd.
Vitamin group Important examples Important natural International Recommended adult

sources Units (IU) daily intake
Folic acid Pteroyl-glutamic acid, Cereals, liver, 400 I'g

(B.-group, Bc ' M) pteroyl-diglutamic meat, milk,
acid, vegetables
pteroyl-trigIutamic
acid
Vitamin B12 group Cyanobalamin (B 12 ), Meat, liver, milk,
hydroxycobalamin bacterial flora
(B I2b) ,
nitrosocobalamin,
(B 12c )
Vitamin B13 Orotic acid
Vitamin C group Ascorbic acid Citrus fruit, fruit, 1 IU = 50 I'g
(vitamin q, vegetables, L-ascorbic acid ,(not for animals
dehydro-ascorbic acid potato except, primates,
cavia, etc.)
Lipoic acid Yeast, meat
Inositol Yeast, meat,
vegetables
Choline (B7' J) Egg yolk, meat, 50-600mg
Para-aminobenzoic acid hops
(PABA) (Bx' H', H 2 ) Yeast
Gibberellins Plants, fungi
The vitamin content was originally and still often is expressed in Interna-
tional units (IU), which relate to the biological activity of a certain amount of
pure vitamin towards a specific test animal.
Presently, it is common practice to express the vitamin content as mg or f-tg
per 100 g of material; conversion factors for IU values into weight units are also
given in Table 1. Recommended daily intake per person per day (FAO/WHO
data) is also given.
Quantitative assays for vitamin content, in foodstuffs, concentrated mix-
tures, synthetic formulations, tissues, blood, fermentation broths, etc., are
very important and ever more sophisticated techniques (e.g. HPLC) are
introduced; for several vitamins, i.e. B 12 , biotin, etc., a microbiological
bioassay is still the method of choice (Freed, 1966; Berg & Behagel, 1972; De
Leenheer et al., 1985).
3 PHYSIOLOGICAL FUNCTIONS
Vitamins have a catalytic role in the body in enabling optimal synthesis,

conversion and degradation of macromolecules such as nucleic acids, proteins,
lipids and carbohydrates.
The biochemical (physiological) function of most water-soluble vitamins is
well known: they are part of co-enzymes and thus co-responsible for specific
biochemical reactions (Machlin, 1984; Diplock, 1985). A survey is given in
Table 2.
Table 2
Water-Soluble Vitamins and their Corresponding Coenzymes
Vitamin Coenzyme Group Transfer
Nicotinamide Nicotinamide-adenine Hydrogen
(B3 or PP) dinucleotide (NAD+)
Nicotinamide-adenine Hydrogen
dinucleotide phosphate
(NADP+)
Riboflavin Flavine adenine mono- Hydrogen
(B2) nucleotide (FMN)
Flavine adenine di- Hydrogen
nucleotide (FAD)
Pyridoxine Pyridoxalphosphate Amino-group, decar-
(B6) (PLP) boxylation
Folic acid Tetrahydrofolic Formyl
(Bg) acid
Biotin Biotin (biocytin, Carboxyl
(H) E-N-biotinyllysine)
Pantothenic Coenzyme A Acyl
acid (Bs)
Thiamine Thiaminepyrophos- Cz-aldehyde, decar-
(B 1 ) phate (TPP) boxylation
Cyanocobalamin B12-coenzyme Carboxyl
(B 12)
As to the function of fat-soluble vitamins and vitamin C, much controversy

still exists; their involvement can often be traced down to specific biochemical
processes (Table 3), although their exact function is not yet known in all cases
(De Luca, 1978; Diplock, 1985).
Much debate also exists about the positive effects of high (mega) doses of
certain vitamins, (e.g. vitamin C) on human and animal physiology; on the
other hand, several hypervitaminoses, (e.g. A, D, K) are well known
(Machlin, 1984).
Some vitamins display different activity toward man or animal, i.e. ergocal-
ciferol (D 2 ) is poorly active in poultry, while in other animals and man it is
equally active as cholecalciferol (D3)' This observation has great practical
repercussions on feed formulations. Others are essential solely for man, (i.e.
vitamin C), while most animals (except primates, cavias) can synthesise them.
Compounds which specifically counteract the functioning of vitamins are also
known and are named antivitamins or vitamin antagonists (Machlin, 1984).
Their negative action can be based on degradation of the vitamin (thiaminase,
ascorbase, etc.), or on the complexation of the vitamin into a non-resorbable
complex, (i.e. avidin plus biotin). N5-HydroxY-L-arginine is a vitamin B12
antagonist (Perlman et al., 1974). Dicoumarin excludes vitamin K from the
prothrombin synthesis system and amethopterin is an antagonist of folic acid;
however, both antivitamins are medically important for the treatment of
6 E. I. Vandamme
Table 3
Important Biochemical Actions of Fat-Soluble Vitamins and of Vitamin C
Vitamin Physiological functions
Vitamin A Retinol is part of rhodopsin, the light sensitive

active form: molecule in the eye
ll-cis-retinol Biosynthesis of mucopolysaccharides;
synthesis and maintenance of epithelial cells
Vitamin D3 Regulation in Ca2 + and phosphorous metabolism
(cholecalciferol)
active form:
1,25-diOH-cholecal-
ciferol
Vitamin E: Antioxidant action towards unsaturated com-
( a-tocoferol) pounds, i.e. fatty acids, etc.; membrane
integrity
Vitamin K j : Essential for formation of prothrombine,
(phylloquinone) a blood coagulation factor
Cofactor for carboxylation of protein bound
glutamate residues to form y-carboxyglutamates
Vitamin C Cosubstrate of monooxygenases;
Role in redox reactions and in
hydroxylation reactions of amino acids and
amines (i.e. proline conversion into
hydroxyproline in collagene);
Role in hormone-synthesis, iron
absorption, etc.
thrombosis and leukemia, respectively. Antivitamins present in our daily food

are usually destroyed during processing or cooking.
Excellent information on detailed nutritional, clinical and physiological
aspects of vitamins has been assembled by Machlin (1984), Diplock (1985) and
Friedrich (1988).
4 TECHNICAL FUNCTION OF VITAMINS, PIGMENTS AND

GROWTH FACTORS
In addition to their nutritional, physiological and medical importance, vitamins

and vitamin-like compounds have also found large-scale technical applications
as antioxidants (vitamin C, E), as acidulant (vitamin C), and as pigments
(f3-carotene and analogues) in the food, feed, cosmetic, chemical and
pharmaceutical industry (Machlin, 1984; Diplock, 1985; Florent, 1986).
Apart from the synthetic pigments, most of the natural colorants are now
being extracted from plants and animals, e.g. annatto, grapes, red beets,
paprika, female insects (Coccus cacti). Micro-organisms could be an excellent
source of non-toxic food colorants, but except for the fungal Monascus
pigments (monascin), used in the Orient, and for astaxanthin from yeast
few attempts have been made to produce and introduce them (Lin, 1973;
Palleroni et a/., 1978; Institute of Food Technologists, 1980; Wong & Koehler,
1981). Indeed, in addition to the well-known provitamin-carotenoids, a range
of anthraquinone pigments, chlorophylls and several others have been demon-
strated in bacteria, yeasts, fungi and algae and are attractive as natural
colorant.
In this respect, more emphasis should be given to screening and research on
other natural pigment synthesis via safe micro-organisms; this would open up a
wider application area in agriculture, food, feed, chemicals, cosmetics and
pharmaceuticals.
Fungal gibberellins are already widely used as plant growth-regulators in
agriculture and horticulture and in the brewing industry (Jefferys, 1970;
Palmer, 1974; Brueckner et a/., this volume).
Details about technical applications of certain vitamins, pigments and
gibberellins are given in the corresponding chapters in this volume.
5 PRODUCTION AND APPLICATION
The staple food of man, including cereals, rice, potato, vegetables, fruits, milk,
fish, meat, eggs, forms his basic source of vitamins and growth factors. Adequate
nutrition should thus supply this daily vitamin need, which however increases
with physical exercise, pregnancy, lactation, active growth, reconvalescence,
drug abuse, stress, air pollution, etc. Pathological situations (intestinal
malresorption, stressed intestinal flora, liver/gall diseases, drug, antibiotic or
hormone treatment, enzyme deficiencies), can also lead to vitamin shortages
despite a sufficient intake. Malnourishment in many countries of the world also
asks for direct medical remediations, combined with diet adjustment. Vitamin-
enriched and medicated feed is used worldwide to procure healthy livestock.
However, derived concentrates or extracts from these vitamin-rich natural
food products find relatively little use in the food, feed, pharmaceutical or
cosmetic industry (except for w-3-fatty acids, vitamin E, etc.). Some of the
reasons are:
(a) the average daily intake of vitamins does not always seem to be supplied
solely via these natural products;
(b) the level of the natural plant/ animal source vitamins is usually relatively
low and fluctuates drastically;
(c) their organoleptic presentation and shelf-life is often far from optimal;
(d) vitamins are labile molecules during the process of preservation, storage
or preparation of foodstuffs and are generally very sensitive to pH, heat,
light, oxygen, etc.; water-soluble vitamins are easily lost by aqueous
extraction or manipulation of foods.
These factors have led to the industrial manufacturing of most vitamins and
growth factors. Current world production of important vitamins is given in
Table 4.
At the moment, several vitamins are produced only or mainly chemically
8 E. I. Vandamme
Table 4
Survey of Vitamins and Growth Factors-Production and Application
Vitamin Industrial Application World production

group production (F = food/feed;
(see also (E = extraction; M=medical; tons/year Average Producer
Table 1) C=chemical T = technical) price company
synthesis; (S/kg) or country a
B = biotechnol- (1988)
ogy)
Provitamin A E F 100 450 2, 5, 6, 6a, 15a,

group C M 18, 20, 22, 25
8 (fermenta- T
tion, algal)
Vitamin A C F 2500 70 33,34,44,46
group M
Provitamin D C F 8, 14a, 18, 34
8 (fermenta- M
tion)
Vitamin D C F 25 350
group 8 (fermenta- M
tion)
Vitamin E E F 6800 17 9,11,17,18,
group C M 33,34
8 (algal) T
Vitamin F C F 200,2000 800,50 3, 6, 6a, 10, 18,
group E M (GLA) (EPA, (GLA) (EPA, 26,32,
8 (fermenta- DHA) DHA) 35,37,38
tion, algal)
Vitamin K C M 1·3 1500 9,11,17, 18,
group 34,45
Vitamin 8 1 C F 2000 23 2,18,31,39,
M 40,41
Vitamin 8 2 C F 2000 27 2,5,13,18,
8 (fermenta- M 24,30,41
tion)
Vitamin PP C F 8500 4 2,7,18
(vitamin 8 3 ) M 47
Pantothenic C F 4000 4 6a,18,39,41
acid 8 (fermenta- M
(vitamin 8 s) tion)
Vitamin 8 6 C F 1600 25 18,31, 39, 41
group 8 (fermenta- M
tion)
8iotin C F 3 5000 2, 18, 24, 27,
(vitamin H, (8) M 39,43
8 8)
Folic acid C F 300 115 18,39,41,42,48
(89 ) M
Vitamin 8 12 8 (fermenta- F 5-10 5000 14, 15, 16,21,23,
group tion) M 24,26,33,36
(continued)
Table 4--contd.
Vitamin Industrial Application World production

group production (F = food/feed;
(see also (E = extraction; M = medical; tons/year Average Producer
Table 1) C=chemical T = technical) price company
synthesis; (S/kg) or country a
B = biotechnol- (1988)
ogy)
Vitamin B13 B (fermenta- M 100 21

tion)
Vitamin C C plus F 65000 8 2,4,11,
B (fermenta- M 18,24,28,
tion) T 30,31,41,
44
Lipoic acid E M 30
Inositol C F
E M
T
Choline C F
M
T
PABA C M 18,33,
(B., H', Hz) 41,42,
48
Gibberellins B (fermenta- T 12-15 2000 1,4,12,
tion) 19,21,
24,29,
41,44,
a Listing of vitamin and growth factor producing companies or countries: 1. Abbott (USA); 2. BASF
(FRG); 3. Biocrops Ltd (UK); 4. China; 5. Coors Biotech Inc. (USA); 6. Cyanotech Inc (USA); 6a.
Dainippon Ink & Chemicals (Japan); 7. Degussa (FRG); 8. Duphar (The Netherlands); 9. Eastman-
Kodak (USA); 10. Efamol Holdings Ltd (UK); 11. Eisa Co. (Japan); 12. Eli Lilly (USA); 13. E. Merck
(FRG); 14. Farmitalia-Carlo Erba (Italy); 14a. Fujisawa (Japan); 15. Genex Corp. (USA); 15a. Gist
Brocades (The Netherlands); 16. Glaxo (Sefton Chern) (UK); 17. Henkel (FRG); 18. Hoffmann-
Laroche (Switzerland); 19. ICI (UK); 20. Koor Foods (Israel); 21. Kyowa Hakko Kogyo (Japan); 22.
Martek Inc. (USA); 23. Medimpex (Yugoslavia); 24. Merck S & D (USA); 25. Microbial Products Inc.
(USA); 26. Nippon Oil & Fats Co. Ltd (Japan), 27. Nippon Zeon (Japan); 28. Pao Yeh (Taiwan); 29.
Pierrel (Italy); 30. Pfizer Inc. (USA); 31. Pliva (Yugoslavia); 32. Q. P. Corporation (Japan); 33.
Rhone-Poulenc (France); 34. Riken Vitamin (Japan); 35. RMC (Evening Primrose Oil Company Ltd)
(UK); 36. Roussel-UcLaF (France); 37. Shiseido Co. Ltd (Japan); 38. Sturge Biochemicals (UK); 39.
Sumitomo (Japan); 40. Synergen Inc. (USA); 41. Takeda (Japan); 42. Tanabe-Kongo (Japan); 43.
Tanabe (Japan); 44. USSR; 45. Vanetta (Italy); 46. Western Biotechnology Ltd (Australia); 47. Yuki
Gosei (Japan); 48. Yodo Gowa-Duphar (Japan).
(vitamins A, D 3 , E, K, Bh H, etc.), although microbiological methods exist or

emerge; others are produced (exclusively) via fermentation (D2' B 6 , B 12 , F) or
micro-algal culture (fJ-carotene, F) and for others both chemical and microbial
processes are run industrially (vitamin B2 ), while vitamin C is produced via a
10 E. J. Vandamme
combination of chemical and microbiological reactions (Sakaguchi et al., 1971;

Yamada et al., 1971; Ogata et al., 1976; Periman, 1978; Florent, 1986;
Borowitzka & Borowitzka, 1988). Details about these bioprocesses form the
backbone of this volume!
A broad range of applications now exists for these vitamins and related
compounds in food, feed, cosmetics, technical and pharmaceutical
preparations:
revitamination: restoring the original vitamin level of a foodstuff;
standardisation: addition of vitamin(s) to compensate for natural
fluctuations;
vitamin-enrichment: extra addition of vitamin(s) to a level higher than the
natural one (health food, diet food industry);
vitamination: addition of vitamins to products lacking them (feed,
cosmetics) ;
technical additive (in food, feed, cosmetics), e.g. p-carotene as pigment,
vitamin C and E as antioxidants, vitamin C as acidulant, gibberellins as
growth hormones for plants;
medical applications: to alleviate hypo- or even avitaminoses.
Apart from obtaining these vitamins via a natural way-what microbial and
algal biotechnology is all about-fermentation or bioconversion reactions yield
the desired enantiomeric vitamin compound, while products from organic
synthesis are often racemic mixtures, each displaying a differing biological
activity. Furthermore, vitamin yields in fermentation broths are often very
high-especially when using mutants or genetically engineered strains-as
compared with their natural levels in plants or animals.
In this respect, continued efforts should be made to further explore the
potential and power of microbial and algal biotechnology in the field of
economic natural vitamin, pigment and growth factor production.
REFERENCES
Adrian, J. (1988). Parat Dictionary of Food and Nutrition, VII. Ellis Horwood Series in
Food Science and Technology. Ellis Horwood, Chichester, UK.
Berg, T. M. & Behagel, H. A. (1972). Semi-automated method for microbiological
vitamin-assays. Appl. Microbiol., 23,531-42.
Borowitzka, M. A. & Borowitzka, L. J. (eds) (1988). Micro-algal Biotechnology.
Cambridge University Press, Cambridge, UK.
Brueckner et al. (1989). Fungal gibberellin production. In Biotechnology of
Vitamins, Pigments and Growth Factors, ed. E. J. Vandamme. Elsevier Science
Publishers, London, pp. 383-429
Chytil, P. & McCormick, D. B. (eds) (1986). Vitamins and Coenzymes. Methods in
Enzymology, Vols 122, 123. Academic Press, New York, London.
De Leenheer, A. P., Lambert, W. E. & Deruyter, M. G. M. (eds) (1985). Modern
Chromatographic Analysis of the Vitamins. Marcel Dekker, New York, Basel.
De Luca, H. F. (ed.) (1978). The fat-soluble vitamins. In Handbook of Lipid Research,
Vol. 2. Plenum Press, New York, London.

Diplock, A. T. (ed.) (1985). Fat-Soluble Vitamins. Their Biochemistry and Applica-
tions. Heineman, London.
Florent, J. (1986). Vitamins. In Biotechnology, Vol. 4, Microbial Products II; ed. H.
Pape & H. J. Rehm. VCH, Weinheim, pp. 115-58.
Freed, M. (ed.) (1966). Methods of Vitamin Assay, 3rd edn. John Wiley & Sons, New
York.
Friedrich, W. (1988). Vitamins. Walter de Gruyter, Berlin, New York.
Goodwin, T. W. (1963). The Biosynthesis of Vitamins and Related Compounds.
Academic Press, London. .
Institute of Food Technologists (1980). Food colors. Food Technology, 34,77.
Jefferys, E. G. (1970). The Gibberellin Fermentation. Adv. App/. Microbio/., 13,
283-316.
Lin, C. F. (1973). Isolation and cultural conditions of Monascus sp. for the production
of a pigment in a submerged culture. 1. Ferment. Techno/., 51,407-9.
Machlin, L. J. (ed.) (1984). Handbook of Vitamins: Nutritional, Biochemical and
Clinical Aspects. Marcel Dekker, New York, Basel.
Ogata, K., Kinoshita, S., Tsunoda, T. & Aida, K. (1976). Microbial Production of
Nucleic Acid Related Substances. Kodansha, Tokyo.
Palleroni, N. S., Reichelt, K. E., Mueller, D., Epps, R., Tabenkin, B., Bull, D. N.,
Schnep, W. & Berger, J. (1978). Production of a novel red pigment, rubrolone, by
Streptomyces echinoruber sp. 1. Antibiotics, 31, 1218-25.
Palmer, G. J. (1974). The industrial use of gibberellic acid and its scientific basis-a
Review. 1. Inst. Brewing, 80, 13-30.
Perlman, D. (1978). Vitamins. Economic Microbiology, 2, 303-26.
Perlman, D., Vlietinck, A. J., Matthews, H. W. & Lo, F. F. (1974). Microbial
production of vitamin B12 anti metabolites. I. N5-hydroxY-L-arginine from Bacillus
cereus 439. 1. Antibiotics, 27, 826-32.
Sakaguchi, K., Uemara, T. & Kinoshita, S. (1971). Biochemical and Industrial Aspects
of Fermentation. Kodansha, Tokyo.
Sebrell, W. H. & Harris, R. S. (eds) (1971). The Vitamins, 2nd edn. Academic Press,
New York, London.
Wong, M. C. & Koehler, P. E. (1981). Production and isolation of an antibiotic from
Monascus purpureus and its relationship to pigment production. 1. Food Sc., 46,
589.
Yamada, K., Nakahara, T. & Fukui, S. (1971). Petroleum microbiology and vitamin
production. In Biochemical and Industrial Aspects of Fermentation, ed. Sakaguchi,
T. Uemura & S. Kinoshita. Kodansha, Tokyo, pp. 61-90.
FAT-SOLUBLE VITAMINS AND PIGMENTS
Chapter 2
II-CAROTENE (PROVITAMIN A) PRODUCTION WITH
ALGAE
L. J. BOROWITZKA
Western Biotechnology Ltd, 2-6 Railway Parade, Bayswater, WA 6063,
Australia
&
M. A. BOROWITZKA
Algal Biotechnology Laboratory, School of Biological and Environmental

Sciences, Murdoch University, Murdoch, WA 6150, Australia
1 HISTORICAL
Dunaliella is a unicellular, biflagellate, naked green alga (Chlorophyceae,

Dunaliellales), and the type species of this genus, Dunaliella salina (Dunal)
Teodoresco is often found in natural hypersaline waters where it colours the
brines red (Teodoresco, 1905). This algal species was first recognised as
containing high intracellular concentrations of p-carotene by Mil'ko (1963) and
Aasen et al. (1969). Initial research on the potential of using this alga as a
commercial source of p-carotene began in the Ukraine in the 1960s (cf.
Massyuk, 1966; Massyuk & Abdula, 1969) and it was later also proposed as a
commercial source of glycerol (Ben-Amotz; 1980; Chen & Chi, 1981;
Ben-Amotz & Avron, 1982).
A number of commercial-scale developments on the production of p-
carotene from D. salina have commenced in Australia (Curtain et al., 1987;
Borowitzka & Borowitzka, 1988a), Israel (Rich, 1978) and the USA (Klaus-
ner, 1986), and a number of open-pond and closed reactor experimental units
are in development stages. Pilot projects are also under way in China and
Chile. Commercial quantities of extracted algal p-carotene and dried D. salina
powder rich in p-carotene have been marketed by companies from the US and
Australia since 1985. All are targeting the markets for 'natural' food and
animal feed colourings, and 'natural' p-carotene (provitamin A) nutritional
supplements.
2 CHEMICAL AND PHYSICAL PROPERTIES
p-Carotene is accumulated as droplets in the chloroplast stroma of Dunaliella

salina cells, particularly when culture conditions include high light intensities,
15
16 T. Kutsal & M. T. Ozbas
v
c::
....Vo
L
111
U
I
C!l.
205·071 I
III
c::
111
....
L
I
vv
v iii c::C::
c:: Vv
U
c:: :c 0
0LL
111 1::111 111111
fo 105·931 )( v UU
I I
111 c::
III
.c c:: V c:: c:: c:: v C!l.C!l.
<{ ~
o
N
~ ~ ~
0
-0L I
.- U
I
U\.!!!
c:: 0 0 U M
~ c:: c:: c:: 111
~ U Ol~
c:: ~ ~
c:: c::
c::
jj
I
:> :> :>
:> d
6790~==~==~=====;~~====~~==~==~~~~====t=========
o 5 10 15
Time (min)
Fig. 1. Typical HPLC analysis of D. salina J3-carotene extract.
high temperature, high salinity and deficiency of nitrogen source (Lerche,

1937; Mil'ko, 1963; Mironyuk & Einor, 1968; Semenko & Abdullayev, 1980;
Ben-Amotz & Avron; 1983; Borowitzka et at., 1984). p-carotene contents of
up to 14% of dry weight have been reported for D. salina (Ben-Amotz et at.,
1982; Borowitzka et at., 1984). The p-carotene does not appear to act as a
light-harvesting photo-accessory pigment, but rather as a photo-protective
'sunscreen' to the chlorophyll and cell DNA in the high light intensities which
characterise the normal salt-lake environments where this alga grows
(MacKinney & Chichester, 1960; Ben-Amotz, 1980). Borowitzka & Borowit-
zka (1988a) have also proposed that p-carotene acts as a 'carbon sink' in D.
salina for excess combined carbon produced when photosynthesis continues
under conditions where growth is limited.
The p-carotene in the chloroplast droplets is a mixture of the cis- and
trans-isomers. A typical analysis from D. salina (also called D. bardawil) gives
the following composition as percentages of total p-carotene: 15-cis-p-
carotene, 10%; 9-cis-p-carotene, 41%; all-trans-p-carotene, 42%; other iso-
mers, 6% (Ben-Amotz et at., 1982).
For analytical purposes, total p-carotene may be calculated from its
extinction in acetone extracts (m~::. at 452 nm is 2592; Bauernfeind, 1981).
HPLC is required to separate the isomers, and to characterise the small
quantities of other carotenoids also present in typical D. salina extracts (Nelis
& de Leenheer, 1983). Figure 1 shows an HPLC analysis of a typical
commercial batch of 20% p-carotene suspension in peanut oil produced by
Western Biotechnology Limited from D. salina.
3 STRAIN IMPROVEMENT, SELECTION AND MAINTENANCE

Dunaliella salina production systems, like those used by Western Biotechnol-
ogy Ltd (Fig. 2), have large open ponds situated in, or near, salt lakes or solar
{3-Carotene Production with Algae 17
Fig. 2. Aerial photographs of Western Biotechnology's Dunaliella production facility at

Hutt Lagoon, WA, Australia.
salt works. These ecosystems and the ponds are inhabited by wild-type D.
salina, non-carotenogenic Dunaliella species, predatory protozoa, halophilic
bacteria and brine shrimp (Borowitzka et al., 1986; Moulton et al., 1987b). In
the presence of this mixture of organisms, maintenance of cultures dominated
by the carotenogenic D. salina is the first priority of pond management. In an
environment dominated by the wild-type D. salina, introduction of an
18 L. J. Borowitzka & M. A. Borowitzka
'improved' D. salina strain is extremely difficult, and has not been reported on
a commercial scale. In the large, open-air ponds used for the commercial
culture of D. salina the major control of the culture available to the operator is
salinity (Borowitzka et al., 1986), although some additional control may be
exercised by manipulating nutrient concentrations.
In more isolated systems, as for example, concrete or plastic-lined raceway
ponds and tubular photobioreactors, wild-type D. salina and other competing
organisms may be excluded and the introduction of improved strains can be
achieved, at the cost of higher investment in plant and equipment.
Strain improvement, both in terms of growth characteristics and product
yield, can be achieved using mutagenesis and selection programmes. Improved
strain breeding using mating strains as in Chlamydomonas, a closely related
alga, is also possible (Craig et al., 1988). Further into the future, and awaiting
additional research, is the use of cell fusion techniques or the genetic
engineering manipulations developed for yeast cells (e.g. Spencer & Spencer,
1983).
In the laboratory, D. salina may be isolated into unialgal and axenic culture
either by repeated streaking on agar plates or by isolating clonal cultures from
single cells. The most commonly used medium for D. salina and related algae
is modified Johnson's medium (Borowitzka, 1988), although a range of other
media can also be used, as long as the salinity is appropriately adjusted with
NaCI.
Laboratory cultures are usually maintained on agar slants or in liquid
cultures, and sub-cultured every 1-2 months. Care must be taken to avoid
excessive drying out of the high salinity media. We have also successfully
maintained Dunaliella species, frozen in liquid nitrogen in our laboratory for
periods in excess of 12 months. In the field, stock cultures are best maintained
at saturating salinities where the likelihood of contamination by other
organisms is minimised.
4 BIOSYNTHESIS AND REGULATION
There is an inverse relationship between growth rate and carotenogensis in D.

salina; conditions which enhance accumulation of p-carotene include high light
intensity, high temperature, high salinity, nitrogen deficiency, and these same
conditions tend to decrease growth rate. This is a typical pattern for secondary
metabolite production in stationary phase cultures of algae. Table 1 sum-
marises the environmental factors which influence growth and carotenogenesis
in D. salina.
The effect of salinity on carotenogenesis is best shown in experiments which
investigated the induction of carotenogenesis. In these experiments the NaCI
concentration was increased from 15 to 25% (w/v) and the p-carotene content
of the D. salina cells increased linearly from <10 to 260 mg (g cell protein)-t,
over 5 days, while growth ceased (Borowitzka et al., 1984).
fJ-Carotene Production with Algae 19
Table 1
Summary of the Influence of Various Environmental Factors on
the Biomass and Jj-Carotene Content of Dunaliella salina. + =
Stimulating Effect; - = Inhibitory Effect; 0 = No Effect
Factor Biomass Jj-carotene
Increase in salinity +++

Decrease in salinity + (-t
N deficiency +
P deficiency +
Increase in [C02] supply + 0
Increase in light 0 ++++
Decrease in light 0
Increase in temperature 0 +
Decrease in temperature
Increase in [0 2] 0
a Rate of decrease is very slow.

Fortuitously, if salinity is reduced by a similar amount, as for example when
rain enters open ponds, the decline in p-carotene content of the cells is very
slow (Borowitzka et al., 1984) as the p-carotene is not broken down, but
rather the cell content is reduced as the cells divide.
5 BIOMASS PRODUCTION
Open-pond D. salina production is perhaps more closely related to farming

than to fermentation techniques. Open cultures are influenced by weather
(temperature, light, rainfall, wind), seasons, and input of contaminants from
the environment. Enclosed ponds, tank or tubular photobioreactors and
fermentor systems can reduce the influence of these external factors on the
culture; however, these have, as yet, not been applied to D. salina culture on a
commercial scale.
Dunaliella salina cultures may be operated either in batch or continuous
mode. A two-stage process, using lower salinity conditions for optimum
growth rate and biomass production and then changing to high salinity stress
conditions favouring carotenogenesis has also been piloted and the disadvan-
tages of this two-stage process are discussed in Borowitzka et al. (1984). A
single-stage, semi-continuous process at an intermediate salinity appears to be
the best at this stage, and this is the process used by Western Biotechnology
Ltd (Fig. 3).
In all D. salina culture systems the following are necessary for good growth
and carotenogenesis:
(a) Light. Light is the only energy source for metabolism as D. salina is an
obligate photoautotroph. The light source may be sunlight or high
intensity artificial white light. Growth and carotenogenesis respond
20 L. 1. Borowitzka & M. A. Borowitzka
HIG H SALINITY BRINE •

EV APORATIVE MAKE-UP •
GROWTH PONDS
INO CULUM • (50 Ha)
NUTRIENTS •
1
I HOLDING PONDS
I I HARVESTERS
I
1
1
CULTURE MEDIUM
BIOMASS
(BRINE)
I
1
l DRY EXTRACT fJ-CAROTENE
!
CONCENTRATE
1
PURIFY
1
MILL
1
Dunaliella salina FORMULATION
powder containing
3% fJ-Carotene Suspensions in
Vegetable oil
(2%, 10%,20%,30%)
Water Dispersibles
Water Solubles
--- Final
Quality control
-- .....
PACKAGING
J PACKAGING
J
Fig. 3. Flow chart of the Dunaliella salina fJ-carotene production process used by
Western Biotechnology Ltd.
differently to particular wavelengths of light (Mil'ko, 1963), and high

photon-flux densities favour fj-carotene accumulation.
(b) Temperature. Dunaliella salina is tolerant of a wide range of tempera-
tures for growth, from below O°C (Siegel et al., 1984) to above 40°C
(Wegmann et al., 1980). The optimum growth temperature lies between
21 and 40°C, and is dependent on the strain and light intensity (Gibor,
1956; Federov et al., 1968; Borowitzka, 1981). Temperatures close to
40°C and higher stimulate carotenogenesis at the expense of growth
(Mil'ko, 1963). In practice, the combination of light intensities, day
length and temperatures between 30 and 40°C give optimum growth and
fj-carotene production at Western Biotechnology's Australian plant in
the summer months, although growth and fj-carotene production are
sustained throughout the year.
(c) Carbon source. Dunaliella salina is a photoautotroph; it can use only CO2
and possibly bicarbonate (HC03 ) as carbon source. Growth of D. salina
is stimulated by addition of NaHC0 3 or bubbling with COz, provided
there is no precipitation of carbonates (Massyuk, 1965; Drokova &
Dovhorouka, 1966). Limited availability of inorganic carbon (C0 2 and
HC0 3 ) is the most common growth limiting factor in the high salinity,
high pH and temperature conditions used for D. salina culture.
Inorganic carbon equilibria and uptake by D. salina under typical
growth conditions are discussed by Borowitzka & Borowitzka (1988a).
(d) Nitrogen and phosphorus sources. The best nitrogen source for D. salina
is NaN0 3 (Mil'ko, 1962; Grant, 1968); ammonium salts, urea and
nitrites are less effective (Gibor, 1956; Mil'ko, 1962; Borowitzka &
Borowitzka, 1988b). Care must be taken to maintain culture pH; despite
natural buffering conferred by the inorganic carbon equilibrium
(CO~-~HC03~C02)' nitrate uptake can cause alkalinisation, and
ammonium uptake acidification of the growth medium, leading to cell
death (Borowitzka & Borowitzka, 1988b). The optimum phosphate
concentration for growth is approximately 0·02 j.lg litre- 1 K 2HP0 4
(Gibor, 1956); higher concentrations can inhibit growth.
(e) NaCI. All known marine and halophilic species of Dunaliella have a
specific requirement for sodium; D. salina has the broadest reported
NaCI tolerance range, from 1 to 35% (w/v) NaCI (saturation) (Loeblich,
1972). Selection of the appropriate NaCI concentration for the commer-
cial culture depends on growth or carotenogenesis rate required, and the
need (if any) to control predators, competitors or bacterial numbers
(Borowitzka et al., 1986). Normal salinities used for growth range from
20 to 30% (w/v) NaCI, i.e. about 9-10 times the salinity of sea-water.
(f) Other requirements. Dunaliella salina requires Mg2+, Ca2+, Na+, CI-,
SO~-, chelated iron and trace elements for growth. Optimum pH is
between 7 and 9. A discussion of the literature reports on concentration
and ratios of these components is given in Borowitzka and Borowitzka
(1988a). No requirement for vitamins has been demonstrated.
22 L. 1. BOTowitzka & M. A. BOTowitzka
6 PRODUCT RECOVERY AND PURIFICATION
Commercial D. salina production is a young industry, only 3 years old, and

companies in development and production phases guard the confidentiality of
their processes. In particular, because harvesting and extraction represent
major cost areas, technical advantages in these areas are eagerly sought and
strongly guarded. Harvesting D. salina is more difficult and costly compared to
most commercial algae. Dunaliella salina is a single cell, with no protective cell
wall, approximately 20 x 10 /lm in size, and neutrally buoyant in a high specific
gravity, high viscosity brine. Cell densities in large cultures tend to be about
1 g litre- 1 and very large volumes therefore have to be processed. Efforts to
centrifuge or filter the algae from the brine generally shear-damage the cells
leading to tJ-carotene loss by oxidisation. The cells also distort and pass
through filters with pore sizes less than 10 /lm. Corrosion of all metal
equipment by the brine is also a major problem.
These problems have led to development and patenting of several biological
and chemical methods for harvesting the cells, or for pre-concentration of cells
prior to use of more conventional harvest methods. Patents issued for
Dunaliella harvesting include high pressure filtration using diatomaceous earth
(Ruane, 1974a), exploitation of salinity-dependent buoyancy properties in
stationary and moving gradients (Bloch et al., 1982), exploitation of the
phototactic and gyrotactic responses of the algae (Kessler, 1982, 1985)
salinity-dependent hydrophobic adhesion properties of Dunaliella cells (Cur-
tain & Snook, 1983) and flocculation (Sammy, 1987). The harvesting of
micro algae is reviewed by Mohn (1988).
The process for extraction of tJ-carotene from the biomass depends in part
on the harvesting procedure used, and on the market requirements. Extraction
using conventional organic solvents is efficient (Ruane, 1974b), but may not be
acceptable to customers seeking a 'natural' product. More acceptable alterna-
tive extraction methods use hot vegetable oil (Nonomura, 1987; Potts, 1987) or
liquid or supercritical gas solvents.
Harvested biomass may be dried, (i.e. spray-drying) rather than extracted,
and is marketed as a tJ-carotene-rich supplement for human health or animal
feed (Ben-Amotz et al., 1986; Avron et al., 1987; Curtain et al., 1987).
By-products of tJ-carotene production could include glycerol, which can
make up 30% of biomass dry weight (see Chen & Chi, 1981, for process
proposal and cost analysis) and high quality protein meal remaining after
extraction. The amino acid analysis of several Dunaliella spp is summarised in
Table 2.2. in Borowitzka and Borowitzka (1988a).
7 BIOLOGICAL PROPERTIES
tJ-Carotene is marketed mainly as an orange colouring, for foods such as

margarine, baked goods, and beverages (KUiui, 1981). A smaller market exists
for use of {:1-carotene as a pigment supplement in the commercial feeds for

crustaceans, such as prawns, which convert it to astaxanthin, the natural red
colouring of the shell and flesh.
{:1-Carotene is provitamin A, and provides the main source of vitamin A in
human diets. More recently, interest has increased in the role played by
{:1-carotene as an anti-oxidant in human nutrition and correlations have been
reported between high dietary {:1-carotene intake and low incidences of some
cancers (Mathews-Roth, 1982; Schwartz et aZ., 1986). Large-scale trials are
underway in the US (National Cancer Institute) and in Europe to confirm the
'anti-cancer' activity of {:1-carotene.
Trials with cattle have also indicated an increase in fertility attributable to
supplements of {:1-carotene.
8 CHEMICAL SYNTHESIS
The first commercial synthesis of f3-carotene was established by Hoffman-La

Roche in 1956. Since then, the process has been improved by Roche and other
companies; patents describe the key steps in the synthesis. Syntheses of
{:1-carotene are reviewed by Mayer & Isler (1971).
9 FORMULAnONS AND APPLICA nONS
{:1-Carotene is a lipid- and oil-soluble product. As suspensions or solutions in

vegetable oil (Fig. 2) it finds applications in colouring margarine, baked goods
and some prepared foods. Conversion to water-soluble or water-dispersible
formulations, by forming emulsions or microencapsulated beadlets extends the
food applications to beverages including orange drinks, and to confectionery
and further prepared foods.
Nutritional supplements can be prepared by encapsulation of oil suspensions
or solutions, or by tableting using beadlet forms. Current recommended daily
doses for {:1-carotene are around 6 mg; 20-25 mg doses are in use in the
'anti-cancer' trials in the US and Finland. Dried Dunaliella powder is also
prepared for use in animal feed supplements.
10 ECONOMICS
Just as the companies in the new industry guard the confidentiality of their
processes, costs of commercial production are not available. Moulton et aZ.
(1987a), however, present a bioeconomic model for the {:1-carotene production
process using D. salina.
Synthesised {:1-carotene is sold for a minimum of US$300 per kg {:1-carotene,
and at higher prices depending on the formulation. 'Natural' {:1-carotene
24 L. I. Borowitzka & M. A. Borowitzka
commands higher prices, with the highest price attainable for the nutritional
supplement application. Market price for 'natural' IJ-carotene is volatile and
in 1987-88 world market demand exceeds supply.
If a price range for formulations of natural IJ-carotene of US$500-1000 is
assumed, and IJ-carotene represents 10% of D. salina biomass, production
costs for D. salina should not exceed US$50-100 per kg. In fact, costs must be
significantly lower, to account for losses at each processing step, capital
expenses, marketing, packaging and distribution costs.
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Ben-Amotz, A., Katz, A. & Avron, M. (1982). Accumulation of p-carotene in
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from Dunaliella bardawil (Chlorophyceae). J. Phycol., 18,529-37.
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Dunaliella bardawil as a source of retinol. Br. Poult. Sci., 27,613-9.
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& Perath, A. (1982). Oil products from algae. US patent no. 4.341 038.
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Hydrobiologia, 81, 33-46.
Borowitzka, M. A. (1988). Algal growth media and sources of algal cultures. In
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University Press, Cambridge pp. 456-65.
Borowitzka, L. J., Borowitzka, M. A. & Moulton, T. P. (1984). The mass culture of
Dunaliella salina for fine chemicals: from laboratory to pilot plant. Hydrobiologia,
116/117, 115-2l.
Borowitzka, L. J., Moulton, T. P. & Borowitzka, M. A. (1986). Salinity and the
commercial production of beta-carotene from Dunaliella salina. Nova Hedwigia,
Beih., 83, 224-9.
Borowitzka, M. A. & Borowitzka, L. J. (1988a) Dunaliella. In Microalgal
Biotechnology, ed. M. A. Borowitzka & L. J. Borowitzka. Cambridge University
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Borowitzka, M. A. & Borowitzka, L. J. (1988b) Limits to growth and carotenogenesis
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Craig, R., Reichelt, B. Y. & Reichelt, J. L. (1988). Genetic engineering of
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Drokova, I. G. & Dovhorouka, S. I. (1966). Carotene-formation in Dunaliella salina
Teod. under the effect of some carbon sources. Ukransk. Bot. Zhour., 21, 59-62.
Federov, V. D., Maksimov, V. N. & Kromov, V. M. (1968). Effect of light and
temperature on primary production of certain unicellular green algae and diatoms.
Fiziol. Rast., 15, 640-5l.
Gibor, A. (1956). The culture of brine algae. Bioi. bull., Woods Hole, 3,223-9.
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nitrate and nitrite and assimilation by Dunaliella tertiolecta, J. Gen. Micro., 54,
327-36.
Kessler, J. O. (1982). Algal cell harvesting. US patent no. 4324067.
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KI1tui, H. (1981). Industrial and commercial uses of carotenoids. In Carotenoid
chemistry and Biochemistry, ed. G. Britton & T. W. Goodwin. Pergamon Press,
Oxford, pp. 309-28.
Klausner, A. (1986). Algaculture: Food for thought. Biotechnology, 4,947-53.
Lerche, W. (1937). Untersuchungen iiber die Entwicklung und Fortpflanzung in der
Gattung Dunaliella. Arch. fur Protistenk., 88, 236-9.
Loeblich, L. A. (1972). Studies on the brine flagellate Dunaliella salina. PhD thesis,
University of California, San Diego.
MacKinney, G. & Chichester, C. O. (1960). Biosynthesis of carotenoids. In
Comparative Biochemistry of Photoreactive Systems, ed. M. B. Allen. Academic
Press, New York, pp. 205-15.
Massyuk, N. P. (1965). Carbonate and bicarbonate as stimulators of growth and
carotene accumulation in Dunaliella salina Teod. Ukransk. Bot. Zhour., 22, 18-22.
Massyuk, N. P. (1966). Mass culture of the carotene-bearing alga Dunaliella salina
Teod. Ukransk. Bot. Zhour., 23, 12-19.
Massyuk, N. P. & Abdula, E. G. (1969). First experiment of growing carotene-
containing algae under semi-industrial conditions. Ukransk. Bot. Zhour., 26,21-7.
Mathews-Roth, M. M. (1982). Medical applications and uses of carotenoids. In
Carotenoid Chemistry and Biochemistry, ed. G. Britton & T. W. Goodwin.
Pergamon Press, Oxford, pp. 297-307.
Mayer, H. & Isler, O. (1971). Total syntheses. In Carotenoids, ed. O. Isler.
Birkhauser, Basle, pp. 328-575.
Mil'ko, E. S. (1962). Study the requirement of two Dunaliella spp. in mineral and
organic components if the medium, Moscow University Vestnik. Biologya, 6, 21-3.
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the alga Dunaliella salina. Mikrobiologiya, 32,299-307.
Mironyuk, V. I. & Einor, L. O. (1968). Oxygen exchange and pigment content in
various forms of Dunaliella salina Teod. under conditions of increasing NaCl
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Mohn, F. H. (1988). Harvesting of micro-algal biomass. In Micro-algal Biotechnology,
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395-414.
26 L. J. Borowitzka & M. A. Borowitzka
Moulton, T. P., Borowitzka, L. J. & Vincent, D. J. (1987a) The mass culture of

Dunaliella salina for p-carotene: from pilot plant to production plant,
Hydrobiologia, 151/152,99-105.
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Competition between Dunaliella species at high salinity. Hydrobiologia, 151/152,
107-16.
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1196-7.
Chapter 3
PRODUCTION OF CAROTENOIDS WITH FUNGI

E. CERDA-OLMEDO
Departamento de Genetica y Biotecnia, Universidad de Sevilla, Apartado 1095,
E-41080 Sevilla, Spain
1 INTRODUCTION
Humans, like most other animals, need carotenoids but cannot synthesize
them. Our main suppliers are the fruits and vegetables, with lesser, mostly
indirect contributions from fungi, algae, and bacteria. Carotenoid production
is rarely the main objective of a culture, but colourful products rich in
carotenoids are obtained from various plants and algae. The oldest example is
saffron (Crocus sativus) , still planted in La Mancha, Spain, and elsewhere for
the stigmas of its flowers. Many fields of central Mexico become bright orange
in the summer with the flowers of cempasuchil (Tagetes ten uifolia , a kind of
marigold), whose dried petals are fed to chicken for meat and egg yolk colour.
Annatto is extracted from the seeds of a tropical tree, Bixa orellana. The most
familiar carotenoid-rich product is probably paprika, a powder made by
grinding red peppers (Capsicum annuum).
The carotenoids may be scarce in the diets of humans and animals for
various reasons. Many staple foods and feeds, including most of the cheap
sources of carbohydrates and fat, are poor in carotenoids, as witnessed by their
lack of colour. The long winters of many countries halt the natural production
of carotenoids, and these are relatively unstable and may not survive in stored
foods and feeds.
Organic chemists have devised several complete syntheses, which now
supply considerable amounts of f3-carotene and other purified carotenoids. The
current trend towards natural food colours and the discovery of salutary effects
of the carotenoids, beyond their important role as provitamin A, stimulate the
demand and press towards a biological production.
The fungi can hardly be considered as traditional food colourants, but the
relative ease of cultivation and the many possibilities for physiological,
genetical, and industrial manipulations make them attractive to biotech-
nologists. Several species are potential sources of carotenoids, and a large-
scale process has been based on Blakeslea trispora.
27
28 E. Cerda-Olmedo
It seems that the Soviet Union is the only country that uses Blakeslea
trispora for the industrial production of carotenoids; the yearly production is
estimated at about 200 kg purified fJ-carotene and another 4 Mg fJ-carotene in
the form of mycelia used as an additive to animal feed (A. A. Dmitrovskii,
pers. comm., 1988).
The abundant literature on carotenoids has been the subject of many books
and reviews. The monumental and indispensable treatise edited by Isler (1971)
is complemented by newer reviews (Feofilova, 1974; Goodwin, 1976, 1980,
1988). The practical aspects are explained in great detail by Bauernfeind
(1981). Updates are provided by the International Symposia on Carotenoids,
published every 3 years as a special issue of Pure and Applied Chemistry.
There are specialized and recent reviews on the influence of external factors on
carotenogenesis in the Mucorales (Lampila et al., 1985a), on Phycomyces
(Cerda-Olmedo & Lipson, 1987), on the regulation of carotenogenesis
(Bramley & Mackenzie, 1988), on the metabolism and metabolic effects of the
carotenoids (Goodwin, 1986), and many other subjects.
2 FUNGAL CAROTENOIDS
Most fungi produce no carotenoids at all and many of the others contain a
single major carotenoid. Although the pathway intermediates may be present
in amounts widely variable with the strain and the culture conditions, the
carotenoid composition of the fungi is usually much simpler than that of the
photosynthetic organisms. Here are a few selected examples of fungal
carotenoids. fJ-Carotene predominates in the Mucorales Blakeslea trispora,
Phycomyces blakesleeanus and Choanephora cucurbitarum, in the yeast
Rhodotorula aurea, in Aspergillus giganteus (EI-Jack et al., 1988) and various
species of Penicillium; neurosporaxanthin is the end-product of Fusarium
aquaeductuum (Bindl et al., 1970); neurosporaxanthin and fJ-carotene, those
of Gibberella fujikuroi (Avalos & Cerda-Olmedo, 1987), Neurospora crassa
and Verticillium agaricinum (Valadon & Mummery, 1973); torularhodin and
fJ-carotene, those of the yeasts Rhodotorula rubra, R. minuta, and
Rhodosporidium diobovatum; fJ-carotene, lycopene, and fJ-zeacarotene are
found in different strains of the smut Ustilago violacea (Will et al., 1984);
canthaxantin in the mushroom Cantharellus cinnabarinus; astaxanthin, in the
yeast Phaffia rhodozyma. See Goodwin (1980) for other fungi and additional
references.
Figure 1 shows the structure of the carotenoids that have just been
mentioned, with their likely biosynthetic pathways. The carotenoids, like all
terpenoids, are synthesized from hydroxymethylglutaryl-coenzyme A, which is
first converted to mevalonic acid. The specific part of the pathway begins with
the condensation of two molecules of geranylgeranyl pyrophosphate to form
phytoene, a colourless carotene. Four dehydrogenations transform phytoene
into lycopene, the pigment of red tomatoes. Two cyclisations convert lycopene
----.. ------ ~ ~
Ivcopene
~-/
V-carotene torulene
J
5'
;:
COOH
~
f) - carotene " torularhodin a~
~
;:
c
~
;
~
~
~.
.DH
"
Fig. 1. Some fungal carotenoids with their possible biosynthetic pathways. The horizontal arrows indicate dehydrogena-
tions, the vertical arrows, cyclisations, and the tilted arrows, oxidative reactions.
~
30 E. Cerda-Olmedo
into p-carotene. In Phycomyces these reactions are carried out by an enzyme

aggregate that contains four copies of a dehydrogenase and two copies of a
cyclase (review, Cerda-Olmedo, 1987). In Giberellafujikuroi the production of
torulene requires a fifth dehydrogenation by the same enzyme; the final break
and oxidation to neurosporaxanthin appear to be carried out in a single step
(Avalos & Cerda-Olmedo, 1987). On the other hand, several dehydrogenases
have been postulated for Ustilago violacea (Will et al., 1984). The detection of
some minor intermediates indicates that the order of the reactions is
sometimes altered in these organisms, and may be different in others.
Canthaxantin, a likely intermediate in the biosynthesis of astaxanthin in the
crustaceans (Castillo, 1980), was not detected in Phaffia rhodozyma
(Andrewes et al., 1976).
Many carotenoids found in plants and algae are unknown in the fungi, but
most of the fungal carotenoids are also found in plants or algae, and this is
fortunate for fungal biotechnology, because the traditional ingredients of our
diet are more acceptable to the health authorities and to the consumers than
new products. A few carotenoids are exclusive of certain fungi: torulene,
neurosporaxanthin, torularhodin, plectaniaxanthin, phillipsiaxanthin, p-y-
carotene. The astaxanthin from Phaffia rhodozyma has the opposite chirality
(3R, 3'R) to that of the astaxanthin responsible for the admired colours of
salmon and lobster (Andrewes & Starr, 1976).
Research towards industrial production has concentrated on the mucorales
Blakeslea trispora and Phycomyces blakesleeanus, whose main and nearly only
carotenoid is all-trans p-carotene. Commercial advantages of p-carotene are its
colour, its high provitamin A activity, its wide distribution in nature, and its
many current applications. A disadvantage is its inability to pigment some
avian and fish products.
3 NATURAL VARIATIONS IN CAROTENOID CONTENT
Many fungi attracted the attention of carotenoid chemists because of their

bright colours, but the strains used in the laboratories may not be optimal for
carotenoid production.
Thus, an impressive polymorphism was found in natural isolates of Ustilago
violacea (Garber et al., 1978). Fourfold differences in carotenogenesis were
found in a few natural isolates of Phycomyces blakesleeanus (Goodwin &
Griffiths, 1952). A large systematic search for better starting strains should
be rewarding, as it was for many other biological products.
The carotenoid concentrations are usually too low for commercial applica-
tion. The accumulation of carotenoids in the fungi is influenced by many
environmental variables; their study is a first step in yield improvement. While
so doing, one should bear in mind that the fungi are very distantly related
organisms, that the regulations of carotenogenesis are not universal, and
therefore, that observations on a species need not apply to others.
Production of Carotenoids with Fungi 31
3.1 Light
Blue illumination stimulates the production of carotenoids in many fungi, as it
does in various bacteria, algae, and plants (reviews, Harding & Shropshire,
1980; Rau, 1983; Rau & Schrott, 1987). This phenomenon (photo-
carotenogenesis) does not occur in many fungi, for example, in Blakeslea
trispora (Sutter, 1970).
Photocarotenogenesis presumably protects the cells against the deletereous
effects of very strong illuminations and avoids waste in the dark or in dim light,
but is of limited interest to biotechnologists, because bright illumination is not
practical in large cultures and the final concentrations of carotenoids are not
very high. The examples given in Table 1 prove this point, but should not be
compared with each other, because they were obtained under very different
conditions, most under non-saturating light exposures.
3.2 Sexual Interaction
In many species of the Mucorales the contact zone of mycelia of opposite sex
becomes bright yellow (Blakeslee, 1904). The sexual stimulation is easily
observed in mated cultures (mixed cultures of strains of opposite sex) (Barnett
et al., 1956; Anderson et al., 1958). The beta-carotene content increases 5-15
times over the level found in separate cultures, according to various reports.
Sexual stimulation in the Mucorales occurs in the absence of physical contact
when the interacting hyphae are separated by a membrane (Burgeff, 1924). An
enhanced carotenogenesis occurs in strains of Blakeslea trispora grown under
such conditions (Barnett et al., 1956). A race between chemists of several
major biotechnological companies led to the isolation of chemicals, the
trisporic acids, present in mated cultures and able to stimulate carotenogenesis
in single cultures (Caglioti et al., 1966). The trisporic acids are industrially
useless because the increases in p-carotene content do not compensate their
cost.
Table 1
Examples of Photocarotenogenesis in some Fungi
Name of Coloured carotenoids Reference

fungus in illuminated cultures
(Ilg/gt Main component
Aspergillus giganteus 170 tJ-Carotene EI-Jack et al., 1988
Fusarium aquaeductuum 213 ~eurosporaxanthin Bindl et al., 1970
Gibberella fujikuroi 159 ~eurosporaxanthin Avalos & Cerda-Olmedo, 1987
Neurospora crassa 152 ~eurosporaxanthin Harding et al., 1969
Phycomyces blakesleeanus 550 tJ-Carotene Bergman et al., 1973
Rhodosporidium diobovatum 700 tJ-Carotene Vaskivnyuk & Getman, 1984
Rhodotorula minuta 150 Torulene Tada & Shiroishi, 1982
Verticillium agaricinum 445 Torulene Valadon & Mummery, 1973
a The numerical values are the total concentrations of the carotenoids detected in
illuminated cultures, in Ilg/g dry mycelial weight.
32 E. Cerd4-01medo
Since trisporic acids are made from p-carotene (Austin et a/., 1970), the
activation of carotenogenesis by trisporic acids in the Mucorales provides a
regulatory loop for increased sexual stimulation. Another likely role of the
newly synthesized p-carotene is to make sporopollenins, tough polymers found
in the cell walls of the zygospores (Gooday et a/., 1973) and of other
structures. Lack of trisporic acids and sporopollenins explains the sexual
impotence of mutant Mucorales devoid of p-carotene (Sutter, 1975; Khabrova
& Zhdanov, 1979), but not that of the carS mutants, which overproduce it. The
trisporic acids have no effect on carotenogenesis or morphogenesis in fungi
other than the Mucorales.
3.3 Culture Conditions
The accumulation of carotenoids is influenced by all sorts of variations in the

culture conditions, but usually only to a minor extent. The best temperatures
for carotenogenesis in B/akeslea trispora and Phycomyces b/akes/eeanus are
about 26°C. Acetate, leucine, and other amino acids increase the p-carotene
content of Phycomyces, presumably because they can be converted directly to
hydroxymethylglutaryl-coenzyme A (Lilly et al., 1960; Cerda-Olmedo, 1987).
Many of the conditions tried by Lilly and co-workers allowed Phycomyces to
exceed 2 mg p-carotene per g dry weight.
The optimization of the media for the industrial production of p-carotene
with B/akes/ea trispora was the central concern of a series of successful
investigations (Anderson et a/., 1958; Ciegler et al., 1959a, b, 1962). The best
media are rich in various grain products, vegetable oils, and hydrocarbons
(kerosene, preferably deodorized).
Other fungi have received much less attention. A medium with tomato
pressings increased the astaxanthin content of Phaffia rhodozyma to 0·8 mg/g
(Johnson & Lewis, 1979). An appropriate carbon-to-nitrogen ratio and a
surface-active agent allowed Neurospora crassa to make over 2·5 mg caroten-
oids per g dry weight (Krzeminiski & Quackenbush, 1960).
4 CHEMICAL STIMULATION
Mycelial colour changes facilitate the detection of chemicals that influence

carotenogenesis in the fungi. The many inhibitors and the weak activators do
not interest us now. The accumulation of p-carotene in the Mucorales is
stimulated by p-ionone (Mackinney et a/., 1952, 1953; Reyes et a/., 1964) and
retinol (Eslava et a/., 1974; Feofilova & Bekhtereva, 1976). These molecules
are identical to p-carotene in the p-ring and the adjacent atoms, but they are
not incorporated into it (Engel et a/., 1953; Eslava et a/., 1974). Some
variations of these structures are active, and others are not (Mackinney et a/.,
1952; Engel et a/., 1953; De la Concha & Murillo, 1984; Bejarano et a/., 1988).
p-Ionone and retinol are kept away from industrial fermentors by their high
prices. In the production of fJ-carotene by Blakeslea trispora, fJ-ionone may be

replaced by citrus pulps (alkali-heated peels, seeds, and rags) (Ciegler et al.,
1963b) and by limonene and citrus oils that contain limonene (Ciegler et al.,
1963c).
Many aromatic compounds activate carotenogenesis in Phycomyces. These
compounds stimulate the pathway and inhibit phytoene dehydrogenase to
different extents. Predominance of the stimulation (observed with dimethyl
phthalate, 1,2-dimethoxy-4-propenylbenzene, 4-bromoveratrol, veratrol, and
others) results in vast accumulations of fJ-carotene. Mixtures of fJ-carotene and
partially desaturated acyclic intermediates result when both activities are
prominent (cinnamic alcohol, eugenol, safrol, 1,3-dimethoxybencene, 4-
methoxyphenol, etc.) (Cerda-Olmedo & Hllttermann, 1986). These intermedi-
ates are undesirable in the industrial production of fJ-carotene, but may be of
interest for research and other uses.
The simple comparison of the structural formulae is insufficient to establish
functional analogy between chemical activators. A genetic test for functional
analogy has been made possible by the availability of many mutants of
Phycomyces blakesleeanus with various defects in carotene synthesis and its
regulation. If mutants of a certain gene are activated by a chemical but not by
another, the mechanisms of action of the two chemicals differ in their need for
the product of that gene. The activators were thus classified into four groups:
light, trisporic acids, fJ-ring containing chemicals (retinol, fJ-ionone), and
phenols (dimethyl phthalate, veratrol) (Bejarano et aI., 1988; Bejarano &
Cerda-Olmedo, 1989). It is interesting to note that fJ-ionone, trisporic acids,
and phenols are not analogues of each other.
A more classical way to establish functional groups is to determine
synergisms. Thus, the joint application of activators belonging to different
groups leads to higher carotene contents than may be attained with either
alone (Govind & Cerda-Olmedo, 1986; Bejarano et al., 1988).
2,6,6-Trimethyl-1-acetylcyclohexene and some related compounds satisfac-
torily replace fJ-ionone in industrial media for Blakeslea. Other chemicals
(isoniazid, iproniazid) are synergistic with them (Ninet et aI., 1969). Many
herbicides are inhibitors of carotenogenesis, but propanil (N-(3,4-
dichlorophenyl)propanamide) is an activator (Feofilova et al., 1982).
The addition of microbial biomass to mated Blakeslea cultures stimulates
carotenogenesis (Ciegler et al., 1964). About equally effective are the spent
Blakeslea mycelia, after the extraction of fJ-carotene in previous fermenta-
tions, and cells of various bacteria, yeasts, and fungi.
Although over 100 chemical activators of carotenogenesis are known, most
are relatively ineffective, or too expensive, or too toxic for practical applica-
tion. The search for activators is far from complete, and the spot test
(Cerda-Olmedo & Hllttermann, 1986) should permit the rapid screening of
new ones. The results for one fungus are often invalid for another: fJ-ionone
stimulates carotenogenesis in Blakeslea and Phycomyces, but inhibits it in
Gibberella (Avalos & Cerda-Olmedo, 1986); isoniazid is active for Blakeslea
but not for Phycomyces.
34 E. Cerda-Olmedo
5 GENETICAL STIMULATION
5.1 Phycomyces
The genetic analysis of carotenogenesis in Phycomyces blakesleeanus (review,

Cerda-Olmedo, 1987) was made possible by the study of its sexual cycle and its
genetics (review, Eslava, 1987), the isolation of numerous car mutants with
alterations in the biosynthesis of carotene and its regulation (review, Cerda-
Olmedo, 1985) and the design of procedures to bypass their sexual distur-
bances (Roncero & Cerda-Olmedo, 1982).
Sexual stimulation can be obtained in a single mycelium with a simple
genetic trick. Mycelia containing a mixture of nuclei of opposite sex (inter-
sexual heterokaryons) accumulate about 10 times more p-carotene than
mycelia containing one or the other kind of nuclei (homokaryons) (Murillo &
Cerda-Olmedo, 1976). Intersexual heterokaryons express full sexual stimula-
tion of carotenogenesis: their p-carotene cannot be further increased by the
addition of trisporic acids (Govind & Cerda-Olmedo, 1986). When the
constituent nuclei have different genetic backgrounds, the intersexual hetero-
karyons tend to sector out, i.e. to produce mycelia of various nuclear
proportions and even pure homokaryons. Consaguineous strains, such as the
ones developed by Alvarez & Eslava (1983), reduce this danger, but the best
solution is a system of balanced lethals, that is, the induction of a recessive
lethal mutation in each of the nuclei. This is much easier than it sounds
(Murillo et al., 1978).
Attempts to block carotenogenesis in Phycomyces result in a general
activation of the pathway that tends to maintain the concentration of the final
product, p-carotene. Thus, the addition of an enzyme inhibitor (such as
diphenylamine, which blocks phytoene dehydrogenation) causes an accumula-
tion of intermediates (phytoene and other partially desaturated acyclic
carotenes); the p-carotene content is affected only at high inhibitor concentra-
tions. The same is true for inhibitors of lycopene cyclization (nicotine,
2-(4-chlorophenylthio)triethylamine), with accumulation of lycopene and
y-carotene, and for mutations that reduce either enzyme activity. Full chemical
or mutational blocks cause a 50-fold increase in carotene content; the product,
of course, is no longer fJ-carotene, but the untransformed intermediate,
phytoene or lycopene (Eslava & Cerda-Olmedo, 1974; Murillo & Cerda-
Olmedo, 1976; Murillo et al., 1981).
This end-product inhibition of the pathway is mediated by p-carotene and
two gene products (Cerda-Olmedo, 1987; Bejarano et al., 1988), as sum-
marized in Fig. 2. Retinol and beta-ionone, which compete with beta-carotene,
and recessive carS mutations, which destroy a dispensable gene product,
abolish the inhibition and lead to high p-carotene contents. The best carS
mutants contain 100 times more fJ-carotene than the wild type.
Other superproducing strains, containing up to 20 times more p-carotene
than the wild type, carry recessive mutations in another gene, carD. Mutations
phytoene
~
p8
p8
.. \
pB carRA carB
pB I
pR
pR .. ~
f)-carotene
~ \
TRANSCRIPTION
I
I
,
\
\ I
I
~" ~ ~ ///
/
y
pS pA
I carD
---C±l-PD~
--8-PC~
9=1===F
care
carS
Fig. 2. The genetics of carotenogenesis in Phycomyces blakesleeanus (Cerda-Olmedo,
1987). Four copies of phytoene dehydrogenase (the product of gene carB) and two
copies of lycopene cyclase (one of the products of gene carRA, closely linked to carB)
convert phytoene into beta-carotene (above). The product of gene carS and a product
of gene carRA combine with beta-carotene to repress the synthesis (center). Two other
genes, care and carD, regulate in opposite ways the action of carS (below).
in gene carS are epistatic over those in gene carD: the double mutants do not
surpass single carS mutants and are, therefore, of no practical value. There are
superproducing strains that considerably exceed the J3-carotene content of the
carS mutants, but they have not yet been published.
The highest published J3-carotene level in Phycomyces blakesleeanus
corresponds to intersexual heterokaryons with carS mutations and balanced
lethals (Murillo et aI., 1978). They contain 25 mg J3-carotene per g dry weight,
some 500 times more than the wild type. Their genetic make-up provides
constitutive sexual activation and removal of end-product inhibition, and
therefore, they do not respond to retinol or J3-ionone. They do not respond to
light or dimethyl phthalate either, although no provision is made for
endogenous activation of the mechanisms normally triggered by these agents.
36 E. Cerda-Olmedo
When certain Phycomyces strains are grown in the dark, most of the
fJ-carotene appears in a red complex, similar to the main carotenoprotein of
many plants, which stabilizes fJ-carotene and changes its colour in an attractive
way. These strains contain a carS mutation and a cytoplasmic carE mutation
(De la Concha & Murillo, 1984). They accumulate about 100 times more
fJ-carotene than the wild-type and no attempt has been made to improve
their yield.
Some genetic constructs contain considerable amounts of pathway inter-
mediates. Lycopene is accumulated by carR mutants, defective in lycopene
cyclase. Constitutive sexual activation and partial removal of end-product
inhibition led to strains with about 15 mg lycopene per g dry weight (Murillo et
al., 1978). A high phytoene content (9 mg/g dry weight) was obtained by
growing a carB mutant, defective in phytoene dehydrogenase, with dimethyl
phthalate in the light (Bejarano & Cerda-Olmedo, 1989). Other intermedi-
ates can only be produced as part of carotene mixtures: phytofluene and
~-carotene in some leaky carB mutants (Eslava & Cerda-Olmedo, 1974;
Bejarano et al., 1987); y-carotene in certain heterokaryons (De la Guardia et
al., 1971).
5.2 Other fungi
The genetic analysis of carotenogenesis is feasible in many fungi, but has been
carried out in few, and only to limited extents (see, for example, Khabrova &
Zhdanov, 1979, for Blakeslea trispora; Harding & Turner, 1981, for
Neurospora crassa; Will et al., 1984, for Ustilago violacea; Avalos &
Cerda-Olmedo, 1987, for Gibberella fujikuroi). Certain mutants of Gibberella
are deep orange even when grown in the dark and contain up to 1.7 mg
neurosporaxanthin per g dry weight, but they are not analogous to Phycomyces
mutants because there is no end-product regulation in Gibberella.
6 INDUSTRIAL PRODUCTION
6.1 Blakeslea
The only fungal production of carotenoids that has been developed to the
industrial stage is that of fJ-carotene with mated cultures of Blakeslea trispora.
After detailed studies of media and additives, Ciegler and his co-workers at the
US Department of Agriculture scaled up their cultures to a volume of 11 litres
(Ciegler et al., 1963a). The process has been reviewed in detail (Ciegler, 1965;
Hanson, 1967). The production phase (Ciegler et al., 1963a) consists in the
joint culture at 28°C for 3 days of mycelia of two wild types of opposite sex in
liquid medium (20 g ground maize, 40 g cotton-seed meal, 30 g white grease or
vegetable oil, 30 g deodorized kerosene, 50 g citrus molasses, and 0·2 mg
thiamine·HCI per litre). The fermented product contains about 17 mg fJ-
carotene per g solids or about 1 g/litre medium. An antioxidant, ethoxyquin, is

added at the end to stabilize the carotene. The two strains are cultivated
separately before inoculation into the production vessels.
Several industry-affiliated research groups have improved the process
(review, Ninet & Renaut, 1979, and the patents referred to therein). The
procedure presented by a Rhone-Poulenc team in France (Ninet & Renaut,
1979) has two successive mated cultures. First, 0·4litres of separate cultures of
each sex are inoculated into 120 litres of medium (70 g corn steep, 50 g corn
starch, 0·5 g KH2 P04, 0·1 g MnS04, 10 mg thiamin· HCI per litre tap-water)
and incubated for 40 h at 26°C with agitation and aeration. Then, 32 litres of
the first culture are inoculated into 320 litres of another medium (70 g distillers
solubles, 60 g cornstarch, 30 ml soya oil, 30 ml cotton-seed oil, 20 ml kerosene,
0·35 g ethoxyquin, 0·6 g isoniazid, 0·2 g MnS04, 0·5 mg thiamine·HCI, per litre
at pH 6·3) and incubated at 26°C for 185 h with agitation and aeration. After
48 h, 1 g p-ionone, 5 ml kerosene, and 42 g glucose are added per litre (the
glucose solution is continuously injected to the end of the culture). The
production should be about 30 mg p-carotene per g dry weight or about
3 g/litre.
Other sources of carbon and nitrogen have received consideration. One of
them is whey, a subproduct of cheese manufacture (Lampila et al., 1985b,c).
Blakeslea allows the production of lycopene and other intermediate products
in the presence of appropriate enzyme inhibitors (Ninet et al., 1969; Hsu et al.,
1972; Ninet & Renaut, 1979).
6.2 Phycomyces
Production has been studied only under laboratory conditions. The genetic
work was carried out on minimal agar; the wild type contains about 0·040 mg
p-carotene per g dry weight and the superproducing strains described above,
about 25 mg (Murillo et al., 1978). The mycelial dry weight is 10 g/litre in both
cases. We have observed that various simple media, some composed of cheap
subproducts of biological industries, increase the biomass to about 40g dry
mycelia per litre and maintain its p-carotene concentration. Carotenogenesis
suffers if the cultures are agitated, whether they are wild type (Lilly et al.,
1960) or superproducers, and therefore surface cultures are the most
productive.
6.3 Application
Industrial purification of p-carotene (Ninet et al., 1979) is complicated and

expensive, and may not be necessary in many practical applications. For use in
animal feeds, the carotene-rich mycelium may be dried, blended with various
polymers, extruded into filaments, and powdered; the p-carotene in such
preparations survives storage at 41°C for 1 year (Gogek, 1968).
38 E. Cerda-Olmedo
7 THE PROSPECTS
The p-carotene content of Blakeslea and Phycomyces mycelia has been

enormously increased through optimization of culture conditions and genetic
breeding, respectively. Both lines of research are far from exhausted and
should lead to further production gains.
The production of xanthophylls (oxidated carotenoids) would be feasible
with certain fungi, particularly after yield improvements brought about by new
research. Phaffia rhodozyma offers a good start (Johnson et al., 1978; Johnson
& Lewis, 1979) and effectively gives the desired pigmentation to pen-reared
salmonids (Johnson et al., 1977).
Genes for carotenoid biosynthesis may eventually be inserted and expressed
in colourless, but genetically malleable organisms, such as Saccharomyces
cerevisiae. Carotenoids would then become important subproducts of well-
established industries. More likely is the application of molecular genetics to
strain improvement in organisms that already produce carotenoids.
Neurospora crassa offers, of course, magnificent prospects for all sorts of
genetic manipulations. Efficient transformation of Phycomyces blakesleeanus
with exogenous DNA (Arnau et al., 1988; Suarez & Eslava, 1988) may now
be combined with the more traditional approaches.
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Vol. 1, ed. H. J. Peppler & D. Perlman. Academic Press, New York, pp.
529-44.
Ninet, L., Renaut, J. & Tissier, R. (1969). Activation of the biosynthesis of carotenoids
by Blakeslea trispora. Biotechnol. Bioeng., 11, 1195-210.
Rau, W. (1983). Photoregulation of carotenoid biosynthesis. In Biosynthesis of
isoprenoid compounds, ed. J. W. Porter & S. L. Spurgeon. John Wiley, New York,
pp. 123-57.
Rau, W. & Schrott, E. L. (1987). Blue light control of pigment biosynthesis-
Carotenoid biosynthesis. In Blue Light Responses, Vol. I, ed. H. Senger. CRC
Press, Boca Rat6n, Florida, pp. 43-63.
Reyes, P., Chichester, C. O. & Nakayama, T. O. M. (1964). The mechanism of
beta-ionone stimulation of carotenoid and ergosterol biosynthesis in Phycomyces
blakesleeanus. Biochim. Biophys. Acta, 90, 578-92.
Roncero, M. I. G. & Cerda-Olmedo, E. (1982). Genetics of carotene biosynthesis in
Phycomyces. Curro Genet., 5, 5-8.
Suarez, T. & Eslava, A. P. (1988). Transformation of Phycomyces with a bacterial gene
for kanamycin resistance. Molec. Gen. Genet., 212, 120-23.
Sutter, R. P. (1970). Effect of light on beta-carotene accumulation in Blakeslea trispora.
1. Gen. Microbiol., 64, 215-21.
Sutter, R. P. (1975). Mutations affecting sexual development in Phycomyces blakes-
leeanus. Proc. natn. Acad. Sci. USA, 72, 127-30.
42 E. Cerda-Olmedo
Tada, M. & Shiroishi, M. (1982). Mechanism of photoregulated carotenogenesis in

Rhodotorula minuta. I. Photocontrol of carotenoid production. Plant Cell Physiol.,
23,541-7.
Valadon, L. R. G. & Mummery, R. S. (1973). Effect of certain inhibitors of
carotenogenesis in Verticillium agaricinum. Microbios, 7, 173-80.
Vaskivnyuk, V. Y. & Getman, E. I. (1984). Biosynthesis of carotenoids in light in
continuous cultures of the yeast Rhodosporidium diobovatum (in Russian). Priklad.
Biokhim. Mikrobiol., 20, 480-83.
Will, O. H., Ruddat, M., Garber, E. D. & Kezdy, F. J. (1984). Characterization of
carotene accumulation in Ustilago violacea using high-performance liquid chroma-
tography. Curro Microbiol., 10,57-64.
Chapter 4
MICROBIAL PRODUCTION OF CAROTENOIDS OTHER

THAN Ii-CAROTENE
H. J. NELIS & A. P. DE LEENHEER
Laboratories of Medical Biochemistry and Clinical Analysis, Faculty of
Pharmaceutical Sciences, State University of Ghent, B-9OOO Ghent, Belgium
I GENERAL ASPECTS
1 HISTORY
1.1 Discovery of Carotenoids in Nature
The early discovery of carotenoids in living organisms is obviously attributable

to the conspicuous appearance of these yellow, orange or red pigments. In
1831 Wackenroder prepared 'carotene' in crystalline form (Isler, 1971),
whereas xanthophylls were first isolated from autumn leaves by Berzelius in
1837 (Isler, 1971). Following the main-classical chromatographic experiments
of Tswett at the beginning of this century (Isler, 1971), it was soon realized
that there existed a complex group of carotenoids in nature. The next decades
indeed saw the isolation and structural elucidation of a large variety of new
derivatives. More recently, the advent of modern spectrometric techniques and
the refinement of separation methods have resulted in the ongoing discovery of
more new structures, the determination of their absolute configurations and
the establishment of routes for their partial or total synthesis. Concurrently,
the biochemistry of carotenoids in plants and animals has gradually been
unravelled (Goodwin, 1980, 1984).
1.2 Early Observations on the Occurrence of Carotenoids in

Micro-organisms
The occurrence of carotenoids in micro-organisms was first reported about 100

years ago, when a number of pigmented non-photosynthetic bacteria (quoted
in Ingraham & Baumann, 1934) and fungi (quoted in Valadon, 1976) were
isolated. A more systematic survey of carotenoid-producing non-
photosynthetic bacteria was conducted in the 1930s (Ingraham & Baumann,
43
44 H. J. Nelis & A. P. De Leenheer
1934). Pioneering work on photosynthetic bacteria was carried out by Van Niel
& Smith (1935).

(Davies, 1976; Moss & Weedon, 1976)
Chemically, carotenoids are polyenes consisting of a number of isoprenoid

units (usually 8) arranged in such a way that the two central methyl groups (20
and 20', Fig. 1) are in the 1,6 position, while all remaining substituents are
positioned 1,5 relative to each other. A series of conjugated double bonds
constitutes a chromophore of variable length, resulting in the characteristic
yellow to red colors (absorption maxima 400-500 nm). Figure 1 presents the
five basic (no substituents) structures of which all compounds pertinent to this
chapter are derived. Full structures of the latter are depicted in Fig. 2, together
with their trivial (common), semi-systematic and systematic names (Table 1,
legend to Fig. 2).
Unsubstituted carotenoid hydrocarbons, e.g. lycopene, {:I-carotene, and
torulene, are commonly designated as 'carotenes'. The collective term xanth-
ophylls covers oxygenated derivatives, including alcohols (e.g. lutein and
...
1
20
r~
2
20
,.'
.' 3'
~ 2'
B 3
20'
~. 2' 4
f
Fig. 1. Basic structures of carotenoids of which the compounds of interest (Fig. 2,

Table 1) are chemically derived. Systematic IUPAC names in parentheses. 1, lycopene
('IjI,'IjI-carotene); 2,y-carotene (p,'IjI-carotene); 3,p-carotene (p,p-carotene); 4,a-
carotene (P, e-carotene); 5, retrodehydro-p-carotene (4'5' -didehydro-4,5'-retro-p, p-
carotene).
Microbial Production of Carotenoids 45
1 ""
•• OH
2
HO
3
-",
HO
4
0 0
5
0
OH
6
HO
9
OCH3
10
11 ...,
0
Fig. 2. Structural formulae of carotenoids of interest. See Table 1 for explanation.
zeaxanthin), epoxides (e.g. violaxanthin), ethers (e.g. spheroidene), ketones

(e.g. canthaxanthin, astaxanthin, spheroidenone) and acids such as torular-
hodin. Rhodoxanthin is an example of a retrocarotenoid, in which all single
and double bonds of the conjugated polyene system have shifted by one
position. The modern IUPAC systematic nomenclature (IUPAC, 1971) is
based on a designation of the end groups of the molecules, using such prefixes
as 1jJ (acyclic end-group), f3 or E (cyclohexene end groups) (indicated in Fig. 1).
Most natural carotenoids possess the all-trans configuration, although the
genuine presence of cis-isomers has been documented, e.g. in photosynthetic
bacteria (Koyama et al., 1983). If a compound contains chiral centers (as in
zeaxanthin and astaxanthin) the absolute configuration of the optical isomer(s)
found in nature is also mentioned in Table 1.
Table 1 ~
Structures and Nomenclature of Carotenoids of Interest.
Structure Trivial name Basic structure Semi-systematic name Systematic name (IUPAC)
(Fig. 1)
1. Compounds of immediate biotechnological interest (produced by the micro-organisms listed in Table 2, I)

A. Carotenes
1 Lycopene 1 Lycopene 1/J, 1/J -Carotene
B. Hydroxy-xanthophyUs
2 Lutein 4 3,3' -DihydroxY-ll'-carotene (3R, 3'R, 6'R)-jJ,E-Carotene-
3,3' -diol. ~
3 Zeaxanthin 3 3,3' -Dihydroxy-{:j -carotene (3R, 3'R)-jJ,jJ-Carotene-3,3' -diol ~
C. Monocyclic ketocarotenoids
~
4 2 4-lCeto-y-carotene jJ,1/J-Caroten-4-one ~
D. Bicyclic ketocarotenoids Ro
5 Canthaxanthin 3 4,4' -Diketo-jJ-carotene jJ,jJ-Carotene-4,4' -dione ~
6 Astaxanthin 3 3,3' -Dihydroxy-4 ,4' -diketo- 3,3' -Dihydroxy-jJ,jJ-carotene-4,4'- ~
jJ-carotene dione (3S, 3'S or 3R, 3'R)
E. Retrocarotenoids ~
t"-
7 Rhodoxanthin 5 3,3' -Diketoretrodehydro- 4' ,5' -Didehydro-4,5' -retro-jJ,jJ-ca- §;:-
jJ-carotene rotene-3,3' -dione
2. Compounds of possible future biotechnological interest (produced by the micro-organisms listed in Table 2,11)
~
8 Torulene 2 3' ,4' -Dehydro-y-carotene 3' ,4' -Didehydro-jJ, 1/J-carotene
9 Torularhodin 2 16' -Carboxyl-3' ,4' -dehydro- 3' ,4' -Didehydro-jJ, 1/J-caroten-16'-
y-carotene oic acid
10 Spheroidene 1 1-Methoxy-1 ,2,7' ,8' -tetrahy- 1-Methoxy-3,4-didehydro-1 ,2,7' ,
dro-3,4-dehydrolycopene 8' -tetrahydro-1/J, 1/J -carotene
11 Spheroidenone 1 1-Methoxy-2-keto-1,2,7'8'-te- 1-Methoxy-3,4-didehydro-1,2,7' ,8'-
trahydro-3 ,4-dehydrolycopene tetrahydro-1/J, 1/J -caroten-2-one
The extended chromophore in the carotenoid structure points to a biological

significance for these pigments in micro-organisms, by suggesting photosensi-
tive and antioxidant properties. In algae and photosynthetic bacteria, caroten-
oids participate as secondary light-harvesting compounds in the photosynthetic
process (Mathis & Schenck, 1982) and exert photoprotective effects as well
(Burnett, 1976). As antioxidants they protect vulnerable tissues against the
deleterious effects of singlet oxygen and free radicals (Krinsky, 1979). Similar
(photo )-protective effects have been invoked in non-photosynthetic bacteria
(Mathews & Krinsky, 1965; Mathews-Roth & Krinsky, 1970; Mathews-Roth et
al., 1974), although conflicting evidence exists (Schwartzel & Cooney, 19740;
Dieringer et al., 1977).
4 INDUSTRIAL APPLICATIONS
The economic significance of carotenoids has been extensively reviewed (KHiui

& Bauernfeind, 1981; Marusich & Bauernfeind, 1981; Simpson et al., 1981;
KHiui, 1982). There is a continuously growing market for carotenoids as food
colorants (KHiui & Bauernfeind, 1981) and pigmenters in animal feeds
(Marusich & Bauernfeind, 1981; Simpson et al., 1981). Poultry feeds based on
agricultural waste products have to be supplemented with pigments in order to
impart a desirable yellow-orange color to egg yolks and/or the skin of broiler
chickens (Marusich & Bauernfeind, 1981). Optimal yolk color ensues from the
combination of a yellow base pigment (e.g. lutein, p-apo-8'-carotenoic acid
ethyl ester) with a small quantity of an orange-red one, preferably a
ketocarotenoid (canthaxanthin) or zeaxanthin.
The addition of ketocarotenoids (astaxanthin or canthaxanthin) to fish feeds
also intensifies the reddish colour of the flesh of the salmon and the trout
(Simpson et al., 1981). So far, the application of natural sources of carotenoids
remains confined to animal feeds (Marusich & Bauernfeind, 1981; Simpson et
al., 1981). Industrially produced natural carotenoids are mainly derived from
vegetable sources, including marigold flowers, alfalfa, red peppers and annatto
(Marusich & Bauernfeind, 1981).
5 PRODUCING MICRO-ORGANISMS
There is a wealth of literature on the occurrence of carotenoids in algae, fungi

and bacteria, the emphasis being on structural studies and the elucidation of
the pertinent biosynthetic pathways. This information has been exhaustively
reviewed in an authoritative book by Goodwin (1980).
Our survey of carotenoid-producing organisms is limited to those species
which show biotechnological potential, i.e. which could be considered for the
development of a commercially exploitable, natural source of pigments. The

selection of useful organisms (Table 2) was made on the basis of three criteria:
(a) The presence in the organism of carotenoids of interest (cf. the
limitative list in Table 1, p-carotene excluded).
(b) The already established industrial use of a given strain. So far this has
only been the case for green algae. However, most organisms included
in Table 2 have not (yet) found such application, but do show promise to
be developed for this purpose. Specifically, this could mean that
attempts have already been made, are currently being made or are likely
to be made in the future to optimize the pigment levels in these
organisms. In order to justify the efforts, the carotenoids involved
should be particularly valuable, difficult to synthesize and be absent or
rare in cheaper and more accessible natural sources. The recent
commercial exploitation of micro-algae as a source of p-carotene proves
that there is a potential market for natural carotenoids, even if the
(cheaper) synthetic equivalents exist (cf. Chapter 2).
(c) The current interest in a number of carotenoid containing organisms for
reasons other than their pigment composition. Alternative applications
include the production of biomass, single cell oil or lipid, (other) fine
chemicals, or the purification of waste waters. However, the presence of
often high quantities of carotenoids in those particular organisms could
make them attractive for commercial production of these useful 'by-
products' as well.
6 BIOSYNTHESIS (for a review, see Spurgeon & Porter, 1983)
A first series of steps in the formation of carotenoids belongs to the

well-known mevalonate pathway, the general biosynthetic scheme of all
isoprenoid compounds. Starting from acetate (acetyl CoA), activated isoprene
units undergo a sequence of head-to-tail condensations, leading to geranylger-
anyl pyrophosphate (Cw). From this point on, all subsequent steps are unique
to the biosynthesis of carotenoids. Head-to-head condensation of 2 C20 units
yields the key intermediate phytoene, the first colorless carotenoid. Through a
series of stepwise dehydrogenations, phytoene is converted to lycopene, from
which all other carotenoids can eventually be derived. The biosynthetic
interrelationship of the derivatives pertinent to this chapter is illustrated in
Fig. 3.
7 REGULATION OF BIOSYNTHESIS
The biosynthesis of carotenoids by micro-organisms is under genetic control,

but is also influenced by various environmental factors (Spurgeon & Porter,
1983).
Table 2
Micro-organisms of Potential Practical Interest for the Production of
Carotenoids
Species Carotenoid(s) produced
1. Micro-organisms specifically designed as pigment sources
1. Sources of carotenes
A. Fungi
Blakeslea trispora Lycopene
B. Non-photosynthetic bacteria
Streptomyces chrestomyceticus, Lycopene
subsp. rubescens
2. Sources of xanthophyllsa
A. Green algae
Spongiococcum excentricum Lutein
Chlorella pyrenoidosa Lutein
B. Fungi
Dacrymyces deliquescens Lutein
C. Non-photosynthetic bacteria
Flavobacterium sp. Zeaxanthin
Streptomyces chrestomyceticus, Unidentified xanthophylls
var. aurantioideus
Mycobacterium phlei Unidentified xanthophylls
3. Sources of monocyclic ketocarotenoids
A. Non-photosynthetic bacteria
Deinococcus radiophilus-radiodurans- Derivatives of 4-keto-
radiopugnans y-carotene
Mycobacterium smegmatis Derivatives of 4-keto-
y-carotene
4. Sources of bicyclic ketocarotenoids
A. Cyanobacteria
Anabaena variabilis Canthaxanthin
Aphanizomenon flos-aquae Canthaxanthin
Nostoc commune Canthaxanthin
B. Green algae (N-deficiency)
Dictyococcus cinnabarinus Canthaxanthin
Haematococcus pluvialis Astaxanthin
C. Fungi/yeast
PhajJia rhodozyma Astaxanthin
D. Non-photosynthetic bacteria
Brevibacterium KY -4313 Canthaxanthin
Rhodococcus maris / Mycobacterium Canthaxanthin
brevicale 32-MCT
Mycobacterium lacticola Astaxanthin
Brevibacterium 103 Astaxanthin
5. Sources of retrocarotenoids
Pseudomonas extorquens Rhodoxanthin
II. Micro-organisms designed for applications other than
pigment production
A. Fungi/yeast
Rhodotorula, Rhodosporidium sp. Torulene,
(red yeasts) torularhodin
B. Non-photosynthetic bacteria
Methylotrophs Miscellaneous
C. Photosynthetic bacteria
Rhodobacter capsulatus Spheroidene,
Spheroidenone
a Ketocarotenoids excluded.
OCH,
~
1 IS'
~ JA
lc .--e-~
3
Fig. 3. Biosynthetic relationship between the carotenoids of interest. 1, All-trans-

phytoene (actual precursor of lycopene may be 15,15'-cis-phytoene); 2, lycopene; 3,
spheroidene; 4, spheroidenone; 5, y-carotene; 6, 4-keto-y-carotene; 7, torulene; 8,
torularhodin; 9, /J-carotene; 10, zeaxanthin (3R,3'R); 11, rhodoxanthin; 12, canth-
axanthin; 13, astaxanthin (3S, 3'S); 14, a-carotene; 15, lutein (3R, 3'R, 6'R). Reactions
involved: A, stepwise desaturation (dehydrogenations). B, water addition at 1,2 double
bond + dehydrogenation (C-3 and C-4) + methylation of hydroxyl group at C-l. C, keto
group addition at C-2. Mechanism not established. 0, cyclization of 1 or 2 terminal
end(s) of lycopene, yielding 1/J-end group (/J, tp: y-carotene, 5), 2/J-end groups (/J,/J:
/J-carotene, 9) or 1/J- and Ie-end group (/J,e: a-carotene, 14), respectively. E,
dehydrogenation at C-3' and C-4'. F, terminal oxidation (-CH3--CH20H--
CHO--COOH). G, keto group addition at C-4. Probably hydroxylation followed by
dehydrogenation. H, hydroxylation at C-3 and C-3'. Requires molecular oxygen.
Mechanisms unknown. I, Keto group addition at C-4 and C-4'. Probably hydroxylation
followed by dehydrogenation. J, Mechanism uncertain. Probably attack of OH+ on
zeaxanthin at C-5, loss of H+ at C-4', loss of water and dehydrogenation of hydroxyl
groups at C-3 and C-3'.
7.1 Environmental Factors
Both the pigment patterns and the amount of carotenoids in the cells may be
altered by changes in the composition of the growth medium and by the
influence of physical factors such as light, temperature and oxygen. Variation
of the growth conditions of an organism with the aim to optimize its carote-
noid content sometimes has a rational basis (e.g. generation of biosynthetic
intermediates), but very often is rather empirical. From the numerous effects
reported in the literature (for a compilation, see Goodwin, 1980) only a few
examples have been selected to illustrate how the carotenoid biosynthesis in

certain algae, fungi and bacteria responds to changes in the environment.
7.1.1 Algae
(A) COMPOSITION OF THE GROWTH MEDIUM

Under optimal conditions for proliferation, green algae produce the typical
chloroplastidic carotenoids ~-carotene, lutein, zeaxanthin and various epox-
ides. However, nutritional imbalance, particularly the lack of nitrogen, often
induces the synthesis of secondary extraplastidic (keto )carotenoids, so that the
cultures turn orange or red.
(8) PHYSICAL FACTORS

Under autotrophic conditions, light has in general little or no effect on the
carotenoid production by algae. An interesting effect of oxygen is noted in
some organisms. Anaerobic conditions exclusively lead to carotene formation,
whereas in the presence of oxygen synthesis is primarily directed towards
xanthophylls.
7.1. 2 Fungi and yeast

The nature of the carbon and nitrogen source affects carotenoid formation in
fungi in an unpredictable manner. In general, a high carbon to nitrogen ratio
promotes carotenogenesis. The stimulatory effect of leucine and valine can be
explained in that these amino acids are precursors of ~-hydroxy-~-methyl
glutaryl-CoA, a key intermediate in isoprenoid synthesis. Fluoride is thought
to exert its positive influence at the enzymatic level: this toxic chemical
preferentially inhibits certain phosphatases, which catalyze the hydrolysis and,
hence, cause the depletion of essential phosphorylated intermediates from the
isoprenoid pathway.

A strange effect of temperature on the qualitative carotenoid composition has
been noted in certain red yeasts. At low temperatures, ~- and y-carotene
predominate, while a temperature increase reportedly favors the formation of
torulene and torularhodin. Strict photoregulation of carotenogenesis occurs in
a few, thoroughly investigated species (Rau, 1983). The latter only contain
negligible amounts of carotenoids when grown in total darkness. A brief
exposure to light, in the presence of oxygen, induces de novo synthesis of
carotenogenic enzymes and, accordingly, carotenoid production. The exact
nature of the photoreceptor is still uncertain and the whole process is under
genetic control (see below).
7.1. 3 Non-photosynthetic bacteria

Carotenoid formation is common among hydrocarbon assimilating bacteria.
However, the carotenoid pattern may be different according to whether they
are grown on hydrocarbon or non-hydrocarbon media. Like in fungi, high
carbon to nitrogen ratios commonly increase the pigment yield. Ammonium
salts are preferred nitrogen sources. The concentration of thiamine in the
medium may both affect the nature of the carotenoids formed (cyclic vs
acyclic) and their cellular concentration.

Photoinduction, as outlined for fungi (see Section 7.1.2(B» has been reported
in a number of strains, particularly belonging to the genus Mycobacterium
(Rau, 1983). Illumination of cultures may also alter the qualitative carotenoid
composition, by converting carotenes, normally produced in the dark, into
xanthophylls.
Z 1. 4 Photosynthetic bacteria

Few data are available regarding the influence of nutrients in the medium on
the carotenoid formation by photosynthetic bacteria. A specific example of the
effect of iron will be discussed below (see Section 111,4).

Oxygen and light are key factors controlling carotenogenesis in photosynthetic
bacteria. In general, anaerobic conditions favor the formation of less-oxidized
derivatives (e.g. spheroidene), as opposed to ketocarotenoids (spheroide-
none), which are produced in the presence of oxygen.
7.2 Inhibitors (Ninet et aI., 1969; Spurgeon & Porter, 1983)
Compounds inhibiting the normal biosynthesis of carotenoids can be classified

in two groups. The first group consists of inhibitors that cause the accumula-
tion of early intermediates in the pathway, particularly the colorless phytoene.
These compounds, e.g. diphenylamine, are very useful academically to
elucidate the biosynthetic schemes, but obviously of no interest for practical
application. Other inhibitors preferentially block later reactions in the path-
way, notably the cyclization of lycopene. Examples of these selective inhibitors
include nicotine and CPTA (2-(4-chlorophenylthio )-triethylamine) and other
substituted amines, as well as nitrogenous heterocyclic bases (e.g. imidazole).
7.3 Genetic Control
The biosynthesis of carotenoids is under the control of different genes that

code for the individual enzymes in the pathway. The basis of the photoregula-
tion in fungi and non-photosynthetic bacteria is also genetic; the actual control
probably takes place at the transcription level in the protein synthesis (Rau,
1983). Illumination may either cause the inactivation of a repressor (derepres-
sion) or, alternatively the production of an inducer. Similarly, repression and
derepression may also be involved in the environmental control of
carotenogenesis.
8 STRAIN IMPROVEMENT
Various bacterial mutants have been prepared that have a defect in the
structural gene for one of the carotenogenic enzymes (Spurgeon & Porter,
1983). Three types of mutants with altered carotenoid biosynthesis are of
potential practical interest.
8.1 Blocked Mutants-Mutation to Non-production of Undesirable

Carotenoids
Mutants blocked at the level of cyclization of lycopene to fJ-carotene could be

useful when overproduction of the former is aimed at. The undesired cyclic
compounds will not be formed and consequently lycopene will accumulate.
8.2 Mutational Biosynthesis-New Carotenoid Formation
Mutation may lead to an altered and, from a practical standpoint more

interesting pigment pattern (qualitative strain improvement). Saperstein et al.
isolated various colored mutants following treatment of a Corynebacterium
michiganense strain with uranium salts (Saperstein et al., 1954). While the
predominating pigments in the yellow parent strain were reportedly fJ-
cryptoxanthin and lycopene, an orange mutant and a red back-mutant were
found to contain mostly canthaxanthin and lycopene, respectively. In both
cases the 'uninteresting' fJ-cryptoxanthin was replaced by more valuable
compounds.
8.3 Mutants with Enhanced Pigment Levels
Few examples are known of systematic mutation programs directed towards

increasing the carotenoid content of a given species. This may be in part
attributed to the lack of objective criteria for the selection of mutants with
enhanced pigmentation. It may seem obvious to carry out such selection on the
basis of visual inspection. However, quantitative differences in carotenoid
54 H. 1. Nelis & A. P. De Leenheer
content may not always be so easy to recognize. The subjectivity of the

interpretation of color differences is exemplified by the results of Russian
workers who prepared carotenoid overproducing mutants of Mycobacterium
phlei (Voznyakovskaya & Daraseliya, 1972). The parent strain was described
as 'yellow-orange' (carotenoid content of 1·7 mg/ g), whereas two more
pigmented mutants were judged as 'orange' (4·4 mg/ g) and 'clear orange'
(7·9 mg/g), respectively.
Alternatively, it could be assumed that mutants with enhanced carotenoid
content are more resistant to photodynamic killing. If a correlation indeed
exists between pigmentation and the extent of photoprotection, these mutants
would supposedly show a higher survival rate upon illumination, e.g. in the
presence of a photosensitizer. However, as mentioned before (see Section 3), a
correlation does not necessarily exist between pigment content and the degree
of photoprotection supposedly conferred upon the organism by the carotenoid.
9 FERMENTATION OF CAROTENOID-PRODUCING
MICRO-ORGANISMS-GENERAL
The production of 'interesting' carotenoids (cf. Fig. 2 and Table 1) through

fermentation of selected organisms (Table 2) will be surveyed in the light of
the criteria listed in Section 5: a basic distinction will be made between
organisms which have been or could be specifically designed for pigment
production, and those which are applied otherwise, but may become attractive
as pigment sources as well. How important fermentative production of a given
carotenoid really is may be judged from the availability of alternative natural
sources (briefly discussed in General). For each organism details will be
provided on fermentation conditions, optimization strategies to increase the
pigment content and the overall yields (cell mass, pigment level, fermentation
time). Most of the data are taken from the scientific or the patent literature,
except for part of our own unpublished work with hydrocarbon-utilizing
bacteria. The study of the carotenoids of Rhodobacter capsulatus is part of a
collaborative project coordinated at the State University of Ghent (Professor
Dr W. Verstraete, Faculty of Agricultural Sciences), in which the authors of
this chapter were responsible for the analytical monitoring of the carotenoid
levels in the course of the optimization work.
II FERMENTATION-MICRO-ORGANISMS SPECIFICALLY
DESIGNED FOR PIGMENT PRODUCTION
1 CAROTENES: LYCOPENE
1.1 General
Lycopene, the major carotenoid in tomatoes is mainly used as a food colorant

(KHiui & Bauernfeind, 1981). However, there is evidence indicating that its
presence in poultry feeds enhances the color of the egg yolk (Marusich &
Bauernfeind, 1981).
1.2 Fungi
As discussed in this book (Chapter 3), the fungus Blakeslea trispora (Muco-
rales) synthesizes massive amounts of p-carotene when the two sexual forms of
this species are mixed in the same culture. However, there are several
approaches to block the p-carotene biosynthesis at the lycopene level.
1. 2.1 Composition of the Growth Medium

A medium with a neutral to alkaline pH favors lycopene production by
Blakeslea, whereas at more acidic pH's p-carotene is formed as the pre-
dominating pigment (Ciegler, 1965; Ninet & Renaut, 1979). In a 1963 patent, a
yield of 0·15 g lycopene per liter was claimed, using a broth containing glucose,
oleic acid, fish stick liquor and sodium carbonate (Ciegler, 1965; Ninet &
Renaut, 1979). The latter was added to maintain the pH above 6·6.
1. 2. 2 Use of Inhibitors
As mentioned above, substituted amines and various nitrogenous heterocyclic
compounds block the cyclization of lycopene to p-carotene (Ninet et al., 1969;
Spurgeon & Porter, 1983). The addition of CPTA to the growth medium
indeed results in the accumulation of lycopene in Blakeslea trispora (Hsu et al.,
1972). The maximum yield achieved so far, in a patented 5-day fermentation
process, was 0·7 g lycopene/liter (Ninet & Renaut, 1979). In a less efficient
process using the more readily available triethylamine as an inhibitor, 0·4 g
lycopene/liter was produced in a medium based on cotton-seed, cornflour and
soybean oil (Ninet & Renaut, 1979). Ninet et al. obtained about 1 g lycopene
per liter by adding 0·1 % piperidine to a medium containing, amongst others,
soybean oil, cotton-seed oil, kerosene and p-ionone (Ninet et aI., 1969).
1. 2. 3 Mutants
Mutants could be prepared which lack the ability to convert lycopene to
p-carotene. However, Blakeslea trispora lends itself reluctantly to mutation
(Ninet & Renaut, 1979).
1.3 Non.Photosynthetic Bacteria

Lycopene production (0·5 g/liter in 6 days) by a mutant of Streptomyces
chrestomyceticus, subsp. rubescens has been reported in the patent literature
(Ninet & Renaut, 1979). The medium used contained starch, soybean flour
and ammonium sulfate.
2 XANTHOPHYLLS
2.1 General
Among the xanthophylls, the hydroxylated derivatives lutein and zeaxanthin in
particular are of high interest as key components in natural pigmenters for
poultry (Marusich & Bauernfeind, 1981). Important vegetable sources of this

pigment include alfalfa meal, corn gluten meal and marigold flowers (Marus-
ich & Bauernfeind, 1981). Zeaxanthin acts synergistically with lutein in that it
intensifies the egg yolk color obtained with the yellow base pigment. Some
micro-organisms containing xanthophylls of unspecified nature have also been
tested as pigmenters in poultry feed. However, legal considerations may
restrict their practical application.
2.2 Green Algae
In the early 1960s, alga meals were extensively studied for broiler and yolk
pigmentation, as alternatives to other xanthophyll sources like com and alfalfa
meal. So far the green alga Spongiococcum excentricum represents the only
example of a micro-organism industrially exploited for the production of
natural xanthophyll (lutein). For some time, the dried fermentation meal of
this species, containing 1·6-2·4 g of xanthophylls per kg dry mass was
marketed in the US under the name of A-Zanth (Grain Processing Co.,
Muscatine, Iowa) (Anonymous, 1962). The alga was grown under hetero-
trophic conditions, at 28°C, in a medium containing glucose, com steep liquor,
urea and mineral salts (Ciegler, 1965; Hanson, 1967). Incremental feeding of
nutrients was required because Spongiococcum does not tolerate high sugar
concentrations. Another relative disadvantage is that this alga grows slowly in
liquid medium so that ample time must be allowed for inoculum development
(Hanson, 1967). Lutein was produced at a rate of 294 mg/liter in 9 days.
Of all other green algae tested Chlorella sorokiniana (pyrenoidosa, 7-11-05)
in particular showed great promise for further development. Cell yield and
xanthophyll content of this species, grown under heterotrophic conditions in
30-liter fermenters, were optimized by Theriault (Ciegler, 1965; Theriault,
1965; Hanson, 1967). Dry cell weights in excess of 100 g/liter and a total
xanthophyll content of 467-512 mg/liter were obtained from 230 to 260 g
glucose/liter, in 168 h illuminated batch-type fermentations (25°C). Apart from
glucose, the medium constituents were urea, phosphate, MgS0 4 ·7H2 0 and
trace elements. The yield was further increased in continuous feed runs to
302 g cells/liter and 650 mg xanthophyll/liter, obtained from 520 g/liter of
glucose. Again, maximum concentrations of xanthophylls resulted from using
low initial levels of nutrients, followed by incremental feeding of additional
medium ingredients.
In a patent assigned to F. Hoffmann-La Roche, another, thermophilic strain
of Chlorella pyrenoidosa (A 119 Lagos, ATCC 14860) grown at 35°C was used.
The culture medium consisted of cerelose, crude invert molasses, ethanol,
potassium nitrate, amino acids, guanidine nitrate, MgS04 ·7H2 0, phosphates,
acetate, EDTA salts and thiamine (Wendall et ai., 1964). Although the total
carotenoid content amounted to 7·4 mgt g dry weight, the cell yield (28 g/liter
in 4 days) was lower than in the process of Theriault.
2.3 Fungi
Compared to algae, certain fungi belonging to the Dacrymycetaceae family are

inferior sources of xanthophylls (Ciegler, 1965; Ninet & Renaut, 1979). In a
patented process, the yield did not exceed 40 mg/liter broth (4 mg/g dry
biomass, fermentation time 5 days). A typical medium contained glucose,
glycerol and corn steep liquor as main ingredients. External illumination was
found obligatory for optimal pigment formation, the wavelength determining
the ratio of xanthophylls to total carotenoids and the yield of xanthophylls.
2.4 Non-photosynthetic Bacteria
In view of the relative shortage of zeaxanthin in higher plants, microbial

production of this valuable carotenoid is of high interest. Only few bacterial
species are known to produce free (non-glycosidic) zeaxanthin, among them
certain methylotrophs (Urakami & Komagata, 1986) and marine Flavobacteria
(McDermott et al., 1973). The latter in particular could be candidates for
industrial application. When no special measures are taken, fermentation of a
Flavobacterium sp. in a medium containing glucose and corn steep liquor
generates approximately 10-40 mg zeaxanthin per liter (Ninet & Renaut,
1979). The yield was increased to 335 mg/liter by supplementation with
palmitic esters, methionine, pyridoxine, ferrous salts, by continuous addition
of the F nutrients and reduction of the temperature (Ninet & Renaut, 1979).
Some actinomycetes produce high amounts of mostly unidentified xanth-
ophylls. Streptomyces chrestomyceticus var. aurantioideus reportedly yields
0·5 g pigment/liter (Ninet & Renaut, 1979). Addition of the dry mycelium to
poultry feed was found to 'improve pigmentation' (Anonymous, 1969-1970a).
Even higher yields (up to 0·9 g/liter) were obtained with Streptomyces
mediolani, grown in a medium based on dextrin, casein and corn steep liquor
(fermentation time 5 days) (Ninet & Renaut, 1979). The predominating
carotenoids produced were allegedly isorenieratene (an aromatic carotene) and
its oxygenated derivatives. However, the efficiency of these compounds as
pigmenters in animal feeds has not been established.
Mycobacterium phlei deserves a special mention in that this species produces
carotenoids in quantities far exceeding those normally found in non-
photosynthetic bacteria. Unfortunately, there is some confusion about its exact
pigment composition, possibly because different strains have been investigated.
Schlegel reported the presence of mainly glycosides of oxycarotenoids in
amounts exceeding 10 mg/g dry cell weight (Schlegel, 1959). The media used
consisted of peptone, glucose, glycerol, sodium citrate, magnesium sulfate and
phosphates. Ingraham, in a 1935 study (Ingraham & Steenbock, 1935), found
that the addition of glycerol and various alcohols and glycols to a similar basic
medium markedly stimulated carotenogenesis, although the pigments isolated
were different. Other studies reported isorenieratene (leprotene), various
carotenes and some xanthophylls as the major pigments (Goodwin &
Jamikorn, 1956; Hochmannova et al., 1968). More recent patents describe the
production of 0·3 g 'oxycarotenoids' /liter in a medium containing beet
molasses and urea (Anonymous, 1966-1967, 1969-1970b; Ninet & Renaut,
1979).
3 MONOCYCLIC KETOCAROTENOIDS
3.1 General
Monocyclic, mono-ketocarotenoids have not been systematically studied so far
for application as pigmenters in animal feeds. One of the reasons may be that
they are not widespread in nature. However, the color (Amax 470-480 nm) and
chemical properties (keto group in conjugation with the polyene chain) of
these compounds resemble those of their bicyclic counterparts, e.g. canthaxan-
thin. Hence they could theoretically become useful if a sufficient supply could
be secured.
Keto-derivatives of y-carotene and dehydro-y-carotene have been demon-
strated in radio-resistant pink tetracocci, e.g. Deinococcus radiophilus and
radiodurans (formerly designated as Micrococcus) (Bamji & Krinsky, 1966;
Lewis & Kumta, 1973). Our own (unpublished) investigation of Deinococcus
radiopugnans ATCC 19172 (formerly Micrococcus roseus) also suggested the
presence of monocyclic ketocarotenoids in this related species. A systematic
study has been devoted to optimizing the growth during fermentation of
tetracocci (Shapiro et al., 1977). The iron content of the medium proved to be
a crucial factor in this regard. Various compounds increasing the availability of
this element, e.g. hydroxamic acids and hemin, acted as growth promoters.
Although no quantitative data on the pigments are available, the mere
presence of these unique ketocarotenoids and the exotic nature of the bacteria
appear to warrant further study.
Similar monocyclic ketocarotenoids have been reported in a Mycobacterium
smegmatis strain, grown on hydrocarbons (Tanaka et al., 1968a,b». The effect
of various nutrients and added compounds (amino acids, detergents) on
growth and pigmentation was examined. The optimal medium contained 2% of
an alkane (preferably n-hexadecane), a mixture of ammonium sulfate and
urea, or, alternatively, ammonium phosphate, as a nitrogen source, a number
of mineral salts and trace elements as well as histidine. However, the
maximum carotenoid yield was disappointingly low (0·8 mg/liter). A non-
specific part of this consisted of 4-keto-y-carotene and its mono-hydroxy- and
mono-methoxy derivatives.
4 BICYCLIC KETOCAROTENOIDS
4.1 General
Canthaxanthin and astaxanthin are widely used as pigmenters in poultry and
fish feeds (see above). Although both compounds are manufactured syntheti-
cally, their high market value makes any suitable natural source potentially
attractive. Canthaxanthin is found in substantial quantities in Crustacea
(Castillo et al., 1982) and the feathers of birds (Brush, 1981), but not in higher
plants. The fungus Cantharellus cinnabarinus, the first discovered natural
source of canthaxanthin (Haxo, 1950) can be dismissed from a practical point
of view. Astaxanthin occurs in the feathers of birds (Brush, 1981) and flower
petals of Adonis annua (Seybold & Goodwin, 1959) and is abundant in
Crustacea (Castillo et aI., 1982) and fish (Simpson et al., 1981). Apart from
crustacean products (shrimp meal, crayfish waste), no producing organism has
been used commercially (Marusich & Bauernfeind, 1981; Simpson et al.,
1981).
Although strictly speaking the retrocarotenoid rho do xanthin also belongs to
the ketocarotenoids, this compound is discussed in a separate paragraph (see
Section 5).
4.2 Cyanobacteria
In view of the revival of interest in the biotechnological potential of

Cyanobacteria (formally named blue-green algae) (Lem & Glick, 1985) the
large-scale ketocarotenoid production from certain species belonging to the
Nostocaceae (Table 1) could theoretically be envisaged. No systematic study
of the usefulness of Cyanobacteria as industrial sources of canthaxanthin has
been conducted so far. However, the secretion of deadly toxins by certain
Nostocaceae (Lem & Glick, 1985) could obviously be prohibitive for any
practical application.
4.3 Green Algae
As mentioned in Section I, 7.1.1(A), many green algae, which normally

contain the xanthophylls typical of photosynthetic tissues, synthesize secondary
(keto)carotenoids at the end of their growth phase, when certain essential
nutrients, particularly nitrogen, become depleted (Czygan, 1968). The nature
of the ketocarotenoid( s) varies with the type of algae and not· all species
synthesize them (Czygan, 1968). Astaxanthin is the most common secondary
pigment, mostly accompanied by canthaxanthin and echinenone. The obvious
practical potential of secondary carotenoid formation is probably outweighed
by the slow nature of the process and the low levels obtained. The two-phase
culturing on laboratory scale first involves proliferation of the algae in a
mineral medium containing an ample amount of a nitrogen source, e.g.
potassium nitrate. Production of ketocarotenoids starts after replacement of
the growth medium by a 'production medium' low in nitrate. Heterotrophic
conditions (addition of carbohydrates) accelerate the process indirectly by
stimulating growth (and primary carotenoid formation), thus causing the
nitrogen supply to be exhausted sooner.
4.4 Fungi and Yeast
4.4.1 Importance of Phaffia rhodozyma

The red yeast Phaffia rhodozyma is unusual among the Deuteromycetes in
many respects. Unlike other genera like Rhodotorula, Rhodosporidium and
Sporobolomyces, it has the property of fermenting various carbohydrates
(Andrewes et al., 1976). In addition, it contains none of the pigments typical of
red yeasts (p-carotene, y-carotene, torulene, torularhodin), but rather as-
taxanthin (Andrewes et al., 1976). Apart from the genus Peniophora
(Hymenomycetes) (Arpin et al., 1966), this carotenoid has never been
demonstrated in fungi. The occurrence of astaxanthin is even more unique in
that its absolute configuration (3R, 3' R) is opposite to that of astaxanthin from
other natural sources (3S,3'S) (Andrewes & Starr, 1976). In general, the
chirality of a carotenoid is indeed considered not to be source dependent
(Andrewes & Starr, 1976).
Several observations indicating the efficiency of Phaffia rhodozyma as a
pigmenter in salmonid and crustacean diets (Johnson et al., 1977, 1980a) and
poultry feeds (Johnson et al., 1980b) created much excitement about this
organism. It proved superior to crustacean waste products as a feed additive
because of its higher nutritional value and pigment content. These promising
results appeared to warrant a thorough feasibility study of Phaffia rhodozyma
as a potential industrial source of natural astaxanthin.
4.4.2 Fermentation of Phaffia rhodozyma

The goal of such a feasibility study was 3-fold: optimization of pigment
production, a search for cheaper media and the improvement of the extrac-
tability of astaxanthin from the yeast cells.
(A) OPTIMIZATION OF THE GROWTH MEDIUM (Johnson & Lewis, 1979)

A 'standard medium' for the propagation of Phaffia rhodozyma contained
cerelose, ammonium sulfate, KH2 P04 , MgS0 4 ·7H2 0, CaClz·2H2 0 and yeast
extract. Essentially, astaxanthin formation is growth-associated, but its pro-
duction still continues when growth has stopped upon exhaustion of glucose.
An optimum pH of 4·5 yielded maximum cell weight, growth rate and
astaxanthin levels. Of all carbon sources tested cellobiose afforded maximum
astaxanthin yield but less biomass and a lower growth rate than glucose.
However, increasing concentrations of the latter as well as reduced aeration,
both suggestive of fermentative conditions, were inhibitory to astaxanthin
production but rather caused less oxygenated precursors like echinenone and
p-carotene to accumulate. This is consistent with the fact that cellobiose,
which can only be utilized aerobically, is stimulatory. Unlike the carbon
source, the nature of the nitrogen source affected growth and carotenoid
formation only to a very limited extent. Light was totally without effect. The
addition of yeast extract to a vitamin-free medium increased pigmentation by a
factor of about 4. Carotenogenesis was strongly promoted (astaxanthin level of
8141lg/g dry weight) in the presence of tomato waste. This can be rationalized
in terms of the uptake by the yeast cells of carotenoid precursors. Typical
astaxanthin yields are of the order of 2 mg/liter, obtained in a 6O-h
fermentation.
(B) USE OF CHEAP MEDIA

Attempts to grow Phaffia rhodozyma on cheap food-processing waste products
have been restricted so far to the use of alfalfa residual juice as a substrate for
yeast propagation (Okagbue & Lewis, 1984a). This material supports appreci-
able growth but totally inhibits astaxanthin formation, which has been
attributed to the presence of saponins (Okagbue & Lewis, 1984b). Saponin
presumably disturbs the integrity of the plasma membrane of the cells. The
effect is indeed partially reversed by unsaturated acids and sterols.
(C) EXTRACfABILITY OF ASTAXANTIUN

A factor seriously limiting the efficient utilization of the astaxanthin from
Phaffia rhodozyma by fish and poultry is the high resistance of its cell wall. No
pigment is absorbed from intact cells (Johnson et al., 1978). Mechanical
rupture is not feasible for large-scale extraction and chemical hydrolysis of the
cell wall destroys the astaxanthin. Autolysis of the yeast cells by incubation in
distilled water or a citrate buffer has been proposed as a method to facilitate
extraction (Okagbue & Lewis, 1984c). Enzymatic digestion using the ex-
tracellular enzymes produced by Bacillus circulans is another useful alternative
(Johnson et al., 1978; Okagbue & Lewis, 1981, 1985). This can be accom-
plished in a 2-step method, involving the cultivation of Phaffia rhodozyma,
followed by heat-inactivation of the yeast cells, pH-adjustment and inoculation
of Bacillus circulans (Johnson et al., 1978). However, during heat treatment
part of the astaxanthin is lost and readjustment of the medium pH is laborious
and impractical. These drawbacks are overcome by growing the two organisms
in mixed culture (Okagbue & Lewis, 1981, 1985). Carbohydrates cheaper than
glucose, e.g. molasses can be used as carbon sources for this purpose
(Okagbue & Lewis, 1981). Strict pH control remains however critical to
survival and lytic activity of the bacillus (Okagbue & Lewis, 1985). After
growth, the biomass is directly amenable to extraction with acetone, resulting
in astaxanthin yields of over 90%. Unfortunately, the overall production of
astaxanthin is partly suppressed in mixed culture (maximum yield 1·2-
1·5 mg/liter) (Okagbue & Lewis, 1981, 1985). A practical advantage of the
mixed culture approach is the possibility for re-use of the cell-free culture
liquid (Okagbue & Lewis, 1985). After fortification with nutrients, this
medium supports growth of Phaffia rhodozyma and at the same time retains
the lytic activity required to modify its cell walls. Alternatively, this culture
fluid can also be used to digest the walls of yeast cells previously grown in pure
culture. A scheme both including mixed culture and recycling of culture filtrate
was developed for the enzymatic large-scale processing of Phaffia rhodozyma
for inclusion in animal diets (Okagbue & Lewis, 1981, 1985).

The phenomenon of carotenoid production by members of the CMN complex
(Corynebacterium-Mycobacterium-Nocardia) grown on hydrocarbons has
been recognized for a long time (Haas et al., 1941; Haas & Bushnell, 1944;
Davis, 1967). The ketocarotenoids astaxanthin and canthaxanthin occur in a
limited number of those hydrocarbon utilizing bacteria.
4.5.1 Brevibacterium KY-4313: Canthaxanthin
(A) BASIC WORK

Brevibacterium KY-4313 contains canthaxanthin and its biosynthetic precur-
sors echinenone and p-carotene as the predominating carotenoids (Sakurai et
al., 1971). This organism has been used as a model to study the biosynthesis of
canthaxanthin (Hsieh et al., 1974). However, Japanese investigators, who
apparently had designed the bacterium mainly for petrochemical applications,
became aware of its industrial potential as a natural source of canthaxanthin.
Thus, a systematic optimization of the pigment level through variation of the
composition of the growth medium was undertaken (Tanaka et al., 1971).
Growth and pigment production were found to be intimately related. Optimal
growth (maximum cell yield about 3·5 g/liter) and maximum canthaxanthin
levels (0·6 mg/g, 1-2 mg/liter) resulted from using a basic medium containing
2-4% octadecane, 0·25% NH4 H 2 P04 , phosphates, magnesium sulfate and
minerals (pH 7), supplemented with vitamin B 12 , a natural nutrient, e.g. malt
extract (0·1 %) and a small amount of a non-ionic detergent (0·01 % Tween
40). High carbon to nitrogen ratios are known to favor the production of
cellular lipid in hydrocarbon fermentation (Ratledge, 1970). Accordingly,
formation of canthaxanthin was also promoted under these conditions. Light
did not affect carotenogenesis, while strong aeration was slightly inhibitory.
(B) OPTIMIZATION STRATEGY (Nelis & De Leenheer, unpublished)

Hydrocarbon fermentations suffer from a number of drawbacks (Einsele,
1983), including poor yields (low cell weights and slow growth rate), high costs
when pure alkanes are used and complex processing of the product (see
Section IV, 1). Pigment production by bacteria grown on paraffins is said to be
'very low' (Ninet & Renaut, 1979). Yet the figures cited above for canthaxan-
thin are not far below the astaxanthin levels obtained in Phaffia rhodozyma, an
organism which continues to be advocated as commercially attractive (Ok-
agbue & Lewis, 1985). In our hands Brevibacterium KY-4313 tended to yield
more pigment in hydrocarbon than in non-hydrocarbon media. Therefore, a
systematic study was devoted to optimizing the canthaxanthin levels and
promoting the bacterial growth during hydrocarbon fermentation.
(a) Stimulation of carotenogenesis:

1. Optimization of the growth medium: Variation of the basic medium
constituents only marginally increased the canthaxanthin yield. Minor positive
effects were noted from combining the optimal carbon source, octadecane or
(cheaper) paraffin mixtures enriched (50-90%) in this compound, with certain
branched alkanes (pristane) or hydrocarbon mixtures rich in cycloalkanes.
Ammonium phosphate, ammonium acetate and sodium nitrate were nearly
equally effective as nitrogen sources. Yeast extract was slightly superior to malt
extract as a growth factor. The incorporation in the medium of chemicals
supposedly acting as precursors of carotenoid biosynthetic intermediates had
little or no effect. Examples of such compounds include acetate, pyruvate,
farnesol, geraniol, leucine and various sources of acetyl CoA. In addition,
Brevibacterium KY-4313 proved refractory to the action of a variety of
chemicals, which are reportedly carotenogenic in other micro-organisms,
particularly Blakeslea trispora: nitrogenous heterocyclic compounds, vegetable
oils, /3-ionone, retinol, aromatics. However, in non-hydrocarbon medium
(Brain Heart Infusion Broth) a number of substances were found to promote
substantially carotenoid biosynthesis, particularly alcohols (propanol and
isopropanol), glycerol and retinol. The latter caused accumulation of /3-
carotene rather than canthaxanthin. Vegetable oils greatly stimulated growth
but suppressed carotenoid formation.
2. Preparation of mutants: Following treatment of Brevibacterium KY-4313
with the mutagen NMU (Voznyakovskaya & Daraseliya, 1972), we isolated a
colony that distinguished itself from the bulk of the orange ones by its reddish
hue. This 'new', presumably mutant strain, contained about twice as much
canthaxanthin as the parent strain, but also more echinenone. Retreatment of
the 'new' strain with the same mutagen again permitted the isolation of a
slightly more pigmented colony. The latter was subsequently used for all
optimization work.
(b) Growth promotion: In view of the existing positive relationship between

growth and pigmentation, growth promotion was crucial to increase the overall
productivity of canthaxanthin. Two major factors are growth-limiting in
hydrocarbon fermentation, i.e. the availability (dispersion) of the insoluble
alkane (Einsele & Fiechter, 1971) and the secretion of toxic metabolites
(Einsele & Fiechter, 1971; Yamada et al., 1971). The effective dispersion of
octadecane, a liquid at the working temperature (30°C) was facilitated by
thorough mechanical stirring and the addition of small amounts of a non-ionic
detergent.
A major breakthrough came from removing the toxic metabolites, sup-
posedly carboxylic acids, produced in the course of the bacterial growth. In
one fermentation on gaseous hydrocarbons, detoxification was accomplished
using continuous dialysis culture (Coty, 1968). As a result, the cell yield in this
particular example dramatically increased from 4 to 47 g/liter. However,
dialysis represents technical problems when liquid alkanes are used. We
mimicked its effect by regularly (each time when the pH of the medium
dropped to a constant minimum value) renewing the aqueous component of
the medium. The separation of the aqueous broth from the hydrocarbon layer,
containing the cells, is very convenient because, after discontinuing the

stirring, the latter is floating on top of the medium. This semi-continuous
approach yielded dry cell weights of the order of 8-14 g/liter, as opposed to
3-4 g in the absence of the aqueous medium renewal. Concurrently, the
ketocarotenoid yield increased to 13 mg/liter (1-1·5 mg/g dry weight), of
which 70% was canthaxanthin and the remainder echinenone, obtained from
50 ml of hydrocarbon (working volume of the fermenter, 1·25 liters) in a 7-day
run. This figure compares favorably with the astaxanthin yield obtained from
Phaffia rhodozyma (1·3-1· 5 mg/liter in 60 h using the mixed culture conditions
described above).
4.5.2 Rhodococcus maris/Mycobacterium brevicale

As part of an investigation of hydrocarbon-oxidizing marine bacteria (Koro-
nelli et al., 1981), Russian workers isolated a pigmented Mycobacterium
brevicale strain (32-MCT) (Koronelli et al., 1982), later found to be synony-
mous with Rhodococcus maris (Koronelli et al., 1987). This organism contains
a dark orange peptidolipid with canthaxanthin as the main carotenoid
(Koronelli et al., 1987; Pachlavuni, 1987). Details on culturing conditions were
not available at the time of writing, except that the organism was grown in a
mineral medium containing hexadecane as a carbon source (Pachlavuni et al.,
1987). Rhodococcus maris / Mycobacterium brevicale 32-MCT is apparently not
equivalent with the Rhodococcus maris type strain described by Nesterenko
(Nesterenko et al., 1982). The latter also grows on various hydrocarbons and
produces canthaxanthin as well (personal observation, unpublished). However,
pigment levels were lower than in Brevibacterium KY-4313 and reproducibility
of carotenoid formation and growth on hydrocarbons was poorer (personal
observations, unpublished).
4.5.3 Miscellaneous Hydrocarbon-Utilizing Bacteria

Mycobacterium lacticola was the first bacterium reported to form astaxanthin in
hydrocarbon medium (Haas & Bushnell, 1944). Remarkably, the pigment was
absent when the organism was grown on nutrient agar. Brevibacterium 103 is
the second known example of a hydrocarbon utilizing bacterium that produces
astaxanthin (Iizuka & Nishimura, 1969). A mineral medium containing 8%
kerosene yielded approximately 3 g cells/liter, with a pigment content of
30 ",g/g dry cell weight. In view of this poor yield and the availability of Phaffia
rhodozyma, there appears to be no future for either of the above bacteria as a
useful source of astaxanthin.
4.5.4 Micrococcus roseus: a controversy

The presence of canthaxanthin and other ketocarotenoids has also been
reported, by one particular research group, in Micrococcus roseus (ATCC 516)
grown in a variety of (non-hydrocarbon) media (Cooney et al., 1966; Ungers &
Cooney, 1968; Schwartzel & Cooney, 1970, 1972, 1974a,b; Ascenzi & Cooney,
1975; Dieringer et al., 1977; Cooney & Berry, 1981). However, despite this
extensive evidence, we as well as others (unpublished results) have repeatedly

failed to confirm this finding. We re-investigated the pigment composition of
Micrococcus roseus ATCC 516 and of the original strain, donated by Cooney,
using HPLC and photodiode array detection (Nelis & De Leenheer, 1987).
Although a chromatographic peak corresponding in retention to canthaxanthin
was observed, its absorption spectrum, recorded with the aid of a photodiode
array detector (see Section IV, 2) was in no way consistent with this structure.
None of the pigments separated displayed an absorption spectrum indicative of
any ketocarotenoid. The reason for this extreme discrepancy of results has
remained obscure so far.
5 RETROCAROTENOIDS: RHODOXANTHIN
5.1 General
By virtue of its deep red color, rhodoxanthin, which has been approved as a
food additive in certain countries, might be envisaged as an alternative to
astaxanthin. The compound occurs in autumn leaves (Ida, 1981), fish (Kat-
suyama & Matsuno, 1979) and the feathers of birds (Brush, 1981).
5.2 Non-Photosynthetic Bacteria
Rhodoxanthin was tentatively identified as the main carotenoid in the

methylotroph Pseudomonas extorquens (Downs & Harrison, 1974) (obviously
equivalent with Protomonas extorquens (Urakami & Komagata, 1986». The
pigment was formed during growth in a mineral medium consisting of
ammonium sulfate as a nitrogen source and methanol or glycerol as a carbon
source. Oxygen and magnesium limitation caused a marked increase in cell
pigmentation, suggesting that conditions of growth restriction in general tend
to divert the available carbon to pigment synthesis. However, no attempts
have been made to quantitate or to optimize the actual carotenoid content.
III FERMENTATION-MICRO-ORGANISMS DESIGNED FOR

APPLICATIONS OTHER THAN PIGMENT PRODUCTION
1 INTRODUCTION
Three selected examples will be briefly discussed of carotenoid-containing

micro-organisms whose primary biotechnological potential lies in a field other
than pigment production. The carotenoids present in those bacteria and yeasts
have in general not been approved yet as additives for feeds or foods.
However, this situation could obviously change in the future if these organisms
gain acceptance for large-scale biomass or single lipid production and, hence,
addition to animal feeds.
2 FUNGI: RED YEASTS
Various red yeasts belonging to the genera Rhodotorula and Rhodosporidium

(Basidiomycetes) have been considered as industrial sources of single cell
protein (Lichtfield, 1979; Costa et al., 1984) and microbial fat (Ratledge,
1982). These organisms can be grown on cheap substrates, e_g. domestic
seawage, potato hydrolysate, whey, molasses, etc_ (Lichtfield, 1979; Ratledge,
1982).
Concurrently with fat accumulation, enhanced carotenoid synthesis can be
reasonably expected_ The major carotenoids in Rhodotorula and
Rhodosporidium are p-carotene, j'-carotene, torulene and torularhodin (Simp-
son et al., 1971; Goodwin, 1972). Supplementation of poultry feeds with
preparations of Rhodotorula mucilaginosa were found to promote egg yolk
pigmentation (Schwarz & Margalith, 1965). This yeast was propagated in
glucose-peptone-yeast extract media or, alternatively, in chemically defined
media containing a carbohydrate (5-10% glucose, beet molasses), ammonium
sulfate, urea and/or ammonium lactate as nitrogen sources, as well as minerals
and growth factors (yeast extract, thiamin). The total carotenoid yield on a dry
weight basis was as high as I-Smg/g (corresponding to 49mg/liter). Unlike in
Blakeslea trispora, the addition of p-ionone or kerosene inhibited carotenoge-
nesis, whereas non-ionic detergents were only slightly stimulatory. However, a
disadvantage of using Rhodotorula mucilaginosa biomass as an additive for
poultry feed was the appearance of a violet hue in the egg yolk, caused by the
presence of torularhodin (Margalith & Meydav, 1968). Therefore, subsequent
efforts were directed towards reducing the torularhodin/torulene ratio. This
could be accomplished by adding diphenylamine, ethanol or isopropanol to a
glucose-ammonium lactate medium (Margalith & Meydav, 1968). Unlike
diphenylamine, the latter two additives also promoted the total carotenoid
yield, which however remained below 0-4 mg/g dry weight. The minimal
torularhodin/torulene ratio was 0-33, vs 1-00 in the absence of additives. Other
workers obtained a value of 0·48 using a glucose-asparagine medium
(Peterson et al., 1958). Both figures contrast with the unacceptably high ratio
of 6-12, resulting from the use of sucrose-ammonium sulfate (Villoutreix,
1960)_
3 NON-PHOTOSYNTHETIC BACTERIA: METHYLOTROPHS
A number of methylotrophic bacteria are known vitamin B12 producers

(Toraya et al., 1975; Sato et al., 1977; Shimizu et al., 1982; Dumenil et al.,
1983) and potentially useful sources of single cell protein (Lichtfield, 1979,
1980). The commonly encountered pink color of these bacteria is due to the
presence of, mostly unidentified, carotenoids (Shimizu et al., 1982; Dumenil et
al., 1983; Urakami & Komagata, 1986), sometimes of a rhodoxanthin-like
nature (Downs & Harrison, 1974; Urakami & Komagata, 1986) (see also
Section 11,5). Attempts have been made to optimize the carotenoid yield
from selected methylotrophs by altering the culturing conditions. A
Corynebacterium sp. XG was found to form most pigment on methanol,
dimethyl amine and methylamine as carbon sources (Dumenil et al., 1983).
Other medium ingredients included ammonium sulfate, minerals and yeast
extract. Light, pH variation and the nature of the growth factor did not
appreciably affect pigmentation. Protaminobacter ruber, another facultative
methylotroph, produced 2 mg/liter of an unidentified pink carotenoid in a
medium containing 1,2-propanediol as a carbon substrate and methionine and
riboflavine as moderately effective carotenogenic agents (Shimizu et al., 1982).
Both examples demonstrate the interest in methylotrophic bacteria for their
carotenoid composition. Although their primary biotechnological potential
clearly lies in the production of vitamin B12 and biomass, the presence of
useful pigments again adds extra potential value to these organisms.
4 PHOTOSYNTHETIC BACTERIA
4.1 General
Purple photosynthetic bacteria, particularly Rhodobacter capsulatus (formerly

Rhodopseudomonas capsulatus, Imhoff et al., 1984) are useful sources of single
cell protein (Kobayashi & Tchan, 1973; Shipman et aI., 1977; Kobayashi &
Kurata, 1978; Lichtfield, 1979, 1980, 1983; Driessens et aI., 1987). Their
ability to grow on waste solutions makes them attractive for the purification
of polluted industrial effluents, giving highly nutritious cells as a valuable
by-product (Kobayashi & Tchan, 1973). Apart from its general nutritional
value, as evidenced from its amino acid profile (Kobayashi & Kurata, 1978;
Driessens et al., 1987) Rhodobacter capsulatus reportedly also promotes egg
production in chickens and increases the survival of fish (Kobayashi, 1972;
Kobayashi & Tehan, 1973).
Although the nature of carotenoids in Rhodobacter capsulatus has been
established for a long time (for reviews see Liaaen-Jensen, 1978; Schmidt,
1978; Goodwin, 1980), Driessens & Verstraete were the first to recognize the
potential of this organism as a useful natural source of pigments (Driessens et
al., 1987 and unpublished results). Spheroidene is the predominating caroten-
oid in Rhodobacter capsulatus when grown anaerobically, whereas under
aerobic conditions this compound is further oxidized to the ketocarotenoid
spheroidenone (Goodwin 1980; Spurgeon & Porter, 1983). Because of its
favorable color characteristics, the latter in particular is interesting from a
practical! economical standpoint. In the course of a thorough study of optimal
biomass production by Rhodobacter capsulatus, the co-production of caroten-
oids was also examined (Driessens et al., 1987).
4.2 Production of Carotenoids by Rhodobacter capsulatus
4.2.1 Batch culture

Initially, when Rhodobacter capsulatus was grown photo heterotrophically in
batch culture (lactic acid as carbon source, ammonium sulfate as nitrogen source,
anaerobic conditions and illumination) high pigment contents (5-10 mg/g)
were obtained. Spheroidene represented, as predicted, the major carotenoid
(>85%), whereas spheroidenone was only present in amounts ranging from 1
to 5%. Subsequent attempts were, for unknown reasons, less successful.
Under anaerobic conditions, the maximum levels of spheroidene and sphero-
idenone were 1 and 0·3 mg/g, respectively. Corresponding values obtained in
aerobic culture were 0·05 and 0·4 mg/g, respectively. Unexpectedly, in one
anaerobic fermentation cells containing 0·6 mg spheroidenone and virtually no
spheroidene were produced.
However, batch fermentations of Rhodobacter capsulatus are associated with
low cell yields (1-2 g/liter) (Kobayashi & Kurata, 1978; Lichtfield, 1983).
4.2.2 Culture in a continuous flow reactor (Driessens et aI., 1987)

Culture of Rhodobacter capsulatus in a continuous flow-through reactor, under
external illumination and microaerophilic conditions, yielded 5-10 times
higher biomass production rates. The photobioreactor used consisted essen-
tially of a glass tube having a PVC decanter on top and two ports at the
bottom, one for the removal of excess biomass and one for the entry of
(recirculated) medium. The PVC decanter contained ports for effluent
removal, gas outlet, medium recirculation and nitrogen flushing (when
required). Two non-axenic feed solutions were supplied separately, by means
of a peristaltic pump. The first one contained the buffered carbon source
(preferably calcium lactate) and the second one the nitrogen source (am-
monium sulfate) plus minerals, growth factors as well as selective inhibitors of
sulfate reducing bacteria (sodium molybdate) and algae (chloroxuron). The
bacteria readily flocculated in the reactor, which permits easy harvesting.
The quality of the resulting biomass did not differ from the one obtained in
batch culture. Under conditions of iron excess (4 mg Fe2 + /liter) about 1 mg
pigment per g dry weight was produced, of which 0·7 mg was spheroidene and
0·3 mg spheroidenone. However, because of their high toxicity, the com-
pounds added to suppress competing micro-organisms had to be eliminated, in
order to make the end-product nutritionally acceptable. To maintain the
non-axenic conditions and yet to avoid contamination the iron content of the
medium had to be drastically lowered (down to 251lg Fe2 + /liter). This did not
appreciably affect the growth, but dramatically altered the carotenoid profile.
A number of unidentified, red lycopene-like compounds were now formed in
amounts ranging from 0·4 to 0·8 mgt g, but neither spheroidenone or sphero-
idene were present. A compromise in the iron concentration (with incremental
addition) still resulted in a similar carotenoid pattern. Although the cells, when
added to pOUltry feed, improved the color of the egg yolk without any signs of
toxicity (provided the sodium molybdate had been eliminated), the uncertainty
about the exact pigment composition precludes, for the time being, the use of
Rhodobacter capsulatus as a pigmenter for chickens.
IV PROCESSING-ANALYSIS-APPLICATION AND ECONOMICS
Because carotenoids in micro-organisms are localized intracellularly, their

recovery essentially comes down to the isolation of the biomass. Well-
established approaches for the harvesting of cells include filtration, centrifuga-
tion and coagulation/flocculation (Belter, 1979). However, hydrocarbon fer-
mentations pose a number of additional recovery problems due to the presence
of residual alkanes (Levi et al., 1979; Einsele, 1983). After separation of the
aqueous and the organic phases by centrifugation or flotation, often in
conjunction with specialized techniques (Various patents, 1973), the residual
hydrocarbon, adsorbed on the cells, has to be removed. Solvent extraction or
treatment with surfactants have been suggested for this purpose (Einsele &
Fiechter, 1971). However, this purification will cause a very substantial part of
the carotenoids to leach from the cells and to dissolve in the hydrocarbon
(paraffin) or the extraction solvent (personal observations). The resulting
'clean' cells will not only be defatted, but also be devoid of most of the
carotenoids. The separation of the carotenoids from the hydrocarbon itself at a
later stage is unfeasible. Therefore, in hydrocarbon fermentation a basic
incompatibility appears to exist between the recovery of pure biomass and the
quantitative isolation of the intracellular carotenoids.
If the dry biomass does contain the pigments (as in non-hydrocarbon
fermentation) it could probably be directly added to animal feed, provided the
carotenoid concentration is sufficiently high and no toxic compounds are
present. In view of the instability of carotenoids, conditions for drying (spray-
or flash-drying) should be as mild as possible, to avoid oxidation and cis /trans
isomerization. The addition of antioxidants (BHT, ethoxyquin) might be
desirable (Wood et aI., 1983). Alternatively, if the pigment quantity in the
biomass is too dilute, solvent extraction is required for its isolation (useful
organic solvents are mentioned in Section IV, 2).
2 ASSAY METHODS
Traditionally, the determination of carotenoids in biological materials, includ-

ing micro-organisms, has always relied on spectrophotometry (Davies, 1976;
Britton, 1985). A typical sample pretreatment consists of a total lipid
extraction using such polar organic solvents as acetone or methanol, saponifi-
cation to remove triglycerides, partition between hexane/methanol (separation
of non-polar epiphasic from more polar hypophasic derivatives) and open

column chromatography (Davies, 1976; Britton, 1985). Each colored band
eluting from the column is subjected off-line to spectrophotometric measure-
ment at its Amax, with calculation based on the molar absorption coefficient of
the (tentatively) identified compound of interest. Often, this complex purifica-
tion of total lipid extracts is omitted when only one pigment is presumably
present. That this may lead to errors is exemplified in the spectrophotometric
assay of astaxanthin in Phaffia rhodozyma, where the interference of colored
substances adsorbed from the medium onto the cells led to an overestimation
of the astaxanthin content (Okagbue & Lewis, 1984a). This shows that a
chromatographic fractionation is an essential step in carotenoid assays.
As a separation method, modern liquid chromatography on microparticulate
materials (HPLC) has now superseded open column chromatography because
of its superior resolving power, higher sensitivity and improved speed. HPLC
has been used for the profiling and quantitation of carotenoids in algae
(Braumann & Grimme, 1981; Mantoura & Llewelynn, 1983; Bowles et ai.,
1985) and bacteria (Gillan & Johns, 1983; Kester & Thompson, 1984; Korthals
& Steenbergen, 1985; Nelis & De Leenheer, 1987).
A recent development is the use of photodiode array absorbance detection
(Miller et ai., 1982) in connection with the HPLC of carotenoids (Gillan &
Johns, 1983; Mantoura & Llewelynn, 1984; Ruedi, 1985; Nelis & De
Leenheer, 1986). This versatile technique permits a (tentative) identification of
each chromatographic peak, based on its on-line recorded absorption
spectrum. The need for such a second identity criterion (aside from com-
parison with a standard compound) was clearly evidenced from the work with
Micrococcus roseus (see Section III, 4.5.4).
One issue that is poorly addressed in the literature is the problem of the
complete carotenoid extraction from micro-organisms. We observed that even
repeated treatment of several bacteria, e.g. Brevibacterium KY-4313 and
Micrococcus roseus with acetone or methanol failed to release all the pigment
(Nelis & De Leenheer, 1986, 1987). Also, from streptococci, carotenoids were
reportedly poorly extractable using a variety of organic solvents and even
alcoholic potassium hydroxide (Merritt & Jacobs, 1978). In the latter case,
efficient extraction was accomplished using a rather unusual mixture of
glycerol-fenol. Other investigators added trichloroacetic acid (Downs &
Harrison, 1974) or potassium hydroxide (Taylor, 1980) to promote the
liberation of carotenoids from bacteria. In our hands a brief pretreatment of
the cells with pure liquefied fenol was found to make them spectacularly more
amenable to extraction with methanol: this approach resulted in a total
bleaching of the residue, even for the most refractory species (Micrococcus
roseus). Fenol can be removed from the extract by partitioning between
diethyl ether and an alkaline water phase or, alternatively, by column
chromatography on microparticulate C 1S mini-columns (Bond Elut, Sep-Pak).
The ether or the column eluate is subsequently evaporated to dryness and the
residue is redissolved in the chromatographic solvent. An aliquot (50-100 Ill)
of the final mixture is injected on a reversed phase column, eluted with a

non-aqueous mobile phase (Nelis & De Leenheer, 1983, 1986, 1987). When, as
in the case of Brevibacterium KY-4313, hydrocarbons are co-extracted, a
chromatographic step on a Bond Elut Alumina mini-column is included to
separate the carotenoids (canthaxanthin) from the hydrocarbon.
3 CHEMICAL SYNTHESIS--BIOCATALYSIS
The profitability of the biotechnological production of carotenoids has to be

judged in the light of the availability of alternative chemical routes for their
preparation. Virtually all derivatives can be prepared by total synthesis in
economically feasible processes (for an extensive review, see Mayer & Isler,
1971). This applies in particular to the carotenoids of high commercial interest,
i.e. f3-carotene, canthaxanthin, astaxanthin, f3-apo-8' -carotenal and f3-apo-8'-
carotenoic acid ethyl ester. Leading European manufacturers are F. Hoffmann-
La Roche (Basle, Switzerland) and BASF (Ludwigshafen, FRG).
Basically, the C 40 carotenoid skeleton is synthesized by combining two or
three building units in symmetrical (e.g. C 20 + C 20 , C 19 + C 2 + C 19 , CiS + C lO +
CiS) or asymmetrical schemes (e.g. ~i + C 19 , C 2S + CiS). The two most
frequently used methods are the Wittig reaction (condensation of a carbonyl
compound with a trimethylphosphonium halide) and the Grignard reaction
(condensation of a carbonyl compound with a metal acetylide). New develop-
ments continue to appear, particularly with respect to the total synthesis of
optically active carotenoids (Mayer, 1979; Muller et aI., 1982). This is a new
1 2
Fig. 4. Reduction reactions involved in the preparation of an optically active key
intermediate in the synthesis of (3R,3/R)-zeaxanthin. A, enantioselective hydrogena-
tion of double bond, catalyzed by baker's yeast; B, chemical reduction; C, reduction of
keto group, catalyzed by baker's yeast (unwanted side reaction). 1, desired reaction
product (4R, 6R); 2, unwanted reaction product (4S, 6R).
___• (>Yo + ?ir' OH
HO~ HO~
OH 0
Fig. 5. Enantioselective reduction catalyzed by baker's yeast as a first step in the

synthesis of (3S, 3'S}-astaxanthin.
area in which micro-organisms have found large-scale application. Synthetic

procedures for (3R,3'R)-zeaxanthin and (3S,3'S)-astaxanthin indeed involve
the use of micro-organisms as biocatalysts for the introduction of chiral
centers, by taking advantage of the stereoselective course of microbially
catalyzed reactions (Leuenberger, 1985). Thus the optically active synthon for
the synthesis of (3R,3'R)-zeaxanthin has been prepared by a combined
process consisting of enantioselective reduction of oxo-isophorone (hydroge-
nation of double bond) with baker's yeast (Fig. 4A) and chemical hydrogen-
ation (Fig. 4B). However, special precautions have to be taken in the
microbial process to avoid reduction of the 4-keto group of oxo-isophorone
(Fig. 4C). This secondary reaction leads to the unwanted S configuration at
C-4. A synthetic route for the preparation of (3S,3'S)-astaxanthin also
involves the use of baker's yeast to carry out an enantioselective reduction. In
this particular reaction the 3-keto group of 4-hydroxy-6-oxo-isophorone is
specifically attacked to yield a tautomeric mixture of optically active ketodiols
(Fig. 5). Both reaction products can be converted chemically (acetonization) to
a ketal, a key intermediate in the further synthesis of (3S, 3'S)-astaxanthin.
Another type of useful reaction catalyzed by micro-organisms is microbial
resolution of racemates. In an improved synthetic process for (3S,3'S)-
astaxanthin a Bacillus sp. selectively hydrolyzes the 3R enantiomer of racemic
3-acetoxy-4-oxo-fl-ionone, but leaves the desired S-enantiomer untouched
(Fig. 6). The latter is subsequently isolated in high yield by physico-chemical
methods.
Fig. 6. Microbial resolution by a Bacillus sp. of racemic 3-acetoxy-4-oxo-tJ-ionone, a

key step in the synthesis of (3S,3'S}-astaxanthin. 1, unwanted reaction product (3S)
(hydrolyzed); 2, desired reaction product (3R) (untouched).
The efficiency of microbially catalyzed reactions can be enhanced by

immobilization of the cells, the use of two-phase biocatalysis (for water
insoluble substrates such as carotenoids) or via recombinant DNA technology.
4 APPLICATIONS AND ECONOMICS-FUTURE PROSPECTS
So far there are no examples of microbiological fermentations of carotenoids

that are competitive with their total synthesis or their isolation from plants. A
limited number of processes, e.g. the production of lutein from green algae
have a mere historical value, as their development and use have been
discontinued since the sixties. The production of lycopene from fungi is, to the
best of our knowledge, not further developed at present. The most suitable
candidates for fermentation are carotenoids with chiral centers, which are
more difficult to synthesize than others. A process for the production of
(3R,3'R)-zeaxanthin from Flavobacterium sp. appears to approach the stage
of commercialization. The same presumably applies to Phaffia rhodozyma as a
source of astaxanthin, the absolute configuration of which is, however,
opposite to the desirable one (3R, 3'R instead of 3S, 3'S). To a lesser extent
there may be a future for a fermentative process for canthaxanthin prepara-
tion, although no really useful organisms have been found yet. Still ketocarot-
enoids remain valuable compounds because of their efficiency as pigmenters in
poultry and fish feeds. The future will tell whether a market for 'natural'
canthaxanthin and astaxanthin will develop as an alternative to their synthetic
counterparts.
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5 NOTE ADDED IN PROOF
Several biotechnology companies are presently (May 1989) conducting exten-

sive research to improve the production of astaxanthin from Phaffia rhod-
ozyma. One of them (Gist Brocades) has now marketed the organism as a
pigmenter for salmonid diets. Recent papers on Phaffia rhodozyma are by N.
F. Haard (1988, Biotechnol, Lett., 10, 609-14) and G.-H. An (1989, Appl.
Environ. Microbiol., 55, 116-24). Maximum astaxanthin levels obtained by
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2.5 mg/g (mutant strain). Corresponding yields on a volume basis were
15 mg/liter and not specified, respectively.
Chapter 5
VITAMIN 0: THE BIOTECHNOLOGY OF ERGOSTEROL

P. MARGALITH
Department of Food Engineering & Biotechnology, Technion-Israel Institute of
Technology, Haifa, 32000, Israel
1 INTRODUCTION
Among the vitamins, vitamin D is outstanding because of its chemical nature,

it being the only vitamin with a steroid structure. Vitamin D is necessary for
the proper metabolism of minerals and bone formation, in mammals and birds.
Biotechnologically speaking, however, one usually refers to the provitamin,
ergosterol (1) the main sterol in yeasts and mycelial fungi. The provitamin may
be converted to the proper vitamin (D 2 ) which undergoes further metabolism
before exerting its hormone like activity in controlling the processes involved in
mineral metabolism.
24
HO HO
1 2
Ergosterol is not the only provitamin suitable for antirachitic treatment.

7-dehydrocholesterol (2) as produced in the mammalian body, may also be
transformed into vitamin D (D3) with somewhat different chemical and
physiological characteristics. Since the latter vitamin has certain advantages in
use, ergosterol as a source for vitamin D no longer occupies the same position
in the manufacturing of this vitamin.
Under natural circumstances, the human body produces its vitamin D from
7-dehydrocholesterol upon exposure to sunlight. In many geographical areas
81
82 P. Margalith
where clouded skies prevail throughout most of the year not enough vitamin D
is biosynthesized. The migration into cities and the increase in indoor
professions led to a decline in the synthesis of the natural vitamin and created
a demand for a dietary supplement that would protect the human body from
rickets.
In rickets, the organic matrix of new bone is not mineralized. This is due to
the body's inability to calcify the collagene matrix of the growing bone and
results in large areas of uncalcified bone (osteoid). The resultant lack in
rigidity of bones leads to the ends becoming entwisted and bent. Also, tooth
enamel formation may be affected. Children are specially prone to this disease.
Even with an optimal intake of calcium and phosphorus in vitamin D deficient
children, the concentration in the blood serum of these minerals is very low.
Vitamin D increases the calcium and phosphate absorption and restores the
mineral balance which results in the appropriate calcium phosphate deposition
in the bone.
The use of liver oil as a prophylactic against rickets, as well as the
importance of light as an antirachitic factor, have been recognized since before
CH3
HO HO
7-Dehydrocholesterol Ergosterol
1 1
./"
HO HO
Cholecalciferol (D3) Ergocalciferol (D 2 )
Fig. 1.
The Biotechnology of Ergosterol 83
the end of the 19th century. Steenbock & Black (1924) showed that irradiation
of foods led to the generation of the vitamin. We now know that irradiation
may lead to two types of vitamin 0: (1) By irradiation of ergosterol,
ergocalciferol (or O 2 ) is formed while (2) irradiation of 7-dehydrocholesterol
leads to the production of cholecalciferol (or D 3 ), see Fig. 1. This chapter will
deal chiefly with the production of ergosterol, the provitamin O 2 •
Ergosterol is a fat-soluble vitamin with a typical steroid structure. It consists of

4 rings (the cyclopentanoperhydrophenanthrene system, comprising 3 fused
6-membered rings and 1 5-membered ring) and a side chain, in all 28 carbon
atoms. Ergosterol differs from cholesterol, the best known sterol of animal
origin, in additional carbon (methyl group) at C-24 in the J3-position (referring
to the position on the axis of the side chain), as well as additional double
bonds between carbons 7: 8 and 22: 23. Like most steroids, ergosterol also has
2 angular methyl groups at carbon 10 and 13 in the J3-position (perpendicular
to the plane of the ring). Both sterols have a hydroxyl group at carbon 3 in the
J3-position. Many of the chemical and physiological characteristics of these
sterols can be attributed to this group.
Contrary to the saponifiable lipids, hydrolysis of steroids with alkali will not
make them water-soluble. They are therefore classified as unsaponifiable
lipids. Like most 3J3-hydroxysteroids, ergosterol can also be precipitated with
digitonin, a steroidal saponin. Although digitonin may be employed for the
precipitation of such sterols, the quantitative determination by gravitational
methods is usually not sensitive enough. Colorimetric methods are now widely
employed, most of which are based on the Liebermann-Buchard reaction. A
sterol in chloroform solution is treated with acetanhydrid and sulfuric acid at a
suitable temperature. With time a color develops with characteristic absorp-
tion. Ergosterol, on the other hand, may be determined spectrophotometri-
cally by absorption in UV. The characteristic spectrum of the 5,7 steroid in
absolute alcohol has peaks at 271, 282 and 293 nm. For the quantitative
determination of ergosterol it is advisable to employ a suitable separation
procedure (TLC or GLC) to avoid the interference of related compounds. The
molar extinction of ergosterol at 282 nm is 11 900.
3 OCCURRENCE AND PRODUCING ORGANISMS
Although yeasts are known as producers of ergosterol, the sterol was first
isolated from rye ergot (sclerotia of C/aviceps purpurea). It is from this plant
disease that the name ergosterol was derived (Tanret, 1889). Many organisms
were later screened and examined for their sterol content. While procaryotes
were found to be practically devoid of true sterols, eucaryotic micro-organisms
84 P. Margalith
could be divided into a number of categories with regard to the role sterols
play in their cell biology:
(1) Organisms in which sterols are an important constituent of the cell
material:
(a) Organisms that synthesize their own sterols.
(b) Organisms unable to synthesize their sterols, their requirements
being met by environmental sources.
(2) Organisms which do not require sterols for vegetative growth, but
need such compounds for the differentiation of certain sexual or
asexual reproductive organs.
(3) Organisms which have no known requirements for sterols but which
may incorporate such compounds if suitably exposed to them (Mar-
galith, 1986).
Screening for organisms that are good producers of ergosterol was obviously
directed to the first category of organisms that synthesize their own sterols.
Although sterols are widespread among photosynthesizing plants including the
fast-growing microalgae, phytosterols of green plants usually do not comprise
ergosterol. The search was therefore limited to eucaryotic heterotrophs,
amongst these the Mucorales (Zygomycotina), the Ascomycotina (including
the yeasts and yeastlike fungi), the Basidiomycotina (in the mushrooms) and
many of the Fungi Imperfecti.
In most cases ergosterol is not the only sterol to be found, it is usually
accompanied by other related sterols such as zymosterol (3) fungisterol (4) or
ergosta-5,7,22,24(28)-tetraenol (5). We shall return to these sterols when
discussing the biosynthesis of ergosterol. The quantitative aspects of ergosterol
formation and distribution have been also studied. There seems to be no great
variation in the sterol content of fungi. According to Appleton et al. (1955),
molds have an average of c. 0·6% sterols on a dry weight basis. Exceptional
higher values were recorded for Penicillium westlinghi (2·2%).
Not surprisingly, yeasts have been the object of many workers interested in
the biotechnology of ergosterol and its biosynthesis. The ease with which
yeasts can be grown and the familiarity of many microbiologists with these
organisms in various food industries, in addition to their being an excellent
source for most vitamin Bs, have made yeasts the organism of choice for the
production of provitamin D.
The first to study the ergosterol content of yeasts were Bills et al. (1930).
They examined 29 strains from 13 species of 5 genera: Endomyces, Nadsonia,
Mycoderma, Saccharomyces, and Zygosaccharomyces. Values of 0·2-0·3%
(dry weight) have been recorded. Saccharomyces carlsbergensis (strain ATCC
2345) had the highest ergosterol level (2·4%). Usdin & Burell (1952) found
2·7% in Rhodoturula gracilis. A systematic study for ergosterol in yeasts was
carried out by Dulaney et al. (1954) who examined 146 cultures of the
following genera: Candida, Diploascus, Hansenula, Nematospora,
HO
3
HO
4
HO
5
Saccharomyces, Schwanniomyces, Torulopsis, Cryptococcus, Endomyces,
Kloeckera, Pichia, Saccharomycodes, Sporobolomyces, Debaryomyces,
Endomycopsis, Nadsonia, Rhodotorula, Schizosaccharomyces, Taphrina. With
the exception of Saccharomyces, levels of ergosterol never exceeded 0·4%.
The best organism was Saccharomyces cerevisiae for which 3·9% was reported.
Additional studies were made by Appleton et al. (1955) who reached similar
conclusions. They confirmed the advantage of the genus Saccharomyces for the
formation of ergosterol. There seems to be considerable variation between
species or even strains. However, generally speaking, brewer's yeast had the
highest level of sterols, followed by baker's yeast. A method for the
production of high sterol yeasts (10% dry weight) was patented by Dulaney
(1957).
Not all reports deal with the nature of the sterols found in yeast. In most
cases where this was done the ratio of ergosterol to other sterols was 8: 2.
4 MEDIA AND ENVIRONMENTAL FACTORS
Sterol production by yeasts is greatly affected by environmental conditions.

High concentrations of assimilable carbohydrates promote the production of
ergosterol, while nitrogen-rich media usually lead to decreased levels of sterol
biosynthesis (Dulaney et al., 1954; Gal'tsova et al., 1959).
Non-sugar carbon sources for the production of sterols have also been
examined. Acetate seems to have a promoting effect when added to sucrose
(Starr & Parks, 1962). Ethanol can also serve as a carbon source. An old
method for increased sterol production consisted of exposing yeast cells to
vapors of ethanol. It was claimed that by this method a sterol level of c. 10%
86 P. Margalith
could be obtained. The most important environmental factor in yeast growth

and sterol productivity is, however, the availability of oxygen.
Although fermenting yeasts have a mechanism for the attainment of
workable energy (ATP) under anaerobic conditions, it has been known for
some time that Saccharomyces yeasts would not proliferate under strictly
anaerobic conditions (Nordheim, 1966). An explanation for this incongruity
was first suggested by Andreasen & Stier (1953) in the classical papers on the
anaerobic nutrition of S. cerevisiae. It was established that sterols (as well as
unsaturated fatty acids) cannot be synthesized under anaerobic conditions and
only if added to the media may they replace in part oxygen for growth under
these conditions. Since growth cannot take place under anaerobic conditions,
these are totally unsuitable for sterol production. Growth under semi-
anaerobic conditions, i.e. without aeration, yielded only 1/10 of sterols in
comparison to fully aerated yeasts (Klein, 1955). There seems to be a linear
relationship between oxygen availability and sterol biosynthesis. However,
there appear to be no detailed studies describing such a relationship and
whether there are any detrimental effects on sterol production due to
overaeration.
Earlier workers (Maguigan & Walker, 1940) suggested a relationship between

sterols and the total lipid composition, the former comprising 18-19% of the
total lipids. This was later found to be inaccurate since yeast strains with high
sterol content and low lipid composition were also found (Maugenet & Dupuy,
1964).
Since sterols belong to the lipid fraction of an organism, it is not surprising
that both sterols and fatty acids employ acetate as precursors. Indeed, label
studies showed that acetate, both the methyl and carboxyl carbons, serves as a
source for all the carbons of ergosterol, with the exception of C-28.
From acetate to lanosterol: the pathway by which the 2 carbon acetate
molecule is converted to the 5 carbon isoprene molecule, involves the
condensation of 3 acetate residues with the elimination of 1 carbon atom as
CO2 • From 3-hydroxy-3-methylglutaric acid (HMG) , isopentenyl pyrophos-
phate (IPP) and dimethylallylpyrophosphate (DPP) are formed. A stepwise
addition of C s units leads eventually to the formation of farnesylpyrophosphate
(FPP)-C 1s ; 2 FPPs forming by tail-to-tail condensation squalene, the open
chain C 30 of sterols. The cyclization of squalene is the second major step in
sterol biosynthesis. This is a complex reaction involving an oxygenated
intermediate:2,3-oxidosqualene which is formed in presence of oxygen only.
Obviously, anaerobically grown yeasts cannot form this intermediate (Fig. 2).
Cyclization of the epoxide is initiated by a specific enzyme (squalene cyclase)
leading to a series of hydrogen and methyl migrations giving rise to a cyclic
triterpenoid having the sterol nucleus (Schechter et aZ., 1970) known as
ACETATE
MEVALONATE
1
1
FARNESYLPYROPHOSPHATE
1
SQUALENE
1
SQUALENE EPOXIDE
1
LANOSTEROL
1
ZYMOSTEROL
I
FECOSTEROL
1
EPISTEROL
1
ERGOSTA-7 ,22,24(28)TRIENOL
1
ERGOST A-5,7 ,22,24(28)TETRAENOL
!
ERGOSTEROL
Fig. 2.
lanosterol (6). Interestingly, this compound is also formed during sterol
synthesis in animal tissues and is not found in green plants which synthesize the
related cycloartenol (7) as the first cyclization product.
HO HO
6 7
88 P. Margalith
Demethylations and alkylations: The tetracyclic lanosterol differs from a

true sterol in its 3 'extra' methyl groups at positions 4,4' and 14. In addition,
lanosterol shows 2 double bonds at C-24 (25) and C-8 (9). In order to become
ergosterol, the ultimate sterol of most fungi and yeasts, de methylation
reactions, reduction and rearrangements of double bonds in addition to the
alkylation of C-24 have to take place. Lanosterol as such cannot take the place
of ergosterol in anaerobic growth of yeast (Nes et al., 1978). This can be
explained by the bulky nature of lanosterol. The demethylation reactions
similar to squalene oxygenation require the presence of oxygen (for the
elimination in the form of CO2 ) and cannot proceed under anaerobic
conditions. The de methylation reactions lead to the formation of the first true
sterol compound:zymosterol (3).
Zymosterol differs from ergosterol in a number of features. The double
bond is still at 8: 9 and 24: 25, and C-24 must be alkylated with the C-28 methyl
group. We have already pointed out that the C-28 carbon is not derived from
acetate. The methyl group is derived from S-adenosylmethionine by a
transmethylation reaction (24-methyltransferase) yielding fecosterol (8) to be
followed by double bond migration from 8: 9 to 7: 8 giving episterol (9); the
introduction of an additional double bond (ergosta-7 ,22,24(28)-trienol); the
formation of the tetraenol (with the 5: 6 double bond) and the final reduction
of C-24(28) to ergosterol (1).
HO HO H 9
The pattern described above is not the only possible pathway for the
production of yeast ergosterol. Sterol synthesis, in general, does not follow a
rigid pattern but rather a more flexible framework where many alternative
pathways may take place. (Fryberg et al., 1973). Because of the complexity of
sterol biosynthesis and the diversity in the timing of certain key reactions (in
some strains (A~ B~ C, while in others A~ C~ B), the regulation of sterol
synthesis has not been sufficiently studied. Of the many enzymic reactions
potentially capable of a regulatory function, hydroxy-methyl-glutaryl-CoA
reductase is probably the rate-controlling enzyme of sterol biosynthesis
(Qureshi et al., 1980).
6 FERMENTATION
Very few publications deal with the ergosterol fermentation. In the 1950s a
number of papers were published dealing with the production of ergosterol by
various yeasts and fungi. Since yeasts with strong fermentative metabolism
were found to be the best producers of ergosterol, most of the studies
concentrated on the fermentation of S. cerevisiae strains. Using a molasses-
cornsteep medium, cells with an ergosterol content of 7-10% could be
obtained. Cell yields of 30-40 g/liter medium and a sterol content of
3-4 g/liter broth were found in shake flask experiments after 4 days of growth
at 28°C (Dulaney et al., 1954). Good yields were also obtained with S.
fermentati (EI Refai & EI Kady, 1969). Little has been published about
fermentation in larger volumes. Nor has the patent literature revealed
important new information on the industrial fermentation of the provitamin.
Since the early patent of Dulaney (1957) a number of patents were granted to
several companies in various countries. Most of these deal with extraction
procedures for the isolation and crystallization of ergosterol from the biomass
(e.g. Nelson, 1959). Few patents describe a modification of the fermentation
procedure by the incorporation of precursors to the medium. The addition of
isopentenol (0·92%) and Na-polyphosphate (0·3%) to the growth of S. uvarum
UFO 1264 at 26°C for 96 h, yielded 2·1 % ergosterol (dry weight) (Japanese
Patent 8158496, 1981). Organisms described in the patent literature comprise:
S. cerevisiae, S. carlsbergensis, Candida tropicalis, Fusarium spp and
Trichoderma spp. Yields (ergosterol on basis of dry weight or volume of
medium) described in the patent literature are generally far lower than those
reported in scientific publications. A number of patents have been granted for
the growth of yeasts in medium containing hydrocarbons as sole carbon source,
e.g. Candida petrophilum ATCC 20226 (Horiguchi et al., Japanese Patent
7392586, 1973). Also here ergosterol yields were rather low (see Table 1 for
list of patents).
Isolation of ergosterol from biological sources usually involves the extraction
of the fatty component, saponification and re-extraction with an ether. Direct
extraction of yeast is not sufficient, usually a preliminary digestion with hot
alkali is included prior to the extraction.
Provitamin D3
The equivalent of ergosterol in animal tissues is 7-dehydrocholesterol (2) which

can be produced commercially by the bromination of an esterified cholesterol
followed by dehalogenation to give the 7 -dehydrocholesteryl ester.
7 THE FORMATION OF VITAMIN D
Both ergosterol and 7-dehydrocholesterol can be transformed into previtamin

D by UV irradiation (280-300 nm). Sufficiently low temperatures are main-
tained to prevent the formation of side products. This results in the opening of
the B ring. The 5,7 conjugated diene is activated yielding the previtamin (10),
which undergoes a thermal equilibration to vitamin D. Fission of the 9: 10
8
Table 1
Some Patents for the Production of Ergosterol
Name Country Patent No. Year Organism SJlecialfeatures

Ergosterol containing yeast USA 2817624 1957 S. cerevisiae
NRRL strains
Ergosterol from yeast Britain 801390 1958 Recovery
Extraction of sterols USA 2837540 1958 Recovery ~
Production of ergosterol Japan 49792802 1971 C. troJlicalis N-alkane as sole
(pk233)
~
by yeasts from hydrocarbons carbon source ~
Q
Ergosterol production from Japan 7392486 1973 C. JletroJlhilum n-Paraffins and ::::
petroleum (ATCC 20226) acetone in medium :r
Fermentative production of Japan 7886092 1978 Aureobasidium
sterols Jlullulans
(FERM + P3496)
Cultivation of yeast Czechoslovakia 196950 1981 S. cerevisiae Medium supplemented
producing ergosterol with L-methionine
Production of Japan 8158496 1981 S. uvarum Isopentenol
ergosterol (UFO 1264) supplementation
bond leads to the formation of the secosteroid. Ergosterol thus becomes

Vitamin D2 (ergocalciferol) and 7-dehydrocholesterol becomes vitamin D3
(cholecalciferol). The biological activity of 0·025 f.lg of pure cholecalciferol is
considered the international unit (IU) of vitamin D.
R
HO
10
8 METABOLIC TRANSFORMATIONS
Vitamin D formed in the skin or absorbed by ingestion through the gut wall, is
transported by the bloodstream into the liver where it is hydroxylated at C-25
to yield 25-hydroxycalciferol which is the main circulating form of Vitamin D.
It is transported on a specific a-globulin to the kidney where further
hydroxylation takes place at C-1 or C-24, apparently in response to the calcium
levels in the blood. Low phosphate levels also stimulate 1,25-
dihydroxycholecalciferol (11) production which, in turn, enhances calcium
absorption. The C-1 hydroxylation is the critical step which enables the vitamin
to assume its hormonal activity in regulating the calcium transport as well as
bone calcium mobilization. This important reaction is carried out in kidney
mitochondria by a mixed function mono-oxygenase involving cytochrom P-450.
OH
/;k
HO
11
Regulation of vitamin D metabolism appears to be controlled by the levels
of calcium, phosphate and the parathyroid hormone in blood serum. Below
9·5 mg/ml of Ca2 +, 25-dihydroxy vitamin D is formed, above that level
24,25-dihydroxycalciferol is produced with the discontinuation of the la-
hydroxylase activity. The enzyme is also stimulated by the homeostatic
parathyroid hormone which causes phosphate diuresis in the kidney and
enhances 1,25-dihydroxycholecalciferol production. The parathyroid hormone
and 1,25-dihydroxycholecalciferol act at the bone site cooperatively in the
stimulation of calcium mobilization from bone. Other metabolites of vitamin D
92 P. Margalith
are also formed. However, they are less potent and their metabolic significance
is not yet fully understood.
9 APPLICATION AND ECONOMICS
While vitamin D is used for the fortification of many foodstuffs (margarine,

milk), large amounts are added to the animal feeds, mostly for swine, chicken
and dairy calves. Vitamin D2 and D3 have the same biological activity in rats.
However, in chicken, vitamin D2 is only 1/10 or less active in comparison to
vitamin D 3. In man cholecalciferol is apparently also more active than
ergocalciferol. The difference is probably due to the C-24 methyl group and
not the C-22 double bond since 22-dihydroergosterol is as effective as
provitamin D 2. Considering these facts led to the obvious preference of
cholecalciferol produced synthetically. Vitamin D3 is sold at about 1 $/1(t IU;
or 10 $/kg of a product of 400 000 IU/g(Hirsch, 1984). Considering the
worldwide demand for vitamin D at about 1750 X 1012 IU (1985), neither the
quantities required nor the price obtained on the market have given great
impetus to the improvement in the biotechnological production of provitamin
D2•
Nevertheless, some efforts have been made to produce biotechnologically a
precursor that could be employed for the production of vitamin D 3. Avruch et
al. (1976) suggested to inhibit the 24-methyltransferase, the enzyme
responsible for the C-24 methylation, characteristic to ergosterol. By growing
S. cerevisiae in the presence of 25, aza-24,25 dihydrozymosterol at 1·3 J.lM
concentration, it was possible to inhibit the transmethylation reaction without
affecting the growth of the yeast. Under these conditions little ergosterol was
formed but the accumulation of zymosterol (3) and cholesta 5,7,22,24(25)-
tetraenol (12) took place in considerable quantities. The cholestatetraenol was
suggested as a substitute for the more expensive 7-dehydrocholesterol provita-
min. The conversion of this compound to the corresponding cholecalciferol by
irradiation would be expected. However, no additional information has
become available about this modified process.
HO
12
REFERENCES
Andreasen, A. A. & Stier, T. J. B. (1953). Anaerobic nutrition of Saccharomyces

cerevisiae. I. Ergosterol requirement for growth in deficient medium. J. Cell.
Physiol., 41, 23-36.
Appleton, G. S., Kieber, R. J. & Payne, W. J. (1955). The sterol content of fungi II.
Screening of representative yeasts and molds for sterol content. Appl. Microbiol., 3,
249-51.
Avruch, L., Fischer, S., Pierce, H. D. & Oehlschlager, A. C. (1976). The induced
biosynthesis of 7-dehydrocholesterols in yeast: potential sources of new provitamin
D3 analogs. Can. J. Biochem., 54,657-65.
Bills, C. E., Massengale, O. N. & Prickett, P. S. (1930). Factors determining the
ergosterol content of yeast. I. Species. J. BioI. Chem., 87, 259-64.
Dulaney, E. L. (1957). Preparation of ergosterol containing yeast. US Patent 2817624.
Dulaney, E. L., Stapley, E. O. & Simpf, K. (1954). Studies on ergosterol production
by yeasts. Appl. Microbiol., 2,371-9.
EI-Refai, A. E. M. & EI Kady, I. A. (1969). Utilization of some industrial by-products
for the microbiological production of sterols from Saccharomyces fermentati. J. Bot.
UAR, 12, 55-66.
Fryberg, M., Oehlshiager, A. C. & Uorau, A. M. (1973). Biosynthesis of ergosterol in
yeast. Evidence for multiple pathways. J. Am. Chem. Soc., 95,5747-57.
Gal'tsova, R. D., Novichkova, A. T. & Vakina, I. P. (1959). Effect of glucose on
ergosterol synthesis by yeasts. Mikrobiologiya, 28, 502-6.
Hirsch, A. (1984). Vitamin D. In Kirk Othmer's Encyclopedia of Chemical Technol-
ogy, 3rd edn, Vol. 20. Wiley-Interscience, New York, pp. 186-213.
Klein, H. P. (1955). Synthesis of lipids in resting cells of Saccharomyces cerevisiae. J.
Bacteriol., 69, 620-7.
Klein, H. P., Eaton, N. R. & Murphy, S. C. (1954). Net synthesis of sterols in resting
cells of Saccharomyces cerevisiae. Biochim. biophys. Acta, 13, 591.
Maguigan, W. H. & Walker, E. (1940). Sterol metabolism of microorganisms. I. Yeast.
Biochem. J., 34,804-13.
Margalith, P. (1986). Steroid Microbiology. C. C. Thomas, Illinois.
Maugenet, J. & Dupuy, P. (1964). Synthese des sterols par la levure. Ann. Technol.
Agric., 13,329-54.
Nelson, H. A. (1959). Recovery of ergosterol. US Patent 2 874 171.
Nes, W. R., Sekula, B. C., Nes, W. D. & Adler, J. H. (1978). The functional
importance of structural features of ergosterol in yeast. J. Bioi. Chem., 253,
6218-25.
Nordheim, W. (1966). Metabolic behaviour of fermenting yeasts under cytostatic
conditions. Brauwiss., 19, 67-9.
Qureshi, A. A., Burger, W. C., Prentice, N., Bird, H. R. & Sunde, M. L. (1980).
Suppression of cholesterol and stimulation of fatty acid biosynthesis in chicken livers
by dietary cereals supplemented with culture filtrate of Trichoderma viride. J. Nutr.,
110, 1014-22.
Schechter, I., Sweat, F. W. & Bloch, K. (1970). Comparative properties of 2,3
oxidosqualene-Ianosterol cyclase from yeast and liver. Biochim. biophys. Acta, 220,
463-8.
Starr, P. R. & Parks, L. W. (1962). Some factors affecting sterol formation in S.
cerevisiae. J. Bacteriol., 83, 1042-6.
Steenbock, H. & Black, A. (1924). Fat soluble vitamins XVII. The induction of growth
promoting and calcifying properties in a ration by exposure to ultra-violet light. J.
BioI. Chem., 61,405-22.
Taoret, C. (1889). Sur un nouveau principe immediat de L'ergot de seigle,
l'ergosterine. C. R. Seances Acad. Sci., 108,98.
Usdin, V. R. & Burrell, R. C. (1952). Preliminary study of the lipids of Rhodotorula
gracilis. Archs. Biochem. Biophys., 36, 172-7.
Chapter 6
ALGAL AND MICROBIAL PRODUCTION OF VITAMIN E

Y. TANI
Research Center for Cell and Tissue Culture, Faculty of Agriculture,
Kyoto University, Kyoto 606, Japan
1 INTRODUCTION
The fat-soluble vitamin, vitamin E, has received great attention for its
usefulness as an antioxidant in clinical and nutritional applications. The
demand for the vitamin has increased year after year. The supply of
tocopherols has been limited to the chemically synthesized racemate of
a-tocopherol or a mixture of a-, f3(y)- and 6-tocopherols extracted from
vegetable oils.
Tocopherols are indispensable components of the lipid bilayer of biological
membranes; decrease in their content brings about structural and functional
damage to the membranes. It is generally accepted that the stabilizing effect of
tocopherols on the membranes is mainly related to three functions: (1)
reaction with lipid peroxide radicals, (2) quenching of reactive molecular
oxygen, and (3) ordering (i.e. restriction of molecular mobility) of the
membrane lipid bilayer by formation of tocopherol complexes with fatty acids.
The highest physiologically active form among the various tocopherols and
tocotrienols so far identified is D-a-tocopherol.
Since a preliminary survey on the occurrence of tocopherols in micro-
organisms (Green et al., 1959), its presence has been demonstrated especially
in chlorophyll-containing organisms, though not in all of them (Taketomi et
al., 1983). Early, a tocopherol-like antioxidant was found in yeasts (Forbes et
al., 1958) and a small amount of a-tocopherol was detected in cells of baker's
yeast (Diplock et al., 1961). a-Tocopherolquinol and a-tocopherolquinone
were found in the algae, Euglena gracilis, in bacteria and in yeasts (Hughes &
Tove, 1982). Ruggeri et al. (1985) reported enhancement of a-tocopherol
production in temperature-stressed cells of E. gracilis Z grown photo-
heterotrophically. However, a study on industrial production of these com-
pounds has not been performed so far.
Recently, Tani & Tsumura (1989) have initiated a study aiming towards
95
96 Y. Tani
the development of new microbial processes to produce tocopherols, especially

a--tocopherol.
2 CHEMISTRY AND ASSAY OF VITAMIN E

Vitamin E was found as a dietary factor in animal nutrition, considered in the
1920s to be especially important for normal reproduction. a-- and f3-
Tocopherols were isolated from wheat germ oil and characterized in 1936.
Soon afterwards the third active factor, y-tocopherol, was found in cotton-seed
oil. In 1947, the fourth tocopherol, named ~-tocopherol, was isolated from
soybean oil. In the ensuing years, the investigation of several vegetable oils
disclosed the existence of four tocotrienols. Thus, four tocopherols and four
tocotrienols are now known to occur in nature (Fig. 1).
Tocopherols and tocotrienols are white to pale yellow oils. These are soluble
in organic solvents such as ethanol and ether, and miscible with other oils.
They are easily oxidized to a dark color. The esters are stable. The naturally
occurring form is the D-form, and D-a--tocopherol has the 2R, 4'R and 8'R
configuration. The absorption maxima are 290-298 nm.
Determination of tocopherols in cells has been described by Tani &
Tsumura (1989). Cells were homogenized in ethanol and extracted at
50-60°C for 20 min. After an equal volume of n-hexane and one and a half
volumes of water were added to the ethanolic extract and mixed, the n-hexane
layer was obtained by centrifugation. The n-hexane extract was employed for
high performance liquid chromatography. Column systems used were an M&S
packed column (CIS 4·6 x 150 mm) with methanol-dichloromethane (9: 1, v Iv)
as the mobile phase or a YMC packed column (Sil, 6 x 150 mm) with
n-hexane-dichloromethane-isopropanol (70: 30 : O· 2, by volume). The flow
rate was 1 ml/min. Tocopherols were detected at 292 nm.
Rl R2
CH J CH J a- Tocopherol
CH J H B-Tocopherol
H CH J y- Tocophero 1
H H 0-Tocophero 1
Rl R2
CH J CH 3 a-Tocotrienol
CH J H B-Tocotrienol
H CH J y- Tocotri eno 1
H H 6-Tocotrienol
Fig. 1. Chemical structure of vitamin E group.

Vitamin E Production 97
3 BIOSYNTHESIS OF VITAMIN E
It has been accepted that tocopherols are synthesized through two routes, the
tocopherol route and tocotrienol route. Homogentisic acid, a precursor of
tocopherols, which was derived from the shikimate pathway (the biosynthetic
pathway of aromatic amino acids), incorporates phytyl pyrophosphate or
geranylgeranyl pyrophosphate. When phytyl pyrophosphate is incorporated,
tocopherols are formed and the following additional methylation using
S-adenosyl-L-methionine as the methyl donor occurs to form various types of
tocopherols (tocopherol route). When geranylgeranyl pyrophosphate is in-
corporated, tocotrienols are formed with a similar methylation. Subsequently,
tocotrienols can be transformed to tocopherols. NADPH is the reducing
coenzyme for the reduction of the side chain (tocotrienol route). In Spinach
chloroplasts, the enzymes involved in the a-tocopherol synthesis were found in
the chloroplast envelope fraction. The prenyltransferase and methyltransferase
activities were found to be tightly bound to the chloroplast envelope
membrane. Thus, it is strongly suggested that the tocopherol biosynthesis is
related to the occurrence of chloroplasts or plastids.
4 FERMENTATIVE PRODUCTION OF TOCOPHEROL
4.1 Screening of Tocopherol-Producing Micro-organisms
The distribution of tocopherol-producing ability was extensively studied with

162 strains from 26 genera of ascosporogenous and asporogenous yeasts, 74
strains from 15 genera representing molds of the Phycomycetes, Ascomycetes
and Fungi Imperfecti, 41 strains from 12 genera of Basidiomycetes, 4 strains of
algae belonging to genera of Euglena, Chlorella and Chlamydomonas, and 5
strains of isolated but unidentified algae. Though occurrences of tocopherol in
yeast (Diplock et al., 1961) and a-tocopherolquinone and its quinol in
eucaryotic and procaryotic micro-organisms (Hughes & Tove, 1982) were
reported, no tocopherol was detected in cells and culture filtrates of yeasts,
molds and Basidiomycetes under the cultured and detection conditions
employed. On the other hand, all algae tested were found to contain
tocopherols, mainly the a-type.
Euglena gracilis Z was the best producer of a-tocopherol among all algae
tested. It appeared on high performance liquid chromatograms that the
tocopherol detected corresponded to the retention time of a-tocopherol and
that it was the dominant component, with only a small amount of fJ(y)- and
6-tocopherols. Shigeoka et al. (1986) demonstrated during a study on the
intracellular localization of tocopherols in E. gracilis Z, that a-tocopherol
constituted more than 97% of total tocopherols.
98 Y. Tani
4.2 Isolation of a-Tocopherol from EuglelUl gracilis Z CeUs
Cells of E. gracilis Z weighing 2·1 g as dry matter were obtained from 80 ml of

culture broth. The cell paste was suspended in 100 ml of ethanol and kept at
50°C for 30 min. The colorless cell debris were filtered and re-extracted once in
the same manner. The combined extract was partitioned between equal
volume of sodium chloride-saturated water and 150 ml of n-hexane. The
n-hexane phase was dried by anhydrous sodium sulfate and then concentrated
at 50°C under reduced pressure. Resultant lipid residues dissolved in 20 ml of
isooctane were passed through Sep-Pak (silica cartridge, Waters Associates)
with a flow rate below 0·5 ml/ml and then 5 ml of isooctane was passed to
eliminate non-polar carotenoids. After the elution with 15 ml of isooctane-1,4-
dioxane (98:2, v/v), the eluate was concentrated, dissolved in n-hexane and
then applied on a thin layer chromatography (Kieselgel60F254 ). The thin layer
chromatography was developed with benzene. The band appeared as dark blue
against yellow background under UV light (Rf = 0·62) was scraped off and
extracted with n-hexane-ethanol (5: 1, v/v). Pale yellow oil, 3·6 mg, was
obtained in the overall recovery of about 34% by evaporation.
The isolate dissolved in ethanol showed the typical absorption spectrum of
Amax at 292 nm and corresponded to the authentic a-tocopherol. The peak of
the retention time of the isolate on a high performance liquid chromatography,
4·5 min, corresponded with that of a-tocopherol. Therefore, the oily substance
isolated from E. gracilis Z cells was identified as a-tocopherol.
4.3 Production of a-Tocopherol by Euglena gracilis Z
When E. gracilis Z was grown on a conventional algal medium, Koren-Hutner

(KH) medium, the maximum amount of a-tocopherol, 1·98 mg/liter culture
broth and 0·26mg/g dry cells, was obtained at 9th day of cultivation. To
improve the productivity and simplify the medium composition, components of
culture medium and their concentrations were investigated using KH medium
as the basal medium. As a result, a medium was derived as an a-tocopherol
production medium consisting of glucose (2%), peptone (1·2%), inorganic
salts, thiamin and cyanocobalamin, pH 5·0. The amount of a-tocopherol in the
culture broth increased with increase of the growth and chlorophyll levels in
the cells, and reached a maximum after 10 days of cultivation. The amount of
a-tocopherol was 9·9 mg/liter culture broth and 1·1 mg/ g dry cells.
The production of a-tocopherol, however, was much higher under light
conditions than in the dark, indicating that chloroplasts whose differentiation is
induced by light could be related to a-tocopherol productivity.
The effect of additions of a-tocopherol biosynthesis-related compounds on
a-tocopherol production was investigated. L-Tyrosine, L-phenylalanine, iso-
pentenyl alcohol, AMP, 4-hydroxyphenylpyruvate and homogentisate ap-
parently increased the a-tocopherol productivity. Since L-tyrosine and L-
phenylalanine share the biosynthetic pathway with tocopherols, their addition
might repress their own biosynthesis and thus increase the biosynthetic flow to
a-tocopherol. Homogentisate is a direct precursor of the tocopherol biosynth-
esis. Among prenyl alcohols, which might be involved in the tocopherol
biosynthesis in the form of pyrophosphate to be incorporated to the phytyl side
chain, isopentenyl alcohol was slightly effective on a-tocopherol production.
When these compounds were added simultaneously to the a-tocopherol
production medium, a combination of 0·1 % homogentisate and 0·01 %
L-tyrosine was the best for a-tocopherol production. Moreover, it was found
that higher productivity can be obtained when this mixture was added to the
culture medium only at 5th day of cultivation rather than at the beginning.
Feeding of ethanol to the medium together with peptone, when glucose was
almost consumed, gave higher production of a-tocopherol than that of
glucose-peptone in spite of the same cell yield.
Supplementation with ethanol and peptone, and homogentisate and L-
tyrosine during cultivation was performed finally. As shown in Fig. 2, the
specific production of a-tocopherol increased markedly when the mixture was
fed at 5th, 7th and 9th days of the cultivation. On the other hand, when only
ethanol and peptone were fed, the amount of a-tocopherol in the culture broth
increased according to increase in cell mass but the intracellular content of
a-tocopherol was almost the same as that of the non-supplemented culture.
(8) (C)
,
100
+ ,
,
a;-
...
....
......
~'"
.....
20 .~
..
50
a... ......
.&:
'"
a. "0
0
u 10 .~
....
0
I
>.
"
0
0 5 10 15 0 S 10 lS 0 5 10 15
Cultivation Time (day)
Fig. 2. a-Tocopherol production in a batch culture. During the cultivation on the

a-tocopherol production medium (A), 1% ethanol and 0·6% peptone (B), and 1%
ethanol, 0·6% peptone, 0·1% homogentisate and 0·01 % L-tyrosine (C) were added at
periods indicated by arrow. Concentrations indicated are the final concentration (w/v)
in the medium.
Y. Tani
Table 1
Progress of a-Tocopherol Production by E. gracilis Z
Medium Cell yield a-Tocopherol produced

(g /liter)
(mg/liter) (mg/g dry cells)
KH medium 7·0 2·0 0·3
a-Tocopherol production medium 9·0 9·9 1·1
Added L-tyrosine 10·7 14·8 1·3
Added homogentisate 11·9 17·0 1·4
Added L-tyrosine, homogentisate
and ethanol 11·4 49·8 4·3
Fed L-tyrosine, homogentisate,
ethanol and peptone 28·4 143·6 5·1
The amount of a-tocopherol produced in the culture reached 143·6 mg/liter

culture broth and 5·1 mg/g dry cells at 20th day of cultivation.
The increase in intracellular production of a-tocopherol by E. gracilis Z
during the optimization study on culture conditions is summarized in Table 1.
The final productivity in this medium was 71·8-fold that of the original KH
medium and the intracellular content was increased 19·6-fold.
5 BIOCONVERSION REACTIONS FOR VITAMIN E SYNTHESIS
As represented in Fig. 1, a-tocopherol is composed of a chroman ring and an

aliphatic side chain. The naturally occurring molecule has three chiral centres
Phytol
"'co~
~CHO +
Wittig reaction
hydrogenation
hydrolys"
Ho4c
Fig. 3. Synthesis of (2R, 4'R, 8'R)-a-tocopherol by coupling optically active chroman
aldehyde with a C I5 phosphonium salt derived from natural phytol.
R
I
I •
(4* :C 1 : (4- (5
Fig. 4. Construction of an optically active C 14 side chain from S-fJ-hydroxyisobutyric
acid.
with the (2R, 4'R, 8'R)-configuration. Chemical synthesis of vitamin E-which

is now industrially applied-yields a racemic product. In this respect, several
attempts are made to synthesize the natural optically active compound
(Leuenberger, 1985).
One example of such synthesis involves the coupling of optically active
chroman aldehyde with a CIs-phosphonium salt derived from natural phytol
(Wittig reaction), followed by double bond hydrogenation and hydrolysis of
the protecting group. The natural side chain precursor, phytol, is the source of
the two chiral centres (Fig. 3).
Recently, the total synthesis of the optically active side chain has been
reported. The formation of such side chains starts from small optically active
building blocks (C: or Cn, which are produced via microbial reactions. In this
respect, microbiologically formed S-( + )-fJ-hydroxyisobutyric acid (a C:-
synthon) has been used to produce a C l4-vitamin E side chain according to the
j
~COOH
HO I
~COOR
CD/
HO~OH
Fig. S. Microbial reactions affording optically active S- or R-fJ-hydroxyisobutyric acid

(fJ-HIBA). 1. S-fJ-HIBA; Pseudomonas putida or other micro-organisms. 2. S- or
R-fJ-HIBA; depending on micro-organisms used. 3. R-fJ-HIBA; Gluconobacter roseus.
4. R-fJ-HIBA; Candida humicola.
102 Y. Tani
I B' I B
A'~ -- A~
-
hyctroly."
cyclzatlon
Fig. 6. Construction of an optically active C I5 side chain.
scheme: C 5 + C; + C i + C; (Fig. 4). Several examples of microbial bioconver-

sion leading to this optically active S- or R-tJ-hydroxyisobutyric acid are given
in Fig. 5.
Another possibility for the synthesis of an optically active C 15 side chain is a
C; + C; + C; condensation (Fig. 6). The optically active C5 -synthon can be
produced from a suitable unsaturated precursor via enantioselective microbial
reduction (Fig. 7). Saccharomyces cerevisiae and Geotrichum candidum are
useful in this respect and formed the C 5 -synthon, (S)-3-methyl-y-butyro-
lactone, in high yield from suitable precursors.
The optically active chroman ring has also been synthesized using (S)-( + )-
citramalic acid to introduce chirality; asymmetric hydration of mesaconic acid
into the desired (S)-( + )-citramalic acid has been performed with Clostridium
tetanomorphum (Fig. 8). This synthon has also been used to form the vitamin
D 3 -metabolite, (25S, 26)-dihydroxycholecalciferol.
A more detailed description of the use of microbially obtained optically
active synthons in vitamin E synthesis is given by Leuenberger (1985).
i/ i
c~
Fig. 7. Bioconversion leading to optically active C 5 -synthons.
HaOC) Clostridium tetanomorphum Haoc~

~
leaoH HO COOH
HO*
I ----
~ OH
I
I
,
I
I
I
I
I
I
t'
H~
¥O~OH
Fig. 8. Synthesis of the optically active chroman moiety from trimethylhydroquinone
and (S)-citramalic acid.
REFERENCES
Diplock, A. T., Green, J., Edwin, E. E. & Bunyan, J. (1961). Tocopherol,

ubiquinones and ubichromenols in yeasts and mushrooms. Nature, Lond., 189,
749-50.
Forbes, M., Zilliken, F., Roberts, G. & Gyorgy, P. (1958). A new antioxidant from
yeast. Isolation and chemical studies, J. Am. Chern. Soc., SO, 385-9.
Green, J., Price, S. A. & Gare, L. (1959). Tocopherols in microorganisms, Nature,
Lond., 184, 1339.
Hughes, P. E. & Tove, S. B. (1982). Occurrence of a-tocopherolquinone and
a-tocopherolquinol in microorganisms. J. Bacteriol., 151, 1397-402.
Leuenberger, H. G. W. (1985). Microbiologically catalyzed reaction steps in the field of
vitamin and carotenoid synthesis. In Biacatalysts in Organic Synthesis, eds. J.
Tramper, M. C. Van der Plas & P. Linko. Elsevier, Amsterdam, pp. 99-118.
Ruggeri, B. A., Gray, R. J. H., Watkins, T. R. & Tomlins, R. I. (1985). Effects of
low-temperature acclimation and oxygen stress on tocopherol production in Euglena
gracilis Z. Appl. Env. Microbial., 50, 1404-8.
104 Y. Tani
Shigeoka, S., Onishi, T., Nakano, Y. & Kitaoka, S. (1986). The contents and
subcellular distribution of tocopherols in Euglena gracilis. Agric. BioI. Chern., 50,
1063-5.
Taketomi, H., Soda, K. & Katsui, G. (1983). Results of screening test in tocopherols in
microbial realm. Vitamins (Japan), 57, 133-8.
Tani, Y. & Tsumura, H. (1989). Screening of tocopherol-producing microorganisms
and a-tocopherol production by Euglena gracilis Z. Agric. Bioi. Chern., 53, 305-12.
Chapter 7
MICROBIAL PRODUCTION OF POLYUNSATURATED

FATTY ACIDS (VITAMIN-F GROUP)
& H. YAMADA
S. SHIMIZU
1 INTRODUCTION
The classical investigations performed by Burr & Burr in 1929-1930 clearly

demonstrated that animals cannot survive without an exogenous source of fat.
Subsequent studies have shown that supplementing a fat-free diet with certain
essential fatty acids prevents the occurrence of symptoms of fat deficiency. It is
now well established that both the n-6 and n-3 polyunsaturated fatty acids
(PUFAs) are essential in human nutrition. Current information indicates that
they have important roles in the structure and function of biological mem-
branes. They have recently attracted great interest due to their unique
biological activities, such as lowering of plasma cholesterol level, prevention of
thrombosis, and so on. In addition, they are precursors for the biosynthesis of
a variety of a large family of structurally related C-20 compounds, such as
prostaglandins, prostacyclins, thromboxanes and leukotrienes. Such current
status of PUFAs suggests that they are highly important substances in the
pharmaceutical, medical and nutritional fields. These substances are sometimes
referred to as the vitamin-F-group.
In this chapter, we summarize current progress in the biotechnology of
PUFA production. Emphasis will be placed on possible advantages of
micro-organisms as practical sources of PUFAs. Several reviews summarize
other various aspects of PUFAs and provide excellent background information
(Wagner & Folkers, 1964; Rahm & Holman, 1971; Brenner, 1974; Fluco,
1974; Holloway, 1983; Numa, 1984; Dyerberg, 1986; Needleman et al., 1986;
Horrobin & Huang, 1987).
2 NATURALLY OCCURRING POLYUNSATURATED FArry

ACIDS
The n-6 and n-3 families of PUFAs are distinguished by differing positions of
the double bond closest to the methyl terminal group of the fatty acid chain
105
106 S. Shimizu & H. Yamada
n-7
c:::::::.~ --~ ~~~~ c::::::~~ ~~
16:1 16:2 18:2 18:3 20:3
c:::::::;:.--~~ ~~- c:::::::;:

n-9
18:1 18:2 20:2 20:3

n-6
~
18:2
-c::;::::; -- c::;:;:;~- c:::;::;:::
18:3 GLA 20:3DHGLA 20:4 ARA
n-3
C:::::::::=H ~C::::::::H - ~~.. ~~. C::::::: OOOH
18:3 18:4 20:4 20:5 EPA 22:6 DHA
Fig. 1. Pathways for the biosynthesis of PUFAs of the n-7 (palmitoleic), n-9 (oleic),
n-6 (linoleic) and n-3 (a-linolenic) families. PUFAs include the n-7, n-9, n-6 and n-3
families, which are defined by the position of the double bond closest to the methyl end
of the fatty acid molecule. Desaturation occurs toward the carboxyl end of the molecule
and chain elongation at the carboxyl end, leaving the methyl end unaltered. Usually,
PUFAs of each family are not inconvertible. GLA, r-linolenic acid; DHGLA,
dihomo-r-linolenic acid; ARA, arachidonic acid; EPA, eicosapentaenoic acid; DHA,
docosahexaenoic acid.
(see Fig. 1). Linoleic acid (9,12-cis-octadecadienoic acid, 18: 2 n-6) and
a-linolenic acid (9,12,15-cis-octadecatrienoic acid, 18: 3 n-3) are found abun-
dantly in plants. They are synthesized de novo in higher plants and together
represent nearly all the polyenoic fatty acids present. Linoleic acid comprises
about 50% of the total fatty acids in corn, soybean, sunflower-seed and
cotton-seed oils and over 70% of the fatty acids of safflower oil. a-Linolenic
acid represents over 50% of the total fatty acids of linseed and perilla oils. In
Oenothera biennis (evening primrose) and O. lamarckiana, however, linoleic
acid is further desaturated to form y-linolenic acid (6,9,12-cis-octadecatrienoic
acid, 18: 3 n-6), which comprises about 10% of the total fatty acids in their
seeds. Mosses (Hartmann et al., 1986), algae (Seto et al., 1984) and protozoa
(Erwin & Bloch, 1964), some fungi (Gellerman & Schlenk, 1979; Yamada et
al., 1987, Shimizu et al., 1988a,1989a) and some marine bacteria (Yazawa et
al., 1988) can further add a 2-carbon unit to these C-18 PUFAs to form C-20
PUFAs.
Animals cannot synthesize fatty acids with double bonds at either the n-3 or
n-6 positions, and therefore are dependent upon dietary sources for these fatty
acids. Most animals, however, possess enzyme systems for further desaturation
and chain elongation at the carboxyl end of these fatty acid molecules. The
resulting further desaturated or longer-chain derivatives such as y-linolenic
acid, dihomo-y-linolenic acid (8,1l,14-cis-eicosatrienoic acid, 20: 3 n-6),
arachidonic acid (5,8,1l,14-cis-eicosatetraenoic acid, 20: 4 n-6), eicosa-
pentaenoic acid (5,8,1l,14,17-cis-eicosapentaenoic acid, 20:5 n-3, or EPA)
and docosahexaenoic acid (4,7,10,13,16,19-cis-docosahexaenoic acid, 22: 6
Microbial Production of PUFAs 107
n-3, or DHA), are important as essential fatty acids in human nutrition or as

structural components of cellular membrane phospholipids.
Most aquatic plants can synthesize the longer chain n-3 fatty acids including
EPA and DHA from common metabolites. Fish, shellfish and marine
mammals at the apex of the aquatic food chains contain relatively large
quantities of these n-3 fatty acids, and therefore provide the human diet with a
direct source of preformed EPA or DHA. Terrestrial food chains also contain
some n-3 PUFAs, but they are dominated by the n-6 PUFA family because
most land plants synthesize linoleic acid rather than a-linolenic acid.
A variety of PUFAs other than those of the n-6 and n-3 families have been
detected in living organisms. Some of them are also listed together with the
PUFAs of the n-6 and n-3 families in Table 1.
Table 1
Several Naturally Occurring PUFAs and their Dietary Sources and Biological Functions
Fatty acid families and Major dietary Biological function,

their major members sources tissue distribution
and other remarks
n-6
Linoleic acid Most vegetable oils Essential fatty acid, minor
9,12-cis-18: 2 component of most tissues
y-Linolenic acid (GLA) None Major fatty acid of evening
6,9,12-cis-18: 3 primrose seed; used as an addi-
tive of feed, cosmetics, etc.
Dihomo-y-linolenic acid None Precursor of 1-group of prosta-
8, 11,14-cis-20 : 3 glandins
Arachidonic acid Meat, liver, brain Major component of most mem-
5,8,11 , 14-cis-20 : 4 brane phospholipids; precursor
of 2-group of prostaglandins
Docosapentaenoic acid None Frequently found in tissues
4,7, 10,13, 16-cis-22 : 5 deficient in n-3 PUF As
n-3
a-Linolenic acid Some vegetable oils Minor component of tissues
9,12,15-cis-18: 3 (soy, linseed), leafy
vegetables
Eicosapentaenoic acid Fish, shellfish, algae Precursor of 3-group of prosta-
(EPA) 5,8,11,14,17- glandins; prevent thrombosis;
cis-20:5 used as pharmaceuticals and a
feed additive
Docosahexaenoic acid Fish, shellfish, algae Minor component of membrane
(DHA) 4,7,10,13,16,19- phospholipids in retinal photo-
cis-22: 6 receptors, sperm, etc.
n-9
Oleic acid Animal and vegetable
9-cis-18: 1 fats Major component of many tissues
Eicosatrienoic acid None Accumulated in total essential
5,8,11-cis-20: 3 fatty acid deficiency
n-7
Palmitoleic acid None Prevent apoplexy
7-cis-16: 1
3 BIOSYNTHESIS
The biosynthesis of linoleic acid and a-linolenic acid in higher plants, lower
plants and some micro-organisms takes place via common metabolic C-18 fatty
acids, i.e. stearic acid and oleic acid, from acetate fragments produced during
the catabolism of carbohydrates (Fluco, 1974). Linoleic acid and a-linolenic
acid are usually the terminal products of fatty acid synthesis in higher plants.
These two fatty acids are converted to two distinct families of long-chain and
more unsaturated fatty acids through the n-6 and n-3 routes, respectively, as
shown in Fig. 1. In these routes, the successive chain elongation and
desaturation take place toward the carboxyl end of the fatty acid molecule.
Thus, the members of the n-6 family have six carbons after the last double
bond in the molecule, and the members of the n-3 family have three carbons in
the terminal structure. The first step of the biosynthesis of PUFAs is the
desaturation at the ~6-position of linoleic acid or a-linolenic acid to yield
y-linolenic acid or 6,9,12,15-cis-octadecatetraenoic acid (18: 4 n-3),
respectively (~6-desaturation). The ~6-desaturation is followed by the chain
elongation to the respective C-20 PUFAs, i.e. dihomo-y-linolenic acid and
8,1l,14,17-cis-eicosatetraenoic acid (20:4 n-3). The ~5-position of dihomo-y-
linolenic acid in the n-6 route is then desaturated to yield arachidonic acid
(~5-desaturation). Usually, arachidonic acid is the terminal product of the n-6
route. The ~5-desaturation on 20: 4 n-3 fatty acid in the n-3 route results in
the formation of EPA, which is followed by chain elongation and desaturation
to yield DHA. It is usually the end product of the n-3 route.
The n-6 and n-3 routes have been suggested to share the same enzymes or
enzyme systems for the transformations of C-18 PUFAs to C-20 PUFAs
(Brenner, 1974). Therefore, linoleic acid and a-linolenic acid compete with
each other for the same enzymes. In animals, the n-6 family is usually favored
in this competition, forming arachidonic acid. Neither isolation nor detailed
characterization of enzymes responsible for the desaturation and chain
elongation reactions involved in both routes has been performed yet except for
~6 desaturase from rat liver (Okayasu et al., 1981) . Desaturation reactions
require CoA, A TP, Mi+ and NAD(P)H as cofactors, suggesting that
desaturation enzymes are a kind of oxygenase and that only fatty acyl-CoA
derivatives are substrates for the enzymes. Elongation enzymes have also been
suggested to utilize only acyl-CoA derivatives as substrates.
The members of these two families of fatty acids constitute nearly all the
PUFAs in most living organisms. However, small amounts of different families
of fatty acids have been detected. 5,8, ll-cis-eicosatrienoic acid (20: 3 n-9) is
derived from oleic acid (9-cis-octadecenoic acid, 18: 1 n-9) through the n-9
route which involves the following successive reactions: desaturation at the ~6
position of oleic acid to form 6,9-cis-octadecadienoic acid (18: 2 n-9), followed
by its carbon chain elongation to 8,1l-cis-eicosadienoic acid (20:2 n-9) and
desaturation at the ~5-position of the latter to 20: 3 n-9 fatty acid (Fig. 1).
7,10,13-cis-eicosatrienoic acid (20: 3 n-7) is derived from palmitoleic acid
(9-cis-hexadecenoic acid, 16: 1 n-7) through the n-7 route, as shown in Fig. 1.
These data indicate that there are at least four families of PUFAs in
organisms.
4 PRODUCTION OF POLYUNSATURATED FATTY ACIDS

The available lipid sources relatively rich in C-18 (y-linolenic acid) and C-20
PUFAs are seeds of evening primrose, and animal tissues, algal cells and fish
oils, respectively. For practical purposes, however, these conventional sources
are not satisfactory, both in their lipid contents and in the PUFA contents of
the resultant lipid products (see Table 2).
Micro-organisms are thought to be very promising lipid sources because of
their extremely high growth rates in simple media and the simplicity of their
manipulation. Erwin & Bloch (1964) suggested that lower classes of organisms
including micro-organisms can be classified into several groups based on their
ability to produce PUFAs, as shown in Fig. 2. Show (1965) pointed out that
some fungi belonging to the Mucorales can accumulate relatively large
Table 2
Sources of PUFAs
Source PUFA FAa content Potential

type PUFA
Total PUFA productivity
(mg/g) (% in (mg/g) (g/Iiter)
total FA)
Fungi
Mortierella isabellina 18:3 553 10 55 5·0
Penicillium cyaneum 20:4 90 3-11 1 -g
M. elongata lS-5 20:4 88 25 22 1·0

M. alpina lS-4 20:3 456 6·6 28 0·6
M. alpina lS-4b 20:3 463 23·1 107 2·17
M. alpina lS-4 20:4 474 58 275 4·3
M. alpina 20-17c 20:5 243 10·9 27 0·5
M. alpina 20-17d 20:5 585 7·1 42 1·35
Others
Chlorella minutissima 20:5 35-40
Porphyridium cruentum 20:4 30-220 5-36 20-80 0·07
Euglena gracilis 20:4 5-15 0·1-0·5
Seed oile 18:3 6-10
Porcine liver 20:4 65 2-8 0·5
Fish oil' 20:5 50-150 10-15 20
a FA, fatty acid.

b grown with sesame oil extract.
C grown at 12°e.
d grown with linseed oil at 28°e.
e Oenothera biennis.
f Scomber scrombrus.
g not reported.
Metazoa; Des; y-lin Metaphyta;X;a-lin
----- "I
Ciliates;Des;y-lin Ameba;Desjy-lin I
Green Algae;X;a-lin
Phytomonads;X;a- and y-lin

Yeasts and Fungi; - - - - - .
Des;a-lin .... ~somonads;Des;a- and y-lin Euglenids;Des;X;
_____ a-and r-lin
Cryptomonads;Des(?);a-lin
Red Algae;Des;a-lin
..-----
Eubacteria;
An;Des;O
Actinomycetes;Des;O
Photosynthetic Bocteria;An;O
Blue-green Algae;Des;a-lin
Hypothetical Primitive Organisms
Fig. 2. Presumed phylogenetic relationships between groups of existing protists, and

patterns of fatty acid biosynthesis observed in these groups. An, Anaerobic mechanism
for the synthesis of monoenoic acids; Des, oxidative de saturation for the synthesis of
monoenoic acids; X, unknown ('plant') mechanism for the synthesis of monoenoic
acids; 0, absence of di- and polyunsaturated fatty acids; a-lin, the n-3 route for the
synthesis of PUFAs; y-lin, the n-6 route for the synthesis of PUFAs. For details, see
Erwin & Bloch (1964).
amounts of y-linolenic acid in their mycelia. Based on these early observa-

tions, several workers have only since early 1980 started to screen for
micro-organisms capable of accumulating lipids containing these PUFAs in
order to obtain more suitable sources for large-scale preparation of these
PUFAs.
Recently, Suzuki and co-workers (Suzuki, 1985, 1988; Suzuki & Yokochi,
1986) found several potent producers of y-linolenic acid through their
extensive screening for filamentous fungi. They reported that a strain of
Mortierella isabellina accumulates about 5 g/liter of y-linolenic acid when
grown in a medium containing glucose as a major carbon source. Based on this
finding, commercial production of y-linolenic acid with a Mortierella fungus
started recently in Japan. Another process is in progress in England.
Yamada and co-workers (Yamada et al., 1987, 1988) found that several
fungal strains produce large amounts of lipids rich in arachidonic acid,
dihomo-y-linolenic acid or EPA, or all of these C-20 PUFAs. They are new
and promising sources for C-20 PUFAs.
4.1. "I-Linolenic acid

Suzuki (1985, 1988) reported that several Mortierella strains, belonging to
the subgenus Micromucor produce lipids rich in y-linolenic acid in their
mycelia when grown in a liquid medium containing glucose or sugar cane

molasses as a major carbon source. They can grow rapidly in a medium
containing a high concentration glucose (200 g/liter) under the conditions of
D.O., 2 ppm; pH, 4·0; and cultivation temperature, 30°C, yielding a mycelial
mass of about 80 g/liter. In the case of M. vinacea, the lipid content of the
resultant mycelia was found to be 48%, by weight, which corresponded to
38 g/liter of the lipids. The purified oil obtained from the mycelia contained
myristic acid (0·7%, by weight), palmitic acid (27·2), palmitoleic acid (0,9),
stearic acid (S'7), oleic acid (43,9), linoleic acid (12·0), ')I-linolenic acid (8·3),
arachidic acid (0,6), 11-cis-eicosenoic acid (0·4), behenic acid (0·1) and erucic
acid (0·2). The ')I-linolenic acid content in the oil is comparable to that of
Oenothera biennis seed oil. The low content of linoleic acid in the oil may be
advantageous in obtaining highly pure ')I-linolenic acid preparation in high
recovery.
Recently, M. ramanniana (Hansson & Dostcilek, 1988) and Mucor ambiguus
(Fukuda & Morikawa, 1987) were evaluated for their productivities of
')I-linolenic acid from a viewpoint of biochemical engineering. The latter fungus
immobilized in porous support particles has been shown to excrete lipids
containing ')I-linolenic acid into the culture broth and/or the surface of the cell
wall in the presence of a nonionic surfactant.
4.2 Arachidonic acid
Yamada et al. (1987, 1988) assayed productivities of C-20 PUFAs in about 300
fungal isolates from natural sources which are capable of growing on an agar
plate containing stearic acid or elaidic acid as the main carbon source for
growth. They also tested more than 600 stock strains of a wide variety of
micro-organisms, i.e. bacteria (23 genera), actinomycetes (4 genera), yeasts
(20 genera), filamentous fungi (45 genera) and basidiomycetes (11 genera).
Bacteria, actinomycetes, yeasts and basidiomycetes, in general, did not
produce detectable amounts of C-20 PUFAs intracellularly. Most C-20 PUFA
producers were found to be filamentous fungi belonging to the orders of
Mucorales and Entomophthorales. Through this screening, they found that 60
strains of Mortierella and 4S isolates from natural sources produce large
amounts of C-20 PUFAs of the n-6 family (i.e. arachidonic acid and
dihomo-')I-linolenic acid) together with C-18 PUFAs of the same family
(mainly ')I-linolenic acid). Most of the PUFA-producing isolates were found to
belong to the genus Mortierella. It should be noted that all of these Mortierella
strains found as C-20 PUFA producers belong to the subgenus Mortierella.
Neither the stock cultures nor isolates belonging to the subgenus Micromucor
showed any detectable accumulation of C-20 PUFAs, although they produced
n-6 C-18 PUFAs such as ')I-linolenic acid. The arachidonic acid contents of
most of these strains accounted for more than 1S% of the total extractable
fatty acids. These values represent more than SO% of the PUFAs and are
particularly high when compared with those in the case of C-18 PUFAs (Table
Table 3
Comparison of Arachidonic Acid Productivities in Mortierella Fungi and their Mycelial Fatty Acid CompositionG
Strain Productivityb Fatty acid composition (%)C
Mycelial Total 20:4 20:4 16:0 18:0 18:1 18:2 18:3 20:3 20:4 20:5 others
mass FA content yield
(mg/ml) (mg/g) (mg/g) (mg/ml)
M. hygrophila IFO 5941 7·0 183 32-8 0·23 24·7 2·9 3704 9·5 5·5 1-6 17·9 0 0·5
M. zychae CBS 652·68 7·7 120 19·6 0·15 23·2 12-6 29·3 8·7 6·0 3·3 16·3 0 0·6
M. elongata CBS 121·71 8·1 61 13·2 0·11 15-4 14·0 30·3 6·7 6·0 3·8 21-7 0 z.t
M. elongata IS-5 AKU 3999 6·9 166 39·2 0·27 14·5 8·0 34·0 7-6 6·0 2·6 23·6 0 3·7
M. parvispora 2S-13 AKU 3994 8·0 201 19·7 0·16 7-8 9·0 56·3 5·3 6·9 1-9 9·8 0 0
M. schmuckeri NRRL 2761 8·0 205 25-4 0·20 19·9 12-4 37-1 704 4-9 4·9 12-4 0 1·0
M. alpina IS-4 AKU 3998 9·5 318 151·7 1-44 17-9 5-9 11·3 9·8 4-1 3-3 47·7 0 0
M. alpina 20-17 AKU 3996 9·4 277 109·7 1-03 15-8 5·3 12·0 18·2 4·8 2·3 39·6 0 0·7
M. alpina CBS 250-53 6·2 124 33-6 0·21 14·5 5·9 27-8 11-4 704 4·0 27-1 0 1-9
M. alpina 1-83 AKU 3995 9·4 300 143·7 1·35 18·6 4-8 12·3 8·9 4-1 3-4 47-9 0 0
M. alpina CBS 219·35 5·5 139 31-1 0·17 11·2 4·0 30·5 14-4 10·9 4-1 22-4 0 1-7
G For details, see Shimizu et al. (1988b).
b Values are given in mg/ml culture broth or mg/g dry mycelia.

cValues are given in weight %.16:1, palmitic acid; 18:0, stearic acid; 18:1, oleic acid; 18:2, linoleic acid; 18:3, y-linolenic acid; 20:3, dihomo-y-linolenic
acid; 20:4, arachidonic acid; 20:5, EPA. EPA and other PUFAs of the n-3 family were not detected.
d FA = fatty acid.
Table 4
Production of Arachidonic Acid by Selected Mortierella Strains under Optimal Culture Conditions
~
~
Strain Total Temperature pH Culture Mycelial Total 20:4a 20:4 s·""
glucose (0C) period yield FAa content yield -
used (days) (g /liter) (mg/g) (% in (mg/g) (g/liter) ~
$:I..
(g /liter) total FA) I::
<"I
g.
100 24 5·0 4 22 ;3
M. elongata IS-5 188 25 47·0 1·0
M. alpina IS-4 40 28 6·0 4 16 474 58 275·0 4·3 ~
~
M. alpina IS-4 104 28 6·0 10 22 440 31 136·4 3·0
M. alpina 20-17 20 20 5·5 5 10 485 52 252·5 2·5 ~
).:
'"
a FA = fatty acid; 20:4, arachidonic acid.
~
~
W
3). No detectable amount of free fatty acids was found in lipid fractions
extracted with chloroform-methanol, suggesting that the PUFAs produced are
present as triglyceride and/or phospholipid forms . Through this screening,
they obtained three isolates, which were taxonomically identified to be
Mortierella alpina IS-4 , M. alpina 20-17 and M. elongata IS-5, as potent
producers of arachidonic acid.
Subsequently, they investigated the cultural conditions for arachidonic acid
production with the three Mortierella strains (Yamada et al., 1987,1988). These
three fungi utilize not only glucose but also glycerol, maltose, n-hexadecane
and n-octadecane as carbon source for the arachidonic acid production. As
shown in Table 4, all the strains produced more than 1 g/liter of arachidonic
acid under 5-liter bench scale fermentor conditions. Based on the results of
studies on individual and combined factors affecting arachidonic acid produc-
tion, they selected M . alpina IS-4 as the most promising producer. Using a
2000-liter fermentor, 22·5 g/liter mycelia (dry weight) containing 44·0%, by
weight, of the lipids, in which arachidonic acid comprised 31 ·0% of the total
fatty acids, was produced under the conditions of intermittent feeding of
glucose and 10 days cultivation at 28°C (Fig. 3(a» . They also reported that the
aradchionic acid in the harvested mycelia can be specifically enriched when
the mycelia are allowed to stand for a further few days at room temperature.
The arachidonic acid content of the resultant mycelia reached nearly 70% of
the total fatty acids (Fig. 3(b». Fractionation of the lipids in the mycelia
demonstrated that triglycerides (70·1 %) and phospholipids (23,4%) were their
(s)
(b) 20:3 '8'218~
30 3r-------------A 20:~ \'S'3G '6~
l' ::: ~tlMm
10 I i }
9 § }{
1 ill ,8:1
o 2 , 6 8 to o 50 100
CultlVOtlon time (cloys) FA composition (%)
Fig. 3. Production of arachidonic acid by M. alpina IS-4 under the optimal culture
conditions. Mortierella alpina IS-4 was cultivated in a 2000-liter fermentor containing
1400 liters of a medium containing 2% glucose and 1% yeast extract, pH 6·0 at 28°C.
Glucose was added to the medium as shown in (a). Changes in the mycelial fatty acid
composition during growth are shown in (b). 16 : 0, Palmitic acid ; 18 : 0, stearic acid;
18 :2, linoleic acid ; 18 : 3G, y-linolenic acid ; 20:3, dihomo-y-linolenic acid; 20:4 or
Ara , arachidonic acid.
.Iant meyer SOL lar tormontor

2,OOOL lar termentor
o~L1
n·he,ana n.tocophero'
Na.,SO. I_L~
,I ~';;:I.~I;;·:~§":"
mil' 1
~entr"ug..
mil ' 2
f"ter evaporator
Fig. 4. Flow sheet for the production of fungal oil of high arachidonic acid content.
major components, and about 50% of the arachidonic acid was present in the
phospholipid fraction.
The lipids containing arachidonic acid can be obtained as an oil from the
mycelia of M. afpina 1S-4 in a good recovery (80-90%) through the following
successive steps: separation of mycelia by filtration , drying, crushing by ball
mill, extraction of the lipids with n-hexane , removal of insoluble materials by
centrifugation, decolorization and deodorization with active charcoal , and
concentration . The resultant purified oil contains myristic acid (0·2%, by
weight), palmitic acid (7·0) , palmitoleic acid (0·1), stearic acid (2·8), oleic acid
(6·6), linoleic acid (5·9), y-linolenic acid (3 ·9), 11-cis-eicosenoic acid (0·9),
11 ,14-cis-eicosadienoic acid (1 ·3), dihomo-y-linolenic acid (3·9) and arachido-
nic acid (67·4) . The overall process is outlined in Fig. 4. The arachidonic acid
in the purified oil can be isolated as the methyl or ethyl ester in a good
recovery after successive transesterification, liquid-liquid partition chromatog-
raphy and high-performance liquid chromatography.
4.3 Dihomo-y-Linolenic Acid
Most arachidonic acid-producing fungi can accumulate small amounts of

dihomo-y-linolenic acid, when they are cultivated under the conditions for
arachidonic acid production (Yamada et af. , 1988; Shimizu et af. , 1988a).
However, the ratio of dihomo-y-linolenic acid to arachidonic acid in mycelia
is usually only about 0·2. Yamada et af. (1988) reported that M. afpina
IS-4 accumulated 0·6 g/Iiter of dihomo-y-linolenic acid. Because dihomo-y-lino-
lenic acid is a precursor of arachidonic acid in the n-6 route, the arachidonic
acid accumulated in the mycelia must be produced via dihomo-y-linolenic
I
2.0~
J 1.0 CII
II)
o
o ~
~2.0 "
• .c!J
1 20 :
"';;-1.0
<"c
...J
"o
l:
I
I
I
,
I
I
Time (days)
Fig. 5. Time course of the production of dihomo-y-linolenic acid by M. alpina 1S-4.

Mortierella alpina 1S-4 was cultivated in a 50-liter bench scale fermentor containing 30
liters of a medium containing 2% glucose, 1 % yeast extract and 0·022% of sesame oil
extract, pH 6·0. Glucose was added to the medium at the time indicated by the arrow.
DHGLnA, dihomo-y-linolenic acid; Ara, arachidonic acid.
acid, suggesting that all the arachidonic acid-producing fungi potentially have
the ability to produce large amounts of this fatty acid.
Shimizu et al. (1989a) reported that the mycelial dihomo-y-linolenic acid
content of M. alpina 1S-4 increases, with an accompanying marked decrease in
its arachidonic acid content, on cultivation with sesame oil. They suggested
that this unique phenomenon is due to specific repression of the conversion of
dihomo-y-linolenic acid to arachidonic acid by the oil. The effective factor(s)
causing this phenomenon was found to be present in the non-oil fraction of the
oil, after fractionation of the oil with acetone. In a study on optimization of the
culture conditions for the production of dihomo-y-linolenic acid by M; alpina
1S-4, a medium containing glucose, yeast extract and the non-oil fraction was
found to be suitable for the production. Under the optimal conditions in a
SO-liter bench scale fermentor, the fungus produced 2·17 g/liter of dihomo-y-
linolenic acid (107 mg/ g dry mycelia) (Fig. S). This value accounted for 23·1 %
of the total mycelial fatty acids. The mycelia were also rich in arachidonic acid
(S3·S mg/g dry mycelia, 11·2%). Other major fatty acids in the lipids were
palmitic acid (24·1%, by weight), stearic acid (7·0), oleic acid (20·1), linoleic
acid (6·6) and y-linolenic acid (4·1).
4.4 Eicosapentaenoic Acid (EPA)
4.4.1 EPA -Production under Low Temperature Growth conditions
Yamada and co-workers (Shimizu et al., 1988a; Yamada et aI., 1988) investi-
gated the growth conditions causing compositional changes in mycelial fatty
acids using the arachidonic acid producers obtained through their screening
studies, because they could not find any strains capable of accumulating a
detectable amount of EPA under their screening conditions (see Table 3).
They found that lowering the cultivation temperature caused the additional
accumulation of a PUFA with five double bonds, EPA, by all the arachidonic
acid producers tested, as shown in Table 5. In all cases, cultivation at low
temperature significantly increased the mycelial phospholipid content. For
example, the mycelial lipids extracted from M. alpina 1S-4 grown at 12°C
contained 47·6% (by dry weight) of phospholipids and 35·7% of triglycerides.
More than 60% of the EPA accumulated in the mycelia was found in the
phospholipid fraction. On the other hand, the lipid from the mycelia grown at
28°C contained only 21·6% of phospholipids, the triglyceride fraction compris-
ing 73·5% of the total extractable lipids. It should be noted that most of these
arachidonic acid producers grow well at low temperature (6-16°C), and yield
enough mycelia in simple growth media.
Subsequently, they studied the mechanism involved in this unique phenome-
non. The results of experiments with cell-free extracts of M. alpina 1S-4
demonstrated that the enzyme(s) that catalyzes the formation of EPA is
produced even when the fungus is grown at high temperature, but that the
reaction(s) yielding EPA does not take place at high temperature, as shown in
Table 6. Since all the EPA-producing Mortierella strains accumulate PUFAs of
the n-6 family (i.e. y-linolenic acid, dihomo-y-linolenic acid and arachidonic
acid) in their mycelia and do not accumulate PUFAs of the n-3 family other
than EPA, they suggested that an n-6 PUFA, probably arachidonic acid, may
be a precursor of EPA. If this is the case, an enzyme(s) or enzyme system
catalyzing the methyl-end directed desaturation of arachidonic acid (a17-
desaturation) may be activated on cold adaptation. The resultant EPA may be
necessary for maintaining a proper membrane fluidity in a low temperature
environment (Shimizu et al., 1988a).
Among various arachidonic acid-producing strains, they selected M. alpina
20-17 as the most promising EPA producer. The fungus was found to
accumulate about O· 5 g/liter (26·6 mgt g dry mycelia) of EPA on cultivation
for 7 days at 12°C. This value accounted for 11 % of the total fatty acids in the
extracted lipids. Other major fatty acids in the lipids were palmitic acid (6·4%,
by weight), stearic acid (4·8), oleic acid (3·2), linoleic acid (3·1), y-linolenic
acid (4·5) and arachidonic acid (63·8) (Shimizu et al., 1988b).
4.4.2 Conversion of Linseed Oil to an Oil Containing EPA

Shimizu et al. (1989b) demonstrated that several arachidonic acid-producing
Mortierella strains accumulate detectable amounts of EPA in their mycelia
when grown in media containing a-linolenic acid. This observation suggests
that the n-3 route occurs in the Mortierella fungi as well as animals, although
they are lacking the ability to synthesize a-linolenic acid. This route seems to
be independent of growth temperature, because EPA production takes place
even when they are grown at 28°C. The ability of the Mortierella fungi to
....
....
00
Table 5
Formation of EPA and Changes in Mycelial Fatty Acid Composition in Mortierella Fungi at Various Growth temperaturea
Strain Growth EPA Fatty acid composition (% t

temperature content
CC) (mg/g)b 16:0 18:0 18:1 18:2 18:3 20:3 20:4 20:5 others
M. alpina IS-4 6 6·43 8·0 2·0 14·0 15·0 10·0 3·3 30·3 14·8 2·6 :-->
AKU 3998 12 8·09 8·7 2·2 16·8 14·4 10·1 3·0 28·4 14·9 1·5 ~
16 1·35 9·6 6·7 14·6 3·9 4·4 4·2 50·2 2·4 4·0 §.
20 0 12·0 4·5 12·1 5·1 3·4 3·2 56·2 0 3·5 N'
I::
28 0 15·9 5·9 11·3 9·8 4·1 3·3 47·7 0 2·0 R-
M. hygrophila 12 9·76 16·0 3·8 30·7 8·9 11·3 2·5 13·6 10·4 2·8 ~
IFO 5941 28 0 24·7 2·9 37·4 9·5 5·5 1·6 17·9 0 0·5
M. elongata 12 15·5 13·9 2·5 32·0 7-4 13·1 4·9 12·3 13-9 0 ~
~
IFO 8570 28 0 25·2 4·3 49·1 3·5 2·5 1·0 13·9 0 0·5 I:>
M. exigua 12 5·45 9·8 1·5 22·5 9·9 6·0 3·2 37·5 7·0 2·6 ~
IFO 8571 28 0 22·3 7·2 33·1 10·2 4·7 3·6 18·0 0 0·9
M. parvispora 12 6·97 10·1 4·0 43·3 7·0 8·7 2·7 15·3 5·2 3·7
20-24 AKU 3997 28 0 13·2 3·7 54·6 5·5 5·4 1·7 15·5 0 0·4
a For details, See Shimizu et al. (1988a).
b Values are given in mg/g dry mycelia.
C Values are given in weight %. Any fatty acid of the n-3 family other than EPA was not detected. For abbreviations of fatty
acids, see Table 3.

Table 6
Enzymatic Formation of EPA by a Cell-Free Extract of M. alpina 1S-4
Grown at 28°C'
Omission from Reaction EPA found (nmollml)

reaction mixture time on incubation at
(h)
12°e 28°e
None 0 o (2)b o (2)

5 18 (23) o (4)
10 38 (49) o (3)
22 48 (67) o (4)
Lipid 10 5 (8) o (3)
Cofactors C 10 8 0
Lipid, cofactors 10 1 0
Enzyme 10 0 0
aFor details, see Shimizu et al. (1988a).
bValues obtained with the cell-free extract prepared from the mycelia
grown at 12°C are given in parentheses.
CATP, CoA, NADPH and MgCI 2 •
convert added a-linolenic acid to EPA is very promising from a biotechnologi-

cal viewpoint because there are various kinds of easily available natural oils
containing a-linolenic acid, and it is expected that they can be converted to
oils rich in EPA on incubation with these fungi. They examined the potential
of such natural oils as precursors of EPA and found that linseed oil, in which
a-linolenic acid amounts to about 60% of the total fatty acids, is the most
suitable for EPA production. Under optimal cultural conditions, M. alpina
20-17 converted 5·1 % of the a-linolenic acid in the added linseed oil into
EPA, the EPA production reaching 1·35 g/liter (41·5 mg/g dry mycelia). This
value is 2·8-fold higher than that obtained under low temperature growth
conditions. The resultant lipid is rich in either arachidonic acid or EPA,
Another advantage of this conversion is that it can be carried out under normal
growth temperature conditions (20-30°C). Under such conditions, the fungal
growth is rapid and dense, and the energy costs for temperature control may
be less than those for cooling.
4.4.3 EPA from Algae
Micro-algae belonging to the genera Skeletonema, Phaeodactylium,
Dunaliella, ... were also found to be high EPA-producers. Algal ponds for
EPA-synthesis and other fine chemicals are already operational in Israel and in
the USA (California, Hawaii) (Borowitzka & Borowitzka, 1988).
5 CONCLUSION
Considerable progress has been made in biotechnological production of
PUFAs during the past years. Areas of progress include: (a) the finding of
potent fungal strains rich in y-linolenic acid; (b) determination of optimal

conditions for large-scale submerged culture, which made it possible to operate
industrial-scale production of this fatty acid; (c) the finding that C-20 PUFAs
can be produced by filamentous fungi and algae efficiently; (d) selection of
potent producers of C-20 PUFAs; and (e) determination of culture conditions
for selective accumulation of arachidonic acid, dihomo-y-linolenic acid and
EPA by the selected fungi or algae. These PUFAs may be of considerable
importance in the utilization of these algal cells or fungal mycelia or their oils
as a feed supplement. In addition, they may be used as pharmaceutical
products. Further progress in this field depends on additional technological
development in downstream processes for the large-scale isolation of oils
containing PUFAs and/or pure PUFAs.
In conclusion, it seems to us that the nutritional, pharmacological and
economical potential of micro-organisms as a source of PUFAs are very high.
REFERENCES
Borowitzka, M. A. & Borowitzka, L. J. (1988). Micro-algal Biotechnology. Cambridge
University Press, Cambridge, UK.
Brenner, R. R. (1974). The oxidative desaturation of unsaturated fatty acid. Molec.
Cell. Biochem. 3,41-52.
Dyerberg, J. (1986). Linolenate-derived polyunsaturated fatty acids and prevention of
atherosclerosis. Nutr. Rev. 44, 125-34.
Erwin, J. & Bloch, K. (1964). Biosynthesis of unsaturated fatty acids in micro-
organisms. Science, 143, 1006-12.
FIuco, A. J. (1974). Metabolic alterations of fatty acids. Ann. Rev. Biochem., 43,
215-41.
Fukuda, H. & Morikawa, H. (1987). Enhancement of ')I-linolenic acid production by
Mucor ambiguus with nonionic surfactant. Appl. Microbiol. Biotechnol. 27, 15-20.
Gellerman, J. L. & Schlenk, H. (1979). Methyl-directed desaturation of arachidonic
acid to eicosapentaenoic acid in the fungus, Saprolegnia parasitica. Biochim.
Biophys. Acta, 573, 23-30.
Hansson, L. & Dostalek, M. (1988). Effect of culture conditions on mycelial growth
and production of ')I-linolenic acid by the fungus Mortierella ramanniana. Appl.
Microbiol. Biotechnol., 28, 240-46.
Hartmann, E., Beutelmann, P., Vandekerkhove, 0., Euler, R. & Kohn, G. (1986).
Moss cell cultures as sources of arachidonic acid and eicosapentaenoic acids. FEBS
Lett., 198,51-5.
Holloway, P. W. (1983). Fatty acid desaturation. In Enzymes, 3rd edn., Vol. XVI, ed.
P. D. Boyer, pp. 63-83. Academic Press, New York.
Horrobin, D. F. & Huang, Y.-S. (1987). The role of linoleic acid and its metabolites in
the lowering of plasma chloesterol and the prevention of cardiovascular disease. Int.
J. Cardiol., 17, 241-55.
Needleman, P., Turk, J., Jakschiik, B. A., Morrison, A. R. & Lefkowith, J. B. (1986).
Arachidonic acid metabolism. Ann. Rev. Biochem., 55,69-102.
Numa, S. (Ed.) (1984). Fatty Acid Metabolism and its Regulation. Elsevier,
Amsterdam.
Okayasu, T., Nagao, M., Ishibashi, T. & Imai, Y. (1981). Purification and partial
characterization of linoleoyl-CoA desaturates from rat liver microsomes. Arch.
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Rahm, J. J. & Holman, R. T. (1971). Essential fatty acids. In The Vitamins, Chemistry,
Physiology, Pathology, Methods, 2nd edn, Vol. III, ed. W. H. Sebrell Jr & R. T.
Harris, pp. 303-39. Academic Press, New York.
Seto, A., Wang, H. L. & Hesseltine, C. W. (1984). Culture conditions affect
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Shimizu, S., Shinmen, Y., Kawashima, H., Akimoto, K. & Yamada, H. (1988a).
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Shimizu, S., Kawashima, H., Shinmen, Y., Akimoto, K. & Yamada, H. (1988b).
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65,1455-9.
Shimizu, S., Akimoto, K., Kawashima, H., Shinmen, Y. & Yamada, H. (1989a).
Production of dihomo-y-linolenic acid by Mortierella alpina IS-4, J. Am. Oil Chem.
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Shimizu, S., Kawashima, H., Akimoto, K., Shinmen, Y. & Yamada, H. (1989b).
Microbial conversion of an oil containing a-linolenic acid to an oil containing
eicosapentaenoic acid. J. Am. Oil Chem. Soc. 66, 342-7.
Show, R. (1965). The occurrence of y-linolenic acid in fungi. Biochim. Biophys Acta,
98,230-37.
Suzuki, O. (1985). y-Rinorensan no biseibutsu seisan (Production of y-linolenic acid by
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Suzuki, O. (1988). Production of y-linolenic acid by fungi and its industrialization. In
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110-16.
Suzuki, O. & Yokochi, T. (1986). Production of y-linolenic acid by fungi. J. Am. Oil
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173-7.
Yazawa, K., Araki, K., Okazaki, N., Watanabe, K., Ishikawa, C., Inoue, A., Numao,
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Biochem., 103, 5-7.
Chapter 8
MICROBIAL PRODUCTION OF VITAMIN ~

(MENAQUINONE) AND VITAMIN K, (PHYLLOQUINONE)
Y. TANI
Research Center for Cell and Tissue Culture, Faculty of Agriculture,
Kyoto University, Kyoto 606, Japan
1 INTRODUCTION
Vitamin K, one of the fat-soluble vitamins, is known as an anti-hemorrhagic

factor. As Bentley & Meganathan (1982) pointed out in their review, vitamin
K was the first discovered growth factor for micro-organisms, though it was
named following the classical approach of animal nutrition in 1935. Dam
discovered a new fat-soluble organic compound which was found to possess
blood coagulation capacity, while establishing a bleeding condition in chicks
fed on a diet formulated for the study of sterol metabolism, and he proposed
the name of vitamin K (K for koagulation).
A major function of vitamin K is to act as a cofactor for the carboxylation of
protein-bound glutamate residues to form y-carboxyglutamates. A simplified
scheme of the blood clotting mechanism is shown in Fig. 1. Of these reactions,
only the synthesis of prothrombin appears to be dependent on vitamin K.
Vitamin K is now widely used for various types of bleeding symptoms:
hemorrhagic disease of new-born infants and hemorrhagic symptoms caused by
administration of antibiotics, salicylic acid or warpharin dosages. Mena-
quinone (MK, vitamin K 2 ) has much higher therapeutic activity than phyllo-
quinone (vitamin Kl). However, phylloquinone has been used for a long time
as a medicine, because the chemical synthesis of MK is rather difficult and the
natural sources contain only a small amount of MK.
MK was prepared from bacterially putrefied fish meal as the first crystalline
antihemorrhagic vitamin that was clearly different from that of alfalfa,
phylloquinone. Its function in bacteria centers around its major role as an
electron carrier. Although more information is needed on its distribution in
micro-organisms, a correlation has been found between the taxonomical
classification of bacteria based on other criteria and the type of isoprenoid
quinones found in those cells (Collins & Jones, 1981). However, economic
production of MK by a microbiological process has not been demonstrated.
123
124 Y. Tani
(1) Precursor of Prothrombin ---.r~----~ Prothrombin

CO 2 , O 2
-NH-CH-CO- -NH-CH-CO-
I Vitamin K (red.) I
CH2 CH2
I I
CH2 HC-COOH
I I
COOH COOH
(2) Prothrombin + Thromboplastin + Ca2 + ---~~ Thrombin
(3) Thrombin + Fibrinogen ---~. Fibrin (clot)

Fig. 1. Blood clotting mechanism.
2 CHEMISTRY OF MENAQUINONES
The chemistry of vitamin K was established by the schools of Dam, Doisy and
Karrer, who succeeded in 1939 in isolating two compounds with vitamin K
activity, namely MK-7 (vitamin K 2 (35» from putrefied fish meal and phyllo-
quinone from alfalfa meal (Fig. 2). The structure of phylloquinone was proven
to be 2-methyl-3-phytyl-l,4-naphthoquinone, and that of MK to be 2-methyl-3-
multiprenyl-l,4-naphthoquinone. The multiprenyl side chain of MK is of
variable length from 5 to 70 carbon atoms. A number of derivatives of the
basic structure also occur in nature; this is reflected in the modification of ring
substitutents at the C-2 or C-3 position or both.
Phylloquinone is a yellow viscous oil and MKs are light yellow microcrys-
talline plates. Melting points of MKs vary from 35 to 62°C on the length of the
multiprenyl side chain. These are soluble in ether, petroleum ether, benzene,
n-hexane and acetone, slightly soluble in methanol and alcohol, and insoluble
in water. These are stable to air and heat, but very unstable for alkali and UV
irradiation. The absorption maxima are 243,248,261,270 and 325 nm with the
molecular extinctions coefficient of 18000-19000 at 248 nm.
(a) (b)
Fig. 2. Chemical structure of vitamin K. (a) Menaquinone-n (MK-n); (b) phyllo-
quinone.
Vitamin K2 and Vitamin Kl Production 125
3 BIOSYNTHESIS OF MENAQUINONES
Intense research based on microorganisms during the last two decades has
clearly proven the MK biosynthetic pathway as summarized by Bentley &
Meganathan (1982).
The biosynthetic pathway of MK in bacteria is now believed to consist of the
biosynthesis of demethyl MK by polyprenylation of 1,4-dihydroxy-2-
naphthoate, which is formed as the first naphthalenoid intermediate from
shikimate through chorismate(isochorismate) and o-succinylbenzoate, by ca-
talysis of a membrane-associated transferase and its subsequent methylation.
The methyl group is derived from S-adenosylmethionine. Biosyntheses of MK
and aromatic amino acids share a common route until chorismate. The
immediate precursor, shikimate (chorismate), is incorporated to non-carboxyl
carbon atoms of 2-ketoglutarate to form the naphthoquinone nucleus. The
biosynthetic pathway of MK can now be summarized as shown in Fig. 3, to
which recent experimental results are added.
4 FERMENTATIVE PRODUCTION OF MENAQUINONES
In spite of the increasing need of vitamin K supply, no microbiological process

for MK production has been developed so far. Only recently, work on the
fermentative production of this water-insoluble vitamin by micro-organisms
was initiated by Tani et al. (1984) and is described in this section.
4.1 Screening of Menaquinone-producing Micro-organisms
A number of micro-organisms were aerobically grown on media consisting of

glycerol and peptone as carbon and nitrogen sources, respectively. Washed
cells obtained from culture broth were suspended in acetone, and homogen-
ized with a homogenizer. Lipophilic materials in the extract were absorbed on
SEP-PAK C 18 (Waters Associated, Inc.) and eluted with n-hexane. MK in the
n-hexane eluate was detected with a spectrophotometer by its typical UV
absorption spectrum. The amount of MK was calculated from the absorbance
at 248 nm by using the molar extinction coefficient.
For the determination by high performance liquid chromatography, cells
were homogenized in methanol and then extracted at 50-6O"C for 20 min.
Solid materials were removed by centrifugation. The resultant supernatant was
applied on a liquid chromatograph with 4·6 x 1oo-nm column of Cosmosil 5C18
(Nacalai Tesque Co.). The solvent used was methanol-dichloromethane. MK
was detected with a UV monitor at 248 nm.
Thin-layer and reversed-phase chromatographies were also applied for the
identification of MK. The thin-layer chromatography was performed with
development using a solvent system of benzene-cyclohexane. For the
126 Y. Tani
HO~COOH
,
H O Y 8hlklmata HOOC~H
,
Mavaloneta
HOO'--oOCOOH
HZ~
HO Chorlemeta
6-
OOH
, ~~g
,
Olmethylallyl-PPI l.opentenyl-PPI
Hoar.. OOH
H.<f° O COOH COZt
Z Ieochorleo TPP
meta
, ,.CHZCHZCOOH ~P
..... HO-",
6
TPP
COOH Oerenyt-PPI
2-8ucclnyH-h clroxr-
2,4-cyctohexa
COCil CH eOOH
2 Z
c~rtele
- - , _ I.opentenyt-PPI
,
,
Feme.y""""
,
, _ (n-3)xl.opentenyI-PPI
Polypr...yI-PPI
; Oemethylmenaqulnone-n
Manaqulnone-n
Fig. 3. Biosynthesis of menaquinone.
reversed-phase chromatography, the thin-layer plate was immersed gently in

5% liquid n-paraffin in n-hexane and stood horizontally in the air to allow
evaporation of n-hexane. The reversed-phase chromatography was performed
with development using a solvent system of acetone-water to which a few
drops of liquid n-paraffin had been added. The MK band was detected under
irradiation.
The distribution of isoprenoid quinones was surveyed in about 900 microbial

strains (Tani et al., 1984). The quinone system of molds was tested in 56 strains
of 17 genera representing phycomycetes, ascomycetes and fungi imperfecti. All
of them lacked MK; their quinone system was coenzyme Q (CoQ). The same
result was obtained with 88 strains of yeasts of 32 genera representing
ascosporogenous, asporogenous and ballistosporogenous yeasts, and with 28
strains of Basidiomycetes of 14 genera.
MK was found to be widely distributed in procaryotes. About half of the 112
type strains of bacteria of 27 genera contained MK. The quinone types of the
bacteria tested could be divided into three groups: MK alone, MK and CoQ,
and CoQ alone. MK was found in Gram-positive bacteria, MK and CoQ in
Gram-negative facultative anaerobes, and CoQ in Gram-negative aerobes. The
only exception to this rule were strains of the genus Flavobacterium, which
were Gram-negative facultative anaerobes but had MK mostly or CoQ.
Flavobacterium species showed a higher content of MK than other bacteria.
Therefore, 550 strains of yellow-pigmented bacteria were further isolated.
About 90% of the isolates were MK producers. All actinomycetes tested (40
strains of 6 genera), contained only MK. This coincides well with the
classification of actinomycetes as Gram-positive bacteria.
Actinomycetes, though accounting for a high proportion of potent MK
producers, yield MK with eight or more isoprene units in the C-3 polyprenyl
side chain. In the blood coagulation tests, MK-4, 5 and 6 were shown to have
high biological activity, which decreased with the increase in the number of
isoprenoid units. Consequently, Flavobacterium species was selected to further
study for the improvement of the productivity.
4.2 Menaquinone-6 Production by Wild Type and Mutant Strains of

Flavobacterium meningosepticum
Culture conditions for MK-6 production by the wild type strain of F.

meningosepticum were investigated by Tani et al. (1984). The principal cultural
conditions that can be considered as influencing MK production are carbon
source for growth and the oxygen supply to the medium. Among carbohy-
drates, ribose is closely related to the biosynthesis of MK, sharing the
intermediate of the biosynthesis of aromatic amino acids. In fact, the cellular
content of MK increased when ribose was used in place of glycerol as the
carbon source; but the amount of MK in the culture broth decreased due to
the low cell concentration. When glycerol, glucose or fructose was used as the
carbon source, the growth and the productivity of MK were relatively high.
The cellular content of MK increased and the cell yield decreased with
decrease of aeration.
Prenyl alcohols are involved in MK biosynthesis in the form of pyrophos-
phates, which are incorporated into the polyprenyl side chain. Isopentenol was
the most effective in increasing the cellular content of MK, followed by
dimethylallyl alcohol.
128 Y. Tani
Precursors of the naphthoquinone nucleus, shikimate and 0-

succinylbenzoate, increased the cellular content of MK to some extent.
1-Hydroxy-2-naphthoate (HNA) inhibited the biosynthesis of MK but had
little effect on growth. This suggests that the compound is a specific inhibitor of
the biosynthesis as an analog of 1,4-dihydroxy-2-naphthoate.
The biosynthetic pathways of MK and aromatic amino acids branch at
chorismate after shikimate. The addition of L-tyrosine or its precursor,
p-hydroxyphenylpyruvate, increased the cellular content of MK; however,
L-tryptophan, L-phenylalanine and their precursors, anthranilate and
phenylpyruvate, inhibited the MK production.
Compounds which were found to increase MK production in the experi-
ments described above were examined in various combinations for a possible
synergistic effect. Isopentenol and L-tyrosine formed the best couple. When
isopentenol was fed intermittently to the medium, the amount of MK reached
21·3 mg per liter of culture broth and 3·52 mg per g of dry cells after 3 days of
cultivation.
The MK productivity of F. meningosepticum was improved by mutageniza-
tion with N-methyl-N'-nitro-N-nitrosoguanidine. A mutant strain resistant to
HNA, which was found to be an inhibitor of MK biosynthesis but not of
growth, produced MK more abundantly: 34 mg per liter of culture broth and
5·5 mg per g of dry cells (Tani et al., 1986). The mutant strain was less
sensitive to inhibition by HNA on MK biosynthesis than the wild-type strain.
Mutant strains, which displayed KCN resistance, aromatic amino acid
auxotrophy and no-carotenoid productivity, did not show further increase of
MK productivity.
Isolation of MK-6 from cells of the mutant strain was performed as follows:
to a cell paste weighing about 100 g, 1 liter of acetone-ethyl ether was added
and agitated in a mini-jar fermentor for 2 h. Solid materials were filtered and
extracted again in a similar manner. Combined extracts were evaporated under
reduced pressure to less than 10% in volume. Each 10 ml of the concentrate
was partitioned between 500 ml of diethyl ether and 500 ml of sodium
chloride-saturated water. 'the non-polar lipids fraction obtained was dried with
anhydrous sodium sulfate and then evaporated. Resultant oily materials were
dissolved in chloroform and applied on a thin-layer plate which was prepared
by spreading a silica gel suspension (40 g/80 ml H 2 0) on glass plates at a
thickness of 0·75 mm, followed by activation at 110°C for 2 h after drying
overnight at room temperature. Thin-layer chromatography was performed
with development with a solvent system of cyclohexane-benzene. The MK
band was scraped. MK was eluted with chloroform and crystallized twice from
ethanol. Crystalline MK (220·4 mg) was obtained from cells of the mutant
strain at a yield of 75%. MK loss occurred in the recrystallization step.
4.3 Menaquinone-5 Production by a Mutant Strain of Flavobacterium
meningosepticum
The diversity of the types of isoprenoid quinones present in micro-organisms
has been extensively studied as a result of the recent improvement of analytical
tools. Different types of MK homo logs were dectected in archaebacteria,

mycoplasmas, and in Gram-negative and Gram-positive bacteria with or
without minor component(s) in relation to taxonomic groupings. However, a
change of the type of MK in the same organism has never been noticed.
It was found that an HNA-resistant mutant, strain HNA 12-D, which was
derived from an MK-6 producer, produced an increased amount of MK, due to
the production of another type of MK in addition to MK-6 (Tani et al., 1985).
The newly formed MK was isolated and identified as MK-5. By combined
feeding of isopentenol and L-tyrosine, the total amount of MK produced by
strain HNA 12-D was 55·6 mg per liter of culture broth and 9·19 mg per g of
dry cells in the ratio of MK-6 and MK-5 of 1: 1· 7 after 72 h cultivation,
whereas the parent strain produced 25·3 mg per liter of culture broth and
4·14 mg per g dry cells of MK-6.
The MK system of the wild type strain of the bacterium was composed only
of MK-6 without any minor components. The quantity of MK-5 produced by
the mutant strain was one to two times as much as that of the MK-6. It was the
first observation of a change in the MK pattern within the same micro-
organism. HNA is a structural analog of 1,4-dihydroxy-2-naphthoate, an
intermediate of MK biosynthesis. Whether the change in the specificity of the
enzyme, 1,4-dihydroxy-naphthoate: polyprenyl pyrophosphate transferase,
which catalyzes the prenylation of the naphthalenic intermediate using both
solanesyl pyrophosphate and octaprenyl pyrophosphate as substrates, or that
in the supply of polyprenyl pyrophosphates in strain HNA 12-D extended the
MK spectrum by the acquisition of HNA resistance remains to be determined.
4.4 Menaquinone-4 Production by a Mutant Strain of Flavobacterium

sp.238-7
During further study to develop a new fermentation process for MK

production, a HNA-resistant mutant, strain HNA 250-15, of Flavobacterium
sp. 238-7, which produced MK-6 as the major component of respiratory
quinone with some minor components, showed an increase in MK productivity
due to increased production of a minor component of MK (Tani & Sakurai,
1987). The newly-formed MK was identified as MK-4, which now finds a
clinical use and which is made by a chemical process.
The MK-4 productivity of strain HNA 250-15 was further improved by
making the mutant resistant to usnic acid and menadione. Flavobacterium sp.
238-7 contains MK as the sole quinone in the respiratory chain. Among the
uncouplers and respiratory inhibitors tested, an uncoupler, usnic acid, strongly
inhibited the growth of strain HNA 250-15 at a low concentration. On
subsequent mutagenization of the strain, a mutant, USN r -2, which was
resistant to 4 mg of usnic acid per liter, was found to produce a much larger
amount of MK-4 than strain HNA 250-15, whereas the MK-6 content was
reduced. The productivity of MK-4 was increased more than three times as
compared with that of strain HNA 250-15.
130 Y. Tani
Growth of Flavobacterium was strictly inhibited by addition of menadione,

an artificial derivative of vitamin K, to the culture medium, and the content of
MK was reduced. Derivation of menadione-resistant mutant strains was then
carried out for enhancement of the MK productivity using strain USN r -2 as
the parent strain. Among the mutant strains obtained, strain K 3 -15, which was
resistant to 20 mg of menadione per liter, showed the highest content of MK-4
and MK-6. The total MK productivity was increased about three times as
compared with that of the wild type strain.
The cultural conditions for MK production were investigated with strain
Kr15. Glycerol and peptone were the most effective carbon and nitrogen
sources, respectively, both for growth and MK production. The amount of
MK-4 increased linearly in proportion with the glycerol concentration,
although the amount of MK-6 hardly changed with increasing glycerol
concentration.
A variety of natural oils were supplemented to the culture medium at the
final concentration of 0·05% directly before autoclaving. Among them, cedar
wood oil decreased the growth but significantly increased the MK production.
120 12i
()
0
EJ MK-4 I
a>
100 0 MK-6 100,
E
:::::
a> C
! 80 8 01)
loC C
.. 0
:f ()
loC
60 6
:v I
I
:f
-
III
340
..
I
I 4 !D
:u
20
...
.. ,.
l 2
1:
a>
a:
r1l r I
Fig. 4. Improvement of menaquinone production by Flavobacterium sp. 238-7. The left
and right bars indicate the amount of MK in mg/liter of culture broth and mg/g of dry
celis, respectively, the open and shaded bars denoting MK-6 and MK-4, respectively.
The amounts of MK produced by each strain indicated are the values when grown on
the basal medium, except that K3-1sa was grown in the optimized medium and K3-1S b in
the optimized medium supplemented with cedar wood oil.
Vitamin K2 and Vitamin K) Production 131
MK-4 production increased with increasing cedar wood oil concentration,

whereas the amount of MK-6 decreased. None of the prenyl alcohols increased
the MK production of strain K3-15 at concentrations of 0·5 and 1 mM, in fact
the amount of MK-4 decreased. Among L-tryptophan, L-phenylalanine,
L-tyrosine, anthranilate and phenylpyruvate, only phenylpyruvate somewhat
increased the MK-4 production in contrast with the case of F. menin-
gosepticum.
The amount of MK produced by strain KT 15 was 125·4 mg per liter of
culture broth and 12·8 mg per g of dry cells, in the ratio of MK-4 and MK-6 of
6: 1, under the optimal culture conditions in the presence of cedar wood oil.
The improvement of MK production by Flavobacterium sp. 238-7 on
mutagenization and optimization of the cultural conditions is summarized in
Fig. 4. The MK productivity of strain KT 15 was almost 10 times that of the
wild type strain. It is noteworthy that the increase of MK was due to an
increase in MK-4 but not MK-6. MK-4 was a minor component of the wild
type strain. The physiological role of MK-4 in the mutant strain is still
uncertain. In the course of cultivation, the cellular content of MK-4 still
increased after the growth had reached the stationary phase.
4.5 Extracellular Production of Menaquinone-4 by a Mutant Strain of

Flavobacterium sp. 238-7
It could be considered that the MK productivity of strain K3-15 described in

the previous section, was restricted by the intracellular level causing metabolic
repression of MK biosynthesis. During attempts to further improve the MK
production of the mutant strain, cultivation in a detergent-supplemented
medium resulted in the extracellular production of MK, especially MK-4,
without inhibition of the MK productivity and growth (Tani & Taguchi, 1988).
It was found that a mutant strain, K3-15, of Flavobacterium sp. 238-7
excreted MK into the culture medium supplemented with a detergent. Figure 5
shows the production of MK-4 and MK-6 during the cultivation of strain K3-15
in the basal medium. MK-6, which was the major MK component of the wild
type strain, was produced only in the cells during the cultivation. On the other
hand, MK-4 was also detected in the culture fluid.
A number of detergents (174 in total, i.e. the 110 nonionics, 30 anionics, 21
cationics and 13 amphoterics) were tested as to their induction of MK
excretion into the culture medium. Most of the non-ionic and amphoteric
detergents did not affect the cell growth. On the contrary, many anionic and
cationic detergents inhibited cell growth. A non-ionic detergent, Rikanon VA
5012 (polyoxyethylene oleyl ether), showed highest ability as to induction of
MK excretion into the medium without inhibiting the cell growth. MK-4,
which was newly formed in cells of the mutant strain, was selectively excreted
into the medium, but MK-6, which was the original MK of the wild type strain,
was hardly excreted. In the presence of 0·05% Rikanon VA 5012, the MK-4
132 Y. Tani
10
20
~
eft ~
.
.§ ~
0
'a 0
u .g
..
::a
ie
'a
0
a.
laC C)
::Ii! 10
CultIvatIon tIme (h)

Fig. 5. Menaquinone production by strain K3-15. 0, growth, .. , intracellular MK-6;
V, extracellular MK-6; e, intracellular MK-4; 0, extracellular MK-4; . , total MK-4.
~ 20
£ ~
~
•u:::a
'a 0
0
'a
0
S
a :5
laC
::E 10 5
.
~
0
C)
CultlvaUon time (h)
Fig. 6. Menaquinone production by strain K3-15 in the presence of Rikanon UA 5012.

The symbols are the same as in Fig. 5.
productivity increased about 20% compared to that without the detergent, and
the total amount of MK was 39 mg per liter of culture broth.
Figure 6 shows the fermentation course of MK production on cultivation in
the presence of 0·05% Rikanon VA 5012. The cell mass reached a maximum
level, about 10 g by cell weight per liter of culture broth, after 30 h cultivation,
and then gradually decreased. MK-6 was mainly produced intracellularly and
reached about 12 mg per liter of culture broth after 30 h cultivation. A small
amount of MK-6 was detected in the culture filtrate at the late stage of the
cultivation. On the other hand, the excretion of MK-4 occurred from the
beginning of the cultivation and increased in the later logarithmic phase. The
extracellular production of MK-4 reached its maximum after 48 h cultivation.
The level of intracellular MK-4 was low but increased with increasing
cultivation time. The total amount of MK-4 reached about 25 mg per liter of
culture broth at 48 h cultivation, and the amount of total MK was 39 mg liter.
MK-4 excretion occurred from the onset of the cultivation and the excreted
MK was only MK-4. These facts showed that the excretion of MK-4 was not
due simply to cell lysis. The MK-4leakage might be due to a change in the cell
membrane structure, as a result of exposure to the detergent during growth.
The role of MK-4 in the mutant strain remains unknown, it might be that
MK-4 is not related to the respiratory function, since the growth and growth
rate of strain K3-15 did not decrease when most of the MK-4 had been
excreted.
5 BIOCONVERSIONS FOR VITAMIN Kl (PHYLLOQUINONE)

SYNTHESIS
Natural vitamin Kh [(7'R,1l'R)-phylloquinone] consists of 2-methyl-1,4-

naphthoquinone and the optically active phytyl side chain which is attached to
position 3. (3R,7R)-Hexahydrofarnesol, constructed from either of the micro-
biologically generated optically active lactones [(S)-2-methyl-y-butyrolactone
or (S)-3-methyl-y-butyrolactone] (see chapter 6, p. 102) can be lengthened by
condensation with a trans-Cs-olefin to (7R, llR)-trans-phytol and yields
natural vitamin Kl (Fig. 7).
Fig. 7. Absolute configurations of (7R,1l'R)-phylloquinone.

134 Y. Tani
REFERENCES
Bentley, R. & Meganathan, R. (1982). Biosynthesis of vitamin K (menaquinone) in

bacteria. Microbiol. Rev., 46,241-80.
Collins, M. D. & Jones, D. (1981). Distribution of isoprenoid quinone structural types
in bacteria and their taxonomical implications. Microbiol. Rev., 45, 316-50.
Tani, Y. & Sakurai, N. (1987). Menaquinone-4 production by a mutant of
Flavobacterium sp. 238-7. Agric. BioI. Chem., 51,2409-15.
Tani, Y. & Taguchi, H. (1988). Excretion of menaquinone-4 by a mutant of
Flavobacterium sp. 238-7 in a detergent-supplemented culture. Agric. Bioi. Chem.,
52,449-54.
Tani, Y., Asahi, S. & Yamada, H. (1984). Vitamin K2 (menaquinone):screening of
producing microorganisms and production by Flavobacterium meningosepticum. J.
Ferment. Technol., 62,321-7.
Tani, Y., Asahi, S. & Yamada, H. (1985). Production of manaquinone (vitamin K 2 )-5
by a hydroxynaphthoate-resistant mutant derived from Flavobacterium
meningosepticum, a menaquinone-6 producer. Agric. Bioi. Chem., 49, 111-15.
Tani, Y., Asahi, S. & Yamada, H. (1986). Menaquinone (vitamin K 2 )-6 production by
mutants of Flavobacterium meningosepticum, J. Nutr. Sci. Vitaminol., 32, 137-45.
WATER-SOLUBLE VITAMINS
Chapter 9
MICROBIAL SYNTHESIS OF VITAMIN B, (THIAMINE)
A.IwASHIMA
Department of Biochemistry, Kyoto Prefectural University of Medicine,
Kyoto, Japan
1 HISTORICAL
Thiamine was discovered in the course of a search for an agent that would cure
beriberi. The conquest of beriberi began in 1885 when Takaki (1885)
practically eradicated the disease among the Japanese navy by introducing fish,
vegetables, meat and barley into the diet. In 1897 Eijkman (1897) showed that
an experimental polyneuritis in fowl, which closely resembled the polyneuritic
symptoms of beriberi, could be produced by feeding the birds on a diet of
polished rice. When they were fed on unpolished rice they did not develop the
disease. In 1926 Jansen & Donath (1926) isolated a crystalline hydrochloride of
the antineuritic factor from rice bran.
The structure of thiamine, elucidated in the mid-1930s, was established
by Williams (1936) as 3-(4-amino-2-methyl-5-pyridimidinylmethyl)-5-(tJ-
hydroxyethyl}-4-methylthiazolium chloride hydrochloride (Fig. 1). The name
thiamine derives from the chemical nature of the vitamin, in that it has the
thiazole (sulfur-containing) ring attached to a pyrimidine ring with an amine
group. Thiamine was first synthesized in 1936 by Williams & Cline (1936) from
the pyrimidine and thiazole moieties of thiamine. Within a year, Lohmann &
Schuster (1937) isolated thiamine pyrophosphate (TPP) from yeast and showed
that it was the cofactor for the decarboxylation of pyruvic acid. In addition to
thiamine and TPP, thiamine monophosphate (TMP) and thiamine triphosphate
(TIP) have been found in living organisms. These phosphate esters of
thiamine are also shown in Fig. 1.
The double-salt from thiamine with hydrochloric acid (C 12H 17N 4 0SCI HCI;
molecular weight, 337·28) consists of monoclinic plates in rosette-like clusters
137
138 A. Iwashima
N
I
N
H1C"",,:x
I
NH2
U- 1!:
CH2~~ 4' CH,CH,OR
H,
Thiochrome
R= -H Thiamine
Q
R= -P-OH
I
TMP CH,
OH W:-Ofl
R=
0
II
0II
-P-O-P-OH
(JH OHI
TPP NJ.
- CHi' Nt::
HJCyNyNH'lr--s
""
0 0
~ C,H,O-P-O-P-OH
HJ 6H 6H
0 0 0
R= -P-O-P-O-P-OH TTP Hydroxyethylthlamlne pyrophosphate
ClH elH
OH
Fig. 1. Structures of thiamine and its related compounds.
with slight thiazole odor (Merck Index, 1983). It is soluble in water and
methanol, slightly soluble in ethanol, and practically insoluble in ether,
benzene and chloroform. In aqueous solution thiamine is most stable between
pH 2 and 4, but unstable at alkaline pH (Yurugi et aI., 1979). It is
destroyed by heat if the pH of the solution is above 5·5: the rate of destruction
increases with the pH. Under dry conditions thiamine is stable to heating at
100°C for 24 h. The absorption maximum of thiamine in the UV light is
affected by pH, and it is 243 nm at pH 2·3 (the molar absorption coefficient:
10·6 X 103 ), and 235 and 265 nm at pH 7·4 (Yurugi et al., 1979).
TPP chloride (commercially available in the dried form) is stable when
stored cold in the dark (Yurugi, 1975). In aqueous solution it is stable at
pH 2-6 and O°C for 6 months, but it partially decomposes to TMP and
thiamine when allowed to stand for several months at pH 5 and 38°C. TIP is
stable under dry conditions, whereas it is unstable in aqueous solution,
especially to heat, and decomposes to TMP and TPP. In alkaline solution TIP
decomposes to TMP and inorganic phosphate. TMP, which is obtained by
hydrolysis of TIP and TPP, is quite stable at acidic and neutral pH. Thiamine
and thiamine phosphate esters are quantitatively converted to thiochrome (Fig.
1) and its phosphate esters which are compounds exhibiting intense blue
fluorescence. This property observed in early studies of thiamine oxidation
serves as the basis for the chemical assay for thiamine (Barger et al; 1935).
Hydroxyethylthiamine pyrophosphate (Fig. 1), a TPP-activated aldehyde
intermediate of the enzyme reactions, it is also known to be present in
micro-organisms and mammalian tissues, especially in muscles (Morita et al.,
1968).
Thiamine, as well as other vitamins, is produced by micro-organisms in
extremely small amounts. Usually it is not formed in great excess over the
Microbial Synthesis of Vitamin B 1 139
organism's own requirement. The production of thiamine by growing cultures

of Escherichia coli ATCC 9637 is 1·6 and 1·4 higher than the amount of
thiamine in cells grown in the basal medium (21-23 ng/mg dry weight), when
cysteine and methionine are added at 1 mg per ml of the minimal medium,
respectively (Akagi & Kumaoka, 1963). The addition of some aromatic amino
acids is also stimulatory on thiamine production by growing cultures of the
same organisms: phenylalanine and histidine cause 1·5- and 1·2-fold stimula-
tions of thiamine synthesis at 10 and 1 mM, respectively (Iwashima et al.,
1968). Methionine at 1 mM shows an additional effect of 2·6-fold stimulation of
the thiamine production in the presence of 10 mM phenylalanine. On the other
hand, the addition of purine and pyrimidine bases of nucleic acids to the
growing cultures of E. coli is not effective on thiamine production in the cells.
Thus, the supplement in the growth medium is not very effective to produce
excess thiamine in bacteria and the de novo synthesis of thiamine cannot be
demonstrated in non-growing cells under normal circumstances, because the
control mechanism of thiamine biosynthesis in micro-organisms severely limits
thiamine overproduction in the cells. However, this difficulty can be overcome
by the use of a system that removes the normal control of thiamine over its
biosynthesis, so that the organisms form excess thiamine. Using Salmonella
typhimurium, Newell & Tucker (1966a,b) observed a burst of thiamine
synthesis ensued, increasing the cellular level of thiamine four- to fivefold
(about 200 ng/mg dry weight) when cells were preincubated with adenosine,
washed, and reincubated in minimal medium. Adenosine inhibits the biosynth-
esis of the pyrimidine moiety of thiamine, and the growth of organisms in the
presence of adenosine therefore lowers the cellular concentration of thiamine.
The normal repressible control by thiamine is thereby lost, and the thiamine-
synthesizing enzymes become de repressed to overproduce thiamine. The
thiamine formed occurs almost completely in the form of TPP and is located
intracellularly.
Mutants affecting regulation of thiamine biosynthesis from the pyrimidine
and thiazole moieties were isolated from E. coli K12 as resistant strains to
growth inhibition by pyrithiamine, a thiamine antagonist (Kawasaki and Nose,
1969). Two mutants, PT-R1 and PT-R3, have approximately a threefold higher
cellular thiamine content (90-100 ng/mg dry weight) than the parent strain. Yeast
as well as bacteria do not produce large amounts of thiamine under normal
circumstances (Stieglitz et al., 1974). Recently, however, thiamine-excreting
mutants of yeast have been isolated (Silhankova, 1985a). Two mutants of S.
cerevisiae and four mutants of S. carlsbergensis, which excreted up to 920/-tg
thiamine per liter of the culture medium, were obtained by employing several
rounds of UV mutagenesis and selection. The genetic characterization of these
mutants suggested that thiamine excretion phenotype is recessive and under
the control of nuclear genes (Silhankova, 1985b).
The most effective method for the production of thiamine by micro-
organisms is an enzymatic conjugation of 2-methyl-4-amino-5-hydroxymethyl-
pyrimidine (hydroxymethylpyrimidine) and 4-methyl-5-P-hydroxyethylthiazole
(hydroxyethylthiazole), the preformed pyrimidine and thiazole moieties of
140 A.lwashima
thiamine. Washed cell suspensions of E. coli ATCC 9637 (15-20mg dry cells)
synthesizes 210 ng of thiamine per mg dry weight from 10 IlM each of the two
moieties in the presence of 0·2% glucose at pH 7 for 1 h at 37°C (Iwashima et
al., 1968). With the cells derepressed thiamine biosynthesis by adenine or
thiamine auxotrophs grown in the presence of limited amount of thiamine the
production of thiamine is much more enhanced (Kawasaki et al., 1969). The
cells suspension of baker's yeast also has the capability of joining the
pyrimidine and thiazole moieties of thiamine to form thiamine in the presence
of glucose (Ashida, 1942). In S. cerevisiae, thiamine synthesis from both
moieties added leads to 20-40 times higher contents of intracellular thiamine
in comparison with normal growth conditions (Silhankova, 1985b).
4.1 Biosynthesis
The biosynthesis of thiamine involves the independent formation of the two

ring structures and their subsequent condensation (Fig. 2). The enzymatic
steps involved in the conversion of the pyrimidine and thiazole moieties to
thiamine and TPP were elucidated some years ago (Leder, 1975). Although
the biosynthetic pathway for the formation of hydroxymethylpyrimidine has
not yet been completely elucidated, enough is known to eliminate the
possibility that this pyrimidine and the pyrimidines of nucleic acids do not
share a common biosynthetic pathway (Goldstein & Brown, 1963). An
important contribution to the understanding of the biosynthesis of the
pyrimidine moiety of thiamine in bacteria was the discovery by Newell &
Tucker (1968a,b) of mutants of S. typhimurium with a dual growth require-
ment for purines as well as for hydroxymethylpyrimidine. The dual growth
requirement was satisfied by 5-aminoimidazole ribonucleotide (AIR) alone and
HlC-g:~'NH, H,c:.......,.,jcNH, H,c.......N:rNH,

~ IZ.~ll ----.. r I Q ~ r- I )! 9
N CH,OH N CH,O-P-OH N CH,O-P-O-P-OH
6H 6H 6H
Nl.f1~.'
CH,CH.OH
H.
_7, N
H.
9
CH.CH.O-P-OH
6H
~
PPt
N
N NH, N
Hlcy:x
CHz'~
13: CH,CH.O-f-OH
9
H. OH
Fig. 2. The biosynthesis of thiamine from its pyrimidine and thiazole moieties.
Microbial Synthesis of Vitamin B 1 141
by compounds that precede AIR on the purine pathway, indicating that AIR is
the last common intermediate on the route to the purines and to the
pyrimidine moiety of thiamine. The biosynthetic pathways of hydroxy-
methylpyrimidine in micro-organisms differ between bacteria and yeast. In
bacteria, hydroxymethylpyrimidine is synthesized from AIR, and N-1, C-4 and
C-6 of the pyrimidine are derived from the nitrogen, C-1 and C-2 of glycine,
respectively (Estramareix & Lesieu, 1969; Estramareix, 1970; White &
Rudolph, 1979), and the C-2 from formate (Kumaoka & Brown, 1967;
Estramareix & Lesieu, 1969). These results suggest that the imidazole ring is
opened between C-4 and C-S. The remaining three carbon atoms of hydroxy-
methylpyrimidine (C-S, C-7 and C-8) originate from the ribose part of AIR
which is thus the precursor of all the carbon atoms of this pyrimidine
(Estramareix & Therisod, 1984). Since N-3 and the amino group at C-4 have
been recently shown to be derived from the amide-N atom from glutamine in
E. coli and S. cerevisiae (Tazuya et ai., 1987a), the origin of almost all of the
pyrimidine moiety of thiamine is now known for bacteria, although the
reactions whereby AIR is converted to the pyrimidine remain unknown. In
yeast, on the other hand, C-4 of the pyrimidine originates from formate (David
et ai., 1966) and no significant incorporation of the nitrogen atom and C-2 of
glycine into the pyrimidine is found (Linnett & Walker, 1968; White &
Spenser, 1979). Furthermore, the incorporation of C-3, C-4 and C-S of
pentulose into C-6, C-S and C-7 of the pyrimidine has been recently reported
in S. cerevisiae (Grue-S0rensen et ai., 1986). The origin of C-2, C-8 and N-1 of
hydroxymethylpyrimidine remains to be clarified in the biogenesis of the
pyrimidine moiety in yeast.
Relatively little has been discovered regarding the biosynthetic origins of the
thiazole moiety of thiamine, but some progress has been made in recent years
toward the identification of its precursors. In E. coli and S. typhimurium the
direct utilization of C-2 and the nitrogen atom of tyrosine for the formation of
the thiazole ring was demonstrated (Estramareix & Therisod, 1972; Bellion et
ai., 1976; White & Rudolph, 1978). On the other hand, no incorporation of
C-2 of tyrosine into thiamine is observed in yeast, whereas significant
incorporations of C-2 and the nitrogen atom of glycine into C-2 and N of the
thiazole are shown (Linnett & Walker, 1968, 1969). These observations seem
to indicate that bacteria and yeast use different amino acids as precursors of
the C-2 and nitrogen portion of the thiazole ring, implying that the pathways in
these two micro-organisms are different. Further incorporation studies with
deuterated carbohydrates and related compounds showed that the precursor of
the S-carbon chain (C-4, C-S, C-6, C-7 and C-8) of the thiazole moiety of
thiamine in E. coli is a S-carbon sugar derived from pyruvate and triose
phosphate (White, 1978), which might then react with tyrosine and a sulfur
compound in an undefined series of steps to yield hydroxyethylthiazole. This
has been supported by the incorporation of 1-·deoxY-D-threo-pentulose into the
S-carbon chain of the thiazole without carbon-carbon bond cleavage in E. coli
(David et ai., 1981). In yeast, the incorporation pattern leads to the inference
142 A.lwashima
that the 5-carbon chain may not be derived from pyruvate but from
2-pentulose, which is generated from the hexose precursors by oxidative as
well as non-oxidative pentose phosphate pathway (White and Spenser, 1982).
The precursor of the sulfur atom of the thiazole ring is still not known with
certainty, but several experimental observations suggest that either cysteine or
H 2 S which could be produced from cysteine, is the most likely precursor of the
sulfur atom of hydroxyethylthiazole in bacteria and yeast (Bellion & Kirkley,
1977; Tazuya et al.; 1987b).
4.2 Regulation
As described above, the derepression of thiamine biosynthesis by adenosine

was demonstrated for the first time in S. typhimurium (Newell & Tucker,
1966a,b). Adenine and adenosine, in the form of AMP, exert feedback
control over the synthesis of AIR by inhibiting the formation of 5-
phosphoribosylamine, the first step exclusive to the synthesis of purines and
hydroxymethylpyrimidine. The derepression is unaffected by hydroxy-
ethylthiazole, but does not occur if thiamine or hydroxymethylpyrimidine is
present, or if protein synthesis is prevented during preincubation. Since
derepression by adenosine in a hydroxyethylthiazole auxotroph is prevented by
thiamine, but not by hydroxymethylpyrimidine, it was suggested that hydroxy-
methylpyrimidine influences the state of repression only by being converted to
thiamine. Derepression in the absence of adenosine also occurs when a
hydroxyethylthiazole auxotroph is grown on a limited supply of thiamine.
These studies were then extended to the regulations of the four enzymes in E.
coli that catalyze the synthesis of TMP from hydroxymethylpyrimidine and
hydroxyethylthiazole: hydroxymethylpyrimidine kinase (EC 2.7.1.49), phos-
phomethylpyrimidine kinase (EC 2.7.4.7), hydroxyethylthiazole kinase (EC
2.7.1.50) and thiamine-phosphate pyrophosphorylase (EC 2.5.1.3) (Kawasaki
et al., 1969). These enzymes are derepressed by adenine and repressed by
thiamine. The derepression is also brought about by preincubating the
growing culture with phenylalanine (Kawasaki et al., 1969). This derepression
is reversed by thiamine or hydroxyethylthiazole, but not by hydroxymethylpyr-
imidine. Since thiamine synthesis is inhibited by phenylalanine in a thiamine
regulatory mutant (PT-R1) and the inhibition is reversed by hydroxy-
ethylthiazole (Iwashima & Nose, 1970), this inhibition by phenylalanine
appears to account for the derepression in the wild type. However, two-fold
increase in cellular thiamine levels accompanied by the inhibition of the
thiazole synthesis caused by phenylalanine remains unexplained. In a thiamine
regulatory mutant (PT-R1) hydroxyethylthiazole kinase and thiamine-
phosphate pyrophosphorylase are not subjected to repression by thiamine,
suggesting that it is a constitutive mutant of an operator gene controlling the
synthesis of two enzymes (Kawasaki & Nose, 1969). Several thiamine
regulatory mutants of E. coli affecting other gene loci have been reported
(Kawasaki et al., 1976).
Microbial Synthesis of Vitamin B\ 143
5 ASSAY METHODS
5.1 Microbiological Assays
Assay methods based on use of the thiamine requiring fungus Phycomyces
blakesleeanus and thiamine requiring strains of several bacteria such as
Staphylococcus aureus, E. coli and Lactobacillus species, and S. cerevisiae
have been used (Thomas, 1966).
5.2 Chemical Assays
Chemical assay methods are usually preferred to microbiological methods since
such analyses can be performed rapidly and they are usually more reliable for
routine determination. Although a number of chemical assay methods for
thiamine are known, only the coupling reaction with diazotized p-
aminoacetophenone (Melnick & Field, 1937) and thiochrome reaction have
been developed as precise methods of assay. In recent years, however, the
thiochrome method has been used almost exclusively. As described above,
thiamine in alkaline solution is oxidised quantitatively to thiochrome. As
oxidizing reagents potassium ferricyanide (Hennessy & Cerecedo, 1939) and
cyanogen bromide (Fujiwara & Matsui, 1953) have been routinely used.
Thiochrome formed from non-phosphorylated thiamine is extracted with
isobutanol. Since thiochrome fluoresces intensely under UV illumination, it
can be measured readily in a fluorimeter (excitation 365 nm; emission 430 nm).
Phosphorylated thiamines can be determined after their hydrolysis to thiamine
by phosphatase. Hydroxyethylthiamine gives thiochrome by oxidation with
alkaline ferricyanide, but not with cyanogen bromide. Thiamine gives thio-
chrome by either oxidizing agent, so that this difference in the oxidation
property is used for the simultaneous determination of thiamine and hydroxy-
ethylthiamine (Morita et al., 1968). For the differential determination of
thiamine and its phosphate esters in biological materials electrophoresis, paper
chromatography, thin-layer chromatography and ion-exchange chromatog-
raphy can be used to separate thiamine compounds from each other (Yurugi et
al., 1979). In recent years, high-performance liquid chromatography (HPLC)
has been developed for simultaneous determination of thiamine and its
phosphate esters and assay procedures have been established (Kawasaki &
Sanemori, 1985). The oxidation of thiamine compounds in samples can be
carried out either before the chromatography (precolumn derivatization
procedure) or after the chromatography (postcolumn derivatization proce-
dure). Therefore, the precolumn derivatization procedure is essentially the
HPLC of thiochrome and its phosphate esters. A mixing coil with proportion-
ing pump as an additional equipment for thiochrome reaction is required for the
postcolumn derivatization procedure. The intensity of the fluorescent products
is measured with a spectrophotofluorometer. The minimum detection by these
methods has been reported to be 0·1 pmol or 33·7 pg as thiamine hydro-
chloride (Sanemori et al., 1980).
144 A.lwashima
5.3 Enzyme Assays
TPP has been estimated manometrically by measuring the evolution of CO2

from pyruvate in the presence of the apoenzyme of yeast pyruvate decar-
boxylase (Green et al., 1941), and spectrophotometrically in the same reaction
system by measuring acetaldehyde-dependent reduction of NADH2 using
alcohol dehydrogenase (Kajiro, 1957).
6.1 Metabolism
It has been generally accepted that thiamine transport in micro-organisms

occurs via active transport (Iwashima, 1980). In animal cells, evidence has
been accumulated which shows that thiamine is taken up by Na+-dependent
active process (Iwashima, 1980). In both animals and humans thiamine
transport across the small intestine is biphasic: mainly by active transport at
low or physiological concentrations «2 IlM) and predominantly passive at
higher concentrations (Hoyumpa et al., 1975).
Thiamine must be phosphorylated to TPP, a coenzyme form of thiamine, to
exert its physiological functions. The formation of TPP from thiamine is
catalyzed by thiamine pyrophosphokinase (EC 2.7.6.2) in yeast and animal
tissues. In E. coli thiamine is converted to TPP by two successive phosphoryla-
tions catalyzed by thiamine kinase (EC 2.7.1.89) and thiamine-monophosphate
kinase (EC 2.7.4.16). TPP is further metabolized to TIP by thiamine-
diphosphate kinase (EC 2.7.4.15).
Two thiamine cleavage enzymes have been isolated from various natural
sources (Murata, 1982). Thiaminase I (EC 2.5.1.2) is a transferase which
catalyzes an exchange reaction between a base and a thiazole moiety of
thiamine, and thiaminase II (EC 3.5.99.2) mediates in the hydrolysis of
thiamine.
6.2 Physiological Functions
The physiological functions of thiamine is exerted in the form of TPP in cells.

TPP serves as the coenzyme for a large numbers of enzyme systems in the
metabolism of carbohydrates and amino acids, including pyruvate
dehydrogenase(cytochrome) (EC 1.2.2.2), pyruvate oxidase (EC 1.2.3.3),
pyruvate dehydrogenase(lipoamide) (EC 1.2.4.1), oxoglutarate dehydro-
genase(lipoamide) (EC 1.2.4.2), 2-oxoisovalerate dehydrogenase(Iipoamide)
(EC 1.2.4.4), transketolase (EC 2.2.1.1), pyruvate decarboxylase (EC 4.1.1.1),
benzoyiformate decarboxylase (EC 4.1.1.7), oxalylCoA decarboxylase (EC
4.1.1.8), tartronate-semialdehyde synthase (EC 4.1.1.47), phosphoketolase (EC
4.1.2.9), 2-hydroxy-3-oxoadipate synthase (EC 4.1.3.15), acetolactate synthase
(EC 4.1.3.18) and sulfoacetaldehyde lyase (EC 4.4.1.12).
Microbial Synthesis of Vitamin B) 145
The physiological function of TIP remains enigmatic, although evidence is

emerging that this form of thiamine is involved in nerve membrane function
(Leder, 1975). It is certain that the neuropathy of beriberi is due to thiamine
deficiency and it is probable that the anorexia and cardiac manifestation of
beriberi are caused by decreased activity of the enzymes for which TPP is
coenzyme. However, much remains unclear about the cellular and molecular
mechanism of thiamine action correlated to thiamine deficiency symptoms.
Thiamine has been synthesized in two basic ways (Matsukawa et al., 1970).
The condensation method, first adapted to the synthesis of thiamine by
Williams & Cline (1936), consists of the condensation of the pyrimidine with
thiazole derivatives. In another method, which was devised by Todd & Bergel
(1937), the pyrimidine moiety is synthesized with an appropriate side chain
conductive to the formation of the remaining structure. Matsukawa (1953)
synthesized thiothiamine as an intermediate for thiamine synthesis. This is
formed by the condensation of 4-amino-5-aminomethyl-2-methylpyrimidine,
CS 2 and 3-acetyl-3-chloro-1-propanol. Thiothiamine is a water-insoluble crys-
talline compound and is readily converted by oxidation into thiamine in good
yield.
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Chapter 10
MICROBIAL PRODUCTION OF VITAMIN B2 (RIBOFLAVIN)
T. KUTSAL & M. T. OZBAS

Ankara, Turkey
1 INTRODUCTION
Riboflavin is a water-soluble vitamin which can be produced on a commercial

scale by various fermentation processes using various micro-organisms. Ribo-
flavin is used pure for human nutrition and therapy and in the crude
concentrated form for animal feed supplements. Although it is produced by
both synthetic and fermentation processes, it is believed that the microbial
synthesis should be able to compete quite successfully with the synthetic
chemical processes.
2 HISTORICAL
Riboflavin, also called vitamin B2 (lactoflavine, ovoflavine, uroflavine, hepat-

oflavine, verdoflavine, vitamin G), is a water-soluble vitamin which exhibits a
strong yellowish-green fluorescence.
As early as 1879, A. W. Blyth isolated from whey an impure preparation of
flavine (lactochrome) of a red-orange color. In 1933, P. Gyorgy, R. Kuhn,
and T. Wagner-Jauregg described the isolation of pure, crystallized lactoflavine
and found the vitamin nature of this yellow pigment. The original name of
riboflavin was lactoflavine. Ovoflavine, isolated from eggs; hepatoflavine, from
liver; lactoflavine, from milk; and verdoflavine, from plants, were all identified
as riboflavin. The name riboflavin, indicating that the naturally occurring flavin
is a derivative of o-ribose, was adopted in 1952 by the International
Commission for the Reform of Biochemical Nomenclature (Kirk & Othmer,
1968; Wagner-Jauregg, 1972).
The structure of riboflavin was confirmed in 1934 by synthesis by R. Kuhn
and F. Weygand and P. Karrer and his co-workers. The chemical structure of
vitamin B2 is [6,7-dimethyl-9-(o-1'-ribityl)-isoalloxazine], as shown in Fig. l.
149
l' 2' 3' 4' 5'

CHz-CHOH-CHOH-CHOH-CHzOH
I
H3 C N·N ~O
~91il~
~lOh 43NH
HC N
3 0
Fig. 1. Riboflavin.
Riboflavin (C 17H zoN40 6), crystallizes from 2 N acetic acid, alcohol, water or
pyridine in orange-yellow needles. Its molecular weight is 376·36. The
decomposition point is 278-282°C, and it darkens at about 240°C. The vitamin
is odorless and has a bitter taste. Riboflavin is soluble in water only to the
extent of 10-13 mg in 100 ml at 25-27·5°C, 19 mg in 100 ml at 40°C, and
230 mg in 100 ml at 100°C. The vitamin dissolves in ethanol and is slightly
soluble in amyl alcohol, cyclohexanol, benzyl alcohol, phenol and amyl
acetate, but is insoluble in ether, chloroform, acetone and benzene. Although
alkali dissolves the vitamin well, these solutions are unstable.
Neutral aqueous solutions of riboflavin have a greenish-yellow color. The
absorption spectrum shows characteristic absorption maxima at 475, 446,
359-375, 268 and 223 nm. The absorption in the visible part of the spectrum is
used for quantitative determination of riboflavin (Wagner-Jauregg, 1972).
Neutral aqueous solutions of riboflavin display intense yellowish-green fluores-
cence, with a maximum at 565 nm which can be used for quantitative
determination of the vitamin. The fluorescence vanishes on the addition of acid
or alkali, optimum fluorescence occurs at pH 3-8.
Riboflavin is an amphoteric compound. Its dissociation constants are
Ka = 6·3 X 1O- 1z and KfJ = 0·5 X 10-5 , the isoelectric point corresponds to a
pH of 6·0.
The basic factors affecting the stability of riboflavin in food are heat, light
and the reactions which occur in cells during the storage. Neutral solutions of
riboflavin can be sterilized by autoclaving for a short time: only slight
destruction occurs by heating to 120°C for 6 h. No appreciable destruction of
the vitamin can be observed during the cooking of food since it is relatively
heat stable, but alkali decomposes riboflavin rapidly (Wagner-Jauregg, 1972;
Heimann, 1980). When milk in bottles is exposed to sunlight, 85% of its
riboflavin content is destroyed within 2 h.
Riboflavin is stable against air and most common oxidizing agents, but when
reduced by reducing agents such as sodium hydrosulfite or hydrogen, riboflavin
readily takes up two hydrogen atoms to form the colorless and non-fluorescent
1,1O-dihydro compound, leucoriboflavin, which can be reconstituted by shak-
ing an aqueous solution with air. This reduction and reoxidation is also useful
Microbial Production of Vitamin B2 151
in increasing the specificity of riboflavin assay in food and feeds containing

interfering fluorescent substances (Kirk & Othmer, 1968).
Irradiation of riboflavin in solution in the visible light or ultraviolet range
degrades the side chain, yielding a number of compounds. These compounds
are Lumiflavine (6,7 ,9-trimethylisoalloxazine) , C 13H 12N 4 0 2, and Lumichrome
(6,7-dimethylisoalloxazine), C 12H lON 4 0 2. Irradiation with y rays causes loss of
riboflavin in solution but not in dry mixes. Sterilization of foods by irradiation
or treatment with ethylene oxide may also cause destruction of riboflavin (Kirk
& Othmer, 1968).
4 PRODUCING MICRO-ORGANISMS AND SCREENING
Riboflavin can be produced in significant quantity by various fermentation

processes using a number of micro-organisms and appropriate media. Vitamin
B2 concentrates for the enrichment of poultry and livestock feeds can be
prepared more cheaply by fermentation processes (Smith & Berry, 1975).
Riboflavin can be synthesized by many various types of micro-organisms.
Bacteria, yeasts, moulds, algae and protozoa are the micro-organisms which
can potentially produce riboflavin.
Different natural sources have been used for the production of riboflavin by
fermenting micro-organisms. Lactose fermenting yeasts are especially
Saccharomyces fragilis, Clostridium butylicum, several species of
Lactobacillus, or moulds, e.g., Aspergillus niger, Aspergillus flavus, Peni-
cillium chrysogenum, and species of Fusarium. Whey and other milk by-
products can be employed as lacteal raw materials by these micro-organisms.
Riboflavin was formed in the butyl alcohol-acetone fermentation of cereal
grain mashes or molasses with various strains of Clostridium. Among these
bacteria Clostridium acetobutylicum especially is one of the best producers of
riboflavin (Kirk & Othmer, 1968). A suitable carbohydrate containing mash
cereals, rice, corn, whey and potato starch have been used in these commercial
anaerobic fermentation processes of Clostridium acetobutylicum (Wagner-
Jauregg, 1972).
Most varieties of the yeast species Candida are also able to produce
riboflavin (Goodwin, 1959; Goodwin, 1963a,b). Candida guilliermondia, C.
tropicalis varrhaggi, C. flareri, C. arborea, C. lactis, C. krusei, C. ghoshi, C.
chalmersi, C. lypolytica, C. olea, C. pulcherrima, C. solani and C. melibiosi
were found to synthesize significant amount of riboflavin (Goodwin, 1963a;
Robinson, 1966).
Among these riboflavin producing micro-organisms, two closely related
ascomycetes, Eremothecium ashbyii and Ashbya gossypii (Ainsworth &
Sussman, 1965) are capable of synthesizing very large amounts of this vitamin
under appropriate conditions (Perlman et al., 1952; Hickey, 1954).
A great number of cheap natural materials and industrial wastes have been
used for riboflavin fermentation. Fermentation residues such as grain stillages
from the ethyl alcohol and butyl alcohol-acetone fermentations; sugar

containing substances such as molasses and corn syrup; fish meal, dried yeast,
skim milk, cotton-seed meal, soybean meal, peas, tankage, wheat bran, animal
proteins and malt extract are some of the raw materials employed in industrial
processes for riboflavin production by Eremothecium ashbyii (Hickey, 1954;
Gutcho, 1973).
Ashbya gossypii is also an important ascomycete very similar morphologi-
cally to Eremothecium ashbyii. Corn steep liquor, still ages , proteinaceous
agents from animal sources such as tankage, animal stick liquor and meat
scraps, vegetable proteinaceous agents, such as soybean meals, glutens, linseed
and cotton-seed meals have been used as raw materials for commercial
production of riboflavin by Ashbya gossypii (Goodwin, 1959; Robinson, 1966).
Lipids may be of importance for high vitamin B2 production by these two
ascomycetes (Ozbas & Kutsal, 1986a,b, 1987). The lipids studied included corn
oil, soybean oil, cocoanut oil, lard oil, lecithin, menhaden oil, sunflower oil
and others. (Hickey, 1954; Lago & Kaplan, 1981).
It is known that algae synthesize riboflavin but detailed work has not been
reported in literature (Goodwin, 1963b).
Among the protozoa, Chilomonas paramoecium synthesizes riboflavin when
cultured on a medium containing only acetate and inorganic salts (Goodwin,
1963b).
There are many reports on general factors controlling riboflavin biosynthesis.

Purines stimulate riboflavin over-production in all of the organisms studied.
This holds true not only for complete fermentations, but for cell washed
suspensions as well. Glycine, a known purine precursor, stimulates over-
production in Candida species and in A. gossypii. The stimulatory effects of
purines and glycine in A. gossypii is not due to an effect on growth but is
specific for riboflavin oversynthesis (Demain, 1972). One of the proposed
mechanisms is described as follows.
The biosynthetic pathways for purines, riboflavin, and pteridines are closely
linked. Thus the carbon atoms of glycine are incorporated into these three
classes of compounds in structurally analogous positions. A preformed purine
can be converted directly into riboflavin with loss of only carbon 8 of the
purine ring: other purine carbons and all the ring nitrogens appear finally in
the pyrimidine portion for the riboflavin molecule (Wagner-Jauregg, 1972).
According to some workers who studied the biosynthesis of vitamin,
riboflavin (IV) is formed in E. ashbyii from the purine (I) (9-ribitylxanthin)
t,hrough the unstable 4-ribityl-amino-5-aminouracil (II) and the dioxopteridine
(III) (6,7-dimethyl-8-ribityl-lumazine or DMRL) (Fig. 2). It has been synthes-
ized by condensation of II with diacetyl and the same pathway has been
suggested for the biological synthesis.
R = D-Ribityl
III IV
Fig. 2. The biosynthesis of riboflavin.
That lumazine III alone is the precursor of riboflavin (IV) in organisms such
as E. ashbyii and A. ashbyii has been established by biochemical studies. All
the carbon atoms of the o-xylene moiety of riboflavin are derived from the
lumazine III, two molecules of which are converted by riboflavin synthesis into
one molecule of riboflavin (IV) and 4-ribitylamino-5-amino-2,6-dihydroxy
pyrimidine. In this biochemical reaction the lumazine (III) simultaneously
functions as a donor and acceptor of a C 4 unit which consists of the two methyl
groups and the C atoms 6 and 7. There is also a similar mechanism for
riboflavin formation in plants (Wagner-Jauregg, 1972).
Goodwin et al. showed independently that amino acids provide carbon units
and not nitrogen in the biosynthesis of riboflavin. These workers used E.
ashbyii and found that L-threonine, L-serine or L-tyrosine stimulated riboflavin
synthesis whereas L-glutamate, L-aspartate and L-asparagine stimulated both
growth and riboflavin synthesis; L-cystein on the other hand inhibited both
(Goodwin, 1963a,b; Robinson, 1966).
6 STRAIN IMPROVEMENT
The changes in the culture media and the selection of high riboflavin-producing
mutant cultures cause the most important increases in riboflavin productivity.
In 1950, Pfeifer et al. suggested that some improvement in fermentation
productivity was possible as a result of culture selection. Paralleling the pilot
plant fermentations, a special study was carried out in the laboratory by
Pridham of the NRRL fermentation division to obtain and test natural and
induced variants of Ashbya gossypii NRRL Y -1056 for riboflavin production.
One of the natural variants was chosen as superior to the original strain on the
basis of shaker-flask experiments and was tested in a series of pilot plant
fermentations. This particular strain gave consistently higher yields of
riboflavin than did the standard NRRL Y -1056. The results indicate the
desirability of isolating high-producing strains at regular intervals in order to
maintain riboflavin yields at the highest possible values (Pfeifer et al., 1950).
7 FERMENTATION/BIOCONVERSION PROCESS
Commercial fermentation processes for production of riboflavin or riboflavin

concentrates are relatively recent, having been developed since about 1940.
The Ascomycete processes using Eremothecium ashbyii and Ashbya gossypii
are now assuming the most important position in the riboflavin
manufacturing field. Both organisms are capable of synthesizing very large
amounts of riboflavin under appropriate conditions.
7.1 Inoculum
As reported in Section 4, a great number of cheap natural materials and
industrial wastes have been used for riboflavin fermentation by these Ascomy-
cetes (Hickey, 1954; Goodwin, 1959; Robinson, 1966; Gutcho, 1973; Ozbas,
1985). In order to produce very large amounts of riboflavin, an appropriate
combination of these substrates can be chosen and if necessary, several
minerals can be added to the medium.
The generally accepted inoculating procedure is to use a 0·25-1·0%
inoculum from an actively growing 24-28 h culture (Goodwin, 1959).
7.2 Medium
In laboratories, cultures of E. ashbyii and A. gossypii are grown on solid agar
media in slant tubes and Petri dishes. The composition of the culture medium
is as follows (g/liter): glucose, 20·0; peptone, 5·0; yeast extract, 5·0; malt
extract, 5·0; MgS04 ·7H2 0, 0·2; K2 HP0 4 , 0·2. The -pH of the medium is
adjusted to the desired value with 0·5M H 2 S04 (Ozbas et al., 1984, 1986a,b).
Both liquid and solid media are sterilized at 121°C in an autoclave before
inoculation. The initial substrate concentrations can be changed by making the
amount of initial carbon in the medium equivalent to 20·0 g/liter glucose while
the amounts of other constituents are kept constant.
7.3 Fermentation Course and Parameters

General fermentation conditions that affect micro-organism growth and
productivity are pH, temperature, aeration level, initial substrate concentra-
tion such as glucose, and growth factors such as DL-methionine, inositol,
D( +)-biotin and thiamine.
7. 3.1 Effect of initial pH

An initial pH of 6·0-8·0 for Eremothecium ashbyii and 5·5-7·0 for Ashbya
gossypii were found to be preferable (Hickey, 1954; Goodwin, 1959). The
0.15
.
//:--..-
, \ 3.00
,
L:
-
0
0 E
-~0.10 2.00~
::L.
~o It:
QI
~
0
0.05 .~'~. 1.00 ::
, '~i
0 0
6.0 6.5 7.0 7.5
pH
Fig. 3. Effect of initial pH on the specific growth (I-') and riboflavin production (v)
rates for E. ashbyii and A. gossypi (30°; stirring rate 100 rpm; SGO = 20·0 g/liter; . , E.
Ashbyii; 0, A. gossypii.
effects of initial pH on specific growth and riboflavin production rates with E.

ashbyii and A. gossypii are shown in Fig. 3. The optimum initial pH value is
6·5 for both micro-organisms (Ozbas & Kutsal, 1986a,b).
Riboflavin production by these two ascomycetes fits the third group of the
Gaden classification. In these riboflavin fermentations the pH of the medium
decreases with time and drops from its initial value of 6·5 to approximately 4·5 .
•,.,.__e-- 0.16 9.0
::.
/:#:~_o_o 0.14
0.12
8.0
7.0
~Jo\I// "\
-
0.10 _ 6.0
....
1ot -
0.08 ~ 5.0 ::r:
It: Co
U
0.06 4.0
_
2.0
0.04 3.0
1.0 / \ 0.02 2.0
o 3 /~_'--_--'-_--'-_ _ ",--_.....L..._--' 0 1.0

o 40 80 120 160 200
II hI
Fig. 4. Changes in medium during fermentation (30°; initial pH = 6·5; stirring rate
750 rpm; aeration rate, 220 cc/min; SGO = 20·0 g/liter; 0, riboflavin (C R ); . , dry weight
(X); 0, pH).
0.15 3.00_
.J::.
b
E
t7l
";".c 0.10 2.00-;;::
'~~\~o-r-o
01
1- ",-
e
.,.
I~I .'\.~
0.05 1.00
--. I
0 0 0
20 25 30 35 40
T (oC)
Fig. 5. Effect of temperature on the specific growth and riboflavin production rates for
E. ashbyii (stirring rate, 100 rpm; initial pH = 6·5; 0, SGo = 20·0 g/liter; . , SSo =
20·0 g/liter).
At this point the micro-organisms grow rapidly, but no significant amounts of

riboflavin are synthesized. Then both riboflavin production and pH increase
and riboflavin formation terminates at approximately pH 9·5. Figure 4 shows
characteristic changes in the medium during the fermentation process (Ozbas
et aI., 1984; Ozbas & Kutsal, 1986a, b).
7.3.2. Effect of temperature

The optimum temperature ranges for riboflavin fermentation by Eremothecium
ashbyii and Ashbya gossypii were obtained between 25 and 30°C and 26 and
28°C respectively (Hickey, 1954; Gutcho, 1973).
Ozbas & Kutsal investigated the effects of temperature by utilizing glucose
and sunflower oil as substrate in the range from 20 to 40°C. For E. ashbyii and
A. gossypii maximum specific growth and riboflavin production rates were
obtained at 30°C in all medium compositions. At lower temperatures both
growth and riboflavin yield were reduced to values similar to those observed at
higher temperatures (Figs 5 and 6).
The Ascomycete processes for riboflavin production are aerobic and it is
necessary either to aerate or shake the cultures.
Z 3. 3 Effect of initial substrate

For the growth of both micro-organisms the main carbon source is glucose.
Maximum yields are obtained with an initial glucose concentration of
20·0 g/liter. Glycerol, sunflower oil, whey and several combinations of these
substrates can also be used for riboflavin production. The effects of different
substrates and their initial concentrations on the specific growth and riboflavin
0.30
lei 4.00
0.20
/;or" '\ 300~
:/,/" \ \ 2DO!
0.10 ,./ ~:~ i;
/0. . . . . 0"'0 ~ 1.00
?
'r'-0
1----°
-----0
0~2~0----~2~5~--~3~0----~35~----4~0~0
T (DC)
e
Fig. 6. Effect of temperature on the specific growth and riboflavin production rates for
A. gossypii (stirring rate, 100 rpm; initial pH = 6·5; 0, Sao = 10·0 g/liter, Sso =
1O·0g/liter; e, Sao = 20·0 g/liter).
production rates and riboflavin yields are given in Table 1 (Ozbas & Kutsal,
1986a,b).
7. 3. 4 Fermentor experiments
The effects of agitation and aeration rates on the formation of riboflavin were
observed at the range of 300-1200 rpm and 70-330 cc/min, respectively for
Eremothecium ashbyii. Optimum values were reached as Il = 0·176 h- 1 and
v = 4·45 X 10- 3 g/liter per hat 750-1000 rpm and Il = 0·181 h- 1 and v = 3·69 X
10-3 g/liter per h at 220 cc/min. At these optimum conditions, adding the
sunflower oil to the medium resulting in 5·0 g/liter glucose and 15·0 g/liter
sunflower oil showed Il to be 0·281 h- 1 and v = 5·34 X 10- 3 g/liter per h. These
results suggested that in addition to pH, temperature and initial substrate
concentrations, agitation and aeration are also important control parameters
on the riboflavin production (Ozbas et al., 1984).
7. 3. 5 Effect of growth factors

The effect of some growth factors such as oL-methionine, inositol, 0(+ )-biotin
and thiamine for riboflavin production by E. ashbyii and A. gossypii were
investigated in various media.
In order to determine initial concentration effects of these growth factors,
glucose was used as substrate at the optimum growth conditions. While,
o( + )-biotin and thiamine are ineffective in stimulating either growth or
flavinogenesis, maximum specific growth and riboflavin production rates and
riboflavin yields are obtained in the media containing 0·4 g/liter oL-methionine
and 0·2 g/liter inositol for E. ashbyii. It is also observed that all of these
four compounds are effective in supporting growth and product formation
....
VI
00
Table 1
Effects of Various Substrates on Specific Growth (It) and Riboflavin Production (v) Rates and Riboflavin Yields (YP/SCa)
for E. ashbyii and A. gossypii (30"C, Stirring Rate: 100 rpm, Initial pH = 6·5)
Substrates Initial Ita E. ashbyii YPlSco Ita A. gossypii YPlSco

substrate (h -1) VX 10 3 (h -1) vX 10 3 :-'l
concentration (gR/gmo per h) (gR/gmo per h) ~
(g/liter) l
Roo
Glucose 20·0 0·1854 3·57 3·6 0·1538 1·25 3·1
Glycerol 20·0 0·1733 2·27 3·1 0·1315 0·89 1·3 ~
Sunflower oil 20·0 0·1669 1·45 3·0 0·2245 1·00 2·9 :-'l
Glucose + glycerol 5·0+ 15·0 0·2044 4·16 4·5 0·1979 1·32 1·7 Co
...0-
Glucose + glycerol 7·5 + 12·5 0·2006 4·10 3·7 0·2153 1·5 1·9 ~
Glucose + sunflower oil 5·0+ 15·0 0·2438 5·56 9·2 0·2323 1·49 5·2
Glucose + sunflower oil 10·0+ 10·0 0·2358 4·75 6·8 0·2635 4·05 8·8
Whey 12·5 0·1379 1·48 6·5
Whey 30·0 0·1529 0·24 2·2
a Calculated in the region of exponential growth (Monod kinetic model).

Table 2
Effects of Various Growth Factors on Specific Growth (fJ) and Riboflavin Production (v) Rates and
Riboflavin Yields (YPlScJ for E. ashbyii and A. gossypii (30°C, Stirring Rate: 100 rpm, Initial pH = 6·5)
Substrates E. ashbyii Yp/sco A. gossypii Yp/sco ~
fJ vX 10 3 fJ v X 10 3 "~
(h -1) (gR/g rno per h) (h -1) (gR/g rno per h)
§.:
""
G+M a 0·2900 4·46 4·5 '"tl
~
G+I 0·1957 4·00 3·8 ~
;:
G+S+I 0·1564 4·22 6·3 "::t.c
W+I 0·1386 1·78 7·6 ::
G+Mb 0·1779 3·84 6·4 ~
G+I+B+T 0·2068 3·92 7·5 $
G+S+M 0·1221 0·49 4·9 is
;:
G+S+I+B+T 0·2299 2·64 7·8 s·
W+I+B+T 0·3015 0·48 4·0 t:x:I
N
Initial substrate concentrations were (g/liter):

a Glucose (G), 20·0; G + sunflower oil (S), 5·0 + 15·0; whey (W), 12·5; DL-methionine (M), 0·4;
inositol (I), O· 2.
b G, 20·0; G + S, 10·0 + 10·0; W, 30·0; M, 0·08; I, 0·5; D( + )-biotin (B), 4 x 10- 7 ; thiamine (T), 0·04.
....
VI
\C
160 T. Katsal & M. T. Ozbas
with A. gossypii. Optimum initial growth factor concentrations are found as

0·08 g/liter oL-methionine, 0·5 g/liter isositol, 0·4 Jlg/liter 0(+ )-biotin and
0·04 g/liter thiamine for this ascomycete.
It was observed that the addition of these growth factors to the medium
containing only whey or glucose as substrate caused riboflavin production by
E. asbhyii and A. gossypii to increase (Table 2). Although higher yields were
obtained in the media containing sunflower oil compared to that of glucose
lower yields were attained with these growth factors in glucose and sunflower oil
medium for both micro-organisms (Ozbas & Kutsal, 1987).
7.4 Flow-Sheet
Descriptions of commercial methods for riboflavin production are primarily

limited to the patent literature.
Commercial methods for cultivation of E. ashbyii may be surface methods
exposed to air, or submerged methods in which air is dispersed throughout the
mash, with or without supplementary agitation (Goodwin, 1959; Wagner-
Jauregg, 1972).
The procedures generally adopted in industry by using A. gossypii utilize
aerated and agitated submerged cultures in liquid media. The air flow rate in
liters/min should be at least 0·25 times the volume of the medium in liters.
Excessive agitation which interferes with mycelial development reduces the
riboflavin production. Analysis of the experimental results indicates that
satisfactory yields of riboflavin can be obtained if fermentation variables are
closely controlled. In one of these pilot plant experiments, 2·0% glucose,
1·8-2·1 % suitable corn steep liquor, 1·0% animal stick liquor, and a small
amount of some antifoam agent had been used. After fermentation for
96-120 h at 30°C, the riboflavin content of the liquor was 500-600 y / cc. The
fermented liquor had been evaporated to syrup in conventional evaporators
and dried to a concentrate containing 2·5% riboflavin on steam-heated drum
dryers. In these experiments, inoculum quantity and age, corn steep liquor
from various sources, and use of other nutrients and experimental techniques
are also introduced (Pfeifer et al., 1950; Goodwin, 1959).
The commercial production of riboflavin by A. gossypii is shown in Fig. 7
(Pfeifer et al., 1950).
Fermentation solids containing riboflavin such as residues from butyl alcohol-

acetone fermentation may be recovered for use as animal feed supplements by
drying and powdering to a crude product. Drying is generally accomplished by
drum or spray drying procedures. These food grade products usually contain
other members of the B-complex, along with minerals and proteinaceous
substances.
Laboratory culture
Ashbya gossypii
2 3 4 5
Sulphuric Evaporator
acid
~
~.
~
\)'
s·
8 "tI
-
Fermentor ~
Mixing -._- ~g.
tank . :s
~
~
Water out
10
I·
b:I
N
water in
Air compressor
Fig. 7. Flow sheet for riboflavin production by fermentation (Pfeifer et al., 1950). 1, Glucose; 2, com steep liquor;
3, animal stick liquor; 4, sulphuric acid; 5, soybean oil; 6, caustic soda; 7, inoculum tank; 8, air filter; 9, sterilizer;
10, cooler. 0'1
......
Pure, crystalline riboflavin is obtained from some liquors containing

sufficiently high concentrations of riboflavin, such as the fermentation broths of
E. ashbyii and A. gossypii.
For the purification of riboflavin from the fermentation broths, it is useful to
remove lipids by extraction with ether, in which the vitamin is insoluble. In
some cases, salts and glycogen can be eliminated from riboflavin concentrates
by fractionate precipitation with alcohol or acetone.
Various methods were developed for recovery of riboflavin from fermented
media.
Two methods were described in 1945 by Hines who found first that by
bacteriological reduction, riboflavin in fermentation liquors of E. ashbyii could
be precipitated as a reddish-brown amorphous product by the action of
Streptococci strains (Hickey, 1954; Wagner-Jauregg, 1972; Periman, 1979).
Streptococcus faecaiis, S. liquefaciens and S. zymoge may be used in these
microbiological methods (Brit. Pat. 621,468).
Some chemical methods can be used also for recovery of riboflavin from
fermented mash. Chemical reducing agents, such as sodium dithionite
(Na2S20 4), stannous and chromous chlorides, and certain other reducing
compounds were employed in these chemical precipitation methods. Precipita-
tion could be obtained more rapidly and almost immediately by the use of
chemical reducing agents. Of these, sodium dithionite has economic and low
toxicity advantages over some of the other useable reducing agents, such as
stannous or chromous chlorides (Hickey, 1954; Wagner-Jauregg, 1972).
The chemical precipitation methods operate primarily on solutions contain-
ing at least 20/Jog/ml. In these methods the insoluble matter of mashes is
removed by centrifugation and riboflavin-containing parts are adjusted to
pH 5·0-5·5. A reducing agent such as Na2S204 is added at a temperature of
20-30 C and in a ratio of 5 moles of reducing agent to 1 mole of riboflavin
D
(Brit. Pat. 621,468; US Pat. 2,367,644; US Pat. 2,367,646).

The resulting precipitate is permitted to settle, the supernatant is centrifu-
gated. The residue is filtered. The crude precipitate which is obtained by such
means can be readily converted to crystalline riboflavin. In these processes, the
reddish-brown precipitate is dissolved in a hot polar solvent, such as 75%
aqueous isopropyl alcohol. This mixture is heated to dissolve all the precipit-
ate, except the inert material present, filtered, and the clear filtrate on cooling
gives yellow needlelike crystals of riboflavin which are separated by filtration
or centrifugation. Proportion of solvent to precipitate, and the temperature
employed in obtaining riboflavin are not critical (Brit. Pat. 621 468; US Pat.
2421142; US Pat 2367646).
Riboflavin can be also purified by adsorption methods. Good adsorbents for
riboflavin are Fuller's earth in acid solution, Florisil, Floridin XXF, and
Frankonit in neutral solutions. The vitamin is adsorbed very strongly by
charcoal, but elution is difficult from this adsorbate. A combination of
precipitation and adsorption methods can sometimes be necessary to isolate
pure riboflavin. A general method for the preparation of pure D-riboflavin
from natural sources has been described which is based on adsorption on

Fuller's earth, fractionation with immiscible solvents and acetone, and
crystallization from an aqueous acetone-petroleum ether mixture; aqueous
alcohol solutions have been used for elution of the adsorbates (Wagner-
Jauregg, 1972).
9 (BIO)-ASSA Y METHODS
Riboflavin can be assayed by physico-chemical, microbiological, or biological

methods. The most commonly used physico-chemical method involves meas-
urement of the characteristic, yellow-green fluorescence of riboflavin which
has a maximum at 565 nm at pH 6. For complex mixtures such as foods or
feeds, chemical pretreatment of extracts by column chromatography, oxida-
tion, reduction, or extraction is necessary to remove interfering fluorescent
impurities. Riboflavin can be measured by polarography, and also by photo-
metric or fluorometric measurement of lumiflavine formed by the irradiation of
riboflavin in alkaline solutions.
As with most vitamins, the first assays developed for riboflavin depended
upon the measuring of the biological response of animals. Early studies were
carried out in the rat and in the chicken. Animal assays are time-consuming,
expensive, and least accurate, but have the advantage of being based on a
biological animal response, which is important from the nutritional standpoint.
They are particularly useful for assaying riboflavin derivatives since substituent
groups on the riboflavin molecule frequently depress or eliminate the
biological response.
In animal methods the reference standard is crystalline synthetic riboflavin.
For administration in rat growth assays it is useful to prepare a stock solution,
which must be kept in a dark bottle in the refrigerator.
Since riboflavin is widely distributed in plant and animal tissues, test
substances may occur in a variety of forms. Solutions containing high
concentrations of riboflavin can be administered directly in suitable dilutions.
Samples of relatively low potency are mixed with a small portion of the basal
diet. Test materials with a high fat content may increase the riboflavin
requirement of the rat (Pearson, 1967). A human bioassay based on
comparative urinary excretions of riboflavin following test doses of a standard
vs tablets has been used widely for checking the physiological availability of
riboflavin in tablets (Kirk & Othmer, 1968).
One of the microbiological assays of riboflavin is based on the growth of
some micro-organisms. Not many bacteria have a requirement for riboflavin,
and virtually all of those known are lactic acid bacteria. The classic
microbiological assay for riboflavin with Lactobacillus casei which is measured
by titrating the lactic acid formed is in use in the original or modified form. A
more sensitive assay is afforded by the use of Leuconostoc mesenteroides. This
organism is 50 times more sensitive than L. casei. A method for total riboflavin
in body fluids and tissues suitable for clinical and nutritional surveys is based
on the ciliate protozoan Tetrahymena pyriformis (Pearson, 1967; Kirk &
Othmer, 1968).
The principal forms of riboflavin which exist in living cells are riboflavin
5'phosphate (FMN) and flavin adenine dinucleotide (FAD). Both these
phosphates are protein bound and 60-90% of the total riboflavin in natural
products is present in the form of the dinucleotide. Riboflavin performs its
biological functions in a number of different enzyme systems. Two derivatives
of riboflavin, FMN and FAD, serve as prosthetic groups and combine with
specific protein enzymes which catalyze oxidation-reduction reactions in cells
(Pearson, 1967). The good sources of riboflavin are milk, egg, liver, heart,
kidney, green, leafy vegetables, apricot, tomato, beef and poultry meats
(Wagner-Jauregg, 1972).
Riboflavin can thus be produced by chemical synthesis or by fermentation

processes. Although pure crystallized riboflavin for therapeutic purposes is also
made by chemical synthesis, various commercially competitive fermentation
processes have recently been developed for riboflavin production (Ozbas &
Kutsal, 1986a). Other processes start from D-ribose, which is produced by
fermentation (Chapter 11).
In 1891, O. Kiihling synthesized alloxazines by condensation of o-phenylene-
diamine hydrochloride with alloxan. Using the same principle, in 1934, R.
Kuhn and P. Karrer independently worked out methods for the synthesis of
flavins, based on o-xylene, D-ribose and alloxan as starting materials. Later,
processes of technical importance were developed by employing many refine-
ments in the general synthetic methods of R. Kuhn and P. Karrer (Wagner-
Jauregg, 1972).
Processes employing several chemical procedures have also been patented.
12 FORMULATIONS, APPLICATIONS AND ECONOMICS
Riboflavin is essential for the growth and normal health of animals as well as of
man. A lack of riboflavin in human or animal diets causes the formation of
certain oral, cutaneous and corneal lesions. Vitamin B2 is widely used in the
food enrichment, pharmaceutical and feed supplement industries (Kirk &
Othmer,·1968).
USP riboflavin for therapeutic purposes is administered orally in tablet form
or by injection as a sterile aqueous solution, with niacinamide or other
Microbial Production of Vitamin 8 2 165
solubilizing agent added. All of the vitamin B complex and most of the
multivitamin preparations also contain riboflavin (Kirk & Othmer, 1968).
Sterile, supersaturated solutions of riboflavin in normal saline have been
employed for intravenous administration (Wagner-Jauregg, 1972).
Some of the cereal products such as flour and bread are enriched with iron,
thiamine and niacin as well as riboflavin to compensate for the loss of these
nutrients that occurs in the processing of wheat (Kirk & Othmer, 1968).
Riboflavin can be synthesized by most higher plants, yeasts, and lower fungi,
and bacteria. The tissues of higher animals are unable to synthesize this
vitamin. For the enrichment of poultry and livestock feeds, riboflavin is usually
added at concentrations of 2-8 g/ton, depending on the species, age and
purpose. The supplement of riboflavin improves health, growth, tissue repair,
and reproduction of the animals and also has an economic importance in
poultry raising and egg production (Kirk & Othmer, 1968; Wagner-Jauregg,
1972).
The present world consumption of riboflavin is approximately 1·25 million
kg for human and animal use combined (Lago & Kaplan, 1981).
Commercial fermentation processes for production of riboflavin or riboflavin
concentrates are relatively recent, having been developed in the past 40 years.
In 1946 processes using the ascomycete A. gossypii were started. Manufac-
turers using the microbiological process are Merck and Co., Inc. (USA) and
BASF (FRG). Companies manufacturing riboflavin by chemical synthesis
include Hoffmann-LaRoche Inc. (USA and Switzerland), Takeda Chemical
Industries Ltd (Japan), Pfizer, Inc. (USA), and E. Merck (FRG) (Perlman,
1979).
REFERENCES
Ainsworth, G. C. & Sussman, A. S. (1965). The Fungi, Academic Press, New York,
pp. 37,492.
Brit. Pat. 621,468 (April 11 1949) (to Commercial Solvents Corp.)
Demain, A. L. (1972). Riboflavin oversynthesis, In Annual Reviews of Microbiology
Vol. 26, ed. C. E. Clyton, S. Raffel & M. P. Starr. George Benta Inc., USA, pp.
369-88.
Goodwin, T. W. (1959). Production and biosynthesis of riboflavin in micro-organisms.
In Progress In Industrial Microbiology, ed. D. J. D. Hockenhull. Heywood &
Company, London, pp. 139-177.
Goodwin, T. W. (1963a). Vitamins. In Biochemistry of Industrial Micro-organisms, ed.
C. Rainbow & A. H. Rose. Academic Press, London, pp. 151-8.
Goodwin, T. W. (1963b). Riboflavin and related compounds. In The Biosynthesis of
Vitamins and Related Compounds, ed. T. W. Goodwin. Academic Press, London,
pp.24-35.
Gutcho, S. J. (1973). Chemicals by Fermentation, Noyes Data Corporation, Chemical
Technology Review No. 19, New Jersey, pp. 323-25.
Heimann, W. (1980). Fundamentals of Food Chemistry, Ellis Horwood, Chichester, pp.
212-14.
Hickey, R. J. (1954). Production of riboflavin by fermentation. In Industrial

Fermentations, ed. L. A. Underkofler & R. J. Hickey. Chemical Publishing Co.,
Kirk, R. E. & Othmer, D. F. (1968). Encyclopedia of Chemical Technology, Vol. 17,
John Wiley & Sons, New York, pp. 445-58.
Lago, B. D. & Kaplan, L. (1981). Vitamin fermentations: Bz and B 12 • Adv. in Biotech.,
3,241-6.
Ozbas, T., Kutsal, T. & CagIar, A. (1984). The production of riboflavin by
Eremothecium ashbyii at various media. In Proceedings of the 3rd European
Congress on Biotechnology, ed. D. Behrens, Verlag Chemie GmbH, Weinheim, pp.
389-94.
Ozbas, T. (1~~5). Eremotheciu11J ashbyii Ashbya gossypii Mikroorganizmalari lIe
Riboflavin Uretimi. MSc thesis, Hacettepe University, Ankara, Turkey.
Ozbas, T. & Kutsal, T. (1986a). Riboflavin production by Eremothecium ashbyii in a
batch stirred tank fermenter. Biotechnology Letters, 8,441-44.
Ozbas, T. & Kutsal, T. (1986b). Comparative study of riboflavin production from two
micro-organisms: Eremothecium ashbyii and Ashbya gossypii. Enzyme Microb.
Techno!., 8, 593-96.
Ozbas, T. & Kutsal, T. (1987). Effects of inositol, D( + )-biotin and thiamine on
riboflavin production by Ashbya gossypii. In Proceedings of the 4th European
Congress on Biotechnology, ed. O. M. Neijssel, R. R. van der Meer & K. Ch. A.
M. Luyben. Elsevier Science Publishers B.V., Amsterdam, pp. 273-76.
Pearson, W. N. (1967). Riboflavin. In The Vitamins, ed. P. Gyorgy & W. N. Pearson.
Academic Press, New York, pp. 99-101.
Perlman, D. (1979). Microbial process for riboflavin production. In Microbial
Technology, ed. H. J. Peppler & D. Perlman. Academic Press, New York, pp.
521-7.
Perlman, D., Brown, W. E. & Sylvan, B. L. (1952). Fermentation. Ind. Eng. Chem.,
44, 1996-2001.
Pfeifer, V. F., Tanner, F. W., Vojnovich, C. Jr & Traufler, D. H. (1950). Riboflavin by
fermentation with Ashbya gossypii. Ind. Eng. Chem., 42, 1776-81.
Robinson, F. A. (1966). The Vitamin Co-factors of Enzyme Systems, Pergamon Press,
Braunschweig, pp. 160-63.
Smith, J. E. & Berry, D. R. (1975). The Filamentous Fungi, Vol. 1, Edward Arnold,
London, pp. 67-8, 115-116.
US Pat. 2367644 (January 16 1945), G. E. Hines, Jr (to Commercial Solvents Corp.).
US Pat. 2367646 (January 161945), M. G. W. Millian (to Commercial Solvents Corp.).
US Pat. 2421142 (27 May 1947) J. K. Dale (to Commercial Solvents Corp.).
Wagner-Jauregg, T. (1972). Chemistry. In The Vitamins, ed. W. H. Sebrell Jr & R. S.
Harris. Academic Press, New York, pp. 3-5.
Chapter 11
MICROBIAL PRODUCTION OF D-RIBOSE

K. SASAJIMAa & M. YONEOA b
a Central Research Division, b Corporate Strategy, Takeda Chemical Industries

Ltd, Yodogawa-ku, Osaka 532, Japan
1 HISTORY
o-Ribose and its derivative, 2-deoxy-o-ribose, are components of RNA and

DNA, respectively. It is of interest that these pentoses are components of such
important biopolymers in the hereditary material of organisms whereas other
ubiquitous pentoses, L-arabinose and o-xylose, are usually present in the plant
cell walls. Why the pentose components of nucleic acids are o-ribose and
2-deoxy-o-ribose is an unresolved problem; o-ribose and its derivatives have
been also found widely in nature.
From the viewpoint of commercial application, o-ribose has long served as
the focus for a starting material for the chemical synthesis of riboflavin (Fig.
1), which has been used in large amounts in pharmaceuticals and livestock feed
additives (see also Chapter 10). Various methods have been investigated and
established for the large-scale production of o-ribose.
Investigations on o-ribose started in 1909 when Levene and Jacobs identified
o-ribose as a component of yeast RNA (Levene & Jacobs, 1909). The
subsequent endeavors of many investigators were devoted to efficiently
preparing o-ribose from yeast RNA, e.g. chemical hydrolysis of yeast RNA
(Levene & Clark, 1921; Levene, 1935) and the enzymatic hydrolysis of yeast
RNA (Bredereck & Rothe, 1938; Bredereck et aI., 1940). Along this line, the
production of o-ribose from 5-amino-4-imidazole-carboxamide riboside by
enzymatic hydrolysis with a bacterial nucleosidase has recently been developed
(Sano et al., 1977a,b).
On the other hand, many investigators tried to establish a method for the
chemical synthesis of o-ribose from such a cheap material as o-glucose. These
studies were stimulated by identification of ribitol, the reduced product of
o-ribose, as a component of vitamin B 2 , riboflavin, and by chemical synthesis
of riboflavin in the middle of the 1930s (Karrer et al., 1935a,b; Kuhn et al.,
1935a,b; von Euler et al., 1935). The chemical process has been used as a
167
168 K. Sasajima & M. Yoneda
Fig. 1. Formula of riboflavin.
commercial procedure to produce o-ribose for the synthesis of riboflavin.

Investigations from 1910 to the 1950s on preparing and chemically synthesizing
o-ribose were reviewed succinctly by Jeanlotz & Fletcher (1951) and Overend
& Stacey (1955).
In the early 1950s, the pentose phosphate pathway was established as an
important metabolic pathway other than glycolysis in carbohydrate metabolism
(Wood, 1985). Thus, the biosynthesis of o-ribose was elucidated, which was
very important information for developing a commercial procedure for
o-ribose production using the transketolase (EC 2.2.1.1) mutants (tkt mutants)
of Bacillus species as will be described later.
At the same time, the production of o-ribose by micro-organisms was first
reported by Simonart & Godin (1951). About 10 years later, an unidentified
bacterium was found to excrete both o-ribose and o-ribose 5-phosphate into a
culture medium (Suzuki et al., 1963). A smaller degree of o-ribose accumula-
tion was also described by Saito & Sugiyama (1966).
In the late 1950s, active investigations on the development of nucleotide
flavour enhancers, inosine monophosphate and guanosine monophosphate,
started in Japan. A manufacturing process was established and has been
long used to commercially produce these substances. During the course of
studies on the development of the process, it was found that the mutants of
Bacillus lacking transketolase, a key enzyme of the pentose phosphate path-
way, accumulated a large amount of o-ribose (Sasajima & Yoneda, 1971).
Subsequent development research established an economical manufacturing
process for o-ribose production using these mutants. The commercial produc-
tion of o-ribose for riboflavin synthesis started in the mid-1970s. This process
has now been widely used for 15 years. The characteristic feature of this
process is that mutants of carbohydrate metabolism of Bacillus species are
used. In contrast, wild-type strains of Acetobacter suboxidans, A. xylinum,
etc., are used in L-sorbose fermentation, which is another remarkable
microbial process of sugar production for vitamin C synthesis (Perlman, 1979).
This review focuses on transitions of developments in o-ribose production
Microbial Production of D-Ribose 169
methods and the related basic investigations. In particular, recent microbial

methods will be described in detail.
2 OCCURRENCE OF o-RIBOSE AND ITS DERIVATIVES IN

NATURE
The occurrence of o-ribose and its derivatives in nature is summarized in

Table 1.
o-Ribose was first isolated from yeast RNA and identified by Levene &
Jacobs (1909). Thereafter, it was found to be a component of uric acid in the
blood (Davis et al., 1922); of crotonoside in the croton bean (Croton tiglium
L.) (Cherbuliez & Bernhard, 1932a,b); of coenzymes such as NAD and
NADP from yeasts (von Euler & Vestin, 1935; von El,ller & Schlenk, 1937;
von Euler et al., 1942) and from blood cells (Warburg et al., 1935), FAD
(Ochoa & Rossiter, 1939; Klein & Kohn, 1940), UDP-glucose (Caputto et aI.,
1950), and coenzyme A (Baddiley et al., 1953); of vitamin B12 (Beaven et al.,
1949; Brink & Folkers, 1949; Holiday & Petrow, 1949; Brink & Folkers, 1950;
Brink et al., 1950; Buchanan et aI., 1950a,b); and of poly(ADP-ribose) (Doly
& Mandel, 1967). Other coenzymes such as ADP, CDP, and GDP or many
nucleoside antibiotics (Suhadolnik, 1970) also contain o-ribose. It is also
present in the cell walls of Salmonella typhimurium as a component of
lipopolysaccharide (Kauffman et al., 1962) or of Eubacterium saburreum as a
component of polysaccharide (Hoffman et al., 1977; Hofstad & Lygre, 1977);
in the flavonoids of Phlebodium dictyocallis (Mett.) Gomez as a component of
o-glycosidic moieties (Gomez & Wallace, 1986); and in the coccoliths of
Emiliania huxleyi (Lohmann) Hay and Mohler as a component of an acidic
Ca2 + -binding polysaccharide (Borman et al., 1987).
o-Ribose derivatives such as 2-deoxy-o-ribose, 3-deoxy-o-ribose, 3-amino-3-
deoxy-o-ribose, ribitol, ribonic acid, o-riburonic acid, 5-methylthioribose and
glut amyl o-ribose have also been found as a component of organisms or as a
product of micro-organisms. 2-Deoxy-o-ribose is a component of DNA
(Levene et al., 1930; Chargaff et al., 1949; Chargaff & Lipschitz, 1953) which is
the carrier of genetic information in all organisms except RNA viruses (Avery
et al., 1944; Hershey & Chase, 1952; Watson & Crick, 1953). 3-Deoxy-o-ribose
is a component of an antibiotic, cordycepin, produced by Cordyceps militaris
(Bentley et al., 1951). 3-Amino-3-deoxy-o-ribose is a component of puromycin
produced by Streptomyces alboniger (Baker & Schaub, 1953; Waller et al.,
1953) and an antibiotic, 3'-amino-3'-deoxy-adenosine, produced by
Helminthosporium species (Gerber & Lechevalier, 1962; Guarino & Kredich,
1963). Ribitol (adonitol), the reduced derivative of o-ribose, was first isolated
from Adonis vernalis (Jeanlotz & Fletcher, 1951) and subsequently was found
in the roots of Bupleurum falcatum (Wessely & Wang, 1938), in honeydews
excreted by the genus Ceroplastes (Hackman & Trikojus, 1952), in pneumo-
cocci as a component polysaccharide (Rebers & Heidelberger, 1959, 1961;
Table 1
Occurrence of o-Ribose in Nature
o-Ribose or derivative Source Reference
o-Ribose Ribonucleic acid Levene & Jacobs, 1909

Uric acid Davis et aI., 1922
Crotonoside Cherbuliez & Bernhard,
1932a,b
NAD, NADP von Euler & Vestin,
1935; Warburg et al.,
1935; von Euler &
Schlenk, 1937; von
Euler et al., 1942
FAD Ochoa & Kossiter, 1939;
Klein & Kohn, 1940
UDP-glucose Caputto et al., 1950
Coenzyme A Baddiley et al., 1953
Vitamin B12 Beaven et aI., 1949;
Brink & Folkers, 1949;
Holiday & Petrow, 1949;
Brink & Folkers, 1950;
Brink et aI., 1950;
Buchanan et af., 1950a,b
Poly (ADP-ribose) Doly & Mandel 1967
Lipopolysaccharide of Kauffmann et al., 1962
Salmonella typhimurium
Polysaccharide of Hofstad & Lygre, 1977;
Eubacterium saburreum Hoffman et af., 1977
Flavonoids of Phlebodium Gomez & Wallace, 1986
dictyocallis (Mett)
Coccolith-associated poly- Borman et af., 1987
saccharide of Emiliania
huxleyi (Lohmann)
Hay & Mohler
2-Deoxy-o-ribose Deoxyribonucleic acid Levene et al., 1930;
Chargaff et af., 1949;
Chargaff & Lipschitz, 1953
3-Deoxy-o-ribose Cordycepin Bentley et aI., 1951
3-Amino-3-deoxy-o- Puromycin Baker & Schaub, 1953;
ribose
3'-Amino-3'-deoxy- Waller et al., 1953
adenosine Gerber & Lechevalier, 1962;
Adonis vernalis Guarino & Kredich, 1963
Roots of Bupreum falcatum Jeanlotz & Fletcher, 1951
Capsular of pneumococci Wessely & Wang, 1938
Rebers & Heidelberger,
1959, 1961; Roberts et
ai., 1963
Ribitol teichoic acid of Ward, 1981
Gram-positive bacteria
Lipopolysaccharide of Gmeiner, 1977;
Proteus mirabilis Gmeiner et af., 1977
Lipopolysaccharide of Vibrio Miyano et aI., 1983
parahaemolyticus
Table l--contd.
D-Ribose or derivative Source Reference
D-Ribonic acid Oxidation product of Foster, 1944; Lockwood

D-ribose by pseudomonads & Nelson, 1946; Weimberg,
1961
D-Riburonic acid Oxidation product of D-ribose Amemura et at., 1981
by Rhizobium meliloti
Methylthioribose 5' -Methylthioadenosine Mandel & Dunham, 1912;
Suzuki et aI., 1914,
1924
Glutamyl D-ribose Brain and kidney of a patient Williams et at., 1986
5-phosphate
D-Ribose 5-phosphate Pentose phosphate pathway
D-Ribose I-phosphate Precursor of nucleotides
5-Phosphoribosyl Precursor of nucleotides
I-pyrophosphate
D-Ribose 1,5- Human erythrocyte Vanderheiden, 1970
bisphosphate
Roberts et al., 1963), and in the cell walls of Proteus mirabilis (Gmeiner, 1977;
Gmeiner et al., 1977) and Vibrio parahaemolyticus (Miyano et al., 1983) as a
component of lipopolysaccharides and of various Gram-positive bacteria as a
component of ribitol teichoic acid (Ward, 1981). It is also a component of
riboflavin (Karrer et al., 1935a; Kuhn et al., 1935a; von Euler et aI., 1935).
D-Ribonic acid was the oxidation derivative of D-ribose produced by pseudo-
monads (Foster, 1944; Lockwood & Nelson, 1946; Weimberg, 1961). Another
oxidation product, D-riburonic acid, was found in the cell wall polysaccharides
of Rhizobium meliloti (Amemura et al., 1981). 5-Methylthioribose is a com-
ponent of 5'-methylthioadenosine (Mandel & Dunham 1912; Suzuki et at.,
1914, 1924) which has an important role in the metabolism of adenosylthi-
omethionine (Schlenk & Smith, 1953). Glutamyl D-ribose 5-phosphate was
found as an abnormal storage substance in the brain and kidney of a patient
suffering from renal failure and neurologic deterioration (Williams et al.,
1986). D-Ribose 5-phosphate is an intermediate in the pentose phosphate
pathway (Wood, 1985) which plays an important role in the production of
D-ribose by the tkt mutants of Bacillus species as will be described later.
D-Ribose-l-phosphate and 5-phosphoribosyl I-pyrophosphate take part in
purine and pyrimidine nucleotide biosynthesis (Hartmann, 1970). o-Ribose
1,5-bisphosphate was found in human erythrocytes (Vanderheiden, 1970).
3 CHEMICAL AND PHYSICAL PROPERTIES OF o-RIBOSE
The chemical and physical properties of o-ribose and its derivatives have
already been described in detail by Jeanlotz & Fletcher (1951), Overend &
"O~C"
" " " 0"
" "
Fig. 2. Formula of o-ribose.
Stacey (1955) and Windholz et al. (1983). The formulae of o-ribose is shown in
Fig. 2. The chemical and physical properties of o-ribose and its derivatives are
as follows.
o-Ribose C5 H lO0 5 ; mol. wt 150·13, C 40·00%, H 6·71; 053·29%. Melting
point of crystalline o-ribose is 87°C (Levene & Jacobs, 1909). Crystals are
plates or needles. o-Ribose shows mutarotation, final [«]b - 23'7° (4,5% in
water) (Phelps et al., 1934). Soluble in water, slightly insoluble in alcohol.
Anhydroribose C5 H s0 4; m.p. 229-230°C (Bredereck et al., 1940).
«-Aniline-N-o-ribopyranoside; m.p. 125-127°C, [«m + 63·4°~ + 48·6°
(C = 1·0% in pyridine) (Berger & Lee, 1944; Berger et al., 1944).
Benzylphenylhydrazone ClsH22N204; chrysanthemum-like crystals, m.p.
128°C (Sasajima & Yoneda, 1971).
p-Bromophenylhydrazone; m.p. 166-167°C (van Ekenstein & Blanksma,
1913; Steiger, 1936)
Methyl-o-riboside; crystals from ethyl acetate, m.p. 83-84° C, [«]~ - 113·6°
(p = 3) (Windholz et al., 1983).
Phenylosazone C17H2oN'403; yellow needles from pyridine + water, m.p.
163-164°C.
The infrared absorption of o-ribose from 8 to 15 microns was measured by
Kuhn (1950). A polarographic study of o-ribose was described by Cantor &
Peniston (1940); and crystallographic properties were reported by Keenan
(1926).
4 BIOSYNTHESIS AND METABOLISM OF o-RIBOSE
As already mentioned above, o-ribose is a component of biologically impor-

tant substances such as RNA and coenzymes. After the classical elucidation of
the glycolytic pathway, the pentose phosphate pathway was established as
another important carbohydrate pathway in the early 1950s. The pentose
phosphate pathway and related metabolic pathways are shown in Fig. 3. It has
the following roles; (1) supplement of the o-ribose frame for nucleic acid
biosynthesis (Hartman, 1970); (2) the biosynthesis of o-erythrose 4-phosphate,
one of the essential precursors of aromatic amino acids and vitamins
(Greenberg, 1969); (3) the biosynthesis of C-7 and C-8 carbohydrate com-
ponents of lipopolysaccharide in the cell walls of Gram-negative bacteria
(Eidels & Osborn, 1971; Woisetschlager & Hogenauer, 1986), and (4) the
o-Glucose
o-Gluconate
1
o-Glucose 6-phosphate 1
6-Phospho-o-gluconate Ribitol teichoic acid
~~
1 .---"
o-Xylose o-Ribulose 5-phosphate o-Ribose
L-Arabinose limerase/ ~ Isom' ./
E7 ~rasc /
O-Ribose 1-PhosPhate]
r--_ _ _ _ L__~> o-Xylulose 5-phosphate o-Ribose 5-phosphate ----+ [
5-Phosphoribosyl
1-pyrophosphate
a~
0-
1 §.:
-- --"-- -- ---.- --- I
Glyceraldehyde 3-phosphate Sedoheptulose 7-phosphate
1
Transketolase
1 ~
§o
Purine nucleotide
~
Pyrimidine nucleotide ~
~
s:
o-Fructose 6-phosphate o-Erythrose 4-phosphate A ~
1 ~
Shikimic acid ~
Lipopolysaccharide
1
L-Tryptophan
L-Tyrosine
TCA cycle
L-Phenylalanine
CoO
......
Vitamin K -..J
....,
Folic acid
Fig. 3. Pentose phosphate pathway and related pathwayso
biosynthesis of ribitol, a component of ribitol teichoic acid in the cell walls of

Gram-positive bacteria (Ward, 1981).
The active investigations of two American research groups headed by
Bernard Horecker and Ebraim Racker in the early 1950s, following the
observation of the oxidation of D-glucose 6-phosphate to 6-phospho-D-
gluconate by Warburg & Christian (1936, 1937) and the identification of
D-ribose 5-phosphate as one of the oxidation products by Dickens (1938a,b),
established the pathway scheme. Thus, the biosynthetic pathway from D-
glucose to D-ribose has been elucidated. The roles of the pathway mentioned
above were established through subsequent active investigations. Recently, all
aspects of the pathway were reviewed by Wood (1985). The tkt mutants of
Bacillus species used for commercially producing D-ribose could be isolated on
the basis of the knowledge accumulated about the pentose phosphate pathway.
The catabolism of D-ribose in Escherichia coli has also been studied
extensively. It was elucidated that D-ribose is used as a carbon and energy
source through the pentose phosphate pathway (David & Wiesmeyer, 1970).
D-Ribose catabolism is initiated by transportation into the cells through two
distinct D-ribose transport systems (David & Wiesmeyer, 1970). Intracellular
D-ribose is phosphorylated by ribokinase (EC 2.7.1.15) (David & Wiesmeyer,
1970) and catabolized further through the pentose phosphate pathway. The
high-affinity D-ribose transport system consists of a binding protein (Curtis,
1974; Willis & Furlong, 1974; Galloway & Furlong, 1977) and membrane-
bound components (Iida et aI., 1984; Lopilato et ai., 1984). The D-ribose
binding protein serves not only as a component of the transport system but
also as the attractant receptor for the chemotactic response to D-ribose (Adler,
1969; Hazelbauer & Adler, 1971; Galloway & Furlong, 1977). The gene
encoding the D-ribose binding protein was recently cloned and sequenced
(Groarke et a/., 1983). The D-ribose binding protein of Salmonella typhimu-
rium has also been described (Aksamit & Koshland, 1972, 1974).
The existence of the D-ribose operon (rbs) encoding for the genes of the
D-ribose binding protein, membrane-bound components, and the ribokinase of
E. coli K-12 was first elucidated by Anderson & Cooper (1970). However, the
rbs operon in E. coli B/r was determined to be located at 2 min and expressed
constitutively (Abou-Sabe & Richman, 1973) while, in E. coli K-12, it is
located at 73·5 min (now 83 min) and expressed inducibly (Anderson &
Cooper, 1970). The duplicate rbs operon at 2 min represents a transposition of
the 83-min region to 2 min in strain E. coli B/r (Abou-Sabe et al., 1982). A
silent copy of the 83-min rbs operon is also present in E. coli B/r (Abou-Sabe
et ai., 1982). Recently, the rbs operon was investigated by Lopilato et al.
(1984).
The catabolism of D-ribose in some other bacteria has also been studied.
D-Ribose might be catabolized into the pentose phosphate pathway after being
transported and cleaved into C2 and C3 compounds by phosphoketolase (EC
4.1.2.9) in Lactobacillus species, though the initial reaction was not elucidated
(Bernstein, 1953; Heath et ai., 1958). In Rhizobium meliloti (Duncan, 1981)
and R. leguminosarum (Dilworth et al., 1986), o-ribose is metabolized into the

pentose phosphate pathway after phosphorylation by ribokinase. Pseudomon-
ads oxidized o-ribose into o-ribonic acid (Foster, 1944; Lockwood & Nelson,
1946; Weimberg, 1961). o-Ribose is converted to acetate and propionate in
Bacteroides ruminicola (Caldwell & Newman, 1986) and is converted to
o-ribulose by o-ribose isomerase (EC 5.3.1.20) of Mycobacterium smegmatis
(Izumori et aI., 1975). Recently, the ribokinase gene of Staphylococcus hyicus
subsp. hyicus was cloned (Keller et al., 1984).
In yeasts, there are two metabolic pathways; one initiated by phosphoryla-
tion of o-ribose through ribokinase (Sable, 1950, 1952) and another by
reduction into ribitol (Barnett, 1976), which is oxidized into o-ribulose and,
after phosphorylation, metabolized into the pentose phosphate pathway.
o-Ribose is reduced by aldose reductase (EC 1.1.1.21) from Pachysolen
tannophilus (Morimoto et al., 1987).
50-RIBOSE-PRODUCING MICRO-ORGANISMS
5.1 Wild-Type Micro-organisms
The first o-ribose accumulation was found in the culture medium of Penicillium
brevi-compactum (Simonart & Godin, 1951) during studies on the biosynthesis
of the components of benzene derivatives (Godin, 1953a,b). Since o-ribose
was detected by paper chromatography, the amount of accumulated o-ribose
was unknown. About 10 years later, an unidentified bacterium was reported to
accumulate both o-ribose and D-ribose 5-phosphate (Suzuki et al., 1963).
Pseudomonas reptilivola was also reported to accumulate o-ribose in a culture
medium at the rate of 0·12 mg of o-ribose per ml (Saito & Sugiyama, 1966).
All the micro-organisms used in these studies were wild-type and not much
o-ribose was accumulated. However, the amount of o-ribose produced by the
tkt mutants of Bacillus species was remarkable (Sasajima & Yoneda, 1971) and
the procedure, with subsequent improvement, has been used to commercially
produce o-ribose for riboflavin synthesis for 15 years. A detailed description of
the isolation and improvement of o-ribose-producing tkt mutants of Bacillus
species is given in the following section.
5.2 Transketolase Mutants (tkt Mutants) of Bacillus Species
5. 2.1 Isolation of tkt Mutants of Bacillus Species

Investigations on the production of the nucleotide flavor enhancers, inosine
monophosphate and guanosine monophosphate, by enzymatic degradation of
yeast RNA started in Japan in the late 1950s (Ogata, 1976). The process was
established and has been used for their commercial production ever since.
During this time and subsequently, the production of inosine monophosphate
and guanosine monophosphate or their precursors inosine and guanosine by
fermentation became a research subject in many academic and company

laboratories in Japan. Various types of mutants of purine nucleotide biosynth-
esis were isolated (Ogata et al., 1976) and have been used on the industrial
scale to produce these nucleotides. During the course of studies on improving
inosine-producing mutants of Bacillus species (Nogami et al., 1968), mutants
defective in the pentose phosphate pathway were isolated because it was
assumed that more o-ribose frame would be supplied for purine nucleotide
biosynthesis in such mutants (Sasajima et al., 1970). Contrary to expectation,
inosine productivity was not improved in such mutants. However, it was found
that a large amount of o-ribose was accumulated in the culture medium by the
tkt and o-ribulose 5-phosphate 3-epimerase (EC 5.1.3.1) mutants of Bacillus
species (Sasajima & Yoneda, 1971, 1974b).
Although the reactions catalyzed by transketolase had been elucidated, it
was not simple to isolate tkt mutants. They had never been isolated at that
time because the pentose phosphate pathway is very complicated and,
moreover, plays a role in supplying the precursor of aromatic amino acids such
as L-tryptophan, L-tyrosine and L-phenylalanine and of aromatic vitamins such
as coenzyme Q, vitamin K, and folic acid (Fig. 3). In the beginning, mutants
which could not utilize L-arabinose or o-gluconate that are metabolized
through the pentose phosphate pathway were isolated. No tkt mutant could be
found among them, though aromatic amino acids were complemented in the
culture medium used for mutant isolation. However, a o-ribulose 5-phosphate
3-epimerase mutant was isolated during these studies (Sasajima et al., 1970;
Sasajima & Yoneda, 1974b).
Next, attempts were made to first isolate mutants requiring shikimic acid,
which is an intermediate of aromatic amino acids and vitamins (Fig. 3), and to
secondly select non-utilizers of o-gluconate as a carbon source among them.
Ten shikimic acid-requiring mutants were thus isolated and two of them were
found not to utilize o-gluconate as a carbon source (Sasajima et al., 1970).
These mutant strains were proved to be transketolase-deficient mutants by
enzymatic determinations (Sasajima & Yoneda, 1974a,b). The other shikimic
acid-requiring mutants were assumed to be those deficient in enzymes of
aromatic biosynthesis before shikimic acid. The ratio of 2 to 10 coincided
exactly with that anticipated. These tkt mutants accumulated about 40 mg of
o-ribose per ml in the culture medium (Sasajima & Yoneda, 1971). The
o-ribulose 5-phosphate 3-epimerase mutant produced a large amount of both
o-ribose and o-ribulose at the same time in a two to one ratio.
Josephson & Fraenkel (1969) independently isolated tkt mutants of
Escherichia coli, which did not accumulate any o-ribose (Fraenkel, D.G.,
1973, pers. comm.). Eidels & Osborn (1971) also isolated tkt mutants of
Salmonella typhimurium to elucidate the synthetic pathway of L-glycero-o-
manno-heptose, a component of the lipopolysaccharide in the outer mem-
brane. However, it was not described whether o-ribose was produced by the
tkt mutants or not.
The tkt and o-ribulose 5-phosphate 3-epimerase mutants could not utilize
o-gluconate, o-xylose, and L-arabinose which are catabolized through the

pentose phosphate pathway (Sasajima & Yoneda, 1974b; Sasajima et al.,
1977). The tkt mutants among them, moreover, required aromatic amino acids
such as L-tryptophan, L-tyrosine and L-phenylalanine but, unexpectedly, no
aromatic vitamins for their growth (Sasajima & Yoneda, 1974b; Sasajima et
al., 1977). Aromatic vitamins might be synthesized from these complemented
aromatic amino acids.
As the revertants derived from the tkt mutants did not accumulate o-ribose,
it was concluded that their o-ribose accumulation depended on transketolase
mutations (Sasajima & Yoneda, 1971). The phenomenon was the first example
of the production of carbohydrates by carbohydrate metabolism mutant
strains.
The investigation on the isolation of the tkt mutants of other bacterial strains
such as Bacillus subtilis, B. pumilus, Brevibacterium thiogenitalis, B.
ammoniagenes, Arthrobacter globiformis, Aerobacter aerogenes and
Micrococcus denitrificans revealed that the tkt mutants of only Bacillus species
among them could accumulate o-ribose (Sasajima et aI., 1985b).
A difference in the productivity of pentoses was observed between strains of
Bacillus species. For example, the tkt mutants of a strain of Bacillus subtilis
IFO 3026 accumulated both o-ribose and o-ribulose at the same time (Sasajima
et aI., 1972; Sasajima, 1976; Sasajima & Yoneda, 1984; Sasajima et aI., 1985b).
The tkt mutants of another strain of B. subtilis IFO 12114 accumulated only
o-ribose (Sasajima & Yoneda, 1984). The difference in the kind of pentoses
accumulated might be due to the strain difference in kind or substrate
specificity of the phosphatases which would hydrolize intracellulariy accumu-
lated o-ribose 5-phosphate and o-ribulose 5-phosphate to o-ribose and
o-ribulose during excretion.
Transketolase activity is functional when thiaminepyrophosphate and Mg2+
ion are coexistent (Wood, 1985). It was, therefore, considered that thiamine-
requiring mutants would also accumulate o-ribose. This was shown to be so by
isolating the thiamine-requiring mutants of Bacillus subtilis IFO 12114 but the
amount of o-ribose accumulated was much less than that in the case of the tkt
mutants (Sasajima & Yoneda, 1984; Sasajima et al., 1985b). This is probably
because it is essential to add a small amount of thiamine to the culture medium
for enzymes other than trans keto lase which require it to function. Thus, it is
impossible to make transketolase activity complete!y zero by this mutation,
and consequently they produce a smaller amount of o-ribose than do the tkt
mutants.
5.2.2 Improvement of rrribose-producing tkt mutants

The original tkt mutants accumulated o-ribose at the rate of about 40 mg/ml
(Sasajima & Yoneda, 1971). Improvement of these mutants by subsequent
mutations resulted in isolating high-level o-ribose-producing mutants which
produced about 70 mg of o-ribose per ml in the culture medium (Sasajima et
al., 1972, 1985b; Sasajima, 1976; Sasajima & Yoneda, 1984). Two mutation
steps at which the yield of o-ribose production was remarkably improved were
the following; (1) a mutation of the regulation of D-glucose dehydrogenase
(EC 1.1.1.47) synthesis (Yokota et al., 1979; Yokota & Sasajima, 1981) and
(2) an asporogenous mutation (Sasajima et al., 1972, 1985b; Sasajima, 1976;
Sasajima & Yoneda, 1984)
D-Glucose dehydrogenase in Bacillus species, is known to be synthesized at
stage III of sporulation in parallel with morphological changes during spore
formation (Warren, 1968), exists only in spores (Fujita et al., 1977) and has the
role of supplying energy during germination (Otani et al., 1986). The initiation
of sporulation is also known to occur only after the carbon source is depleted.
In the improved mutant strain, the enzyme was found in vegetative cells while
no morphological changes concerning sporulation occurred (Yokota et al.,
1979; Yokota & Sasajima, 1981). It was presumed that o-glucose was
converted to o-ribose in the mutant strain through two metabolic pathways,
the non-phosphorylated pathway (from o-glucose to the pentose phosphates
via D-gluconate) and the phosphorylated pathway (the oxidative branch of the
pentose phosphate pathway) (Fig. 4). The gluconate pathway, probably,
contributed to the high D-ribose productivity of the improved strain. However,
there was also the possibility of o-gluconate as well as o-ribose accumulation.
The ratio of o-ribose and o-gluconate production depended on the partial
pressure of oxygen during fermentations, i.e. oxygen deficiency led to
D-gluconate rather than o-ribose accumulation (Sasajima & Yoneda, 1984).
D-Ribose accumulation by the mutant strain was about 60 mg/ml (Sasajima et
al., 1972, 1985b; Sasajima, 1976; Sasajima & Yoneda, 1984).
The second-step asporogenous mutation improved the capability of o-ribose
production up to 70 mg/ml (Sasajima et al., 1972, 1985b; Sasajima, 1976;
Sasajima & Yoneda, 1984). In this case, the mechanism for the improvement
was not clear. It might be a mutation by which carbohydrate metabolism is
changed in the direction of higher-level o-ribose accumulation. The high-yield
D-Glucose - - - - - - - o-Gluconate
1
o-Glucose-6-phosphate -
1
6-Phospho-o-gluconate
1
0- Ribulose-5-phosphate
1
0- Ribose-5-phosphate
1
D-Ribose
Fig. 4. Pathways of o-ribose formation from o-glucose in high-yield mutant strains.
mutant strain has been used for 15 years to produce o-ribose for riboflavin
synthesis on an industrial scale.
5.2.3 Pleiotropic properties of D-ribose-producing tkt mutants

As already described, the tkt mutants not only could never utilize carbon
sources which are catabolized through the pentose phosphate pathway but also
required complementation of aromatic amino acids in the culture medium for
their growth. Moreover, it was revealed that the tkt mutants also had another
interesting property, i. e. functions concerning the cell surface changed
pleiotropically. The pleiotropic change is described below.
The first unexpected phenomenon was that the tkt mutants could not utilize
o-glucose as a sole carbon source (Sasajima & Yoneda, 1974b; Sasajima et at.,
1977) though o-glucose is the best carbon source for o-ribose production
(Sasajima & Yoneda, 1984; Sasajima et at., 1985b). The mutant strain seemed
preferentially to utilize other substances in the medium as a carbon and energy
source for growth, and later to become able to metabolize o-glucose for
o-ribose production. Investigation on the phenomenon revealed that the
phosphoenolpyruvate-dependent phosphotransferase system was defective
(Sasajima & Kumada, 1979); especially, the transport function of the Enz n glc
(phosphohistidinoprotein-hexose phosphotransferase; EC 2.7.1.69) of the
PEP-dependent o-glucose phosphotransferase system (Roseman, 1969; Postma
& Roseman, 1976; Saier, 1977; Dills et at., 1980; Postma & Lengeler, 1985),
was defective in the tkt mutant.
Another biochemical change was found in the catabolite repression (Maga-
sanik, 1961). The utilization of o-mannitol, o-fructose and glycerol was
inhibited by o-glucose while utilization of sorbitol was not (Sasajima &
Kumada, 1981a). The synthesis of the enzymes of o-mannitol catabolism, such
as o-mannitol-1-phosphate dehydrogenase (EC 1.1.1.17), and of the 0-
mannitol transport system was found to be hypersensitive to o-glucose
repression. Meanwhile, the synthesis of enzymes of sorbitol catabolism, such
as sorbitol dehydrogenase, and of the sorbitol transport system was resistant to
o-glucose repression. D-Gluconate, o-xylose, and L-arabinose were also found
to repress the synthesis of the enzymes of D-mannitol catabolism. These
changes in the regulation of enzyme synthesis might be caused by a defect in
the membrane.
Subsequent investigations on various functions concerning the cell surface
revealed; (1) the tkt mutant formed chains of cells during the exponential
growth phase; (2) the sensitivity to bacteriophages SPlO and SP01 was altered;
(3) the cell walls of the mutant strain autolyzed during incubation more slowly
than those of the parental strain, and (4) the sporulation frequency of the
mutant strain remarkably decreased (Sasajima & Kumada, 1981b, 1983a). The
mutant strain was non-motile because of a deficiency in flagellation (Sasajima
& Kumada, 1983b). The isolation and characterization of a true revertant
(transketolase reversion) confirmed that these pleiotropic properties were
generated by a single mutation in transketolase (Sasajima & Kumada, 1979,

1983a,b).
The investigation on chemical changes in the membrane composition
revealed that the SDS-polyacrylamide gel electrophoresis pattern of the
membrane proteins and the thin layer chromatography pattern of the
membrane lipids were different in the parental and mutant strains (Sasajima et
al., 1985a).
The tkt mutants of Gram-negative bacteria such as Escherichia coli and
Salmonella typhimurium could not synthesize L-glycero-o-manno-heptose
(Eidels & Osborn, 1971), which is a component of the outer membrane
lipopolysaccharides and essential for a functional cell surface (Ames et al.,
1974; Koplow & Goldfine, 1974; Bayer et al., 1975; Irvin et al., 1975; Smit et
al., 1975; van Alphen et al., 1976). It is of interest that the tkt mutants of both
Gram-negative and Gram-positive bacteria are defective in cell surface
functions, though the mechanisms were probably different. It can be said that
transketolase plays an important role not only in carbohydrate metabolism and
aromatic biosynthesis but also in the cell surface functions of bacteria.
5. 2.4 Production of l-deoxy-ketoses by tkt mutants of Bacillus pumilus
During studies on the relation between o-ribose productivity and transketolase
mutation in various bacteria, it was found that the tkt mutants of Bacillus
pumilus produced a new monosaccharide, 1-deoxy-o-altro-heptulose (Yokota
et al., 1978). Further investigations revealed that 1-deoxy-o-altro-heptulose
7-phosphate was synthesized from o-ribose-5-phosphate and oL-acetoin
(Yokota & Sasajima, 1983). The accumulation of o-ribose 5-phosphate in the
cells of the tkt mutant strains presumably stimulated formation of 1-deoxy-o-
altro-heptulose, which might accumulate in the culture medium after dephos-
phorylation during excretion (Yokota & Sasajima, 1983). Subsequent inves-
tigations revealed that cell-free extracts of various micro-organisms catalyzed
the formation of 1-deoxy-o-fructose, 1-deoxy-o-sorbose, 1-deoxy-o-tagatose,
1-deoxy-o-threo-pentulose, 1-deoxY-L-threo-pentulose and 1-deoxyerythrulose
(Yokota & Sasajima, 1984a, b). It is of much interest that, among them,
1-deoxy-o-threo-pentulose was described as a precursor of the thiazole ring of
thiamine (Therisod et al., 1981). This substance was also described as being
produced by Streptomyces hygroscopicus (Hoeksema & Baczynskyj, 1976;
Slechta & Johnson, 1976). It was elucidated that the formation of 1-deoxy-
ketoses was catalyzed by pyruvate dehydrogenase (lipoamide) (EC 1.2.4.1)
and acetoin dehydrogenase (Yokota & Sasajima, 1986). It was also found that
another similar enzyme, 2-ketoglutarate dehydrogenase (EC 1.2.4.2) produced
2,3-dideoxy-hex-4-ulosonic acid (Yokota & Sasajima, 1985).
6 FERMENTATION
6.1 Inoculation
In the case of Penicillium brevi-compactum (Simonart & Godin, 1951), an
unidentified bacterium (Suzuki et al., 1963), and Pseudomonas reptilivora
Table 2
Inoculum Medium of Bacillus Species
Constituent Concentration (%)

Soluble starch 2·0
Corn steep liquor 2·0
K2 HP0 4 0·3
KH2 P04 0·1
Adenosine 0·01
Guanosine 0·02
(Saito & Sugiyama, 1966), the cells grown on bouillon agar slants were directly
inoculated into the fermentation medium. In the case of the tkt mutants of
Bacillus species (Sasajima & Yoneda, 1971), the inoculum culture incubated at
37°C was used. The inoculum medium composition is shown in Table 2.
Adenosine and guanosine can be excluded from the medium in the case of
high-producing mutant strains whose requirements were compensated by
further reversion mutations.
6.2 Fermentation Media
The composition of the fermentation media used for o-ribose production are
shown in Tables 3, 4, 5 and 6 (Simonart & Godin, 1951; Suzuki et ai., 1963;
Saito & Sugiyama, 1966; Sasajima and Yoneda, 1971).
Suzuki et ai. (1963) described that the Mn2+ ion stimulated o-ribose
accumulation while o-ribose 5-phosphate was accumulated more in the absence
of Mn2+ ion in the case of an unidentified bacterium. Fe2+ and Zn 2+ ions are
also effective in stimulating o-ribose accumulation.
In the case of Pseudomonas reptilivora, o-ribose accumulation from various
carbon sources other than o-glucose was observed (Saito & Sugiyama, 1966).
o-Mannose, o-fructose and o-gluconic acid were found to be as good substrates
as o-glucose.
Table 3
Fermentation Medium of Penicillium brevi-
compactum

o-Glucose 5·0
NaN0 3 0·2
KCI 0·05
KH2 P04 0·1
MgS047H20 0·05
FeS047H20 0·002
Table 4
Fermentation Medium of an Unidentified
Bacterium
D-Glucose 10·0
KHZP04 1·0
K zHP04 1·0
MgS0 47HzO 1·0
CaClz2H2 0 0·01
Urea 0·6
Yeast extract 1·0
In the case of Bacillus species, it was also found that various carbon sources
were useful for D-ribose accumulation as shown in Table 7 (Sasajima &
Yoneda, 1971). D-Glucose, D-mannose, sorbitol, D-mannitol, maltose, and
lactose were good substrates. Various kinds of natural nitrogen sources as well
as dried yeast are useful (Table 8) (Sasajima & Yoneda, 1971). Corn steep
liquor-based medium was used for a large-scale production (Asai et ai., 1978).
It was necessary to complement aromatic amino acids to the medium in the
case where corn steep liquor was used as nitrogen source (Asai et ai., 1978).
Table 5
Fermentation Medium of Pseudomonas
reptilivora

D-Glucose 5·0
Urea 1·0
MgS04 0·05
KH2 P0 4 0·1
Sodium citrate 0·3
Corn steep liquor 0·2
Peptone 0·1
Yeast extract 0·05
Table 6
Fermentation Medium of Bacillus Species
D-Glucose 12·5; 15·0
(NH4)2S04 1·5
CaHP04 0·5
Ca3 (P04 )z 0·5
CaC03 1·0
Dried yeast 2·5
(pH 7·0)
Table 7
D-Ribose Formation from Various Carbon Sources
Carbon source D-Ribose formed (mg/ml)

Strain Shi5" Strain Shi7"
D-Glucose 23·7 20·0
D-Mannose 17·3 18·1
Sorbitol 28·0 7·7
D-Mannitol 25·8 6·1
Maltose 22·4 18·6
Lactose 12·3 14·2
Glycerol 9·6 3·4
Dextrin 9·7 9·7
Soluble starch 11·4 7·0
"Strains SHi5 and Shi7 are tkt mutants of a Bacillus
species (Sasajima & Yoneda, 1971).
Table 8
Efficiency of Various Nitrogen Sources for D-Ribose
Formation
Nitrogen source D-Ribose formed (mg/ml)

1·5% 2·5%
Dried yeast 24·4 23·7

Pharmamedia 31·1 23·3
Meat extract 19·4 30·2
Com steep liquor 23·2 9·3
Yeast extract 30·1 25·3
Peptone 17·6 15·6
Cotton seed meal 20·8 21·6
Cotton seed flour 12·1 26·5
Com gluten meal 22·6 15·4
Soy bean meal 25·1 21·4
Strain S27, a transformant from strain Shi7 which com-
pensated for both adenine and xanthine requirements
(Sasajima & Yoneda, 1971). D-Glucose was used as the
carbon source.
6.3 Fennentation Course
In the case of Bacillus species tkt mutants, D-ribose accumulation started at 1

or 2 days in proportion to D-glucose assimilation and ceased at 4 days
(Sasajima & Yoneda, 1971). The cell population reached about 1010 cells/ml in
2 days. Thus, D-ribose was accumulated during the stationary phase. This
implied that only the aged cells excreted D-ribose into the medium.
A large-scale fermentation course using a high-level producing strain is
7
pH 6 pH
5
"'C
C
..,~
"'C ;f. 10
QI ~
~ QI
-
::> '"
0
E v D-Ribose
::>2
VC)
:;: I
QlO
.8"iii
.- ::>
5
a::"'C
I .;;;
0::'
L -_ _ _ _L...'-_ .. _........--.----1r-
o 50 100
Time (hr)
Fig. 5. Time course of n-ribose fermentation.
shown in Fig. 5 (Sasajima et al., 1972, 1985b; Sasajima, 1976; Sasajima &
Yoneda, 1984).
As already described in the section on 'Producing micro-organisms', the
improved strains have the ability to produce n-gluconate as well as n-ribose.
n-Ribose is made from n-glucose through two pathways: one is a non-
phosphorylated pathway via n-gluconate and the other is a phosphorylated
pathway via n-glucose 6-phosphate (Fig. 4). Both n-gluconate and n-glucose
6-phosphate are converted to 6-phospho-n-gluconate, the common intermedi-
ate of both pathways, and finally converted to n-ribose.
Appropriate culture conditions are required for the smooth conversion of
n-gluconate to 6-phospho-n-gluconate. Otherwise, n-gluconate accumulates in
the culture medium resulting in a reduced production of n-ribose (Asai et al.,
1978; Sasajima & Yoneda, 1984). Effective factors are the pH of media, an
oxygen concentration in the medium enough for three oxidative reactions in
the pathway from n-glucose to n-ribose (Fig. 4) (Sasajima & Yoneda, 1984),
and the temperature of the culture (Asai et al., 1978). The maximum yield of
n-ribose was noted at 36·6°C when exponential growth cells were used as the
inoculum (Asai et al., 1978).
7 n-RIBOSE RECOVERY AND PURIFICATION

In the course of preparing n-ribose by chemical or enzymatic hydrolysis of
RNA, n-ribose was first converted to the p-bromophenylhydrazone (van
Ekenstein & Blanksma, 1913; Steiger, 1936) or the arylamide riboside (Berger
& Lee, 1944; Berger et al., 1944). After subsequent purification, o-ribose was
recovered by hydrolysis (Jeanlotz & Fletcher, 1951; Overend & Stacey, 1955).
In the case of preparing o-ribose by enzymatic hydrolysis of 5-amino-4-
imidazole-carboxamide-riboside with a nucleosidase, o-ribose was separated
and purified from the other product, 5-amino-4-imidazole-carboxamide, by
column chromatography with ion-exchange resin Dowex 50 (H+) (Sano et al.,
1977b). Finally, o-ribose was obtained as the lyophilized form.
Saito & Sugiyama (1966) purified o-ribose from the culture medium of
Pseudomonas reptilivora by first separating microbial cells by centrifugation
and subsequent column chromatographies with chromatopile and strong-base
anion resin (borate form). Finally, they obtained o-ribose in crystalline form
from the syrup.
Sasajima & Yoneda (1971) purified and isolated o-ribose from the culture
medium of Bacillus species tkt mutant by removing microbial cells, column
chromatography with carbon, yeast treatment to assimilate the residual
substrate o-glucose and excluding metal and anionic ions with ion exchange
resins, amberlite IR120 and amberlite IRA400. o-Ribose was crystallized by
adding ethanol to the syrupy solution.
Hough et al. (1948, 1949) described the separation of o-ribose from
coexisting sugars by partition chromatography with a powdered cellulose
column.
8 ASSAY METHODS OF o-RIBOSE
o-Ribose can be determined by reagents for pentoses, e.g. orcinol reagent

(Bial, 1902, 1903; Mejbaum, 1939; Miller et al., 1951; Dische, 1962b), carbazol
reagent (Dische, 1962a) and phloroglucinol reagent (Dische, 1962b ). By
orcinol reagent o-ribose in the culture medium is overestimated because of
coexisting substances in the culture medium (Sasajima & Yoneda, 1971).
o-Ribose can be also assayed by densitometry after paper partition chromatog-
raphy (Sasajima & Yoneda, 1971), and can be determined by gas-liquid
chromatography (Laine & Sweeley, 1973; Petersson, 1974).
The preparative method of o-ribose by chemical synthesis was first described

by van Ekenstein & Blanksma (1913). In 1936, Steiger repeated this process
(Fig. 6). The starting material was prepared from calcium o-gluconate
according to the method of Hockett & Hudson (1934). The process was later
modified by Berezovskii and Rodionova (1953).
Another method of o-ribose synthesis was described by Gehrke & Aichner
(1927). This process was improved by Austin & Humoller (1934) in L-ribose
CHO COOH COOH

HOtH HOtH H60H
I I. boiling aqueous I
HCOH electrolyticaloxidation; HCOH _..<:.pyn<.:;"=di::.:.:ne,--+) HCO H __
HtOH HtOH HtOH

tH2 0H ~H20H 6H 20H
o-arabinose o-arabonic acid o-ribonic acid
CHO
I
sodium
HCOH
amalgam .. I
HCOH
I
HCOH
I
CH20H
o-ribono-y-Iactone o-ribose
Fig. 6. Chemical synthesis of D-ribose according to the method of van Ekenstein &
B1anksma (1913).
Hr[l
--
Zn dust
CH
I
acetic aCid) HCOAc 0
H10
CH
AC
2
I
o-arabinose triacetyl-J3-o- diacetyl-o-arabinal
arabino-pyranosyl
bromide
Hr[l
CH
I H 0
HCO
CHO
I
HCOH
I
...:.pe_r_be_nz_oi_c_ac_id+)
HCOR
I
H10H
CH 2
I
HCOH
I
CH 20H
o-arabinal o-ribose
Fig. 7. Chemical synthesis of D-ribose according to the method of Gehrke & Aichner
(1927).
CH2 0H
HtOH
HOtH
reduction I
HC-O mel"period"le)
II ""
HCOH/CHC6H s
.
H 2 C-O
4,6-benzylidene-o-glucose 4,6-benzylidene-o-glucitol 2,4-benzylidene-o-erythrose
3,5-benzylidene-l-deoxy-l- I-nitro-l-deoxy- D-ribose

nitro-o-ribitol o-ribitol
Fig. 8. Chemical synthesis of D-ribose according to the method of Sowden (1950).
H 2 C-O" /CH 3 H 2 C-O, /,CH 3

I I "CH .
-
CH
HC-O/' "---CH 3 HC-O/' "CH 3
I I .
HOCH - CH 3S0 2 0CH
I I
HCOH HCOH
I I
HC-O....... y,,/CH 3 HC-O, £H3
I /CH I "Cli .
H 2 C-O "---CH 3 H 2 C-O/ "CH 3
1,2: 5,6-diisopropylidene-o-glucose 3-0-mesyl-l,2: 5,6-diisopropylidene-o-glucose
CHO CHO CHO
I I I
-
HCOH CH 3S0 2 0CH HCOH
I . I I
CH 3S02 0CH HCOH - HCOH
. I I J
HCOH HCOCHO HCOH
I I I
HCOH CH 20H CH 2 0H
I
CH 20H
3-0-mesyl-o-glucose 2-0-mesyl-4-0-formyl-D-arabinose o-ribose
Fig. 9. Chemical synthesis of D-ribose according to the method of Smith (1955).

preparation and was used to prepare o-ribose for the chemical synthesis of
riboflavin (Karrer et al., 1935b; Kuhn et al., 1935b). The process includes
arabinal as an intermediate as shown in Fig. 7.
The formation of o-ribose from o-glucose was described by Sowden (1950)
and Smith (1955) as shown in Figs 8 and 9.
Stroh et al. (1964) compared the above four methods and concluded that the
best yield was obtained by the method of Smith (1955). The overall yield was
24%.
10 APPLICATION AND ECONOMY
o-Ribose has long been prepared for the chemical synthesis of riboflavin from
o-glucose by one of the chemical processes described above. However, a
microbial production process was developed in the early 1970s (Sasajima et al.,
1972, 1985b; Sasajima, 1976; Sasajima & Yoneda, 1984). The procedure, using
the tkt mutant of Bacillus species is economical and prevents environmental
pollution by avoiding the use of mercury, which is essential for the electrolytic
reduction required in the chemical process (Harris et al., 1972). The industrial
microbial production of o-ribose for the chemical synthesis of riboflavin started
in 1973; it has given substantial results for about 15 years. Some companies in
the world have been using this process to produce o-ribose for the synthesis of
riboflavin, which is used not only for pharmaceuticals but also for animal feed
additives worldwide in large quantities.
o-Ribose itself has been also used to treat some cases of heart diseases. It
was suggested that administered o-ribose bypasses the pentose phosphate
pathway and increases the size of the phosphoribosylpyrophosphate pool
resulting in stimulation of nucleotide synthesis, maintenance of adenine
nucleotide levels, and protection of the myocardium in various heart diseases
(Zimmer et al., 1984). Exercise-induced muscle pain and stiffness due to
myoadenylate deaminase deficiency was also successfully treated with o-ribose
(ZOllner et al., 1986).
D-Ribose is a natural component of biologically important substances such as
nucleic acids and coenzymes. It has been also found in many nucleoside
antibiotics. Nucleoside analogs have been developed as antiviral agents. It is
hoped that o-ribose can be used in the chemical synthesis of drugs to treat viral
and bacterial infectious diseases in the future.
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Chapter 12
PANTOTHENIC ACID (VITAMIN 8 5 ), COENZYME A AND

RELATED COMPOUNDS
S. SHIMIZU & H. YAMADA
1 INTRODUCTION
Pantothenic acid (R- or D-( + )-N-(2,4-dihydroxy-3,3-dimethyl-1-oxobutyl)-~

alanine; for chemical formula, see Table 1) was first isolated in the 1930s from
liver and found to be an essential growth factor for yeasts (Williams and
co-workers, 1943). During this same period, it was also identified independ-
ently with the chick anti-dermatitis factor, the filtrate factor, the chick
anti-pellagra factor and an essential growth factor for lactic acid bacteria. Later
these activities were shown to be identical with pantothenic acid. Since the
factor could be obtained from a variety of plants and animal tissues, it was
designated pantothenic acid, meaning 'from everywhere' by Williams. The
compound is also referred to as vitamin B 5 • It was independently synthesized
by two groups in 1940.
Pantothenic acid occurs in all types of living organisms in a free form and in
conjugated forms such as coenzyme A, pantetheine (Lactobacillus bulgaricus
factor) and 4'-phosphopantetheine (Acetobacter suboxydans factor) (see Table
1). The coenzyme form of the vitamin is coenzyme A. It was discovered as an
essential cofactor for the acetylation of sulfonamide in the liver and the
acetylation of choline in the brain by Lipmann and co-workers (1953). Since
that time, it has been identified with 'active acetate' and has been found to be
essential for a variety of biochemical transacylation reactions. 4'-
Phosphopantetheine is also a coenzyme form of pantothenic acid. It functions
as a prosthetic group of the acyl carrier protein of fatty acid synthetase, citrate
cleaving enzyme and enzymes involved in the synthesis of peptide antibiotics.
These early studies have been reviewed by several authors (Williams, 1943;
Lipmann, 1953; Wagner & Folkers, 1964; Vagelos 1973; Vandamme, 1981;
Kleinkauf & von Dohren, 1983).
2 CHEMISTRY
Pantothenic acid can be obtained as a colorless, viscous oil by drying under

high vacuum in P 2 0 5 . It is an acid and has a marked tendency to absorb water
199
Table 1
Pantothenic Acid and its Naturally-Occurring Derivatives
o-Pantothenic acid Unstable, viscous oil. Extremely hydroscopic, easily decomposed by

acids, bases, heat. Soluble in water, ethyl acetate, dioxane, glacial
CHlOH acetic acid; moderately soluble in ether, amyl alcohol; insoluble in
I I
HOCH2C-CHCONH(CH2)2COOH benzene, chloroform. Solutions are stable between pH 5 and 7.
I [aHi' + 37·5°. Stiller et al. (1940).
CHl
4H17NOs MW: 219·23
Calcium o-pantothenate White needles. Moderately hygroscopic. Soluble in water, glycerol;

slightly soluble in alcohol, acetone; insoluble in ether, benzene,
CHlOH ), chloroform. Decomposed by bases. Solutions are stable between pH 5
( HOCH2{-lHCONH(CH2hCOO Ca and 7. mp 195-196°C (dec); [aJ~ + 28·2° (c = 5).
CH l 2
CI6H32CaN20to MW: 476·53
Sodium o-pantothenate I White, hygroscopic crystals. Decomposed by acids and bases. Solutions
are stable between pH 5 and 7. mp 122-124°C; [aJ~ + 27·1° (c = 2).
CHlOH For solubility, see calcium pantothenate
I I
HOCH2C-CHCONH(CH2)2COONa
I
CH 3
4H 16NaN0 5 MW: 241·21
Soluble in water; insoluble in ethanol. Unstable to bases. Free acid is
4' -Phosphopantothenic acid (Ba salt) unstable. [a J~ + 9·0° (c = 3· 3). King & Strong (1951); Okada et al.
o CH 3 0H (1967).
/I I I
HO-P-OCH2C-CHCONH(CH2)2COOH
I I
OH CH 3
4H 16NO HP MW: 313·27
Pantothenoyl-L-cysteine (Sa salt) Soluble in water, methanol, moderately soluble in ethanol; insoluble in
ether. Unstable to acids and bases. [ll']D - 14° (c = 2). Ohta et al.
CH,OH COOH
II I (1967).
HOCH2C-CHCONH(CH2hCONHCHCH2SH
I
CH,
C12H22N206S MW: 322·38
4' -Phosphopantothenoyl-L-cysteine (Sa salt) Soluble in water; slightly soluble in alcohol. Unstable to acids and bases.
o CH,OH COOH Easily oxidized in air. [ll']~ 0° (c = 2). Baddiley & Mathias (1954);
II I .I I Nagase (1967).
HO-r-OCH2T-CHCONH(CH2hCONHCHCH2SH
OH CH,
C12H2,N209PS MW: 416·42
Pantetheine Syrup or glass. Soluble in water; slightly soluble in alcohol, insoluble in

ether, benzene, chloroform, ethyl acetate. Unstable to acids and
CH 3 0H
bases. Easily oxidized in air. [ll']~ + 12·2° (c = 3·45). Lactobacillus
HOCHi-~HCONH(CH2hCONH(CH2hSH bulgaricus factor. Shimizu et al. (1965).
I
CH 3
C 1H 22N 20 4S MW: 278·37
Pantethine Disulfide form of pantetheine. Glassy, colorless to light yellow

substance. Unstable to acids. [ll']~ + 17·1° (c = 3·2). For solubility and
CH30H ) ref., see pantetheine.
( HOCH2?-JHCONH(CH2hCONH(CH2hS
CH 3 2
C22H42N405S2 MW: 554·72
4' -Phosphopantetheine (Sa salt) Soluble in water; slightly soluble in ethanol; insoluble in ether. Unstable
o CH 30H to acids and bases. Easily oxidized in air. [ll']~ + 13·3° (c = 2·25);
II I .I Oxidized form, [ll']~ + 12·2°. Acetobacter suboxidans factor. Baddiley
HO-P-OCH 2C-CHCONH(CH2hCONH(CH 2hSH & Thain (1953); Moffatt & Khorana (1961); Nagase (1967).
I I
OH CH 3
CIIH23N207PS MW: 358·35
(continued)
Table.l-(contd.)
Dephospho-coenzyme A (Li salt) Soluble in water, methanol; insoluble in acetone. Unstable to acids and
bases. For ref., see coenzyme A.
o CH,OH
" I . I
HO-P-OCH 2C-CHCONH(CH 2hCONH(CH 2hSH
I I
9 CH, i:NH2
HO-f=0 <N I N
~tJ N
J
OH OH
C21H'5N7012P2S MW: 687·56

Coenzyme A Soluble in water; insoluble in ethanol, ether, acetone. Decomposed to
o CH,OH panthetheine-2' ,4'-cyclic phosphate and 3' ,5'-ADP in 1 N NaOH
II I .I (100°C, 2 min). Decomposed to pantetheine-4'-phosphate and adenine
HO-i-OCH'r-CHCONH(CH2hCONH(CH2hSH in 1 N HCI (1()()OC, 5 min). Easily oxidized in air. Moffatt & Khorana
(1961); Shimizu (1970); Shimizu et al. (1979a).
oI CH .1 NH
. 2
HO-P=O
I
O-CH 2
~IH36N7016P,S
MW: 767·55
o OH
I
HO-P~
HO/~
4' -Phosphopantetheine-S-sulfonate (Ca salt) Soluble in water. [a]~ + 7·2° (c = 1·95). Bifidus factor. Yoshioka &
o CH,OH Tamura (1971).
II I· I
HOrOCH2r-CHCONHCH2CH2CONHCH2CH2SS0,H
OH CH,
CIIHnOlON2S2P MW: 438·41
(continued)
Table l-contd.
4' -O-(!3-glucopyranosyl)-D-pantothenic acid Soluble in water. [c:r]ii - 18· 20 (c = 1·0). Growth factor of Leuconostoc.
Amachi et at. (1971).
CH 20 H lH3?H
f{ o OCH 2-C-CHCONHCH 2CH 2COOH
OH I
HO CH2
OH
C44H2S014N MW: 791·68
OA-6129A (Na salt) Unstable to acids and bases. [c:r]ii + 11.60 (c = 1·0). Antibiotic produced
OH CH 1 by Streptomyces sp. OA-6129. Yoshioka et al. (1983).
I I'
}x£ ;/ SCH2CH2NHCOCH2CH2NHCOCH-r-CH20H
CH 3
COOH .
C2oH31N107S MW: 457·54
Vitamin B s , Coenzyme A and Related Compounds 205
!• 4 '-~tetheine •,
I : ' pantetheine •'
: : - -pantothenic aci.d---\ I
: :-pantoic acid ~ I !
:: : : i
~H <j>H CH.C;>H
0=6-o-~-oCH'-{:-CHCo-NHCH2CH2CO-NHCH2CH2SH
C~HZ~ --~-~~--f- i- S-al.anine-J,..cysteamineJ,

:---S-aletheine I
.~
i
c;> H
Ho-~ ~~~
NHz_L
Fig. 1. The structure of coenzyme A.
from the air. It breaks down into fJ-alanine and pantoic acid by alkaline
hydrolysis. The latter forms a lactone, i.e. D-( - )-pantoyl lactone, readily in
acid solution or upon heating. Acid hydrolysis of pantothenic acid gives
fJ-alanine and pantoyl lactone. Pantothenic acid is soluble in water, ethyl
acetate, dioxane, acetic acid, ether and amylalcohol, but is insoluble in
benzene and chloroform.
The structure of pantothenic acid contains a single asymmetric center, so
that it is optically active; only the natural D-( + )-isomer has vitamin activity.
The absolute configuration of the vitamin has been defined as R (Hill & Chan,
1970). The conformation of the vitamin has also been reported (Fritz & LOwe,
1962).
The calcium salt of pantothenic acid can be obtained as needle crystals from
methanol. It is moderately hygroscopic and is rather more stable to heat, air
and light than the free acid. It is soluble in water and glycerol and slightly
soluble in alcohol and acetone. Reviews by Baddiley (1955) and Wagner &
Folkers (1964) summarize early studies on the chemistry of pantothenic acid.
Table 1 lists several naturally-occurring derivatives of pantothenic acid. They
can be grouped into three types based on their chemical structures, i.e. simple
pantothenate derivatives, pantetheine derivatives in which cysteamine (or its
analogs) attaches by an amide linkage and coenzyme A derivatives in which
the pantetheine is adenosylylated (see Fig. 1).
Pantothenyl alcohol, an alcohol analog of pantothenic acid, is also a
pharmaceutically important unnatural derivative. Unnatural analogs of pan-
tothenic acid and coenzyme A were reviewed by Shimizu (1970).
3 BIOSYNTHESIS
Although pantothenic acid cannot be synthesized by animals, micro-organisms

and plants are generally able to produce it from the precursors pantoic acid
CH,COCOOH 7 ~jH
~ CH"'r-COOH
CH,COCOOH CH,-c=O
pyruvic acid a-aoetolactic acid
yH,OH
HOCH,c-CHCONHCH,cH,aJOH
tlUD-pantothenic acid
\?
H,NCNHCH,CH.a>OH
N-carl:>am:>yl-Il-alanine
- 1 3
dihydrouracil ..L uracil
malonylsemialdehyde
Fig. 2. The pathway for the biosynthesis of pantothenic acid.
and p-alanine, through catalysis of the enzyme pantothenate synthetase (EC

6.3.2.1). Animals, plants and micro-organisms can convert pantothenic acid to
4' -phosphopantetheine and coenzyme A, the metabolically active forms of the
vitamin. The pathway for the biosynthesis of pantothenic acid and coenzyme A
has been elucidated by several workers since the early 1950s and has been
reviewed (Brown & Reynolds, 1963; Plaut et ai., 1974; Abiko, 1975; Brown &
Williamson, 1982). The pathway leading to the vitamin and the coenzymes
from common precursors can be summarized as shown in Figs 2 and 3.
3.1 /I-Alanine
Three routes to p-alanine have been reported. Several micro-organisms have
been reported to form p-alanine by a--decarboxylation of L-aspartic acid (Fig.
2, reaction 1). Confirmatory evidence for this conversion was provided by
Williamson & Brown (1979), who purified (to apparent homogeneity) from
extracts of Escherichia coli an enzyme that catalyzes the a--decarboxylation of
L-aspartic acid to yield p-alanine and CO2 • They also reported that the enzyme
is missing in a mutant of E. coli that requires either p-alanine or pantothenate
as a nutritional factor, but is present in the wild-type strain and in a revertant
strain of the mutant. It has also been suggested that p-alanine is produced by
decarboxylation of N-carbamoyl-p-alanine formed from uracil on the basis of
the observation that mutants of Salmonella typhimurium lacking the ability to
degrade uracil require N-carbamoyl-p-alanine, p-alanine or pantothenate as a
nutritional factor (Fig. 2, reactions 2, 3 and 4) (West et al., 1985). p-Alanine
may also be produced by transamination of malonylsemialdehyde produced
from propionic acid (Fig. 2, reaction 5 or 6), because enzyme activity
catalyzing this conversion was detected in several micro-organisms. However,
there have been no further studies concerning this reaction.
Vitamin B 5 , Coenzyme A and Related Compounds 207
3.2 Pantoic Acid

The route to pantoic acid from pyruvate as shown in Fig. 2 has been
elucidated mainly in E. coli and Neurospora crassa. Two enzymes catalyzing
the conversion of pyruvate to a-ketoisovalerate (Fig. 2, reactions 7 and 8) in
this route are shared by the route for the biosynthesis of the branched chain
amino acids. In E. coli, two enzyme activities have been detected for the
conversion of a-ketoisovalerate to ketopantoic acid (Fig. 2, reaction 9); one is
dependent on tetrahydrofolate and the other is not. The physiological
significance of the tetrahydrofolate-independent activity seemed to be ques-
tionable because of its high Km values for formaldehyde (10 mM) and
a-ketoisovalerate (100 mM). The fact that a mutant mlssmg the
tetrahydrofolate-dependent activity requires pantothenate for growth, whereas
the tetrahydrofolate-independent activity is found in the same amounts in the
same mutant also give concrete evidence supporting the theory that the
tetrahydrofolate-dependent enzyme is the one responsible for the ketopantoate
needed for the biosynthesis of pantothenate. The tetrahydrofolate-dependent
enzyme, i.e. ketopantoate hydroxymethyltransferase (EC 2.1.2.11), has been
purified and characterized in some detail. The observation that pantoate,
pantothenate and coenzyme A are all allosteric inhibitors of this enzyme also
supports this conclusion (Powers & Snell, 1976; Teller et al., 1976).
The reduction of ketopantoic acid to D-pantoic acid (Fig. 2, reaction 10) is
catalyzed by an NADPH-dependent enzyme, ketopanoic acid reductase (EC
1.1.1.169). The enzyme activity has been detected in Saccharomyces cerevisiae
and E. coli. The same reduction is also catalyzed by a-acetohydroxyacid
isomeroreductase (EC 1.1.1.86) which is the responsible enzyme for the
conversion of a-acetolactate to a-ketoisovalerate (Fig. 2, reaction 8) (Primer-
ano & Burns, 1983). Recently, Shimizu et al. (1988a) isolated ketopantoic acid
reductase in a crystalline form from Pseudomonas maltophilia and charac-
terized it in some detail. They also demonstrated that this enzyme is due the
enzyme for D-pantoic acid formation, necessary for the biosynthesis of
pantothenic acid, due to the observation that mutants lacking this enzyme
require either D-pantoic acid or pantothenate for growth and the revertants
regain this activity.
3.3 Coenzyme A
The pathway for the biosynthesis of coenzyme A from pantothenic acid,
L-cysteine and A TP as shown in Fig. 3 was first demonstrated by Brown
(1959a,b) based on his studies with Proteus morganii and the early observa-
tions in the 1950s with pig liver and other organisms. Later, the validity of this
pathway was confirmed by Abiko and co-workers who separated and charac-
terized the enzymes involved in this pathway in rat liver (Abiko, 1975).
The first step in this pathway is the phosphorylation of pantothenic acid (Fig.
3, reaction 1) by pantothenate kinase (EC 2.7.1.33). The enzyme has been
purified and characterized from rat liver (Abiko et al., 1972) and
r---" X
AlP ~ Pantothenic acid
I
: P-Pontothenic acid Pantothenoylcysteine
.~:
: AlP_J2 +Cysteine 6l
:lSi P-Pantothenoylcysteine Pantetheine
.!;!
~,
~lP
~
-g: P-Pantetheine
1!:
,QI L
AlP» "
If': Dephospho-CoA
+-
L5
I
: AlP_
!... __________________ CoA _________ 1I
I
Fig. 3. The pathway for the biosynthesis of coenzyme A from pantothenic acid,
L-cysteine and ATP. For chemical structures of each compound, see Table l.
Abbreviation used: CoA, coenzyme A.
Brevibacterium ammoniagenes (Shimizu et al., 1973). Both enzymes receive

allosteric inhibition by coenzyme A, the end product of the pathway. Since
such feedback inhibition by coenzyme A is observed only at this step and no
other regulation mechanism has been known, this inhibition seems to be the
most important mechanism for controlling the cellular level of coenzyme A.
Pantetheine is also phosphorylated by the same enzyme to yield 4'-
phosphopantetheine (Fig. 3, reaction 7), which can be converted to coenzyme
A.
The condensation of 4' -phosphopantothenic acid with L-cysteine to yield
4' -phosphopantothenoyl-L-cysteine (Fig. 3, reaction 2) is catalyzed by 4'-
phosphopantothenoyl-L-cysteine synthetase (EC 6.3.2.5). The mammalian
enzyme requires A TP as energy for the condensation whereas the bacterial
enzyme preferably utilizes CTP (Brown, 1959a). 4'-Phosphopantothenoyl-L-
cysteine is then decarboxylated to yield 4' -phosphopantetheine (Fig. 3,
reaction 3) by 4' -phosphopantothenoyl-L-cysteine decarboxylase (EC 4.1.1.36).
The enzyme has been shown to be independent of pyridoxal 5'-phosphate.
This step is the only one which does not require A TP among the five steps in
the biosynthesis of this coenzyme.
The pyrophosphate linkage formation between 4' -phosphopantetheine and
ATP to yield 3' -dephospho-coenzyme A (Fig. 3, reaction 4) and the
phosphorylation of it (Fig. 3, reaction 5) are respectively catalyzed by
dephospho-coenzyme A pyrophosphorylase (EC 2.7.7.3) and dephospho-
coenzyme A kinase (EC 2.7.1.24). In rat liver, both enzymes are present as a
complex or a bifunctional enzyme (Suzuki et al., 1967). The reversibility of the
former reaction may be important for controlling cellular levels of coenzyme A
and 4' -phosphopantetheine.
The presence of an alternate route to yield 4' -phosphopantetheine via
pantetheine (Fig. 3, reactions 6 and 7) has been suggested in Acetobacter
Vitamin B 5 • Coenzyme A and Related Compounds 209
suboxydans, Lactobacillus helveticus and others (Brown, 1959b), but confir-

matory evidence for this is not available.
3.4 Control Mechanisms for the Biosynthesis
The allosteric inhibition of ketopantoic acid hydroxymethyltransferase of E.

coli by o-pantoic acid, pantothenic acid or coenzyme A may be involved as a
control mechanism in pantothenate biosynthesis (Powers & Snell, 1976). On
the other hand, such inhibition was not observed in the case of ketopantoic
acid reductase of P. maltophilia (Shimizu et al., 1988a).
In the pathway to coenzyme A from pantothenic acid, the feedback
inhibition of pantothenate kinase by coenzyme A and 4'-phosphopantetheine
has been demonstrated to be involved as a control mechanism in the
biosynthesis (Abiko et al., 1972; Shimizu et al., 1973; Vallari et at., 1987).
Since this inhibition was generally observed regardless of species and the other
four steps following this reaction are not inhibited by coenzyme A or
4' -phosphopantetheine significantly, this may be one of the most important
mechanisms to control cellular levels of coenzyme A. No other mechanism
such as repression has been observed in either pantothenate or coenzyme A
biosynthesis.
Pantetheinase, which specifically degrades pantetheine to pantothenic acid
and cysteamine, may also be an important enzyme, because coenzyme A can
be degraded to pantetheine enzymatically and pantetheine can be reused as a
precursor of coenzyme A after phosphorylation by pantothenate kinase.
Cellular coenzyme A levels may be influenced by competition between
pantetheinase and pantothenate kinase towards their substrate, pantetheine
(Wittwer et al., 1983).
4 CHEMICAL AND MICROBIAL PRODUCTION METHODS
4.1 Pantothenic Add
At present, commercial production of pantothenate depends exclusively on

chemical synthesis. As outlined in Fig. 4, the conventional chemical process
involves reactions yielding racemic pantoyl lactone from isobutyraldehyde,
formaldehyde and cyanide, optical resolution of the racemic pantoyl lactone to
0-( - )-pantoyl lactone with quinine, quinidine, cinchonidine, brucine and so
on and condensation of 0-( - )-pantoyllactone with p-alanine. This is followed
by isolation as the calcium salt and drying to obtain the final product. A
problem of this chemical process apart from the use of poisonous cyanide is the
troublesome resolution of the racemic pantoyl lactone and the reracemization
of the remaining L-( + )-isomer. Therefore, most of the recent studies in this
area have been concentrated on development of an efficient method to obtain
0-( - )-pantoyllactone.
Optical ________
resolution
Fig. 4. Outline of the chemical synthesis of D-pantothenic acid.
To skip this resolution-reracemization step, several microbial or enzymatic

methods have been proposed. They roughly fall into two types based on the
starting substrate used (Yamada & Shimizu, 1988).
Recently, an efficient combined chemi-enzymatic method, which involves an
efficient one-pot synthesis of ketopantoyl lactone as a starting substrate,
followed by stereospecific reduction of it to 0-( - )-pantoyl lactone using
microbial cells as a catalyst was reported (Hata et al., 1987; Shimizu et al.,
1984a, 1987a). As shown in Fig. 5, ketopantoyl lactone is synthesized from
isobutyraldehyde, sodium methoxide, diethyl oxalate and formalin. The
reaction is performed in one step at room temperature with a yield of 81·0%.
Stereospecific reduction of ketopantoyl lactone to 0-( - )-pantoyl lactone is
carried out with washed cells of Rhodotorula minuta or Candia parapsilosis as
a catalyst and glucose as energy for the reduction. About 50 or 90 g/liter of
optically pure 0-( - )-pantoyl lactone can be produced with a molar yield of
nearly 100% by R. minuta or C. parapsilosis, respectively. The enzyme
catalyzing this conversion has been isolated as a crystalline form from C.
parapsilosis cells and characterized. It is a novel carbonyl reductase which
specifically catalyzes the reduction of conjugated polyketone compounds (Hata
et al.,1989a,b; Shimizu et al., 1988b).
Racemic pantoyllactone can also be used as a starting substrate. Shimizu et
ale (1987b) reported that Nocardia asteroides cells specifically oxidize the
L-( + )-isomer in a racemic mixture of pantoyllactone to ketopantoyllactone,
which is then converted to 0-( + )-pantoyl lactone by the reduction with C.
parapsilosis cells as described above. In these two enzymatic steps, the
coexisting 0-( - )-isomer remains without any modification (Fig. 6, reactions 1
and 2). Under suitable conditions, 72 g/liter of 0-( - )-pantoyl lactone was
obtained from 80 g/liter of oL-pantoyl lactone. Similar specific oxidation and
reduction reactions can also be carried out with a single micro-organism as
catalyst. On incubation with washed cells of Rhodococcus erythropolis,
Me Me
~ ~CHg~-~~CHO ~TOOMe
I
MeONa 4MeOH C;::OOEt
COOMe MeONa COOEt
Me MeONii
2 x )CHCHO
Me Me
T'K". [:~CHCH-~~CHO
~COCOOMe
1
~CLHLCHO ~
)CHCOCOOH +
j
Me
Meuet5Me0
'v, HCOOMe + MeOH
HCHO
'CH 0
,.fe 0
Me Me OH
Me
~~/ ___________
-t---\°o Microorganism
•• Me7---\-H
~_?O ~ -alanine • (R)-(+)-pantothenic acid
o 0
Fig. S. The reaction pathway for the chemicoenzymatic synthesis of 0-( - )-pantoyl
lactone.
D-( - )-pantoyl lactone in the reaction mixture reached 18·2 g/liter with a
molar yield of 90·5% (optical purity, 94·4% e.e.). This unique conversion
proceeds through the successive reactions as follows: (1) the enzymatic
oxidation of L-( + )-pantoyl lactone to ketopantoyl lactone (the same enzyme
as that in N. asteroides has been suggested to be the responsible enzyme for
this oxidation); (2) the rapid and spontaneous hydrolysis of ketopantoyl
lactone to ketopantoic acid, and (3) the enzymatic reduction of the ketopantoic
acid to D-pantoic acid. The enzyme catalyzing this reduction seemed to be
ketopantoic acid reductase, because R. erythropolis cells could not utilize
u: eo
OH carbonyl
00
reductase OH
0
(1)
L-PL DH (D-PL forming)
I H
(2)
carbonyl
L-PL° reductase
(L-PL forming} J(PL D-PL°
I f
Spontaneous Chemical
(3)t<PH 7) (HC1)1 (5)
Ho
OH
J(PA
MOH
reductase ,
(4)
OH OH OHOH
KPA D-PA
Fig. 6. Reactions involved in the enzymatic transformation to 0-( - )-pantoyl lactone.
Abbreviations used: L-PL, L-( + )-pantoyllactone; D-PL, 0-( - )-pantoyllactone; KPL,
ketopantoyllactone; KPA, ketopantoic acid; D-PA, o-pantoic acid.
ketopantoyl lactone as substrate, different from C. parapsilosis (see Fig. 6,

reactions 1, 3 and 4) (Shimizu et at., 1987c).
These three enzymatic methods are simple and require no reracemization
step, which is necessary for the conventional chemical resolution. A chemical
reduction of ketopantoyl lactone which uses rhodium complex as a catalyst has
also been reported to yield 0-( - )-pantoyllactone stereospecifically (Ojima et
at., 1978).
4.2 Coenzyme A
The production methods for coenzyme A roughly fall into chemical and
microbial ones. The chemical methods have been reviewed by Shimizu (1970)
and Mautner (1970). They are not practical due to their complexity.
Therefore, commercial production is carried out by microbiological methods.
Extraction of coenzyme A from yeast cells has been performed since the early
1950s. Usually, cells of baker's or brewer's yeasts, which are relatively rich in
coenzyme A, are used as the source. Later, an efficient enzymatic method
using Brevibacterium ammoniagenes cells as the catalyst was developed.
These microbial methods were reviewed by Shimizu & Yamada (1986).
A successful enzymatic method using the biosynthetic route of coenzyme A
from pantothenic acid, L-cysteine and ATP was first reported by Ogata et al.
(1970), who found that B. ammoniagenes has all five enzymes necessary for the
biosynthesis of coenzyme A in high activities. The above three substrates,
when added to a reaction mixture containing the bacterial cells, were
converted to coenzyme A with a satisfactory yield (2-3 g/liter). They also
found that the same organism can accumulate coenzyme A directly in the
culture medium on addition of pantothenic acid, L-cysteine and AMP,
adenosine or adenine in the presence of a surfactant, cetylpyridinium chloride,
and high levels of glucose (usually 10%), K2 HP0 4 and MgS0 4 ·7H2 0. Under
optimal conditions, the amount obtained was 5·5 g/liter. Most coenzyme A in
the medium was present in the disulfide form due to the vigorous shaking
during the reaction. After treatment of the culture filtrate with Duolite S-30,
charcoal and Dowex 1 (Cl-), and the reduction of the disulfide, the very pure
thiol form was obtained in a high yield. The mechanism of this coenzyme A
production has been suggested to be that shown in Fig. 7 (for details, see
Shimizu et at., 1979a,b).
In order to improve the product yield further, the regulation mechanism of
the biosynthesis was investigated. As described in the previous section, the
biosynthesis of coenzyme A in B. ammoniagenes is mainly controlled by the
feedback inhibition of pantothenate kinase by coenzyme A. This was the main
problem in the practical production, because the over-produced coenzyme A
itself stopped the biosynthesis. Two methods to abolish this feedback
inhibition have been reported.
A synthetic scheme in which the reaction is initiated by the condensation of
4' -phosphopantothenic acid and L-cysteine or the transadenosylylation of
Villlmin B s , Coenzyme A and Related Compounds 213
G~( "1TP >kids.~
~ Cysteine
Ar4dhenicacid
PRPP AMP
~ne
Aderiine CoA
Fig. 7. Reaction sequences of coenzyme A production with Brevibacterium am-
moniagenes under A TP-generating conditions. Abbreviations used: PRPP, 5-
phosphoribosyl-l-pyrophosphate; CoA, coenzyme A.
4' -phosphopantetheine was investigated, because these routes do not involve

phosphorylation of pantothenic acid or pantetheine by pantothenate kinase.
Replacement of the enzymatic phosphorylation of pantothenate or pantetheine
with chemical phosphorylation followed by the enzymatic reaction increased
the yield of coenzyme A 10-20 times. Yields from 4' -phosphopantothenic acid
and 4'-phosphopantetheine were 33 g/liter (molar yield based on ATP, 64·5%)
and 115 g/liter (100%), respectively (Shimizu et a/., 1983). This method is
applicable to coenzyme A production under the A TP-generating conditions
(see Fig. 7). 4'-Phosphopantothenic acid (25 g/liter), L-cysteine (15 g/liter),
and AMP (33 g/liter) added to the culture broth of B. ammoniagenes were
converted to coenzyme A with a yield of 23 g/liter (Shimizu & Yamada, 1984).
Another way to improve the yield is to use mutants derepressed for the
feedback inhibition or those showing elevated pantothenate kinase activity. A
mutant of B. ammoniagenes that is resistant to oxypantetheine (the cor-
Fig. 8. Time course of coenzyme A production by an oxypantetheine-resistant mutant

of Brevibacterium ammoniagenes under ATP-generating conditions. (A) Production
from pantothenic acid, L-cysteine and AMP. (B) Production from pantetheine and
AMP. For details, see Shimizu et al. (1984b). 0, Coenzyme A (CoA); . , pantothenic
acid (PaA); D, ATP; .; ADP; D., AMP; .6, pH; x, cell growth.
responding oxygen analog of pantetheine) was found to have an elevated

activity of pantothenate kinase. Under the ATP-generating conditions, the
yields of coenzyme A from pantothenic acid (3-6 g/liter), L-cysteine
(1-8 g/liter) and AMP (6 g/liter) or from pantetheine (5 g/liter) and AMP
(6g/liter) were 9·3 or U·5g/liter, respectively. These values were about
three-fold higher than those obtained with the parent strain, and 70-100% of
the added AMP was converted to coenzyme A (Shimizu etal., 1984b) (Fig. 8).
Continuous production of coenzyme A through five enzymatic steps using
gel-entrapped cells of B. ammoniagenes has also been reported (Shimizu et al.,
1979b; Yamada et al., 1980).
Continuous production of coenzyme A through five enzymatic steps using
gel-entrapped cells of B. ammoniagenes has also been reported (Shimizu et al.,
1979b; Yamada et al., 1980).
4.3 4'-Phosphopantetheine and Other Intermediates in Coenzyme A

Biosynthesis
4' -Phosphopantetheine together with other intermediates in coenzyme A

biosynthesis can be effectively synthesized by using B. ammoniagenes cells as
the catalyst and by modifying the reaction conditions (Shimizu et al., 1979a):
4' -phosphopantothenic acid on omission of L-cysteine from the reaction
mixture, 4'-phosphopantetheine on addition of CTP or CTP and GTP in place
of A TP, because pantothenic acid is phosphorylated in the presence of CTP or
GTP as well as A TP and the condensation of 4' -phosphopantetheine with
nucleoside triphosphate is specific for A TP, and 3' -dephospho-coenzyme A on
treatment of the reaction mixture containing coenzyme A with 3'-nucleotidase.
The amounts of these intermediates obtained by this method are summarized
in Table 2.
5 ASSAY METHODS
Test micro-organisms normally used for the microbiological assay of pan-

tothenic acid are Lactobacillus plantarum ATCC 8014 (L. arabinosus 17-5), L.
casei ATCC 7469 and Saccharomyces uvarum ATCC 9080 (S. carlsbergensis).
Lactobacillus plantarum is suitable for determining unconjugated pantothenate
in samples. It should be noted that pantetheine, when simultaneously present
in a molar ratio to pantothenate of more than O· 5, gives positive errors in the
determination. Saccharomyces uvarum also shows almost specific growth
response to free pantothenate, but fJ-alanine stimulates its growth. Hence, an
assay procedure employing this organism is also the one chosen for determin-
ing the pantothenic acid that occurs in natural products together with other
pantothenate forms. Lactobacillus casei responds not only to pantothenate but
also several conjugated forms of pantothenate. Lactobacillus helveticus ATCC
Table 2
Production of the Intermediates in Coenzyme A Biosynthesis by Brevibacterium ammonillgenes·
Product Substrates Nucleotides Reactions Dried Productivity (mg/ml)

involvedb cells enzyme source
Culture Immobilized
C
broth cells
4' -Phosphopantothenic acid Pantothenic acid ATP 1 3-4 1·5-2·5
4' -Phosphopantothenic acid Pantothenic acid AMP 1 4-5
4' -Phosphopantetheine 4' -Phosphopanto- CfP 2,3 3-4 1·8
thenic acid and
L-cysteine
4' -Phosphopantetheine Pantothenic acid ITP and CfP 1,2,3 2-3 0·3
and L-cysteine
4' -Phosphopantetheine Pantothenic acid GMPandCMP 1,2,3 3-4
and L-cysteine
4' -Phosphopantetheine Pantetheine ITP 7 2-3 0·9
4' -Phosphopantetheine Pantetheine GMP 7 4-5
3' -Dephosphocoenzyme A Pantothenic acid ATP 1,2,3,4,5 1-2
and L-cysteine
a For details, see Shimizu et al., 1979a,b.

b Numbers correspond to those given in Fig. 3.
C In this case, the nucleoside monophosphates added are presumed to be converted to the corresponding nucleoside triphosphates and used for
the reactions in a similar manner as shown in Fig. 7.

12046 and L. bulgaricus Bl are recommended for determination of pan-

tetheine (or pantethine), because both these organisms require more than 100
times as much pantothenic acid as pantethine to give the same response.
Treatment of samples and details of assay procedures have been described
(Bird & Thompson, 1967).
A sensitive enzymatic assay method using pantothenase has been reported
(Airas, 1986), but the enzyme is not commercially available. Chemical and
physical methods have also been reported. They are often used in determining
pantothenic acid in pharmaceutical products. They are not suitable for
determination of natural samples because of their low sensitivity.
6 USE AND ECONOMIC ASPECTS
The current world capacity of calcium pantothenate production and its demand
are presumed to be about 4000 and 3600-4000 tons/year, respectively. It is
mainly used as an additive of animal feed (about 3000 tons/year) and as a
pharmaceutical product (about 600 tons/year). Pantothenyl alcohol is used as a
source of panthothenate activity for pharmaceutical vitamin products. Pan-
tothenyl alcohol itself has no pantothenate activity; in fact, it is a competitive
growth inhibitor of several pantothenate-requiring lactic acid bacteria. How-
ever, it has been demonstrated to be quantitatively converted to pantothenic
acid in the animal body, and to be equivalent to pantothenic acid in man.
Pantethine, the disulfide of pantetheine, and coenzyme A are also used as
pharmaceutical products in several countries. They have been suggested to be
effective in reducing cholesterol level, curing fatty liver, and related diseases.
Some sulfonate derivatives of pantetheine or coenzyme A (Bifidus factors),
such as 4'-phosphopantetheine-S-sulfonate, which were originally isolated
from carrot roots have been shown to be growth factors of Bifidobacterium
(Yoshioka & Tamura, 1971). Addition of the bifidus factors to dried milk for
infants has been suggested to be useful in improving the quality of the milk. A
carbapenem antibiotic, OA-6129A, (Table 1) produced by Streptomyces sp.
OA-6129, may be an interesting example suggesting a new use of the vitamin
as building block for its synthesis (Yoshioka et al., 1983).
ACKNOWLEDGEMENT
Research from the authors laboratory was supported, in part, by a Grant in aid
of Scietific Research from the Ministry of Education, Science and Culture of
Japan.
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Hata, H., Shimizu, S. & Yamada, H. (1987). Enzymatic production of D-( - )-pantoyl
lactone from ketopantoyl lactone. Agric. Bioi. Chem., 51, 3011-16.
Hata, H., Shimizu, S., Hattori, S. & Yamada, H. (1989a). Ketopantoyl lactone
reductase from Candida parapsilosis, Purification and characterization as a conjug-
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Hata, H., Shimizu, S., Hattori, S., & Yamada, H. (1989b). Ketopantoyl lactone
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King, T. E. & Strong, F. M. (1951). Synthesis and properties of pantothenic acid
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Vitamin B 5 , Coenzyme A and Reloted Compounds 219
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Chapter 13
MICROBIAL PRODUCTION OF VITAMIN Ba AND

DERIVATIVES
Y. TANI
1 INTRODUCTION
In 1934, vitamin B6 (or pyridoxine) was discovered by Gyorgy as a rat pellagra

preventive factor. Soon after the determination of its chemical nature the total
synthesis of the vitamin was accomplished. Moreover, clinical investigations
have clarified that the vitamin has therapeutic and prophylactic uses for many
diseases. Since microbiological assays pointed towards several forms of the
vitamin B 6-active compound the enzymatic conversion of various forms of
vitamin B6 into pyridoxal 5 '-phosphate (pyridoxal-P), the active form of the
vitamin in vivo, has been intensively investigated. Pyridoxine, pyridoxamine,
pyridoxal and their 5 -phosphate esters are now recognized as principal vitamin
I
B6 compounds (Fig. 1).

Since Gunsalus & Bellamy first reported in 1944 that pyridoxal-P is the
coenzyme of L-tyrosine decarboxylase, it has become evident that pyridoxal-P
or pyridoxamine 5' -phosphate (pyridoxamine-P) is a coenzyme for various
enzyme systems associated with the metabolism of amino acids, amines,
saccharides, fatty acids, etc. A large number of vitamin B6 enzymes has now
been found, and the mechanisms of vitamin B6 dependent reactions have been
extensively investigated.
Several biotechnological methods for synthesis of vitamin B6 compounds
have been investigated and are described in this chapter. However, the
industrial production is still limited to chemical synthesis processes for
pyridoxine and pyridoxal-Po
2 CHEMISTRY AND ASSAY OF VITAMIN B6
Pyridoxine (3-hydroxy-4,5-dihydroxymethyl-2-methylpyridine) exhibits the

properties of a stable hydroxylated weak nitrogen base. Hydrolytic agents such
221
222 Y. Tani
RJ
HO CH2 0R2
,9'4 5
/""
~ 1 61
CH 3 N
Pyridoxine: R\ = CH 20H, R2 = H
Pyridoxal: R\ = CHO, R2 = H
Pyridoxamine: R\ = CH2NH2, R2 = H
Pyridoxine 5'-phosphate: R\ = CH20H, R2 = P0 3 H 2
Pyridoxal 5'-phosphate: Rl = CHO, R z = P03HZ
Pyridoxamine 5'-phosphate: Rl = CHzNHz, Rz = P03H2
Fig. 1. Chemical structure of vitamin B 6 •
as mineral acids or aqueous alkali, hot or cold, do not affect the vitamin. With
ferric chloride, pyridoxine reacts as a phenolic substance giving a reddish
brown coloration. In alkaline solution, pyridoxine on treatment with 2,6-
dichloroquinone chlorimide gives an immediate blue color fading to reddish-
brown.
Pyridoxine hydrochloride occurs as white platelets, melting point 204-206°C
with decomposition. The free base melts at 160°C. The compound is optically
inactive. Both base and hydrochloride readily sublime without decomposition.
The hydrochloride is freely soluble in water but sparingly in alcohol and
acetone. The base is soluble in methanol. Rapid destruction of pyridoxine by
light occurs in neutral and alkaline solutions. In 0·1 M hydrochloric acid there is
very little destruction.
The tautomeric properties of pyridoxine are well illustrated by the changes
in its UV absorption produced by varying the hydrogen ion concentration. The
single maximum at 292·5 nm at pH 2 diminishes in intensity at pH 4·5, and
concomitantly a new maximum appears at 327·5 nm. This latter band increases
in intensity when the pH is changed to 6·75, and the 292·5 nm maximum
disappears but a new band appears at 256·0 nm. When the pH is further raised
to 10·2, both bands increase in intensity and shift to shorter wavelengths.
The existence of other forms of pyridoxine was recognized as a result of the
comparison of microbiological assays on extracts of natural materials with the
values based on chemical and animal assays.
When pyridoxine was treated with mild-oxidizing agents, a marked increase
in l?io-activity towards the micro-organism, Lactobacillus casei, was observed.
Autoclaving of pyridoxine in the presence of the assay medium or amino acids,
greatly increased the activity of pyridoxine towards the test organism,
Streptococcus faecalis R. The products formed by treating pyridoxine with
animating agents and mild-oxidizing agents were, respectively, the amino and
aldehyde derivatives of pyridoxine. The compounds are pyridoxamine (2-
methyl-3-hydroxy-4-aminomethyl-5-hydroxymethylpyridine) and pyridoxal (2-
methyl-3-hydroxy-4-formyl-5-hydroxymethylpyridine) .
The availability of pyridoxal in synthetic form permitted identification of
pyridoxal-P, the coenzyme discovered as an essential cofactor for the en-

zymatic decarboxylation of amino acids. Pyridoxamine-P was subsequently
discovered as a naturally occurring compound by virtue of its differential
activity in promoting growth of certain lactic acid bacteria. Finally, pyridoxine
5'-phosphate (pyridoxine-P) (although it plays no known essential role in
metabolism) can be formed from pyridoxine by tissue enzymes and further
transformed to pyridoxal-P by other tissue enzymes. It appears therefore to
also be a naturally occurring compound. Direct analysis of many tissue extracts
also indicates its natural occurrence.
A number of microbiological procedures are established for the estimation
of vitamin B 6. The method with Saccharomyces cerevisiae (S. carlsbergensis, S.
uvarum) is widely used for the assay of total vitamin B6 in non-esterified form.
The phosphates of vitamin B6 should be converted to the free form by acid or
enzymatic hydrolysis before applying the bioassay. A differential assay
employs Lactobacillus casei which responds to pyridoxal and Streptococcus
faecalis R which responds to pyridoxal and pyridoxamine. Chemical reactions
with 2,6-dichloroquinone chlorimide and diazotized p-aminoacetophenone are
less sensitive and non-specific but are useful for the detection on paper
chromatograms and paper electrophoregrams. Recent progress in high perfor-
mance liquid chromatography is applied for quantitative and simultaneous
detection of all vitamin B6 compounds by determining UV absorption and
fluorescence.
3 BIOSYNTHESIS AND METABOLISM OF VITAMIN B6
The current knowledge of the biosynthesis and metabolism of vitamin B6 is

summarized in Fig. 2.
Although the de novo biosynthesis of vitamin B6 has been investigated since
the 1940s using micro-organisms, the complete biosynthetic pathway still
remains obscure. Clear evidence of a direct precursor in the vitamin B6
biosynthesis was demonstrated for the first time by Tani & Dempsey (1973). In
a vitamin B6 requiring WG3 strain mutant derived from Escherichia coli B, the
vitamin B6 requirement could be satisfied by glycolaldehyde, and 14C_
glycolaldehyde was incorporated into vitamin B6 without significant dilution.
Subsequently, the biosynthetic route of glycolaldehyde in E. coli (Tani et al.,
1977) and the incorporated position of glycolaldehyde in vitamin B6 (Hill et al.,
1975) were elucidated. Based on these results, a hypothetical sequence of
vitamin B6 biosynthesis, is presented in Fig. 3 in which the condensation of
glycolaldehyde with a 'C4 compound' forming a 'C6 compound' such as
a,p-dihydroxy-p-hydroxymethyl valerate, as well as the following introduction
of a 'C3 compound' are involved. Another route, in which all carbon atoms of
pyridoxine are derived from trioses, is also proposed (Vella et aI., 1980).
As to the biodegradation of vitamin B6, research was performed with
224 Y. Tani
Glycolate
D-Glutamate 2-Ketoglutarate
1
Glycolaldehyde
Pyridoxamine-P • .
Pyndoxal-P •• \. .J. Pyn'doxme-
. P
'-. '-. A ~~i~hOOPhat" ATPt~o'Pha". ~~i~PhO'Pha", A

~ ~
II
""" Pyridoxine • Pyridoxal • Pyridoxamine
Pyridoxine glUCOSi1 IL-Alani[)yrUvate

Pyridoxine glucuronide ~
Isopyridoxal 4-Pyridoxic acid lactone
1
5-Pyridoxic acid lactone
1
4-Pyridoxic acid
1 1
2-Methyl-3-hydroxy-5-formyl-
5-Pyridoxic acid
pyridine-4-carboxylate
1
a-Hydroxymethyl-a' -(N-acetylamino- 1
methylene )succinate 2-Methyl-3-hydroxypyridine-
1
4,5-dicarboxylate
a-Hydroxymethylsuccinate semi al- 1

2-Methyl-3-hydroxypyridine-
dehyde + acetate + NH3 + cO 2
5-carboxylate
1
a-(N -acetylaminomethylene)-
succinate
1
Succinate semialdehyde + acetate
+NH 3 +C02
Fig. 2. Metabolism of vitamin B 6 •
pseudomonads, to which pyridoxine and pyridoxamine were fed as carbon

and/or nitrogen source (Nyns et al., 1969).
The biosynthesis of pyridoxal-P from pyridoxine is considered to occur
through pyridoxine-+ pyridoxine-P-+ pyridoxal-P rather than pyridoxine-+
pyridoxal-+ pyridoxal-P, based on experimental results so far obtained.
Pyridoxine is transformed into pyridoxal by an oxidase or a dehydrogenase.
Pyridoxine oxidase activity was found in an enzyme preparation extracted from
rabbit liver, and the oxidase reaction of pyridoxine to pyridoxal proceeded
only in the presence of a coupling reaction with an aldehyde oxidase which
oxidized pyridoxal to 4-pyridoxic acid. Pyridoxine dehydrogenase requires
NADP as a coenzyme. Both oxidase and dehydrogenase reactions are
CH3
I
CH2
I
C=O CH3
I I
~~~;;~~'::ed) H}r-'-CH'OH C, COrU~d

/C"""
HO COOH
*CHO .. a, f;I-dihydroxy
I f;I-hydroxymethyl
**CH2 0H valerate"
Glycolaldehyde
Fig. 3. Hypothetical biosynthetic sequence of pyridoxine.
reversible, though the reaction equilibria favour the formation of pyridoxine

from pyridoxal.
The phosphorylation of free vitamin B6 is catalyzed by pyridoxal kinase,
which phosphorylates pyridoxine and pyridoxamine as well as pyridoxal in the
presence of A TP to form the corresponding phosphate esters. The enzyme
activity occurs widely in mammalian tissues, bacteria, molds, and yeasts.
Another phosphotransferring reaction using various organic phosphorus
compounds was found in many micro-organisms such as molds, yeasts, and
bacteria, and found to be catalyzed by the phosphotransferring ability of
phosphatase (Tani & Ogata, 1972).
Pyridoxine-P oxidase catalyzes the oxidative deamination of pyridoxamine-P
as well as the oxidation of pyridoxine-Po The coenzyme of the enzyme is FMN.
The enzyme activity is widely present in bacteria and it is strongly inhibited by
compounds phosphorylated at the 5-position of the pyridine ring, but weakly
inhibited by compounds having an aldehyde residue at the 4-position of the
pyridine ring (Yamamoto et al., 1965). The fact that the enzyme is strongly
inhibited by pyridoxal-P (the reaction product) might be attributed to
metabolic regulation of the in vivo biosynthesis of pyridoxal-P. It seems likely
that pyridoxine-P or pyridoxamine-P stored in cells is oxidized to pyridoxal-P
when necessary.
226 Y. Tani
Anaerobic bacteria, clostridia, lack the pyridoxine-P oxidase activity but

have the pyridoxamine-P-2-ketoglutarate transaminase activity which is absent
in aerobic bacteria. The transaminase purified from cell-free extract of
Clostridium kainantoi uses 2-ketoglutarate as the amino group acceptor and
D-glutamic acid as the amino group donor (Tani et al., 1972b).
4 PRODUCTION OF VITAMIN B6 BY DE NOVO SYNTHESIS AND

BIOCONVERSION
4.1 Production of Vitamin 8 6 by Fermentation
Extensive screening for vitamin B6 producers among micro-organisms has been

pedormed. However, the productivity was quite insufficient to establish an
industrial microbiological process of vitamin B6 production. Almost all
micro-organisms synthesize vitamin B6 intracellularly 0·05-0·3 mg per g of dry
cells, and excrete it in amounts of 0·05-0·5 mg per liter of culture medium.
Table 1 is a list of relatively high producers. The extracellular vitamin B6 is the
free form.
The biosynthesis of vitamin B6 in E. coli is known to be repressed by a
feedback control system. However, in a high producer, Flavobacterium sp.
238-7, the amount of vitamin B6 excreted was increased even when cultivated
with the addition of each form of vitamin B6 (Tani et al., 1972a). An enzyme
which reduces glycolate to glycolaldehyde as the first step of the biosynthesis of
vitamin B6 is present in the particulate fraction of Flavobacterium sp. 238~7.
The activity and synthesis of the enzyme was not affected by added vitamin B6
(Tani et al., 1984). From these results, Flavobacterium sp. 238-7 can be
considered to lack this feedback control system for the vitamin B6 biosynthesis,
resulting in the formation of large amounts of vitamin B 6 •
A typical time course of vitamin B6 production by Flavobacterium sp. 238-7
in a glycerol-peptone medium is shown in Fig. 4.
Table 1
Extracellular Production of Vitamin B6 by Micro-organisms
Micro-organisms Vitamin B6 produced (mg/liter)
Bacteria:
Klebsiella sp. 2·0-4·0
Achromobacter cycloclastes 3·0-4·0
Flavobacterium sp. 238-7 20·0
Bacillus subtilis 2·0-5·0
Yeasts:
Candida albicans (n-paraffin-grown) 0·6
Saccharomyces marxianus 2·0
Pichia guilliermondii (n-paraffin-grown) 25·0
!0
-4'
....
""-
9
3
12 0.6 '"...
~
....iii ,.,
01
.......
~
..
""- II)
00
<I
~
"tI
"tI 8 0.4 ....01
~ .......u
....0 ...
'",qI ~
....<I 4 0.2 .c...:-

....I>...
~ 0
"
'-'
0
Cultivation time (h)
Fig. 4. Production of vitamin B6 by Flavobacterium sp. 238-7.
4.2 Production of Pyridoxal-P from Pyridoxine-P and Pyridoxine
As pyridoxine and pyridoxine-P can be chemically synthesized with ease, the

industrial production of pyridoxal-P from pyridoxine and pyridoxine-P by
pyridoxine-P oxidase catalysis with or without enzymatic phosphorylation is
considered to be worthy of study.
Pyridoxine-P is oxidized by a flavoprotein, pyridoxine-P oxidase. It was
found that amino acids accelerate the enzyme reaction due to relief of the
aldehyde inhibition by pyridoxal-P of the enzyme activity, in which these
amino acids combined non-enzymatically with an aldehyde residue of
pyridoxal-P to form a Schiff base. In view of applying this enzyme reaction for
industrial production of pyridoxal-P, a suitable industrial medium for
Alcaligenes faecalis was constructed with corn steep liquor, ammonium sulfate
and glycerol. The cells obtained were autolyzed by addition of toluene or ethyl
acetate and used as the enzyme source. The reaction proceeded adequately on
addition of aspartic acid or glutamic acid, which are relatively cheap, and
resulted in a complete conversion of pyridoxine-P to pyridoxal-P.
An acid phosphatase of Escherichia freundii Kl can phosphorylate pyri-
doxine as the most effective phosphoryl acceptor among free vitamin B6 using
various kinds of organic phosphates as the phosphoryl donor. As E. freundii
had pyridoxine-P oxidase and pyridoxine dehydrogenase activities together
with the phosphotransferase activity, a considerable amount of pyridoxal-P was
formed as well as pyridoxine-P in the phosphotransferring reaction of
228 Y. Tan;
pyridoxine using cells or cell-free extract of the organism (Tani et al., 1969). In
particular, an appreciable amount of pyridoxal-P was formed by use of
phenylphosphate as the phosphoryl donor under alkaline conditions. Under
the optimal reaction conditions, 0·4 g of pyridoxal-P was formed from 2 g of
pyridoxine incubated with phenylphosphate and cells for 24 h.
4.3 Production of Pyridoxal from Pyridoxine
Oxidation of pyridoxine to form pyridoxal is catalyzed by pyridoxine de-

hydrogenase. A biochemical process for production of pyridoxal from pyrido-
xine was studied with the pyridoxine dehydrogenase of dried cells of baker's
yeast (Ogata et al., 1968). The reaction equilibrium of the enzyme lies to the
reduction of pyridoxal to pyridoxine. However, a large amount of pyridoxal
(pyridoxal-carbonyl complex) was accumulated on addition of various car-
bonyl reagents to the reaction mixture. For example, the formed pyridoxal was
non-enzymatically converted into pyridoxal-semicarbazone on addition of
semicarbazide, and the maximum yield of pyridoxal reached 80% based on the
amount of pyridoxine added. Pyridoxal-semicarbazone is highly insoluble in
water and can easily be isolated by crystallization from the reaction mixture.
4.4 Production of Vitamin B6 Derivatives
A number of vitamin B6 derivatives such as vitamin B6 fatty acid esters and

vitamin B6 amino acid compounds are synthesized chemically. On the other
hand, few naturally occurring derivatives have been found: glucuronide of
pyridoxine is found in human urine, sulfates (3-position) of pyridoxine or
pyridoxal are formed by a homogenate of rat liver, and an indole-pyridoxal
complex is found in milk.
It was also found that a new derivative of vitamin B6 was formed in the
culture filtrates of some strains belonging to the genera Sarcina and
Micrococcus, cultivated in medium containing pyridoxine and sucrose, maltose
of phenyl-a-glucoside as carbon source. the derivative isolated from the
culture broth consisted of two components. These were separated by Dowex
1 x 2 (borate form) column chromatography and identified as pyridoxine
4'-a-D-glucoside and pyridoxine 5'-a-D-glucoside (Fig. 5) by comparison with
chemically synthesized samples (Ogata et al., 1969). Under optimal conditions
using Micrococcus sp. No. 431, pyridoxine glucoside, 3·5-3·7 g/liter, was
produced in the culture broth of the organism from 2 g of pyridoxine per liter.
The pyridoxine glucoside-synthesizing enzyme was purified and charac-
terized to be as a-glucosidase having the ability of transglucosidation. The
bioactivity of pyridoxine glucoside using S. cerevisiae as the test organism was
about 20% of that of its molar equivalent of pyridoxine, and it increased with
incubation time, suggesting the gradual hydrolysis of pyridoxine glucoside into
pyridoxine.
_jp~'OHCH,-O
H°fr~OH
I OH
H3 C ::"'N
Pyridoxine 4' -a-D-glucoside
Fig. 5. Chemical structure of pyridoxine glucoside.
Pyridoxine glucoside is relatively more stable than pyridoxine toward heat

and UV irradiation. Pyridoxine glucoside could be transported into rabbit
erythrocytes. The transport of pyridoxine glucoside was about 10-20% of that
of its molar equivalent of pyridoxine (Kawai et al., 1972).
REFERENCES
Gunsalus, I. C. & Bellamy, W. D. (1944). A function of pyridoxal. 1. BioI. Chern.,

155,357-8.
Hill, R. E., Horsewood, P., Spenser, I. D. & Tani, Y. (1975). Biosynthesis of vitamin
B 6 • Incorporation of glycolaldehyde into pyridoxal. 1. Chern. Soc. Perkin, I,
1622-7.
Kawai, F., Yamada, H. & Ogata, K. (1972). Studies on transglycosidation to vitamin
B6 by micro-organisms. VII. Transport of pyridoxine glucoside into rabbit erythro-
cytes. 1. Vitarninol., 18, 183-8.
Nyns, E. J., Zach, D. & Snell, E. E. (1969). The bacterial oxidation of vitamin B6 •
VIII. Enzymatic breakdown of a-(N-acetylaminomethylene) succinic acid. 1. Bio!.
Chern., 244,2601-5.
Ogata, K., Tochikura, T., Kawata, S., Yamamoto, S. & Kawai, H. (1968). Pyridoxal
derivative formation from pyridoxine by baker's yeast, Agric. BioI. Chern., 32,
1479-81.
Ogata, K., Uchida, Y., Kurihara, N., Tani, Y. & Tochikura, T. (1969). Studies on
transglycosidation to vitamin B6 by micro-organisms. II. Chemical structure of
pyridoxine glucoside. 1. Vitarninol., 15, 160-66.
Gyorgy, P. (1934). Vitamin B6 and pellegra-like dermatitis in rats. Nature, 133,
498-9.
Tani, Y. & Dempsey, W. B. (1973). Glycolaldehyde is a precursor of pyridoxal
phosphate in Escherichia coli B. 1. Bacterio!., 116,341-5.
230 y. Tani
Tani, Y. & Ogata, K. (1972). Studies on vitamin B6 metabolism in micro-organisms.

Part IX. Microbial phosphorylation of vitamin B6 through a new phosphotransfer-
ring reaction (5). Phosphorylation of pyridoxine by several phosphatases. Agric.
Bioi. Chem., 36, 173-80.
Tani, Y., Kawata, S., Tochikura, T. & Ogata, K. (1969). Studies on vitamin B6
metabolism in micro-organisms. Part VIII. Microbial phosphorylation of vitamin B6
through a new phosphotransferring reaction (4). Formation of pyridoxal 5'-
phosphatae from pyridoxine. Agric. BioI. Chem., 33,314-20.
Tani, Y., Nakamatsu, T., Izumi, Y. & Ogata, K. (1972a). Studies on vitamin B6
metabolism in micro-organisms. Part XI. Extracellular formation of vitamin B6 by
marine and terrestrial micro-organisms and its control. Agric. Bioi. Chem., 36,
189-97.
Tani, Y., Ukita, M. & Ogata, K. (1972b). Studies on vitamin B6 metabolism in
micro-organisms. Part X. Further purification and characterization of pyridoxamine
5'-phosphate-a-ketoglutarate transaminase from Clostridium kainantoi. Agric. BioI.
Chem., 36, 181-8.
Tani, Y., Morita, H. & Ogata, K. (1977). Glycolaldehyde synthesizing pathway
involved in vitamin B6 biosynthesis in Escherichia coli B. Agric. BioI. Chem., 41,
1749-54.
Tani, Y., Nishise, H., Morita, H. & Yamada, H. (1984). Vitamin B6 biosynthesis and
glycolate reductase in Flavobacterium sp. 238-7. J. Nutr. Sci. Vitaminol., 30,
415-20.
Vella, G. J., Hill, R. E., Mootoo, B. S. & Spenser, I. D. (1980). The status of
glycolaldehyde in the biosynthesis of vitamin B6 • J. BioI. Chem., 255, 3042-8.
Yamamoto, S., Tochikura, T. & Ogata, K. (1965). Studies on vitamin B6 metabolism in
micro-organisms. Part III. Pyridoxine phosphate and pyridoxamine phosphate
oxidation (3). Product inhibition and reactivation with amino compounds. Agric.
Bioi. Chem., 29,597-604.
Chapter 14
MICROBIAL PRODUCTION OF BIOTIN

Y. IZUMI & H. YAMADA
1 HISTORICAL
In 1927, Boas described a skin injury produced in rats by feeding raw egg
white, as well as the occurrence in various foodstuffs of a 'protective factor X'
that prevented and cured this injury (Boas, 1927). Gyorgy et al. (1939) tracked
down this protective factor and called it vitamin H after the German Haut for
skin. Kogi & Tonnis (1936) isolated a yeast growth factor, biotin vitamin B8
from egg yolk in the form of its crystalline methyl ester and Kogi (1937)
determined its empirical formula. Biotin was later shown to be identical to
vitamin H, the protective factor X, and to coenzyme R. Melville et al. (1942)
determined the correct structure of biotin, which was later confirmed by total
chemical synthesis in the Merck laboratories (Harris et al., 1943, 1944a,b,
1945) and verified by X-ray crystallography (Traub, 1956; Trotter & Hamilton,
1966).
Studies on the biosynthesis of biotin started in 1937 with studies on the
nutritional requirements of micro-organisms (Mueller, 1937a, b; du Vigneaud
et al., 1942; Eakin & Eakin, 1942; Dittmer & du Vigneaud, 1944; Dittmer &
du Vigneaud, 1944; Lilly & Leonian, 1944; Stokes & Gunness, 1945).
Subsequently, through radiochemical and genetic-biochemical studies (Tatum,
1945; Pontecorvo, 1953; Ryan, 1956), part of the biosynthetic pathway was
proposed. Thereafter, a hypothetical pathway was established by Okumura et
al. (1962a,b) based on their investigations of the microbial production of
glutamic acid using biotin-requiring bacteria. Ogata et al. (1965a,b) and
Iwahara et al. (1966a,b,c) made extensive investigations of biotin biosynthesis
and verified the main pathway described by Okumura et al. (1962a,b) using
growing and resting cells: pimelic acid - 7-keto-8-aminopelargonic acid
(KAPA)-7,8-diaminopelargonic acid (DAPA)-dethiobiotin (DTB)-
biotin (Izumi & Ogata, 1977). Through the extensive studies of three groups,
Eisenberg (1973), Pai (1971) and Izumi (Izumi & Ogata, 1977), the enzymic
231
232 Y. Izumi & H. Yamada
system involved in the biosynthetic pathway from pimelic acid to DTB had
been established by 1975.
In the 1950s it became clear that biotin was involved as a cofactor in many
biochemical processes, especially a number of carboxyl transfer reactions.
Investigations on the function of biotin in the reaction mechanisms were
obstructed by the fact that biotin is active only when covalently attached to an
e-amino group of a lysyl residue of the enzyme (Kosow et al., 1962), as was
suggested earlier by the isolation of biocytin (e-N-biotinyllysine) from yeast
extract (Wolf et al., 1952).
Wakil et al. (1958) and Wakil & Gibson (1960) showed that highly purified
acetyl-CoA carboxylase, isolated from avian liver extracts, was enriched with
respect to biotin and sensitive to inhibition by avidin, suggesting the tight
binding of the vitamin to the carboxylase and its probable role in acetyl-CoA
carboxylation. Lynen et al. (1959) made the interesting observation that the
A TP- and Mi+ -dependent carboxylation of free biotin was catalyzed by a
bacterial p-methylcrotonyl-CoA carboxylase. This model reaction led to the
formation of an unstable carboxybiotin derivative which was identified as
1'-N-carboxybiotin (Knappe et al., 1961; Lynen et al., 1961). Subsequently, it
was demonstrated with several biotin enzymes that the site of carboxylation of
the enzyme-bound prosthetic group was the same as that of free biotin (Moss
& Lane, 1971).
The existence of separate subsites for the catalysis of each partial reaction
has been unequivocally demonstrated with acetyl-CoA carboxylase of
Escherichia coli (Fall & Vagelos, 1972; Polakis et al., 1974). These investiga-
tions have shown that there are three dissimilar subunits involved in catalysis:
(a) the biotin carboxyl carrier protein (CCP), (b) the biotin carboxylase (BC),
which catalyzes the carboxylation of the CCP biotin, and (c) the carboxyl
transferase (Cf), which catalyzes the carboxyl transfer from the CCP biotin to
acetyl-CoA. Wood's group (Wood & Barden, 1977) has unequivocally
demonstrated with transcarboxylase that each partial reaction is catalyzed by a
separate subunit and that a distinct CCP subunit is present.
Major portions of the amino acid sequence of the CCP from transcar-
boxylase of Propionibacterium shermanii and acetyl-CoA carboxylase from
Escherichia coli have been determined (Wood & Barden, 1977). Up to now,
the sequences of the portions around biotin of the CCP or the corresponding
domains have been elucidated with almost all the biotin enzymes including
chicken liver acetyl-CoA carboxylase (Takai et al., 1987), human pyruvate
carboxylase (Lamhonwah et al., 1987) and human propionyl-CoA carboxylase
(Lamhonwah et al., 1987). Recently, Takai et al. (1988) have deduced for the
first time the complete amino acid sequence of a biotin enzyme, chicken liver
acetyl-CoA carboxylase.

The structure of biotin is shown in Fig. 1. The naturally occurring biotin,
(+ )-cis-hexahydro-2-oxo-1H-thieno-(3,4-d)-imidazole-4-n-valeric acid, is ex-
Microbial Production of Biotin 233
* asymmetric carbon
(a)
Fig. 1. Chemical structure (a) and absolute configuration (b) of ( + )-biotin.
pressed in many ways: d-biotin, d-( + )-biotin, D-biotin and D-( + )-biotin.
Here, we describe it as (+ )-biotin, and unless otherwise stated, the term
'biotin' indicates ( + )-biotin. The structure of crystalline biotin has also been
investigated by X-ray diffraction techniques and is now known precisely
(Traub, 1956; Trotter & Hamilton, 1966; Stallings & de Titta, 1985). X-ray
crystallographic analysis of (+ )-biotin and of the carboxybiotin derivative
described previously revealed that the bicyclic ring system has a boat-like
configuration (Fig. 1(B». The planar ureido ring projects upward at an angle
of 62·0° with respect to the plane (plane A) formed by the four carbon atoms
of the tetrahydrothiophene ring. Another plane comprising S-1, C-2, and C-5
tilts upward at an angle of 37 ·6° with respect to plane A (Bonnemere et al.,
1965). Because of the cis orientation of the ureido ring with respect to the
aliphatic side chain, C 6 resides approximately 2·8 A from the 3' -N of the
ureido ring (Traub, 1956). Repulsion between the 3'-N and C-6 positions is
thought to cause the greater-than-anticipated C 3-Cz-C6 angle of 119°
(Traub, 1956).
There are seven other forms which can be chemically synthesized: (-)-
biotin, (+)- and (- )-epibiotin (cis-form); (+)- and (- )-allobiotin, and
( + )- and ( - )-epiallobiotin (trans-form).
The crystals of ( + )-biotin are colorless, fine long needles: m.p. 232-3°C,
[{1']22=91° (c=1, 0·1NNaOH). The isoelectric point is pH3·5. The pH of
0·01 % aqueous solution is 4·5. Solubility in water is about 22 mg/100 ml at
25°C and it is more soluble in hot water or in dilute alkali. Solubility in 95%
ethanol is about 80 mg/100 ml at 25°C. It is insoluble in other common organic
solvents. It is stable in air, in a wide temperature range, and up to about pH 9.
It is also stable after autoclaving in 6 N H 2S04 at 120°C for 1 h. Accordingly,
most of the naturally occurring biotin bound to protein can be hydrolyzed to
free forms by such autoclave treatments without any problem.
Biotin combines with avidin, a protein in raw egg white, and streptavidin, a
protein produced by an actinomycete to become inactive.
Various chemical reactions with biotin and their products are shown in
Fig. 2.
HNiNH HNiNH HNjlNH
V(CH,>.CONH(CH2,.b:~00H V(01,l.COOH V(CH2'.COOH

biocytin r L,,'-Cu
b
Qlotln sulfoxide
0'" "0
biotin sulfone
ctIHIle
?
Jl.. NH2
?
)l...N
f "MoO,
(HOI
Q(CH24
, COOH
HN~NH ~-<::HCOOOCH, HNt\H , , thienyl·"aleric acid
Q t(CH3J2S0 4
s CH,l.CO'l'H 5 CH,l.COCI A "MoO.
CHCOOH biotin acid '-- HN NH ~ H2N NH2 (HNO,)
N-flIotlnyi ft chloride sOO--- I--l -- H - HOOC(CH2'.COOH
ammo a c l d / CH,N,/
l.A).
5
COC(,
(CH 2'.COOH
l.A).
5 (CH2,.COOH adipic acid
Q NH, JL / biotin diaminoblotln
HN)l...NH HN NH ~ H, (Rane, Ni)
Os -"2'. (t'U \ CONH

2
V(CH \ COOCH
2" 3 HN
jl
NH H N NH HIO
BalOH),
biotin amide bIotln~ methyl ester H 2 H
2 ~HOOC(CH2'5COOH
COCI,
N,H, CH3(CH2'5COOH CH3(CH2'5COOH
Q dethiobiotln ~. pime/Ic acid
HN)l...NH
H HNO, HN
JL NH gonic acid
l...S~CH,l.CO::-- V(CH,>.CONHNH 2
biotin azide biotin hydrazide
Fig. 2. General chemical reactions of biotin and their products.
Using about 1000 strains of molds, bacteria and actinomycetes, Ogata et al.
(1965a,b) and Iwahara et al. (1966a,b) investigated the accumulation of
biotin-vitamers when pimelic acid was added to media as a precursor. They
found a promoting effect of pimelic acid in a large number of strains. On
addition of pimelic acid to the medium of Bacillus sphaericus IFO 3525,
20-200 Ilg/ml of 'total biotin', which is the amount of biotin-vitamer given by
the bioassay using Saccharomyces cerevisiae and includes DTB, KAP A and
DAPA as well as biotin, was accumulated. This was several hundred times
more than has hitherto been reported in micro-organisms. The main com-
ponent of the biotin-vitamers formed from pimelic acid was (+ )-DTB.
Afterwards, Yamada et al. (1983) found this strain also produced relatively
large amounts (0·8-1·1Ilg/ml) of biotin from pimelic acid, as well as from
DTB, under optimized conditions.
Ogino et al. (1974a,b) demonstrated a production method using an
n-paraffin-utilizing bacterium from a new compound, oL-cis-tetrahydro-2-oxo-
4-n-pentylthieno-(3,4-d)-imidazoline (oL-TOPTI), which is a biotin analog
having a methyl group instead of a carboxyl group of the biotin molecule.
TOPTI was chemically synthesized from Nt ,N3 -dibenzyl-oL-cis-tetra-
hydrothieno-(3,4-d)-imidazoline-2,4-dione (compound VII in Fig. 6) via a
Grignard reaction with n-pentyl magnesium bromide, dehydration in the
presence of an acidic catalyst, catalytic hydrogenation, and debenzylation with

concentrated HBr. In this production method, n-paraffin was used as a carbon
and energy source for cell growth with concurrent transformation (co-oxidation
of TOPTI to biotin). First, n-paraffin-utilizing micro-organisms that co-
oxidized TOPTI were selected from natural sources. Three strains, identified
as Corynebacterium sp., were the most excellent producers of biotinol and
biotin from TOPTI. Therefore the conversion of TOPTI to biotin was assumed
to occur via p-oxidation. In a medium containing 2% n-paraffin and 0·2%
urea, with the addition of 50 mg DL-TOPTI/100 ml after 24 h of cultivation, a
maximum conversion of about 60% (0·32 mg (± )-biotin/ml) was obtained
96 h after the addition of TOPTI. However, selective degradation of (+)-
biotin occurred after prolonged incubation, leaving the ( - )-isomer. Thus, to
avoid such degradation, mutants that were incapable of assimilating n-paraffin
and of degrading biotin, but capable of utilizing acetate, were derived. One of
the mutants produced 0·65 mg/ml of ( ± )-biotin from 0·8 mg/ml of DL-TOPTI
(80·5% conversion).
4 BIOSYNTHESIS AND BIODEGRADATION
4.1 Biosynthesis
Biotin can be synthesized by a great number of micro-organisms, while some

organisms which cannot synthesize biotin require it for growth. Biotin is also
known to be synthesized by plants (Watanabe et al., 1982). Almost all the
studies on the biosynthesis of biotin have used micro-organisms. The pathway
of biotin biosynthesis has been established, as shown in Fig. 3. All the enzymes
which are involved in the reaction from pimelic acid to DTB have been
elucidated and were found to be novel types. Table 1 summarizes the
properties of pimelyl-CoA synthetase, KAPA synthase, DAPA aminotrans-
ferase and DTB synthetase (Izumi et al., 1980; Izumi, 1984).
The final step from DTB to biotin through a sulfur introduction has not been
enzymically resolved yet. However, there have been some reports on the
biosynthesis using growing cells and resting cells. Yamada et al. (1983) showed
that resting cells of B. sphaericus IFO 3525 exhibited high activity of biotin
synthesis from DTB. Izumi et at. (1973) also showed that resting cells of
Rhodotorula glutinis which form appreciable amounts of biotin from DTB,
formed biotin from DTB only in the presence of methionine, particularly the
L-form. The isotopic experiment revealed that sulfur contained in one molecule
of L-methionine was incorporated into one molecule of biotin.
Parry & Kunitani (1976), Guillerm et al. (1977), Parry & Naidu (1980) and
Trainor et al. (1980) have developed a stereospecific synthesis of DTB and
examined the mechanism of the conversion of DTB to biotin by using
specifically labelled 3H-DTB and growing cells of Aspergillus niger and E. coli,
respectively. They concluded that the introduction of sulfur at C-1 and C-4
Pimelic acid
Pimelyl CoA
7-Keto-B'amino-
pelargonic acid
(KAPA)
78-0iamino-
pelargonic acid
(OAPA)
Oethiobiotin
(OTB)
Biotin
Fig. 3. Biosynthetic pathway of biotin. CD Pimelyl-CoA synthetase, (6) KAPA synthase,

® OAPA aminotransferase, @) OTB synthetase.
positions of DTB takes place with the loss of two hydrogen atoms at
C-1 and C-4 and without the loss of hydrogen atoms at C-2 or C-3. These
results suggest that unsaturation does not occur at C-2 or C-3.
Table 2 (Izumi et al., 1981) shows that among the strains of bacteria and
yeasts tested for activities of the four biotin biosynthetic enzymes, only B.
sphaericus IFO 3525, a DTB producer as described previously, showed
significant activities for all four enzymes.
Table 1
Properties of Biotin Biosynthetic Enzymes
(a) Pimelyl-CoA synthetase of Bacillus megaterium
Optimum temperature 32°C
Optimum pH 8·0
Km value: pimelic acid 2·7 x 10- 4 M
CoA 5·5 x 10- 4 M
ATP 1·5 x to- 3 M
Mi+ 1·5 X to- 3 M
Substrate specificity Pimelic acid
(Other dicarboxylic
acids were inert)
Nucleotide requirement ATP, ADP
Metal ion requirement Mi+, Mn2+
Inhibitors EDTA, a, a' -dipyridyl,
o-phenanthroline
(b) KAPA synthase of Bacillus sphaericus

Optimum temperature 6O"C
Optimum pH 7·0
Thermostability (10 min) 0-6O"C (100%)
Amino acid specificity L-Alanine
Coenzyme requirement PyridoxaI5'-phosphate
Inhibitors Phenylhydrazine, semicarbazide,
hydroxylamine,o-cycloserine,
oL-penicillamine, isoniazid,
o-phenanthroline, citrate,
L-cysteine, glycine, o-alanine
L-serine, o-,L-histidine
(c) DAPA Aminotransferase of Brevibacterium divaricatum
Molecular weight 24,000
A.max 280, 320, 410 nm
Optimum temperature 37°C
Optimum pH 8·5
Thermostability (10 min) O-60°C (100%)
pH Stability (30 min) 7·0-10·0 (100%)
Amino donor specificity S-Adenosyl-L-methionine
Amino acceptor specificity KAPA (100)
7-Amino-8-ketopelargonate (1)
Coenzyme requirement Pyridoxal 5' -phosphate
Km values: KAPA Pyridoxamine 5' -phosphate
S-adenosyl-L-methionine 0·69 x to- 4 M
pyridoxal 5' -phosphate 0·55 x 10-3 M
pyridoxamine 5' -phosphate 0·83 X to- 6 M
1·2 X to- 6 M
Inhibitors Phenylhydrazine, semicarbazide
hydroxylamine, o-cycloserine,
oL-penicillamine, isoniazid,
p-chloromercuribenzoate, HgCI
iodoacetate; Co 2+, Ass+, KCN
(cOlllinued)
Table l---contd.
(d) DTB Synthetase of Pseudomonas graveolens

Optimum temperature 7·0
Optimum pH 7 ·0-8·0
Thermostability 0-45°C (100%)
Substrate specificity DAPA (100)
ti
NH2 NH2
Biotin diamino-
S COOH carboxylic acid (10)
Nucleotide requirement ATP (100), CTP (20), UTP (10)

GTP (20), ITP (10)
Metal ion requirement Mg2+ Mn2+ Fe 2+
Km values: DAPA 1 X l(j-4 M '
HC03 - 1 X 1O- 2 M
ATP 5 X 10- 5 M
Mi+ 3 X 10- 3 M
Inhibitors EDTA, a, a' -dipyridyl, o-phenan-
throline
4.2 Regulation of Biosynthesis

The biosynthetic regulation mechanism has also been elucidated at the enzyme
level. Eisenberg & Star (1968) have reported that KAPA synthase of E. coli is
almost completely repressed by the addition of 5 ng/ml of biotin to the
medium. Eisenberg & Krell (1969) and Pai (1969) observed similar repression
by biotin of DTB synthetase. Izumi & Ogata (1977) found that KAPA
synthase of B. sphaericus and DAPA aminotransferase of Brevibacterium
divaricatum were repressed by the addition of O·lllg/ml of biotin to the
medium. Moreover, they found that, in contrast to the complete repression of
DTB synthetase in B. megaterium by O·25Ilg/ml of biotin, pimelyl-CoA
synthetase was not repressed by even 1llg/ml of biotin. The biotin synthesiz-
ing reaction by resting cells of B. sphaericus was repressed by 1llg/ml of
biotin. In this way, a strong repressive action of biotin has been demonstrated
on all the enzymes between pimelyl-CoA and DTB and in the biosynthetic
step(s) between DTB and biotin. The corepressor has been found to be
biotinyl-AMP in E. coli (Prakash & Eisenberg, 1979). The repressor protein
(Eisenberg et al., 1982) and biotin operon (Szybalski & Szybalski, 1982) have
also been elucidated.
4.3 Biodegradation
Biotin is known to be degraded by molds, yeasts and bacteria via f3-oxidation
of the side chain of the molecule (Izumi & Ogata, 1977; Tanaka et aI., 1988).
The biotin-degrading bacterium, Mycoplana sp. No. 166, formed bisnorbiotin,
a compound having two less carbon atoms in its side chain than biotin, from
Table 2
Biotin-vitamer Producing Abilitiesa and Biotin Biosynthetic Enzyme Activities of Various Yeasts and Bacteria
Strain True biotin b Total biotin Specific activityc (units/mg protein)

(p.g/ml) (p.g/ml) 1 2 3 4
Yeasts
Saccharomyces kloeckerianus IFO 0016 trt 0·71 tr tr 0·06 0·21
Lipomyces starkeyi IFO 0678 0·015 0·02 tr 2·90 tr 0·07 ~
~.
Sporobolomyces salmonicolar IFO 0374 0·085 0·49 0·23 1·30 tr 0·06
C3
Sporobolomyces salmonicolor IFO 1038 0·068 0·30 0·57 1·80 tr 0·03
Sporobolomyces coprophilus IFO 1442 0·100 0·16 0·14 0·31 tr 0·01
s·0-
Rhodotorula glutinis IFO 0415 0·066 ~
0·12 tr 0·10 tr 0·03
-a
Bacteria t
Escherichia coli AKU 007 0·018 0·40 1-13 2·45 tr 0·03 '"S·
Klebsiella pneumoniae IFO 12059 tr 0·30 tr 0·18 0·08 0·03 :lI
Enterobacter aerogenes IFO 12010 0·021 tr 0·18 0·08 tr 0·02 .l;.
Alcaligenes faecalis IFO 3160 tr 1·58 0·21 0·16 tr 0·06 ~
5·
Bacillus megaterium NI 8100 tr 1·90 4·55 tr tr 0·10 S·
Bacillus roseus lAM 1257 tr 0·87 0·52 tr 0·15 0·01
Bacillus sphaericus IFO 3525 0·067 20·0 1·05 4·42 0·03 1·24
Brevibacterium divaricatum NRRL 2311 tr tr tr tr 1·25 0·03
Pseudomonas graveolens IFO 3460 tr 4·30 tr tr tr 1·44
a The amounts of biotin-vitamers produced by organisms grown with pimelic acid.
b The amount of biotin-vitamer given by the bioassay using Lactobacillus plantarum, which includes biotin and biotin
sulfoxide.
C 1, pimelyl-CoA synthetase; 2, KAPA synthetase; 3, DAPA aminotransferase; 4, DTB synthetase.
dTrace.
~
biotin, as well as j3-hydroxybisnordethiobotin and tetranordethiobiotin from

DTB in a resting cell system (Osakai et ai., 1986a). Biotinyl-CoA synthetase,
the first enzyme involved in biotin degradation, was purified to homogeneity
from Mycopiana sp. No. 166 (Yamada et ai., 1984; Tanaka et aI., 1986, 1988).
The enzyme was a monomer with a molecular weight of 55 000. The enzyme
catalyzes the stoichiometric conversion of biotin, ATP and CoA into biotinyl-
CoA, AMP and inorganic pyrophosphate. DTB is also effective as a substrate.
5 STRAIN IMPROVEMENT AND GENETICS

As described previously, a strong feedback repression by biotin seems to be
one of the main problems that makes microbial production difficult. This
control must be overcome in order to microbially produce large quantities of
biotin and its vitamers. Some potent biotin antimetabolites have already been
found (Fig. 4) (Izumi & Ogata, 1977). Therefore, it has become possible to use
these antimetabolites for the selection of regulatory mutants producing biotin
and its vitamers.
Actithiazic acid a-Dehydrobiotin
o o
~ ~
N N
H
N N
CH3 ~ CH3
H3C (CH2)3CHCOOH lS).(CH2)3 CHCOOH
a-Methyldethiobiotin a-Methylbiotin
5-(2-Thienylhl- Amic/enomycin
valerie acid
H2NO(CH2hCHCOOH CH3
NHCOCHCHCH2 CH3
NHCH3
Stravidin
Fig. 4. Various biotin antimetabolites.
Pai (1975) and Eisenberg et al. (1975) isolated a-dehydrobiotin-resistant

mutants of E. coli, which showed enhanced excretion levels of biotin,
derepressed levels of the biotin biosynthetic enzymes, and resistance to
repression by biotin. However, the level of their accumulation was still quite
low (less than 100 ng/mI). Since actithiazic acid, or acidomycin (ACM) (Izumi
et al., 1981) and 5-(2-thienyl)-n-valeric acid (TVA) (Izumi et aI., 1978) were
found to be biotin antagonists which inhibited the biosynthetic step of biotin
from DTB and the DAPA aminotransferase reaction, respectively, Yamada et
al. (1983) and Tanaka et al. (1988) induced ACM- and/or TV A-resistant
mutants from B. sphaericus IFO 3525. ACM- and TV A-resistant mutant,
AB12, excreted 7·1 times more biotin (5·35Ilg/ml) than the wild type strain.
The TVA-resistant mutant, A16, excreted 11·6 times more total biotin
(400 Ilg/ml). Strain AB12 accumulated 9'5Ilg/ml of biotin under the optimum
reaction conditions using 1 mg/ml of pimelic acid. Strain AB12 was considered
to have enhanced biotin synthesizing enzyme activity, resulting in survival even
in the presence of ACM, whereas the repression of the enzymes by biotin was
still not diminished.
Recently, the production of biotin by bacteria which were transformed by
recombinant DNA has been reported. Hirono et al. (1986) and Ifuku et al.
(1986, 1987) have derived an a-dehydrobiotin-resistant mutant from E. coli.
They transformed E. coli with the biotin operon coding for the biotin
biosynthetic enzymes. The maximum production of biotin by the transformant
was 121lg/ml under the optimized conditions (Hirono et al., 1986). Osawa et
al. (1987, 1989) obtained an ACM- and TVA-resistant mutant of B. sphaericus
IFO 3525, followed by transforming of E. coli strain with the plasmid carrying
under appropriate culture conditions, the transformant excreted 161lg/ml of
biotin from DL-DTB in a 48-h cultivated medium (Osawa et al., 1989). They
have recently determined the nucleotide sequence of the bio B gene of B.
sphaericus and compared the amino acid sequence of its gene product, biotin
synthetase, with that of E. coli, suggesting conservation in the catalytic
mechanism for both enzymes (Osawa et aI., 1989).
Since the acetate produced in the culture medium was found to inhibit the
growth, Ifuku & Yanagi (1988) derived a tluoroacetate-resistant mutant from
the E. coli transformant, which had a level of acetate production 10 times
lower than the parent strain. When the mutant was cultivated in a glucose-fed
system with a controlled dissolved oxygen level in a jar fermentor, the growth
and the accumulation of biotin reached 55 mg (dry cell weight)/ml and
105 Ilg/ml, respectively.
6 FERMENTATION
6.1 Dethiobiotin Production by B. sphaericus IFO 3525 (Ogata, 1970)
Medium: 10 g peptone, 5 g Casamino acids (Difco), 100 g soybean meal, 20 g
glycerol, 1 g K 2HP0 4, 0·5 g KCI, 0·5 g MgS0 4·7H20, 10 mg FeS04·7H20,
10 mg MnSOc6HzO, 200 Ilg thiamine· HCI and 1 g pimelic acid in 1 liter of

tap-water, pH 7·2.
Inoculum and fermentation course: The cells of the bacterium, B.
sphaericus, from a slant culture are incubated in 30 ml of the medium in a
300-ml shaking flask for 4-5 days at 28°C on a reciprocal shaker
(140 strokes/min).
6.2 Biotin Production by an ACM- and TVA-resistant Mutant ABU of B.

sphaericus IFO 3525 (Yamada et al., 1983)
Medium: 20g glycerol, 50g Proteose peptone (Difco), 5g Casamino acids

(Difco, vitamin-free), 1 g KHzP04, 0·5 g KCI, 0·5 g MgS04·7HzO,
10 mg FeS04'7HzO, 10 mg MnS04·4-6HzO and 20llg thiamine·HCI in 1 liter
of tap-water, pH 7·0.
Inoculum and fermentation course: Cells of bacterium AB12 were inocu-
lated into 3 ml of the medium in a test tube and incubated at 28°C with
shaking. After 1 day of cultivation, pimelic acid was added (1 mg/ml) to the
medium and cultivation was continued at 37°C (optimum temperature for
biotin biosynthesis from DTB) for 3 more days. The addition of the precursor
after 1 day of cultivation is important for biotin production in order to avoid
repression by the produced biotin itself of the biotin biosynthetic enzymes (see
Section 4.2).
7 PRODUcr RECOVERY AND PURIFICATION
Biotin and biotin-vitamers accumulated in the culture media are purified by

active carbon treatment and ion-exchange column chromatography (Ogata,
1970). After centrifugation of the culture medium, the pH of the supernatant is
adjusted to about 2-3 with concentrated HCI-15 g of active carbon is added
to 1 liter of the culture medium and stirred mechanically for 4 h at room
temperature. The active carbon is collected on a Buchner funnel and washed
with about 200 ml of water. The active carbon cake is suspended in about
200 ml of 50% ethanol-28% ammonia water (18: 1, v/v) mixture and stirred
for 4 h at room temperature. The active carbon is filtered off onto a Buchner
funnel, and the active carbon cake is washed with ethanol-ammonia mixture
three to four times. The filtrates are concentrated to dryness in vacuo at room
temperature. The concentrate obtained is dissolved in about 40 ml of 90%
ethanol, and insoluble materials are removed by centrifugation; the clear
supernatant solution is again concentrated to a volume of about 5 ml in vacuo
at room temperature. This concentrated solution is subjected to ion-exchange
chromatography. A few milliliters of the solution of biotin-vitamers (contain-
ing about 20-500 Ilg of total biotin, pH 7·0) is quantitatively taken up onto a
Dowex 1 X2-formate column (0·6 cmz x 19 cm). The column is washed with
100 ml of deionized water, which removes the unadsorbed biotin-vitamers such
as DAPA, then the biotin-vitamers (e.g., biotin, biotin sulfoxide and DTB)
are eluted with 0·012 M formic acid, and lO-ml fractions are collected. Biotin
concentrations are quantitatively determined by microbiological assays with
Saccharomyces cerevisiae and Lactobacillus plantarum.
8 BIOASSAY METHODS
There are three methods for the determination of biotin and its vitamers:
microbiological, chemical and enzymatic assay methods. Table 3 summarizes
the characteristics of the three methods. The microbiological assay has been
most commonly used among the three methods. This assay uses the micro-
organisms Lactobacillus plantarum ATCC 8014 (Scheiner, 1985),
Saccharomyces cerevisiae ATCC 7754 (Gyorgy, 1967) and Bacillus subtilis
AKU 236 (Iwahara et al., 1966c). In addition, a biotin-requiring mutant of E.
coli, C162, (bio B-, His-) has also been used as a test of the growth-
promoting activities of biotin-vitamers (Salib et al., 1979; Osakai et al., 1986b).
These micro-organisms have biotin-vitamer specifi.cities for growth, which
enables us to assay them differentially. Table 4 shows the microbiological
activities of various biotin-vitamers when assayed with L. plantarum, S.
cerevisiae and B. subtilis. The microbiological assay also has the advantage of
high sensitivity. However, one of the main disadvantages of the method is that
it requires the time-consuming cultivation of assay organisms and the laborious
preparation of washed cell suspensions. Tanaka et al. (1987a) have developed
improved assay methods for biotin using lyophilized cells of L. plantarum and
E. coli C162 and glycerol-suspended cells of S. cerevisiae. Figure 5 shows the
standard curves by the paper disk plate method and turbidimetric method
using such cells. Those lyophilized or glycerol-suspended cells, which are
preserved at -20°C, can be used for the assay for more than 1 year or half a
year, respectively.
9.1 Biotin Deficiencies in Animals
Feeding a diet either low in biotin or, more frequently, using a diet amended with
with raw egg white, produced biotin deficiencies in the rat, chicken, poultry, pig,
monkey, man and other species (Gyorgy & Larger, 1968). Chicken and poultry,
however, require an outside source of biotin, even when egg white is replaced
by other proteins. The effectiveness of egg white in producing biotin deficiency
is due to the presence of the glycoprotein avidin, which forms a complex with
biotin that renders the biotin unavailable to the host. Avidin also forms tight
complexes when biotin is the prosthetic group of an enzyme, resulting in the
loss of enzyme activity. The biotin-binding capacity is present not only in the
Table 3
Assay Methods of Biotin and Its Related Compounds
Method Principle Characteristics Assay range Reference

Microbiological Growth of L. plantarum, 1. High sensitivity Paper disc (agar Tanaka et al.
S. cerevisiae, B. subtilis 2. High specificity diftUsion) method: (1987a)
and E. coli to various biotin- 0·1-50 llg/ml
vitamers (as (+ )-biotin)
3. Even crude samples can
be assayed. Turbidimetric method:
4. Time-consuming (6-27 h) 0·2-8ng/ml
Chemical
DACA Coloring reaction of the ureido ring 1. Ureido ring-containing 10-100llg/ml McCormick &
method with p-dimethylcinnamaldehyde biotin-related compounds Roth
(DACA) under anhydrous with no microbiological (1970)
conditions (reddish orange to pink activity can be assayed.
A. max = 533 nm) 2. Fast assay
3. Sample should be dried
before assay
Avidin- Avidin is reacted with the dye, 4- 1. Avidin-combinable 0·4-4Ilg Green (1970)
HABA hydroxyazobenzene-2' -carboxylic compounds (0·5Ilg biotin/ml
method acid (HABA) to form the avidin- (ureido ring compounds) gives M = 0·069)
HABA complex (A. max = 500 om), can be assayed.
followed by the addition of biotin. 2. Fast assay
Since the HABA in the complex is
substituted by biotin, the added
biotin is assayed from the
colorimetrical determination of the
remaining avidin-HABA complex.
Avidin- Given amount of avidin, of which 1. Avidin-combinable >0·1 ng Biotin Hood (1979)
3HC 4C)- biotin combinability (a) is known, is compounds (ureido ring (Variable according Dakshinamurti &
biotin reacted with a sample containing compounds) can to the used amounts Allan (1979)
binding biotin(x), followed by the further be assayed. of avidin:
method addition of excess 3H-biotin. x is 2. Fast assay commercially
obtained from the equation x = a - available avidin binds
b, where b is the combined 3H_ 1-13llg biotin)
biotin.
Enzymatic
BCS- The H 20 2 formed through the coupled 1. High sensitivity 10-200 nmol/ml Tanaka et al.
AOD- reactions of biotinyl-CoA synthetase 2. Fast assay (2 h) (1987b)
POD (BCS) and acyl-CoA oxidase (AOD) 3. BCS is stable
method is assayed by the peroxidase (POD)
reaction, which gives a fluorescent
compound.
PC method The enzymatic binding of biotin in situ 1. High sensitivity 5-2000 pg/ml Haarasilta
to the pyruvate carboxylase (PC) 2. Fast Assay (1 h) (1978)
apoprotein of biotin-deficient bakers' 3. The yeast cells should
yeast and the subsequent estimation be prepared just before
of the PC activity by a 14C02 -fixation assay.
method
Table 4
Microbiological Activities of Various Biotin-Vitamers
Biotin-vitamer Activitya toward

L. plantarum s. cerevisiae B. subtilis
Intermediates of
biotin biosynthesis
(+ )-biotin 100 100 100
( + )-dethiobiotin 0 100 100
( ± )-dethiobiotin 0 50 50
7,8-diaminopelargonic acid 0 10 Weak
pimelic acid 0 0 o
Intermediates of
biotin biodegradation
( + )-bisnorbiotin 0 0 100
( + )-bisnorbiotin sulfoxide 0 0 100
( + )-bisnordethiobotin 0 0 100
Naturally occurring
biotin-related compounds
biocytin 0 30 o
( + )-biotin amide 0 100 o
dethiobiotin amide + +
( + )-biotin D-sulfoxide 100 100 100
( + )-biotin L-sulfoxide 5 0·001-0·1
Chemically synthesized
biotin-related compounds
( ± )-oxibiotin 50 10-25
( + )-selenobiotin 100
( ± )-carbobiotin 12 15
diaminobiotin 0 10 100
( + )-biotin methyl ester 100
a Relative activity of equimolar concentration of each compound.
egg whites, but also in the egg yolks of avian species and the turtle (Moss &
Lane, 1971). Since avidin is heat-labile, prolonged heating of egg white
denatures the avidin and destroys its biotin-binding capacity. Another biotin-
binding protein similar to avidin, streptavidin, is produced by a Streptomyces
strain (Chaiet & Wolf, 1964).
Biotin deficiency produces dermatitis and perosis in chicken and poultry
(Gyorgy, 1968); alopecia, seborrheic skin changes, spasticity of the hind legs
and cracks in the feet of pigs. The activities of the biotin-dependent enzymes
are also decreased (Whitehead, 1981). These enzymes are involved in
carboxylation, transcarboxylation, and decarboxylation reactions, and function
in the vitally important metabolic processes of glucose and fat synthesis (Moss
& Lane, 1971; Wood & Barden, 1977; Whitehead, 1981). Among the most
important enzymes are acetyl-CoA carboxylase, pyruvate carboxylase and
propionyl-CoA carboxylase.
It was generally believed that the combination of biotin in the feed
(A)
E
~4
III
c
0
N
.c
i0
bI
03
~
j
is
2
10 50 100 250 1X10 3 5X10 3 20X10 3

Biotin (ng/ml X 71l1/disk)
(B) 5 (e)
2 4
'"ci3
£
.c
i
&2
02468 0.4 1.2 2.0 2.8

Biotin (ng/ml x 100 III/2m!) Biotin (ng/ml X 100 III/2m!)
Fig. 5. Standard curves of microbiological assays of biotin. (A) Paper disc plate (agar
diffusion) method using lyophilized cells of L. plantarum (x) and E. coli (e) and
glycerol-suspended cell of S. cerevisiae (0). (B) Turbidimetric method using lyophilized
cells of L. plantarum. (C) Turbidimetric method using glycerol-suspended cells of S.
cerevisiae (0) and lyophilized cells of E. coli (e). From Tanaka et al. (1987a).
ingredients plus the biotin produced in the intestine by bacteria supplied

sufficient biotin to meet the poultry's requirement. However, since 1966, a
number of reports on biotin deficiency in commercial flocks have appeared
(Scheiner & DeRitter, 1975). Apparent biotin deficiencies in swine under
commercial conditions were also reported (Scheiner & DeRitter, 1975).
In humans, infant seborrheic dermatitis and the related Leiner's disease are
biotin-responsive (Svejcar & Homolka, 1950). Biotin deficiency has been

reported in individuals during prolonged total parenteral nutrition (Bozian et
al., 1981; Mock et al., 1981). Biotin administration has successfully controlled
multiple carboxylase deficiency even in unborn infants (Baumgartener et al.,
1981; Munnich et al., 1981; Thoene et al., 1981; Roth et al., 1982). It has been
suggested that diseases related to biotin metabolism may be more common
than previously thought (Tanaka, 1981).
9.2 Growth·Promoting Activity
Biotin deficiency also causes a marked decrease in the activities of several

glycolytic enzymes in the liver, e.g. glucokinase, phosphofructokinase and
pyruvate kinase. The activities of these enzymes increased rapidly after the
administration of biotin (Dakshinamurti & Cheah-Tan, 1968, 1970; Dak-
shinamurti & Hong, 1970; Dakshinamurti et al., 1970), while other glycolytic
enzymes such as hexose phosphate isomerase were not affected by biotin
administration. Boeckx and Dakshinamurti (1974) showed that biotin ad-
ministration to biotin-deficient rats resulted in increased stimulation, by more
than twofold, of amino acid incorporation into protein, both in vivo and in
vitro in rat liver, pancreas, intestinal mucosa and skin. They also found that
the synthesis of some proteins such as serum albumin, a major product of the
liver protein-synthetic machinery, was stimulated more than twofold, but
others were not stimulated at all. The effect of biotin on protein synthesis was
preceded by stimulation in the incorporation of orotic acid into nuclear and
ribosomal RNA.
Vesely (1982) and Vesely et al. (1984) found that biotin and its analogs at
0·1-1 IJM enhanced soluble guanylate cyclase activity two- to threefold in rat
liver, kidney, colon, cerebellum, and heart. Since cyclic GMP, a product of
guanylate cyclase reaction, is known to increase the growth of fibroblasts and
thymocytes and also to increase RNA and protein synthesis, these results
suggest that the growth-promoting effect of biotin might be mediated by cyclic
GMP. Spence & Koudelka (1984) also found that addition of biotin in the
presence of insulin elicited an increase in the intracellular content of cyclic
GMP, followed by an increase in glucokinase in cultured rat hepatocytes.
10.1 Method of Goldberg et 01.
The industrial synthesis of biotin which is presently carried out is based on a

method developed by Hoffmann-La Roche, Inc. (Goldberg & Sternbach, 1949;
Gyorgy & Larger, 1968). As illustrated in Fig. 6, the synthesis is characterized
by the use of a meso-diaminosuccinic acid derivative as a starting material,
which contains two groups in the same spatial arrangement as the two amino
/t"
° /t"
° /c"
9
ToaH ~r ~r (a) ~HR ~HR (b) R~ RN 7R (e)
~R (d) R~ ~R (e)
CH=CH _ CH-CH - - CH-CH - - HC--CH __ Ht-CH - HC-CH-
tOOH taaH taoH taaH tOOH taoH taaH ot to AcatH)a
R=benzyl "0/ "0
o
• • IV V Ac=CH 3Ca
VI
RN
)<NR
Ht-tH
Hzt Co
"S/
(j)
Resolution
O-Camphorsulfonate
of (+)-IX
XIU XIV
Fig. 6. Flow sheet of one chemical process for the industrial preparation of ( + )-biotin.
(a) Benzylamine; (b) phosgene; (c) acetic anhydride; (d) Zn, a mixture of acetic acid
and acetic anhydride; (e) H 2S, HCI; (f) C2H50(CH2hMgBr; (g) H 2, Raney nickel; (h)
HBr, acetic acid; (i) silver o-camphorsulfonate; (j) isopropanol; (k) sodium di-
ethylmalonate; (I) HBr. From Goldberg & Sternbach (1949).
groups present (in substituted form) in the biotin molecule, i.e. the meso-
configuration in diaminosuccinic acid derivatives corresponding to the cis-
structure of the two amino groups in a ring compound such as biotin.
Moreover, since resolution is carried out at the intermediate stage (XII-XIII)
it permits the direct production of the optically and biologically active biotin,
( + )-biotin.
10.2 Synthesis from Sugars
Efficient stereospecific total syntheses of (+ )-biotin from the sugars, 0-

mannose, o-glucose and o-glucosamine, were achieved by Ohrui & Emoto
(1975), Ogawa et al. (1977) and Ohrui et al. (1978), respectively. The synthesis
from o-mannose is shown in Fig. 7. o-Mannose is first converted to the
aldehyde (II) through isopropyridenylation, benzoylation, selective isopropy-
denylation, and periodate oxidation. The Wittig reaction and hydrogenation of
<" s~. ~
s COOCH3
H~H"
-H COOCH3
---.. H
-H
H_
~
H-·
-H
OOCH3
-H-
~COOCH
"H
-tI -
H 3
(t)-Biotin
o 0 OR bR
'>< IX: R=H
N3 N3 AcNH HNAc
X: R=CHlSO Z
VII XI
Fig. 7. Stereospecific synthesis of ( + )-biotin from D-mannose. From Ohrui & Emoto
(1975).
the aldehyde yield compound (IV) which has a side chain-like biotin. The
treatment of (IV) with NaOCH3 in methanol, followed by the reduction of the
resulting aldehyde (V) with NaBH4 produces (VII). Treatment of (VII) with
NaS affords a tetrahydrothiophene derivative (VIII) which is converted via
(XI) to (X). Treatment of (X) with NaN3 gives a diazido compound (XI).
Catalytic reduction of the azido groups of (XI) in a mixture of methanol and
acetic anhydride gives a diacetoamido derivative (XII). Treatment of (XII)
with Ba(OHb followed by the treatment with phosgene affords ( + )-biotin.
Thus, (+ )-biotin is synthesized from D-mannose in a good yield, because a
five-membered ring consisting of isopropyridene, which protects the hydroxyl
groups of the sugar, is used to fix the molecular conformation during the
intramolecular substitution reactions.
The most practical uses of biotin are as a pharmaceutical and as a supplement

of culture media for amino acid production, where the biotin-requiring
bacteria such as Corynebacterium glutamicum and Brevibacterium ftavum for
glutamic acid and lysine production are used (Aida, 1986). Recently, the
vitamin has attracted increasing interest as a food and feedstuff supplement
(Pearson et al., 1976; Whitehead, 1981; Kornegay, 1985) as can also be seen
from the studies of its growth-promoting effect as described in Section 9.2.
There may be a marked increase in the demand and supply of the vitamin as a
feedstuff supplement if its price becomes lower. At present, the industrial
preparation of biotin is carried out through a chemical process. The present
price of biotin as a biochemical reagent is US $170·00/10 g (price list of Sigma,
1988). Therefore, the price of bulk supplies can be supposed to be much lower
than the reagent price. In order to economically compete with the present
chemical process, the present production levels via fermentation processes
should be greatly improved (the maximum level so far reported is 105 mg/liter
as described in Section 5). If a fermentation process uses a biotin precursor

such as pimelic acid or azelaic acid, the cost of such compounds should also be
taken into consideration.
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Chapter 15
MICROBIAL PRODUCTION OF VITAMIN B12
C. SPALLA, A. GREIN, L. GAROFANO & G. FERNI

1 INTRODUCTION
From the chemical point of view, the term vitamin B12 is synonymous with
cyanocobalamin, which is by far the most important component of the large
family of the cobalt corrinoids. In this review, however, as in most publica-
tions, the word vitamin B12 is given a very broad meaning so as to include all
the cobalt corrinoids of the cobalamin group. Other cobalt corrinoids which
can be considered analogous or precursors of cobalamins are also included.
Discovered in 1948 and identified as the anti-pernicious anemia factor,
vitamin B12 has been thoroughly investigated from several points of view,
including biochemical significance, biosynthesis in micro-organisms, production
in commercial amounts and applications. The B12 biosynthetic pathway in
micro-organisms is now largely known and good production processes are
available and will be described in this review.
Due to its implications in extremely important biochemical reactions,
vitamin B12 originated wide interest in large and useful applications in human
and animal health care. Many analogues and derivatives were obtained and
studied to find new activities with particular emphasis towards B12 molecules
endowed with anti-cancer activity. Unfortunately, none of these substances
were of particular interest and the application of vitamin B12 remained limited
to anti-pernicious anemia activity, polyvitaminic specialties and as a feed
supplement in husbandry. The interest in the development of vitamin B12
reached a peak in the 1960s and then decreased slowly.
At present, a well consolidated but steady market and good production
processes are in the hands of a very limited number of companies. Like other
vitamins, B12 found its niche and from the economic point of view it can now
be considered as a 'mature product'.
2 HISTORICAL
Vitamin B 12 , a natural product endowed with important biological properties,

was isolated in 1948, almost contemporaneously but independently and from
257
258 C. Spalla et aI.
different sources, by three industrial research groups, i.e. by Glaxo Labora-

tories (Smith, 1948a,b) in the UK and by Merck & Co. Laboratories (Rickes et
al., 1948a, b) and Lederle Laboratories (Stokstad et al., 1948) in the USA.
The two first groups found this compound in liver extracts and identified it as
the anti-pernicious anemia factor responsible for the effective control of the
disease. These experimental programs were related to a continuous study over
a 20-year period of the therapeutic effect of whole beef liver in pernicious
anemia.
In their communication Rickes et al. also reported the presence of significant
amounts of vitamin B12 in media fermented by a grisein-producing strain of
Streptomyces griseus as well as in culture broths from fermentations by
Mycobacterium smegmatis, Lactobacillus arabinosus, Bacillus subtilis, Strep-
tomyces roseochromogenes and Streptomyces antibioticus. The vitamin B12
produced was measured by the microbiological assay based on the growth
response of Lactobacillus lactis (Domer strain) devised by Shorb (1947, 1948).
It was also at about the same time that Stokstad et al. (1948) reported the
production by Flavobacterium solare of a growth promotor, active in the
so-called 'animal protein factor' assay in chicks and effective in the treatment
of pernicious anemia in the human body which was identified as vitamin B 12 .
Microbial synthesis was also implicated in the early studies on the presence of
'animal protein factor' in manures and feces (Cary et al., 1946; Hartman and
Cary, 1946; Rubin & Bird, 1946; Lillie et al., 1948). Subsequent investigations,
which showed that the potency of incubated feces and manures was higher
than the freshly voided material, confirmed the earlier hypothesis of the
importance of micro-organisms in this synthesis (McGinnis et al., 1947; Sahashi
et al., 1953). With this background of information, the finding of vitamin B12 in
sewage was not unexpected (Hoover et al., 1951), and some efforts have been
made to exploit this source (Bernhauer & Friedrich, 1954; Friedrich &
Bernhauer 1958).
Although vitamin B12 is present in small amounts in almost every animal
tissue, it originates from micro-organisms. Depending on the nature of their
nutritional habits and digestive physiology, animals obtain the vitamin from
their own intestinal flora or from other animals through their meat diet. An
exogenous supply is mandatory for man. The importance of microbial synthesis
of this group of vitamins has been summarized as follows by Smith
(1950-1951):
It seems probable that the only primary source of vitamin B12 in nature is
the metabolic activity of micro-organisms; there is no convincing evidence
for its elaboration in tissues of higher plants or animals. It is synthesized by a
wide range of bacteria and actinomycetes, though apparently not to any
extent by yeasts or fungi.
Robbins et al. (1950) have concurred and stated:
It appears probable that the synthetic activity of micro-organisms, especially
bacteria and actinomycetes, is the original source of vitamin B12 in nature.
The elucidation of the chemical structure of vitamin B12 is one of the most
outstanding examples of the successful collaboration of the chemist, the X-ray
crystallographer, and the biologist. The chemistry of this complex molecule has
been reviewed by those actively connected with the research and their
interpretation of the X-ray crystallography (Brink et al., 1954; Hodgkin et al.,
1955, 1957) and the degradative studies leading to the confirmation of the
structural formula, proposed by Dr Hodgkin and associates, should be
consulted for a guide to the literature (Folkers & Wolf, 1954; Wolf & Folkers,
1954; Bonnett et al., 1955; Folkers et al., 1957).
Ten years were spent from the first efforts of Woodward and Eschenmoser
until a full chemical synthesis was achieved (Krieger, 1973; Maugh, 1973). It
turned out to be a very difficult and it involved a group in Zurich, and a group
in Cambridge (UK) with more than 100 people. The synthesis requires about
70 steps and is of no value for industrial purposes. Today, vitamin B12 is
exclusively obtained by fermentation processes utilizing high-producing micro-
organisms or, less frequently, sewage.
Vitamin B12 (or cyanocobalamin) belongs to the large family of the cobalt
corrinoids exhibiting the general formula presented in Fig. 1. The molecule of
cobalamins, which are the most interesting cobalt corrinoids, is formed by the
following entities: a macrocycle in planar position, constituted by 4 reduced
pyrrol rings linked through their N -atoms to a cobalt atom in central position
(corrin) and showing 6 side chains (3 acetamide and 3 propionamide residues).
Almost perpendicular to the macrocycle a nucleotide, formed by phosphate,
ribose and 5,6-dimethylbenzimidazole is linked, through a coordination bond,
to the cobalt atom as shown in Fig. 2. The group of the cobalamins includes
cyanocobalamin or true vitamin B 12 , hydroxocobalmin or vitamin B 12 a,
methylcobalamin or mecobalamin and 5'-desoxyadenosilcobalamin or cob am-
mide or coenzyme B12 of Barker (Barker et al., 1958), characterized by a
cyano, hydrozyl, methyl or 5'-desoxyadenosyl radical respectively (Fig. 2).
Other cobaltcorrinoids with different heterocyclic bases (like substituted
benzimidazoles or purines), have been found in various micro-organisms,
either spontaneously or after the supply of the corresponding base to the
fermentation medium. Pseudovitamin B12 and factor III, in which the base is
adenosine and 5-hydroxybenzimidazole respectively, are the most frequently
encountered. They can be considered analogues of vitamin B 12 . A list of these
compounds is reported by Perlman (1959) and by Marvyn & Smith (1964).
Natural analogues designated as 'incomplete' are also known, for instance
factor B which is devoid of the ribose-phosphate moiety. All the known
analogues showed low activity as growth factors for vertebrates and no
therapeutic effect but they are very potent growth factors for the micro-
organisms. Cyanocobalamin, hydroxycobalamin, methylcobalamin and vitamin
260 C. Spalla et al.
NH,COH,C CH,CH,CONH,
CH,
NH,COH,C CH,
~HCOCH,H,C
H,~ '~ ____ ,_

/CH ",,"'" I
H,C '0><' . . . . . . . . 0- I
-----~~P'o OH : x
I
--------------------~
Cobalamins
x y Trivial name
-CN Vitamin Bl2
-OH Vitamin Bua

-CH, Mecobalamin
C obamam ide or
coenzyme Bu:
Fig. 1. General structure of cobaltocorrinoids with references to cobalamins (Florent &

Ninet, 1979).
B12 coenzyme (5,6-dimethylbenzimidazol-5'-deoxyadenosylcobalamin) are the

only compounds of industrial interest.
About the nomenclature, as shown in Fig. 1, the corrinoid molecule without
the nucleotide is named cobinamide or factor B. When only the base is absent,
the compound is called cobamide and finally the complete form, i.e. with the
5,6 dimethylbenzimidazole attached, is named cobalamin.
The cobalamins are water-soluble compounds which crystallize in red
needles and are present in nature in their coenzyme form. Vitamin B12
coenzyme is very unstable to light and only stable at low temperature (-lOOC)
in aqueous solution. At room temperature it is easily transformed to
hydroxycobalamin which has been firstly isolated from Streptomyces aureofaci-
Vitamin B12 V1tamin B12 Coenzyme
Fig. 2. Structures of vitamin BI2 and BI2 coenzyme (Perlman, 1967).
ens. (Pierce et al., 1949, 1950). This compound is also unstable to light and
temperature and in the presence of cyanide originates the more stable, well
known cyanocobalamin. Methylcobalamin is the active form by which vitamin
B12 is involved in methionine biosynthesis in Escherichia coli from which it was
first isolated (Guest et al., 1962). As the other native forms mentioned above,
it is also unstable to light.
The B12 corrinoids show the best stability at pH ranging from 4 to 6; they are
sensitive to ascorbic acid, to thiols, to gamma rays. They are endocellular
products and are extracted from the cells by treatment with water, neutral
buffer solution, lower alcohols or aqueous acetone. When natural corrinoids
like vitamin B12 coenzyme, hydroxycobalamin or methylcobalamin are to be
obtained, the extraction must be performed in the dark. Such a procedure is
not needed when cyanocorrinoids are wanted. In fact, the addition of cyanide
to the cell material transforms the unstable coenzyme forms to the stable
cyano-form. This procedure is usually applied in industry. A comprehensive
review of the chemical and physical properties of vitamin B12 corrinoids has
been written by Friedrich (1975).

As the research on vitamin B12 progressed, it soon became clear that its best
source in nature is represented by micro-organisms. Actually, it is synthesized
by a wide variety of bacteria and actinomycetes, while its production by yeasts,

fungi and tissues of higher plants or animals has not been reported. At first,
vitamin B12 used for human therapy and as a supplement for animal feeds was
obtained as a by-product of antibiotic-producing fermentations. These included
processes for the production of streptomycin by S. griseus (Schindler &
Reichstein, 1952; Smith & Ball, 1953), neomycin with S. fradiae (Jackson et
al., 1951), chlorotetracyclin by S. aureofaciens (Pierce et al., 1950), and
others. As the demand for the vitamin grew, it became profitable to employ
specific production processes in which vitamin B12 was the only product. A
number of processes were tested and some of them operated on a commercial
scale (Periman, 1959). The products of some of these fermentations were used
mainly for the enrichment of feeds poor in animal protein factor, a use
promoted by Ansbacher et al. (1949), while the pure material was isolated to
be used in human therapy.
As already mentioned, fermented feces and manures showed higher vitamin
B12 content than the unfermented material. Hence, sources for isolation of
vitamin B 12-producing micro-organisms became, among others, hen feces
(Stokstad et al., 1948), poultry litter, intestinal contents of animals, like bovine
rumen (Di Marco & Spalla, 1957) as well as sewage sludge. Among the
organisms shown to produce vitamin B12 there are bacteria from the following
genera; Aerobacter, Agrobacterium, Alcaligenes, Azotobacter, Bacillus,
Clostridium, Corynebacterium, Escherichia, Flavobacterium, Methanobacillus,
Mycobacterium, Propionibacterium, Proteus, Pseudomonas, Rhizobium,
Serratia, Streptococcus, Xanthomonas (Periman, 1959). Significant quantities
of vitamin B12 have shown to be produced by the following species of
actinomycetes: Nocardia rugosa, N. gardneri, Streptomyces albidoftavus, S.
antibioticus, S. aureofaciens, S. aureus,S. farinosus, S. fradiae, S. griseus, S.
olivaceus, S. roseochromogenes and S. vinaceus (Periman, 1959).
The isolation of vitamin B12 formed by the above mentioned micro-
organisms was carried out, and, in many instances, examination of the isolated
materials showed that several types of vitamin B12 and of its analogues were
present. It is not unlikely that there are still some undiscovered vitamin
B 12-related compounds present in some cultures. So, for instance, Friedrich &
Bernhauer (1958) reported to have found in a sample of sewage sludge,
besides the identified c5-benzimidazolecobamidecyanide, some 17 other coba-
mides in their extracts.
Numerous micro-organisms have been considered as a potential source of
vitamin B12 for industrial production (Table 1). Because of their rapid growth
and high productivity, Propionibacterium shermanii and Pseudomonas de-
nitrificans were finally selected for industrial purposes.
The usefulness of an industrial process with methane-utilizing bacteria has
still to be proven, while the one based on the use of hydrocarbons does not
seem attractive (Florent & Ninet, 1979).
Improvement of productivity is achieved by screening for spontaneous or
induced mutants obtained with mutagenic treatments. Whatever the strains
Table 1
Vitamin B12 Production by Different Strains
Strain Yield References
(mg/liter)
Micromonospora sp. 11·5 Wagman et al. (1969)

Nocardia rugosa 14 Farmaceutici Italia (1971a)
Propionibacterium freudenreichii 25 Uelaf (1960)
Propionibacterium shermanii 23 Speedie & Hull (1960)
Methanobacillus omelianskii 8·8 Pantskhava & Bykhovsky (1966)
Protaminobacter ruber 2·5 Kojima et al. (1976)
Coryneformbacterium strain XF 0·2 Dumenil et al. (1979; 1981)
Corynebacterium and Rhodopseudomonas 2·3 Nakao et al. (1974)
Nocardia gardneri 4·5 Kyowa Hakko Kogyo Co. Ltd
(1970)
and the culture conditions may be, it is necessary to add to the medium some
elements essential for vitamin biosynthesis, like cobalt ions and frequently
5,6-dimethylbenzimidazole. The addition of potential precursors such as
glycine, threonine, c>-aminolevulinic acid and aminopropanol proved some-
times to be beneficial.
Investigations on the biosynthesis of the vitamin B12 molecule had mainly the
objective of elucidating the biosynthetic pathway leading to the tetrapyrrole
macrocycle and that leading to the incorporation of the nucleotide to form
vitamin B 12 .
With few exceptions all authors agree with the general pathway shown in
Fig. 3. Two molecules of c>-aminolevulinic acid condense to form por-
phobilinogen which is the basic unit of the macrocycle. Latest studies identify
in uroporphyrinogen III the point where the biosynthesis of vitamin B12 and
that of porphyrins diverge. One of the most striking structural differences
between the corrin and the porphyrin ring system is the lack in the former of
a methene bridge between one of the pairs of pyrroline-type rings.
Even if the sequence of methylations in the course of the biosynthesis of the
corrin ring remains unknown, early work from Shemin's laboratory showed
that all of the methyl groups are derived from methionine (Bray & Shemin,
1963; Shemin & Bray, 1964).
Substances endowed with a strong stimulatory effect on the production of
vitamin B12 are betaine and choline. Ansbacher et al. (1949) reported that out
of all the microbial processes available in 1949, the best ones were charac-
terized by the disappearance of choline from the medium.
Miller & Putter, quoted by Demain et al. (1968), discovered that betaine
Glycine
.--Su-C-Cn-y-l--C-oA----,~lle~~%In:id I-I_X_2__ '-I-----,~_'

x4
----------1
CH, units •
C02+
Cobyrinic acid
amlnopropanol
NH,
5' -deaxyadenosine
5,6-0imethyl-
benzimidazole
r
Riboflavin
Fig. 3. General pathway for the biosynthesis of cobalamins (Florent & Ninet, 1979).
and choline stimulate vitamin B12 production by Pseudomonas denitrificans;

this has been confirmed by Miller & Rosenblum (1960) and by McDaniel
(1961). Stern & Friedman (1960) found these two compounds to stimulate by
as much as five-to-sixfold the formation of vitamin B12 by Rhizobium trifolii,
R. meliloti, Agrobacterium sp. and Bacillus megaterium.
The stimulatory effect of betaine and choline on vitamin B12 production by
Pseudomonas denitrificans grown in a chemical defined medium is shown in
Fig. 4 (Demain et al., 1968). The effect of betaine and choline on vitamin B12
production by P. denitrificans in an industrial medium was studied by
Garofano & Merli of these laboratories (unpublished data). The complex
medium currently utilized for industrial production contains high concentration
of sugar beet molasses which, being particularly rich in betaine, is a very good
20
18
16
14
- 12
e
~10
<.
N 8
eli
c: 6
°eItJ
+-
:;: 4
2
00 1 2 3 4 5
Additive (mg Iml)
Fig. 4. EtIect of betaine and choline on vitamin BJ2 production by Pseudomonas
denitrificans (Demain et aI., 1968).
raw material for vitamin Bt2 production. Even in this case, however, addition
to the medium of an extra amount of betaine exerts a stimulatory effect on the
produced vitamin Bt2 which increases from 90 to 140 Ilg/ml. Under the same
conditions betaine can be substituted by choline with similar results provided
the medium be suitably buffered with CaC03 •
As far as the biochemical mechanism· of the stimulation of vitamin B12
production by betaine and choline is concerned, it has been demonstrated that
it is not due to the methyl groups' donation to the corrin ring of vitamin B t2 , as
methionine only is the precursor of these 'extra' methyl groups (Demain &
White, 1971). According to Demain et al. (1968) and White & Demain (1971),
betaine is an absolute requirement also for porphyrin over-production and it is
needed by P. denitrijicans for over-production of both corrins and porphyrins.
Although the site of betaine action in the biosynthetic pathway is unknown,
these data point out that betaine may be a positive effector for some early
common step of corrin and porphyrin synthesis.
6 IMPROVEMENT OF VITAMIN B 12-PRODUCING STRAINS

The industrial production of vitamin B t2 , likewise that of many other microbial
metabolites, is carried out utilizing strains resulting from long lasting and
266 c. Spalla et al.
intense programs of genetic modification directed to improve their characteris-

tics, mainly, but not only, by increasing their productivity (Kyowa Hakko
Kogyo Co. Ltd, 1970; Nakao et at., 1974; Pliva Co., 1964; Pierrel S.p.A.,
1965).
These programs consist essentially of the treatment of the producing
micro-organisms with a mutagenic agent and of the selection of the strains
bringing mutations leading to some practical advantage (higher productivity,
good genetic stability, resistance to higher concentrations of substances present
in the medium, higher rate of growth, etc.). As this kind of mutant is
extremely rare (of the order of 10-5), their selection obtained by direct
examination of all the strains deriving from the mutagenic treatments is a very
tedious and long-lasting task. As soon as knowledge on the biosynthetic
pathway progresses, this essentially random technique is implemented and
more rational and productive techniques are adopted.
Since an inverse correlation had been found between vitamin B12 and
porphyrin productions (Di Marco et at., 1961), porphyrin-less mutants of
vitamin B l2-producing strains were searched. This was done in a very simple
way with Nocardia rugosa, taking advantage of the fact that porphyrins are
excreted in the medium surrounding the colonies on the Petri dishes forming a
reddish halo. Mutants without halo were easily detected by direct examination
of the isolation plates. Out of 180 haloless isolated colonies, about 30 proved
to be really unable to produce porphyrins and two of them showed a significant
increase in vitamin B12 production (Marnati & Spalla, unpublished data).
A more sophisticated technique leading to the same results in
Propionibacterium shermanii has been described by Barrere et at. (1981). The
catalase is an enzyme with a porphyrin moiety. Hence, mutants unable to
synthesize porphyrins cannot produce catalase and, on the other hand, among
mutants lacking catalase activity there are good chances of finding strains
unable to synthesize porphyrins. Catalase-less mutants were easily identified
on Petri dishes because they were unable to develop oxygen bubbles from a
drop of oxygen peroxide put onto the colony surface.
Other techniques consist of looking for mutants resistant to high concentra-
tions of the vitamin BI2 precursor 5,6-dimethylbenzimidazole or to the
antimetabolite ethionine and others. The increase of vitamin B12 productivity,
like that of other metabolites, has been a stepwise process. A significant
example of strain improvement referred to P. denitrificans is shown in Fig. 5.
7 PRODUCTION OF VITAMIN BI2
During the last 30 years, several reviews dealing with research on vitamin B 12
production have been published (Perlman, 1959, 1967; Prescott & Dunn, 1959;
Marvyn & Smith, 1964; Friedmann & Cagen, 1970).
I@ I®
~nloO,<Jg/ml)~(l50,<Jg/ml)
Fig. 5. Mutation and selection program for the improvement of vitamin B12 production
by Pseudomonas denitrificans. The mutagens employed are the following ones: 1,
ultraviolet; 2, ethyleneimine; 3, nitrosomethyluretane; 4-7, N-methyl-N-nitro-N-
nitrosoguanidine; 8,10, ultraviolet-bromouracil; 9,11, psoralene near UV light (Garo-
fano & Merli, unpublished data).
The main objectives of the research work according to Florent & Ninet
(1979) have been:
to acquire a better knowledge of the biosynthetic pathway in order to
support biochemical and genetic studies;
to improve strains which turned out to be the most useful ones (propioni-
bacteria and Pseudomonas);
to replace traditional sugars by more economical nutrients in the media;
to improve extraction and purification techniques.
7.1 Production by Propionibacteria
Several microaerophilic propionibacteria produce cobaltocorrinoids in conven-

tional carbohydrate media supplemented with cobalt and without aeration. For
this process Propionibacterium freudenreichii A Tee 6207, P. shermanii A Tee
13673 and their mutants which can synthesize their own dimethyl benzimidazole
268 c. Spalla et aI.
STOCK CULTURE
Lyophilized with skim milk
1
MAINTENANCE CULTURE
Test tube with medium (a) incubated 4 days at 30°C
1
SEED CULTURE - FIRST STAGE
2-liter Erlenmeyer flask with 0·4 liter of medium (b) incubated 2 days at
30°C without agitation
1
SEED CULTURE - SECOND STAGE
30-liter stainless steel fermentor with 10 liters medium (c), incubated 24 h
at 30°C without aeration and with frequent adjustment of pH to 6·5 with
aqueous NH 4 0H solution
1
PRODUCfION CULTURE
SOO-liter stainless steel fermentor with 340 liters of medium (d) sterilized
40 min at 120°C, inoculated with 7 liters of second-stage seed culture and
incubated at 30°C, during the first 80 h under slight nitrogen pressure (no
aeration) and with slow agitation, then during the next 88 h with agitation
and aeration (2 m3 h): pH adjusted to 7·0 by aqueous NH 4 0H addition
along the whole fermentation
Fig. 6. Vitamin B12 from Propionibacterium shermanii. Semi-pilot plant scale fe~IlI:en
tation process. Medium (a): tryptone, 10 g; yeast extract, 10 g; filtered tomato JUice,
200 ml; agar, 15 g; tap-water to 1 liter; pH adjusted to 7·2. Medium (b): medium (a)
without agar. Medium (c): corn-steep liquor, 20 g; dextrose, 90 g; tap-water to 1 liter;
pH adjusted to 6·5. Medium (d): corn-steep liquor, 40 g; dextrose (sterilized separ-
ately), 100 g; cobalt chloride, 20 mg; tap-water to 1 liter; pH adjusted to 7·0. (Adapted
from Florent & Ninet, 1979).
Microbial Production of Vitamin B 12 269
(DBI) are the most useful ones. Since aeration favours DBI formation, the use
of a two-stage culture is advisable in order to obtain the highest yields. In the
first stage, anaerobic culture is run to almost total depletion of sugar which
promotes the growth of the bacteria and cob amide biosynthesis. Then, in the
second stage, an aeration shift leads to DBI biosynthesis and conversion of
cobamide to cobalamin. The two stages can be carried out batchwise in the
same tank or continuously in two connected fermentors (Speedie & Hull,
1960). Almost no information is available on strain preservation and culture
maintenance conditions.
The fermentation media consist mainly of glucose or inverted molasses
(50-100 g/liter) with small amounts of ferrous, manganous and magnesium
salts in addition to cobalt salts (60-100 mg/liter), of buffering or neutralizing
agents and nitrogenous compounds. Among these, yeast preparations, casein
hydrolysates and other traditional sources are currently used. Corn steep
liquor (50-70 g/liter) is frequently preferred since it supplies some lactic and
pantothenic acids which enhance the growth of the bacteria. Generally, the
temperature of the culture is 30°C and the pH is controlled at around 6·5-7·0.
Reports indicate yields of 25-40 mg/liter of vitamin B12 by propionibacteria.
Figure 6 gives an example of a current process using P. shermanii.
7.2 Production by Pseudomonas
Several Pseudomonas species are vitamin B12 producers but currently the most
used species is P. denitrificans (Miller & Rosenblum, 1960). In contrast to the
propionibacteria fermentation, that of Pseudomonas is characterized by the
fact that its growth parallels cobalamin biosynthesis under aerobic conditions
throughout the whole fermentation process.
Accordingly, fermentation is conducted with aeration and agitation in a
single tank, batchwise or continuously. The strain requires traditional nutri-
ents, such as yeast extract, sucrose and several mineral salts in the growth
medium. In order to enhance vitamin B12 production, the medium has to be
supplemented at the beginning of the culture with 10-25 mg/liter of DBI and
40-200 mg/liter of cobaltous nitrate (McDaniel, 1961; Daniels, 1970).
Likewise, betaine and to some extent choline, have favorable effects in
activating some biosynthetic steps as mentioned before. Glutamic acid
stimulates the growth of bacteria (Daniels, 1966; Koike & Hattori, 1975).
Owing to its low costs and high betaine and glutamic acid content, beet
molasses are preferentially used in industrial fermentations at a 60-120 g/liter
concentration in conjunction with 2-5 g/liter of ammonium phosphate and
some oligoelements. The optimum temperature is 28°C, with a pH around 7·0.
By mutation and selection of proper strains, vitamin B12 production rose
from 5 to 120-140 mg/liter within the last 20 years (Long, 1962; Lago &
Demain, 1969; Ferni & Pennella, unpublished data). The process is outlined in
Fig. 7. In Table 2 the composition of typical media for vitamin B12 production
with selected mutants of P. denitrificans is reported.
STOCK CULTURE
Lyophilized with skim milk
1
MAINTENANCE CULTURE
Test tube with medium A incubated 4 days at 28°C
1
SEED CULTURE-FIRST STAGE
2-liter round-bottomed flask with 0·6 liter of medium E inoculated with a
working culture slants and incubated 48 hr at 28°C on rotating shaker at
120 r.p.m. with 90 mm excursion.
1
SEED CULTURE-SECOND STAGE
40-80 liter stainless steel fermentor with 25-50 liter medium E inoculated
with 1-1·2% seed culture first stage, incubated 25-30h at 32°C, 200
r.p.m. agitation 0·2 bar pressure, 0·5 v/vm' aeration.
1
PRODUCTION CULTURE
500-liter stainless steel fermentor with 300-liter medium P inoculated with
5% seed culture second stage, incubated 140-160 hr at 32°C, 0·2 bar
pressure. Impeller speed regulated in order to assure sufficient oxygen
during the first phase of growth (20-30 hr) and then to maintain constant
the oxygen consumption at the level reached during the first phase. The
fermentation lasts 140-150 h and gives a production of about 120-
130,ug/ml
Fig. 7. Vitamin B12 from Pseudomonas denitrificans. Pilot plant scale production. For
media composition see Table 2. (Femi & Pennella, unpublished data.).
Table 2
Media for the Production of Vitamin B12 with Pseudomonas denitrificans
Media (g/liter)
A E P
Sugar beet molasses 100 70 105
Sucrose 15
Betaine 3
Choline
Ammonium phosphate 2 0·8
Ammonium sulfate 0·25 2 2·5
Magnesium sulfate 0·2 0·2 0·2
Zinc sulfate 0·02 0·02 0·08
5-6 dimethylbenzimidazole 0·005 0·025
Cobalt chloride
Difeo agar 20
Tap water 1000 toOO 1000
pH 7·2 7·2 7·2
An important parameter to follow during the production phase is the level of

dissolved .oxygen in the culture. At the beginning of the process the
concentration of the oxygen must be sufficiently high in order to avoid
limitation in the ratio of growth (p02 > 0). During the production phase, on
the contrary, oxygen becomes the growth limiting factor and this somehow
induces the onset of vitamin B12 production. It is therefore necessary to supply
oxygen (air) in limited amounts so as to maintain the p02levei at a value near
to zero.
8 PRODUCTION OF VITAMIN B12 DERIVATIVES
8.1 Hydroxocobalamin
This compound is produced by cultures of numerous bacteria and streptomy-

cetes and by P. shermanii (Ilieva, 1971). During the extraction, successive
transformations of native cobalamins in their sulfate, nitrate and chloro
derivatives, are required before a final hydrolytic treatment by Amberlite IRA
400 (OH-), to generate hydroxocobalamin (Kaczka et al., 1956; Pierrel
S.p.A., 1963a). The chemical synthesis from cyanocobalamin can also be
performed through sulfite and nitrite derivatives (Pierrel S.p.A., 1963b; Smith,
1965).
8.2 Deoxyadenosylcobalamin (Coenzyme Bu)
Coenzyme B12 is directly extracted from P. shermanii (Chinoin-Gyogyszer,

1971; Ilieva & Popova, 1974), from P. freudenreichii (Sifa, 1964) and from
Nocardia rugosa (Farmaceutici Italia, 1971a), where it can represent up to

80% of the total native cobalamins.
Coenzyme B12 is always endocellular; after harvest of the cells by centrifu-
gation, extraction is performed in the cold by an acetone-water mixture, or at
80-100 C during a short time by a 2% phenol-aqueous solution or an
D
ethanol-water mixture. All operations have to be conducted in the cold, under

subdued light and avoiding any drastic pH condition, in the presence of
cyanide in order to reach a global yield of 80-85% in the extraction process
(Kaken Kagaku, 1965; Chinoin-Gyogyszer, 1971).
9 ISOLATION OF VITAMIN B12
During the last 30 years the extraction processes have been improved many
times; they usually include the following main steps: solubilization of cobala-
mins and conversion to cyanocobalamin and isolation of a crude product, 80%
pure (utilizable directly for animal feeding), followed by purification to a
95-98% level (for medical use). Usually the whole broth or an aqueous
suspension of harvested cells is heated at 80-120 C for 10-30 min at
D
pH 6·5-8·5 in order to extract the vitamin B 12 . The conversion to cyanocobal-

amin is obtained by treating the heated broth or cell suspension with cyanide
or thiocyanate.
The extraction operation units are the classical ones and they are combined
to achieve an efficient and inexpensive process. An extraction process
presently used is briefly outlined in Fig. 8. For further details see Florent &
Ninet (1979).
10 BIOASSAY METHODS
The usefulness and value of microbiological assays in studying biosynthesis of

vitamin B12 and in its detection in natural materials cannot be overemphasized.
Indeed, considering it in retrospect, the vitamin B12 isolation by the American
group of researchers at Merck in comparison to the English group at Glaxo
was due to the possibility of the former one to make use, instead of clinical
tests, of the microbiological method. This was based on the growth response of
Lactobacillus lactis (Dorner strain) to the vitamin as first described by Shorb
(1947, 1948).
Although the sensitivity of this micro-organism to vitamin B12 is high, the
fact that it responds to ribosides and desoxyribosides and that its growth
response is influenced by changes in aeration of the culture as well as by
genetic variability of the culture (Shorb & Briggs, 1948), led to its substitution
with Lactobacillus leichmanii strains ATCC 4797 and A TCC 7830 which gave
more reproducible results (Skeggs et al., 1948). This turbidimetric assay found
general acceptance for the analysis of fermentation samples and other
P. denitrificans Heating 30 min. at 120°C Cooling, adjust-

broth ment of pH to 8·5
Addition of KCN, agitation 16 h at 25°C
Addition of zinc chloride (200 g)
Adjustment of pH to 8·0
Addition of filter-aid (200 g), agitation,
filtration
!
Filtrate Extraction 3 times with 350 ml of a cresol
and carbon tetrachloride mixture (1: 2
ratio)
!
Organic extract I Addition of 500 ml n-butanol
Extraction 3 times with 100 ml water
!
Aqueous extract Extraction 3 times with 30 ml of a cresol
and carbon tetrachloride mixture (1: 2
ratio)
!
Organic extract II Addition of 200 ml acetone and 100 ml
ether
Crude vitamin B12 Dissolution in 10 ml methanol

Chromatography on activated aluminia
(4 g). Elution with 2% acetic acid in meth-
anol. Precipitation with ether
!
Pure vitamin B12
Fig. 8. Crystalline vitamin B12 isolation process (adapted from Florent & Ninet, 1979).
materials, although a number of problems were encountered. Further inves-

tigations showed that all of the vitamin B I2-requiring lactobacilli respond, at
varying degrees, to all of the cobamides but not to cobinamide. These
differences in response have been used as the basis for differential-type assays
but unsatisfactory results have been obtained when samples contained more
than two types of cobamides.
Davis & Mingioli (1950) reported that certain mutants derived by treatment
of E. coli (ATCC 9637) with UV light required either vitamin B12 or
methionine for growth. One of these (strain 113-3) has been, and still is,
widely used as a test organism in bioassay for the determination of the
cobamide content of fermentation materials and other natural products. As
this strain responds to all of the cobamides as well as to cobinamide (Ford &
Hunter, 1955) it is very useful for the detection of all of these factors in
bioautographs of paper chromatograms of such solutions (Ford & Holdsworth,
1952). This strain is cultivated in a very simple medium and can be used in the
agar-diffusion assay as well as in the turbidimetric assay method which is more
sensitive.
The usefulness of the growth response of certain protozoa as a method of
measuring the vitamin BI2 content of samples of natural products was first
reported by Hutner et al. (1949). They found that Euglena gracilis is vitamin
BIZ- and thiamin-requiring. In this case chlorophyll production under light
serves as a measure of the vitamin BI2 content in the samples. All of the
cobamides stimulate the growth of this organism, but some problems in
assaying fermentation samples are encountered as a result of the strain
sensitivity to antibiotics which may be present in them (Robbins et al., 1953).
This test is recommended for vitamin BI2 determination in serum.
Another protozoal species useful in bioassay for cobamides is Ochromonas
malhamensis (Ford, 1953; Hutner et al., 1953) which has been, and still is,
widely used since the finding that only 5,6-dimethylbenzimidazolecobamide
stimulates its growth. Because of this benzimidazole-cob amide specificity, O.
malhamensis is used in substitution of animal tests like chicks and others
where, as in man, only these compounds are active.
The possibility of using chemical or physical methods for the estimation of
the cobamides content in fermentation samples or other crude biological
materials has been attempted. In these cases it is mandatory to perform
preliminary separations of the cobamides from other substances contained in
the samples by thin-layer or column chromatography (Tortolani et al.,
1970a,b; Vogelmann & Wagner, 1973; Tortolani & Mantovani, 1974), or
electrophoresis (Tortolani & Ferri, 1974). Afterwards, several classical meth-
ods are available to assay isolated fractions or pure products; spectrophot-
ometry in the visible spectrum (Farmaceutici Italia, 1971b; Celletti et al., 1976)
or in the IR spectrum (Goldstein et al., 1974), atomic absorption (Whitlock et
al., 1976), potentiometry (Goldstein & Duca, 1976) and enzymatic analysis
(Hermann & Mueller, 1976; Schneider, 1979).
The radioisotope dilution method is widely used (Cooper et al., 1979; Begley
& Hall, 1979; Beck, 1979). Also the application of HPLC for rapid corrinoid
determination has been reported (Jacobsen et al., 1979).
The biological properties of vitamin B12 are many and this has never been
found for any other natural product; interesting biochemical, haematological,
neurological and oncological aspects in man have been reported. This
physiological versatility can be explained by the fact that vitamin B12 is
involved in the functioning of numerous enzymes, which have vital roles in
assuring a normal, undisturbed evolution of the metabolism.
11.1 Biochemical mechanisms
Historically, among vitamin B12-dependent enzyme reactions, the isomeriza-

tion of L-glutamic acid to /3-methylaspartic acid is the most important one
because its study led to the isolation of vitamin B12 coenzyme (Barker et al.,
1958). This enzyme, found in Clostridium tetanomorphum, in Acetobacter
suboxydans (Kato et aI., 1968), in Rhodopseudomonas sphaeroides and
Rhodospirillum rubrum (Ohmori et al., 1971) was also investigated in the
animal system (plasma of the sheep) where it was found to be vitamin
B12-independent (Marston et al., 1961). On the other hand, among biochemi-
cal disorders caused by vitamin B l2-deficient diet in man, a decreased
polyglutamate biosynthesis has been observed (Reed et al., 1979).
The degradation of propionic acid in higher organisms and its formation in
bacteria is performed by methyl-malonyl-CoA which is present in a catalytic
equilibrium with succinyl-CoA and regulated by a specific isomerase. Smith &
Monty (1959) showed that in liver homogenates of vitamin B 1rdeficient rats
the methyl-malonyl-CoA-mutase activity was strongly reduced. Successive
experiments performed on mitochondria from rat liver (Gurnani et al., 1960),
beef liver (Stern & Friedman, 1960), sheep kidney (Lengyel et al., 1960) and
extracts from Propionibacterium shermanii demonstrated the necessary pre-
sence of adenosylcobalamin for the functioning of methyl-malonyl-CoA-
mutase which has been isolated from Propionibacterium shermanii
(Kellermeyer et al., 1964), from liver (Cannata et al., 1965) and more recently
also reported for Rhodospirillum rub rum and Rhodopseudomonas sphaeroides
(Friedrich, 1975).
The presence of methyl-malonyl-CoA-mutase in animal tissues has been
reported by Cardinale et al. (1969) while its determination in leucocytes has
been described by Whitaker & Giorgio (1973). High levels of this enzyme were
observed in leucocytes of vitamin B 12-deficient patients as in rat tissues affected
by the same disorder. Degradation of methyl-malonyl-CoA through succinyl-
CoA is of vital importance because of the toxicity of methylmalonic acid
276 C. Spa//o et aI.
(methylmalonylacidemia). Similarly a defective function of methylmalonyl-

CoA racemase due to vitamin Bt2 deficiency causes accumulation of propionic
acid which is also toxic (propionicacidemia). The ribonucleotide reductase, an
enzyme catalyzing the reduction of ribonucleotides to desoxyribonucleotides
has been demonstrated as vitamin B t2-dependent in Lactobacillus leichmannii
and in Euglena gracilis (Beck, 1968). Its vitamin B12 dependency in animals is
still under discussion (Hopper, 1979).
In many micro-organisms and animals, the methionine synthetase which
necessitates as methyl donor the 5-methylhydrofolic acid, is vitamin B t2-
dependent. The methioninsynthetases (methyltetrahydrofolic acid-
homocysteinmethyltransferase) from Escherichia coli and from liver, are very
similar and have identical coenzyme requirements. Some micro-organisms can
perform this synthesis even through a vitamin Bt2-independent pathway.
Similarly, animals may have an alternative way of methionine synthesis: rats
deficient in methionine, folic acid and vitamin Bt2 respond to betaine and
homocystein utilizing the enzyme betain-homocysteine methyltransferase
which catalyses the CH3 transfer from betain to homocysteine under formation
of methionine (Weissbach & Tylor, 1970).
Today, the mechanism generally accepted which explains the anti-anemic
activity of vitamin Bt2 is based on the interrelationship between the vitamin,
folic acid and methionine (Weissbach & Tylor, 1970). DNA synthesis
necessitates thymidilic acid which is produced by thymidilatesynthetase and
catalyzed by tetrahydrofolic acid derived from CHr-FH4 which in turn is
originated by the vitamin B t2-dependent methioninesynthetase.
In the absence of vitamin B t2 , folic acid is accumulated as CH3-FH4 and the
FH4 amount falls under the permissible level needed for the normal function-
ing of the entire cycle. In this case, vitamin Bt2 plays, in contrast to folic acid,
a secondary role as anti-pernicious anemia factor. The mechanism here
described is known as the 'folate trap'.
Vitamin Bt2 is also involved in the nucleic acid synthesis. In fact,
experimental data from systems where this synthesis is particularly active like
neoplastic mouse cells (Rotherham et al., 1971) and lymphocytes of vitamin
B t2 -deficient patients (Haurani, 1973), showed the existence of the inter-
relationship thymidine-vitamin B t2-folic acid. Vitamin Bt2 participation in the
functioning of methanesynthetase has been reviewed by Stadtman (1967) and
is important insofar as there are production processes of vitamin Bt2 employing
methanobacteria, currently under investigation (Florent & Ninet, 1979;
Dumenil et al., 1979, 1981).
11.2 Absorption, transport, metabolism and elimination
The physiological pathway of vitamin Bt2 transport is rather well known:

intrinsic factor (IF) secreted by the gastric mucosa picks up the minute
amounts of vitamin Bt2 out of the huge bulk of food ingested and mixed
thoroughly in the stomach. The firmly-bound vitamin Bt2 is well protected
Microbilll Production of Vitamin B12 277
against both the greediness of intestinal micro-organisms and loss in the stools
and is safely transported down to the gastrointestinal tract until it reaches a
section of the distal ileum, where it is reabsorbed by the intestinal mucosa.
This process is mediated by specific receptors of the mucosa cells for the
intrinsic-extrinsic-factor complex. Within the intestinal mucosa cells the
intrinsic-extrinsic-factor complex is split, and vitamin Bt2 is now transported
across the cell for reabsorption from its vascular contact side.
The IF (intrinsic factor of Castle; Castle et al. 1930) is a glycoprotein of
55 000-60 000 mol. wt showing variable sugar moieties according to the animal
species bearing it. The IF of pigs and man are very similar and this fact helped
much the elucidation of the absorption mechanism it plays. It is assumed that
IF possesses two receptor sites, one for the vitamin and the other for the
microvilli of the epithelial cells of the ileum. The absorption takes place at a
neutral pH and in presence of Ca or Mg ions (Shinton, 1972). As for the
formation of the IF-vitamin B12 complex, the presence of 5,6-benzimidazole
seems essential (Friedrich, 1975). There are other vitamin Bt2 binder proteins
isolated from the blood serum, named transcobalamins (TC). Among them TC
I and TC II are the most important ones. The biological turnover of vitamin
Bt2 in human liver is much lower than for other soluble vitamins, being its
half-life over 12 months.
Vitamin Bt2 deficiency causes pernicious anaemia, a disease which in the past
usually led to death within 1 month after detection and inevitably within 3 years.
As mentioned before, classical observations and experiments revealed the
cause of the disease in the lack of intrinsic factor produced in the stomach.
This factor was necessary for the reabsorption of vitamin B 12 . The anaemia is
hyperchromic, i.e. there is more haemoglobin available than red cells.
Maturation and division of erythroblasts are disturbed and the resulting cells
are too few and too big, i.e. macrocytic and megalocytic. Similar deficiencies
are seen in the neutrophyls which are hypersegmented and diminished in
number. These anomalies can be reversed by parenteral injection of extrinsic
factor (vitamin B 12) which immediately releases the inhibition of cell matura-
tion and corrects the anaemia in a few days. Patients affected by pernicious
anaemia show a series of disorders like bone marrow depression, transverse
myelitis (myelopathy), peripheral neuropathy and subacute combined de-
generation (Reynolds, 1979). A report on clinical diseases related to de-
ficiencies of vitamin B12 transport proteins is given by Hitzig et al. (1979).
12 FORMULATIONS, APPLICATIONS, ECONOMICS
Vitamin B12 deficiency is revealed best by its serum level, since experiments on
the presence and determination of this compound showed that plasma and
serum are primarily indicative of this disorder, other tissues or organs being
affected only later on (Sullivan, 1970). The normal Bt2 serum level is
200-900 pg/ml and its total binding capacity is about 500-1100 pg/ml so that
the serum is saturated by only about 60% with vitamin B 12 . A serum B12 level
of about 200 pg/ml is considered critical. Clinical deficiency shows serum
concentrations of 150-80 pg/ml. Patients affected by leukemia show significant
higher serum levels due to a TC I increase. The presence of several cobalamins
in the plasma and the prevalence among them of methylcobalamin was
demonstrated first by Lindstrand & Stahlberg (1963) and Lindstrand (1964).
The optimal requirement of a healthy person for vitamin B12 is 0·5-1/-lg daily
according to Herbert (1968). The normal daily turnover of vitamin B12 is
2·5/-lg. In order to have a reabsorbtion of l/-lg of vitamin from an oral dose of
20/-lg, an amount of IF factor is required capable of binding 5-10 /-lg.
A vitamin B12 nutritional-dependent deficiency is rare and observable only
after about 10-20 years; it usually occurs in persons who live on a pure
vegetable diet. Oral treatment of vitamin B12 deficiency caused by the absence
or a defective function of IF allows a low level of reabsorbtion varying from 1
to 3% of a dosage of 100·000 /-lg. Therefore, a maintenance dose of about
300 /-lg daily of cyanocobalamin at intervals of 3-4 days is suggested.
According to Berlin et al. (1968) even higher doses (5OO-1OO0/-lg daily) are
suggested. Tablets of 10 and 100 mg are available. In case of parenteral
treatments in presence of minor vitamin B12 deficiencies a dosage of
500-1000 /-lg of hydroxycobalamin injected 3-4 times at regular intervals
within a year is suggested (Heinrich, 1970). Neuropathies caused by vitamin
B12 deficiency need massive dosages of the order of 1000/-lg daily for the first
week followed by 1000 /-lg twice a week for several months administered by
injection (Friedrich, 1975). The assumption that vitamin B12 lacks side-effects
even at high doses has to be revised.
Vitamin B12 is available in numerous forms and grades. As the US market is
still using almost exclusively cyanocobalamin, the major portion of B12
material in the world keeps being distributed in that form. However, in
Europe hydroxycobalamin is preferred since it shows a better uptake in the
liver, less urinary excretion and a more sustained serum level than cyanocobal-
amin (Heinrich, 1970) and permits a less frequent dosage regimen than needed
for the cyano form. Hydroxycobalamin is often combined with cob amide
(vitamin B12 coenzymes).
The pharmaceutical use of vitamin B12 varies considerably. In the United
States injections of vitamin B12 used as a general boost against tiredness as well
as a remedy for miscellaneous aches and pains are virtually unknown; in many
other countries, particularly in France, Italy and Spain the above-mentioned
indications are the predominant ones. On the other hand, the US is by far the
single largest market on a per capita basis, for vitamin B12 used in dietary
supplement formulations; straight vitamin B12 tablets are available in US stores
in strengths of 100 /-lg. Vitamin B12 finds a fairly large use as feed supplement.
Generally it is dosed into all animal feeds in Europe and the USA with the
exception of ruminants. The dosage levels are of 10-30 mg/ton of feed for
poultry, pigs and for calves as milk replacer.
The overall world market of vitamin B 12 , including both pharmaceutical and
animal feed use, is around 3 tons/year which is not expected to increase in the
near future. The bulk price varies from 3 to 4 US $/g. The main producers of
this vitamin are Rhone-Poulenc and Roussel-Uelaf (France), Glaxo (UK),
Merck (USA) and Medimpex (Yugoslavia).
ACKNOWLEDGEMENT
The help of Anna Tomassoni in the translation and typing of the text is
acknowledged.
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Chapter 16
MICROBIAL PRODUCTION OF OROTIC ACID

(VITAMIN B,3)
K. TAKAYAMA* & A. FURUYA
Kyowa Hakko Kogyo Co, Ltd, Tokyo Research Laboratories 3-6-6, Asahi-
machi, Machida-shi, Tokyo, 194, Japan
ABBREVIATIONS
ATC, aspartate transcarbamylase; AU, 6-azauracil; CPS, carbamyl phosphate

synthetase; CTP; cytidine-trisphosphate. DHO, dihydroorotase; DHOdeh,
dihydrooroatate dehydrogenase; OA, orotic acid; OMP, oritidine 5'-mono-
phosphate; OMPdec, OMP decarboxylase; OPRT, orotate phosphoribosyl-
transferase; OR, orotidine; PRPP, phosphoribosylpyrophosphate; pyr, pyrimi-
dine; UMP, uridine-monophosphate; ura, uracil
1 INTRODUCTION
Orotic acid was first discovered in cow's milk in 1905 and was later found to
accumulate as an intermediate in the biosynthetic pathway of pyrimidine
nucleotides in a variety of mutants of micro-organisms. On the other hand, a
similar substance-which was a growth factor for rats, chickens and lactic acid
bacteria-had been isolated from distillers' dried solubles and had been known
as vitamin B 13 . It was proved in 1953 that orotic acid and vitamin B13 are
identical. Orotic acid is generally formed in mammals, so it is not a vitamin in
a strict sense.
Several reviews have been published on the production of orotic acid
(Furuya, 1976; Enei, 1984; Kuninaka, 1986) and the biosynthesis of pyrimidine
compounds (O'Donovan & Neuhard, 1970; Shiio, 1972).
In this chapter, recent aspects of the microbial production of orotic acid by
the de novo pathway will be described.
2 HISTORICAL
Orotic acid has a complex history. It was isolated from the whey of cow's milk
(Biscaro & Belloni, 1905) and subsequently found to be present in the milk of
* Present address: Central Research Laboratory, Shikishima Baking Co. Ltd, 3-5,
Shirakabe, Higashi-ku, Nagoya, 461 Japan.
285
286 K. Takayama & A. Furuya
various mammals. It was not until 1930 that the chemical structure of orotic
acid was perfectly known (Bachstez, 1930). Further investigations on the
compound were carried out late in the 1940s. Mitchell and co-workers (1947,
1948) observed that one of the pyrimidine-requiring mutants of Neurospora
utilized orotic acid instead of the pyrimidines, while the others, which did not
utilize orotic acid, accumulated large quantities of orotic acid in a culture
medium. Afterwards, isotopic experiments led to the conclusion that orotic
acid was the important intermediate in the biosynthetic pathway of pyrimidine
nucleotides.
On the other hand, a growth factor for rats and chickens, was discovered
from distillers' dried solubles (DDS) and named vitamin B13 (Novak & Hauge,
1948). Moreover, Wright et al. (1950) observed that DDS was also effective in
promoting the growth of Lactobacillus bulgaricus and orotic acid could
substitute for DDS. Subsequently, evidence has been given that vitamin B13
was identical to orotic acid (Manna & Hauge, 1953).
The accumulation of orotic acid by mutants of several species of bacteria was
reported, but the amounts accumulated were very low. In 1961, the accumula-
tion of orotic acid by a uracil-requiring mutant of Micrococcus glutamicus
(synon. Corynebacterium glutamicum) (a glutamic acid-producing micro-
organism), was reported (Tanaka et al., 1961; Kinoshita & Tanaka, 1963;
Konishita et al., 1963). This was the first report aimed at the industrial
production of orotic acid by fermentation procedure. Studies on the microbial
production of orotic acid have mainly been reported by Japanese researchers.
The industrial production of orotic acid by fermentation started in the
middle of the 1970s in Japan.
3 CHEMISTRY AND ASSAY
3.1 Chemistry
Chemical structure and properties of orotic acid (1,2,3,6-tetrahydro-2,6-dioxo-

4-pyrimidinecarboxylic acid; uracil-6-carboxylic acid) and oritidine (3-{J-o-
ribofuranosylorotic acid, 6-carboxyuridine) are shown in Fig. 1 and Table 1.
Data are quoted from the Merck Index (10th edn, 1983). Potassium, sodium,
H~~
~COOH
H~ H
HOH'd HO OH
(a) (b)
Fig. 1. Chemical structure of orotic acid (a) and orotidine (b).
Table 1
Properties of Orotic Acid and Oritidine
Property Orotic acid Orotidine
Molecular weight 156·10 288·22
Melting point 340-45°C Turns brown near 200°C
but failed to melt at 400°C
Absorption max. 282 nm 268nm
E 7680 (pH 4·4-7·2) 9570 (0·1 N HCl)
Solubility Soluble in water Soluble in hot water,
about 1·7 mg/ml lower aliphatic alcohols
magnesium, calcium and ammonium salts of orotic acid are scarcely soluble in
water.
3.2 Assay
Aqueous solutions of orotic acid and orotidine can be easily assayed

spectrophotometrically using the UV absorption property (282 and 268 nm,
respectively). In the case of a culture broth or a mixture solution, UV
detection combined with paper chromatography. TLC or HPLC is a very
useful method for determination. With regard to HPLC, ODS column (reverse
phase) is preferably applicable. Orotic acid can also be assayed selectively by a
microbiological method using Lactobacillus buigaricus 09 (Wright et ai., 1950)
and via a colorimetric method using dimethylaminobenzaldehyde (Adachi,
1963).
Micro-organisms which accumulate orotic acid and/or orotidine through the de

novo pathway are summarized in Table 2. Accumulation of orotic acid was first
reported using a pyrimidine auxotrophic mutant of Neurospora and a maximal
accumulation of 1· 3 mg of orotic acid per ml was obtained (Mitchell et ai.,
1948). Thereafter, accumulation of orotic acid by Aerobacter aerogenes
(Brooke et ai., 1954), Escherichia coli (Yates & Pardee, 1956) and Serratia
marinorubra (Belser, 1961) were reported, but these studies focussed on the
biosynthetic pathways of pyrimidine derivatives and the accumulated amounts
were very low.
Later on, in 1961, it was reported that a uracil-requiring mutant of
Corynebacterium giutamicum accumulated 14 mg/ml in a culture medium
containing 10% glucose (Tanaka et ai., 1961). This strain was subsequently
found to be deleted in orotate phosphoribosyltransferase. Nakayama et ai.
(1965) examined the accumulation of orotic acid and orotidine by uracil-
requiring mutants derived from the genera Corynebacterium, Brevibacterium,
St
00
Table 2
Orotic Acid-Producing Micro-organisms
Micro-organism Genetic character Accumulated Reference

(additive) substance
Bacteria
Aerobacter aerogenes pyr- OA Brooke et al. (1954)
Escherichia coli pyr- OA Yates & Pardee (1956)
Escherichia coli [AUR added] OA,Ura Skoda & Sorm (1958)
Escherichia coli [AUR added] OA,OMP,UMP Handschumacher (1958)
Serratia marinorubra ura- OA Belser (1961) ~
Corynebacterium glutamicum ura- (OPRT) OA Tanaka et al. (1961)
Bacillus subtilis ura- OR Konishi et af. (1963) ~
~
Corynebacterium glutamicum ura- + ade- OA,OR Nakayama et af. (1965) ~
Corynebacterium rathayi ura- OA,OR Nakayama et af. (1965) ""~
Brevibacterium ammoniagenes ura- OA,OR Nakayama et af. (1965) R-
Bacillus subtilis ura- OA,OR Nakayama et al. (1965) ;t.
Escherichia coli amino acid- OA ~akayama et al. (1965)
Brevibacterium ammoniagenes ura- (OPRT) OA Skodova et al. (1969a,b) .,~
Corynebacterium sp. ura- OA Skodova et al. (1969b) ~
Escherichia coli wild (OMP dec) OA Machida et af. (1969,1970) ""
Arthrobacter paraffineus ura- OA,OR Kawamoto et al. (1970)
Streptomyces showdoensis ura- (OMP dec) OA,OR Ozaki et al. (1972)
Escherichia coli ARs (OPRT) OA Shimosaka et al. (1984)
Bacillus subtilis ura- (OMP dec) OA,OR Doi et al. (1988)
Fungi
Neurospora sp. pyr- OA Mitchell et al. (1948)
Penicillum commune [NaF added] OA Sugimoto et af. (1962)
Candida tropicalis ade-/hyp- OA Watanabe et al. (1968)
Candida lipolytica ura- OA,OR Takayama et af. (1971)
ARs , adenosine sensitive
Bacillus and Escherichia. Consequently, they found that C. glutamicum KY

9824 (ura- + ade-) and B. ammoniagenes KY 7349 (ura-) accumulated large
amounts of orotic acid; the maximal amount was 6·0 mg/ml by the latter.
Skodova & Skoda (1969) and Skodova et al. (1969) also reported the
accumulation of orotic acid using a uracil-requiring mutant of B. am-
moniagenes CCEB 394, obtaining 5·8 mg/ml in a medium containing 5%
glucose, and also found a deletion of orotate phosphoribosyltransferase in the
mutant. Watanabe et al. (1968) observed the accumulation of orotic acid by an
adenine or hypoxanthine-requiring mutant of Candida tropicalis and obtained
a maximal accumulation of 7 mg/ml in a medium. Aspartic acid was recognized
to be effective as a precursor. Ozaki et al. (1972) found that a uracil-leaky
mutant of Streptomyces showdoensis (a showdomycin producer) accumulated
orotic acid and orotidine, obtaining 1·2 and 0·8 mg/ml respectively, and
confirmed the considerably decreased activity of OMP decarboxylase.
Research on the accumulation of orotic acid from n-paraffin has also been
carried out. Kawamoto et al. (1970) observed the accumulation of orotic acid
and orotidine, obtaining 6·7 mg/ml and 8·1 mg/ml, respectively, using a
uracil-requiring mutant induced from Arthrobacter paraffineus, a hydrocarbon-
utilizing bacterium. Takayama et al. (1971) also observed the accumulation of
orotic acid and orotidine, obtaining 7·6 mg/ml and 1·0 mg/ml, respectively,
using a uracil-requiring mutant induced from Candida lipolytica, a
hydrocarbon-utilizing yeast.
Machida & Kuninaka (1969) and Machida et al. (1970) reported that
wild-type strains of E. coli K-12 produced orotic acid, but other strains of E.
coli did not. Later, Womack & O'Donovan (1978) investigated this observa-
tion and concluded that this was caused by a mutation in the pyrF gene
encoding OMP decarboxylase. Shimosaka et al. (1984) reported the excretion
of orotic acid by an adenosine-sensitive (20 mM) strain induced from E. coli
C-600, derived from the K-12 strain. They also found that this growth
inhibitory effect was reversed by co-addition of pyrimidines to the medium; the
mutant displayed a lower level (7%) of activity for orotate phos-
phoribosyltransferase than a parent strain.
Recently, Doi et al. (1988) reported the accumulation of orotic acid and
orotidine by a uracil-requiring mutant F-100 induced from Bacillus subtilis IFO
14386; the accumulation was 55 and 27 mM, respectively. Successively, they
derived a uridine-producing mutant from the parent strain IFO 14386 which
was assumed to display a potent pyrimidine de novo pathway.
5.1 Biosynthesis of pyrimidine nucleotides
The de novo biosynthesis of pyrimidine nucleotides has been studied in detail in

bacteria, especially Escherichia coli and Salmonella typhimurium, in fungi and
~
0 0
HN:\ HNJ
o o/I OAN COOH OAN
HO-t°.
~H, 9H, HNACH,
~
I I
HN/ C ~O~ ~O~
o O.. C.... N/CH-COOH O"C'N/CH -COOH ~
/I O)'N:tCOOH ~
NH,-C-OPO,H, H H H OH OH OH OH ~
CO 2 [CPS] [ATC] [DHO] [DHOdeh] [OPRT] [OMPdec]
~ O"1P
~
+ ~ Carbamyl T Carbamyl Di hydro- Orotate • UMP R0-
~H3 1 Phosphate Aspartate oro tate ~
ATP ~
Aspartate t ~Ii:I
Ornithine NH, OR UDP
~ HOOC-CH,-tH-COOH
Cit.rull ine l
NH,
UTP
oII 0 0 O~~
II II N
Arginine HO-P-O-P-O-P-OC~'O
I I I
OH OH OH l
OH OH CTr
Fig. 2. Synthesis of pyrimidine nucleotide.
in mammals. Several reviews have been previously presented (O'Donovan &

Neuhard, 1970; Shiio, 1972; Enei, 1984). The pathway appears to be universal
in all organisms. The de novo pathway of pyrimidine biosynthesis is shown in
Fig. 2.
The atoms of the pyrimidine nucleus are derived from three simple
precursors, CO 2 , NH3 (from ammonia or glutamine) and aspartic acid.
Carbamyl phosphate is an intermediate of pyrimidine and arginine biosynthe-
sis. Orotic acid is formed via N-carbamyl aspartic acid and dihydroorotic acid.
Orotic acid reacts with PRPP to OMP and successively decarboxylated to yield
UMP, which is the starting substance for synthesis of cytidine and thymidine
nucleotide.
Orotic aciduria is a genetic disorder of pyrimidine biosynthesis in man in
which orotic acid accumulates in the blood and is excreted in the urine. The
disorder is alleviated by the feeding of uridine or cytidine.
5.2 Regulation of the biosynthetic pathway of UMP
In Salmonella typhimurium and Escherichia coli, biosynthesis de novo of UMP

is catalyzed by six enzymes encoded by six unlinked genes and operons. The
first enzyme, carbamyl phosphate synthetase (CPS), is essential for both
pyrimidine and arginine biosynthesis. Synthesis of this enzyme is regulated by
cumulative repression exerted by a pyrimidine nucleotide and arginine.
Synthesis of aspartate transcarbamylase (ATC) , orotate phos-
phoribosyltransferase (OPRT) and OMP decarboxylase (OMPdec) are found
to be repressed by uri dine nucleotides, whereas synthesis of dihydroorotase
(OHO) and dihydroorotate dehydrogenase (OHOdeh) are found to be
repressed primarily by cytidine nucleotides.
Carbamyl phosphate synthetase (CPS) is subjected to feedback inhibition by
uridine nucleotides, especially UMP, and this inhibition is reversed by
ornithine. Aspartate transcarbamylase (ATC) is sensitive to feedback inhibi-
tion by CTP and to activation by A TP.
5-Fluorouracil is a powerful antimetabolite of pyrimidine nucleotides. A
mutant of S. typhimurium resistant to a combination of 5-tluorouracil and
5-tluorouridine isolated by Jensen et al. (1982) possessed fourfold elevated
pools of the pyrimidine nucleotide triphosphate. Furthermore, the specific
activities of ATC and OPRT in the mutant were 40-fold and 7-fold higher than
in the parent strain when grown in minimal media and the synthesis of these
two enzymes was not repressed by pyrimidine compounds.
In Bacillus subtilis, the regulatory mechanism appeared to be considerably
different from that of Salmonella (Doi et al., 1989). Synthesis of all six
enzymes, catalyzing UMP biosynthesis, was strongly repressed by uracil
compounds and CPS was inhibited by UMP. In a mutant strain resistant to
uracil analogues such as 2-thiouracil or 6-azauracil, activities of the six enzymes
increased to 16-30-fold higher than in the parent strain. Moreover, the
repression by uracil compounds to the six enzymes and the feedback inhibition
by UMP to CPS were remarkably lost.
It has been observed that great differences exist for the regulation of the
pathway as a whole, especially for ATC, even though a common pathway is
found in all organisms (O'Donovan & Neuhard, 1970).
6 FERMENTATIVE PRODUCTION
6.1 Fermentation
Most orotic acid-accumulating strains require uracil for growth, hence the
concentration of uracil in media severely affects the accumulation of orotic acid
as well as cell growth. Figure 3(a) and (b) shows the remarkable effects of
uracil on the accumulation of orotic acid by C. glutamicum and B.
ammoniagenes, respectively (Nakayama et al., 1965). Maximal accumulations
were obtained at concentrations of uracil which allowed the micro-organisms
to grow to about half-maximal levels. Similar results were obtained with
uracil-requiring mutants of other micro-organisms (Brooke et aI., 1954;
Tanaka et al., 1961; Konishi et al., 1963; Kawamoto et aI., 1970; Ozaki et al.,
1972; Doi et al., 1988).
For the accumulation of orotic acid by a mutant of Candida tropicalis,
addition of aspartic acid to the medium is effective. The amino acid has been
shown to be a metabolic precursor of orotic acid, and the accumulation of
3.0 ( a) (b)
2.8
_ 2.5
e _ 2.4
......
If
-., 2.0
e
..
......
~2.0 1.0
.,
" "
,
~
~ 1.6 0.8
~o 1.5 ..o
o
."
" &... __ -cr--- ___ 00

0.6 ~ 1.2
-'" ,
/ 0.6 :l
~ 1.0 \
~~ p-'/.
~ \ jP',
'0, '~
0.4:; ~0.8
i .~
,I' / "- 0.4 ~
Q
.
V
tJ I ,
E 0.5 )1 '_, 0"0 ~ " 0·.6
o 0.22; 0 0 . 4
o~~~~~ __~__~_ o 0L-~5~0--~1*00n--~I~~AO--2~b~0- 0.2

o 50 100 150
Uracil (pg/ml) Uracil(pg/ml)
Fig. 3. Effect of uracil on orotic acid accumulation. (a) C. glutamicum KY 9824, (b) B.
ammoniagenes KY 7349 . • , Orotic acid, /':,; orotidine; 0, Growth.
orotic acid proceeds in parallel with aspartic acid consumption (Watanabe et

al., 1968). However, such effects of aspartic acid were not observed in the case
of C. glutamicum (Tanaka et al., 1961).
Recently, a mutant resistant to 5-ftuorouracil was derived from C. glutami-
cum ATCC 14275 (a uracil-requiring strain) by Takayama & Matsunaga
(1987). The activities of both carbamyl phosphate synthetase (CPS) and
aspartate transcarbamylase (ATC) in the mutant were recognized to increase,
comparing to the parent strain ATCC 14275 to some extent. By using the
improved strain, about 40-50 mg orotic acid per ml was accumulated in a
culture medium containing 17% glucose or other carbohydrates employing the
technique of submersed culture with stirring and aeration for 3-4 days.
Accumulation of orotic acid and/or orotidine by uracil-requiring mutants
can be ascribed to two genetic blocks, that is, orotate phos-
phoribosyltransferase (OPRT) and OMP decarboxylase. Since nucleotides
permeate poorly through cell membranes, it is assumed that OMP is excreted
only after dephosphorylation to orotidine or further degradation to orotic acid.
Therefore, when accumulation of orotidine in addition to orotic acid is
observed, it is reasonable to assume a deletion of OMP decarboxylase.
However, when accumulation of orotic acid alone is observed, it should be
confirmed which one of the two enzymes is deleted.
6-Azauracil, a pyrimidine analogue, is a powerful inhibitor of OMP
decarboxylase in micro-organisms in vivo. It exerts its effect only after being
Fig. 4. Bacterial cells and crystals of orotate in the orotic acid fermentation broth.
(scanning electron micrograph).
(a)
CO,Et
H,C" NH,
I + I
EtO,CCO
HN-?
CSMe
ro
NH
Ho,clN)"o
H
(b) EtO CH CO
CO,Et
NH
/ + I '--+ EtO,CHC
CO
=f.
0
.
I
NH
--+
CO,H NH,
I
HO,CHC=C~
1--+
_co
, , --- N--":::-O 'N-
H,N H H
Fig. 5. Chemical synthesis of orotic acid.
converted to 6-aza-UMP, inhibits OMP decarboxylase, and causes the ac-

cumulation of OMP, which subsequently inhibits OPRT. As a result, orotic
acid is excreted into the medium.
6.2 Isolation and purification
Orotic acid in the fermentation broth usually exists as crystals of ammonium,

potassium and/or sodium salt due to its limited solubility (Fig. 4). Crude
orotate can be easily separated from the fermentation broth with repeated
decantation of supernatant liquid. After this operation, the resulting precipi-
tates of crude orotate are, for example, dissolved in a solution of hydrochloric
acid or sulfuric acid at an elevated temperature and then the solution is filtered
to remove impurities and an excess of acid. For further purification, treatments
based on active carbon and on ion exchange resin are generally used.
Synthesis of orotic acid involving the condensation of diethyl oxalacetate with

S-methylthiourea is believed to be the most practical method in the laboratory
(Fig. 5(a». Hydantoin sometimes arises in reactions designed to make
pyrimidines. Diethyl oxaloacetate and urea can be converted into orotic acid
under suitable conditions (Fig. 5(b» (Brown, 1984).
8 APPLICATION
Orotic acid has been used as a hepatic drug. However, it may be contrarily
said that large doses of orotic acid result in liver disturbance, because of the
imbalance between purine and pyrimidine compounds.
Considerable quantities of orotic acid are applied in the field of medicine

and in health food in the form of free orotic acid, or its calcium, magnesium,
potassium or iron salt. The demand for orotate is estimated worldwide to be
about 100 tons/year.
Orotic acid is an intermediate in pyrimidine biosynthesis, hence, expansion
of the utilization of orotic acid for the synthesis of various types of pyrimidine
compounds is expected in the near future.
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Belser, W. L. (1961). Uracil biosynthesis in Serratia marinorubra. Biochem. Biophys.
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Biscaro, G. & Belloni, E. (1905). Sur un nouveau constituant du lait. Monit. Sci., 19,
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Escherichia coli K12. Agric. Bioi. Chem., 33, 868-75.
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Manna, L. & Hauge, S. M. (1953). A possible relationship of Vitamin B13 to orotic
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Chapter 17
MICROBIAL REACTIONS FOR THE SYNTHESIS OF

VITAMIN C (L-ASCORBIC ACID)
V. DELlC, D. SUNIC & D. VLASIC

PLIVA Pharmaceutical, Chemical, Food And Cosmetic Industry, Research
1 INTRODUCTION
Microbial conversion of several chemical substances deserves special attention,

mainly due to its economical and ecological advantages as compared to
conventional chemical routes. Industrial production of vitamin C is an example
of sophisticated chemical manufacturing involving highly complex and expen-
sive chemical and microbial technology. In this process only one step is
mediated by micro-organisms. This review is to provide a complete discussion
of results, methods and current ideas of L-ascorbic acid (vitamin C) biosynthe-
sis by micro-organisms, particularly since this field has only rudimentally been
covered in the literature (Razumovskaya, 1962; Kulhanek, 1970; Crawford &
Crawford, 1980).
Many excellent articles have been published on L-ascorbic acid chemical
synthesis, biosynthesis in plants and animals and its biological role in living
organisms (Sebrell & Harris, 1967; Jaffe, 1984). All these interesting issues will
only briefly be covered in this review, the main objective being to focus on
micro-organisms involved in the biosynthetic steps and conditions to L-ascorbic
acid as a final product.
2 HISTORY
L-Ascorbic acid was first isolated by the famous Hungarian biochemist A.

Szent-Gyorgyi in 1928, under the name of 'crystalline hexuronic acid'. He used
natural material such as ox adrenal cortex, orange juice and cabbage juice for
the isolation of a new compound. In the process of conducting this work,
Szent-Gyorgyi and his co-workers established the identity of 'hexuronic acid',
e.g. the presence of an acidic group and the easy reversible nature of its
oxidation. In addition they found that green paprika was a particularly rich
299
300 V. Delic, D. Sunic & D. VlaSic
Table 1
Some Historical, Production and Economical Data on L-Ascorbic Acid Development
Time period Events Production Price References
(years) (t/yr) (US$jkg)
1928 First isolation of 'hexuronic Szent-Gyorgyi, 1928

acid'
1930-1934 Description of the structure. 0·05 7000 Reichstein & Grtissner, 1934
Reichstein's first and second
chemical synthesis. First pro-
ductions of vitamins by
chemical synthesis; microbial
oxidation as a step in
chemical synthesis.
1937-1940 L-Ascorbic acid synthesis on 100-127 Jaffe, 1984
industrial scale.
1945 L-Ascorbic acid from Aspergil- Geiger-Hiiber & Galli, 1945
Ius niger; never reproduced.
Increasing of production c. 17 25·0 Jaffe, 1984.
capacity in USA.
1953 Preparation of intermediates Yamazaki & Miki, 1953
by biosynthesis Yamazaki, 1953a, b, c
1953-1960 Many chemical and technical Crawford & Crawford, 1980
modifications; e.g. electro-
chemical oxidation
Production in USA c. 3000 6·0 Jaffe, 1984
196(J....1966 Increasing production c.5000 4,0-4·5 Jaffe, 1984
c. 14% year. Total Ullmanns, 1983
production in the world.
1966-1973 Biosynthesis of L-ascorbic 3·25-4·25 Isono et al.. 1968
acid intermediates by Okazaki et al.. 1968
micro-organisms. Okazaki et al.. 1969
Kanzaki & Okazaki, 1970
1974 Inflation; increasing in the 7,15-8·30 Jaffe, 1984
raw material and energy Ullmanns, 1983
cost
1981 Total world production. 34 000-36 000 10·40 Ullmanns, 1983
1982 Biosynthesis of 2-keto-L- 12·0 Sonoyama et al.. 1982
gulonic acid by two-stage
fermentation
1984 Biosynthesis of 2-KLG on a Kieslich, 1984
commercial scale in People's
Republic of China.
1985 One step biosynthesis of 2-KLG Anderson et al.. 1985
by genetically modified
Erwinia herbicola.
1987 Total world production. c.60000 11·00
1987-2000 Predictions: (a) growth of nominal
capacity of main world
producers;" (b) intensive efforts on
rDNA techniques; (c) use of bio-
catalysts from modified enzymes
and/or cells and (d) research on
various bioreactors, e.g.
membrane reactors.
" See listing of world producers in Table 4.

Microbial Production of Vitamin C 301
source of vitamin C (Svirbely & Szent-Gyorgyi, 1933). Detailed descriptions of

'hexuronic acid' led to the discovery of the correct structure of vitamin C, later
on named ascorbic acid by Szent-Gyorgyi and Haworth (Crawford & Craw-
ford, 1980).
These data, together with those derived from· the study of L-ascorbic acid
oxidation products, led Hirst et al. (1933) to propose that this compound was
3-keto-L-sorbosone. The early results of L-ascorbic acid structure elucidation are
summarized in several reviews (Haworth & Hirst, 1933; Hirst, 1939; Crawford
& Crawford, 1980).
The importance of L-ascorbic acid in human health care was established soon
after its discovery and caused tremendous interest in all aspects of its scientific
and industrial development. Hence, it is not surprising that up to now
L-ascorbic acid has been and still is one of the major products of interest to the
pharmaceutical, chemical and food industries. The main stages of the history
of L-ascorbic acid over the past 60 years are listed in Table 1.
Its medical importance and the role of L-ascorbic acid in human health have
been very well documented in over 22 000 publications that have appeared in
the literature from 1966 to 1984 (Jaffe, 1984). Since humans and some animals
lack L-gulono-y-lactone oxidase, the key oxidizing enzyme in liver for
L-ascorbic acid biosynthesis, they have to consume vitamin C from exogenous
sources. The main symptoms of L-ascorbic acid deficiency, mainly due to a
dietary lack of fresh fruit and vegetables, are general weakness, fatigue and
listlessness followed by a shortage of breath and aching in the bones. As the
illness-known from ancient times as scurvy-progresses, the skin becomes dry
and rough followed by a swelling of the gums, easy bleeding and a loss of
teeth. Obviously the lack of L-ascorbic acid affects many different metabolic
processes in living cells such as collagen synthesis, amino acid synthesis,
immune response, etc., although the precise mechanisms of its actions are not
clear. L-Ascorbic acid, an important reducing agent functioning as an elec-
tron carrier, plays an important role in the respiration and oxidative reactions
in the cells.
3.1 Nomenclature
Names previously used for L-ascorbic acid were: cevitamic acid, hexuronic
acid, redoxon, scorbutamin, vitamin C and L-xylo-ascorbic acid. Throughout
this review, the name L-ascorbic acid will be used.
In 1965 the trivial name ascorbic acid or L-ascorbic acid was recommended
by the IUPAC-IUB Commission on Biochemical Nomenclature as a name for
vitamin C. The systematic name for L-ascorbic acid is L-threo-hex-2-enonic acid
y-lactone or L-threo-hex-2-enomo l,4-lactone (IUPAC-IUB, 1965).
302 V. Delic, D. Sunic & D. VlaJic
6
H~"
OH
,~
0
-0
H5 0CH0'20H
q HO H H --
HO OH
or 4 __ , =0 (b)
HO OH HH~O'"
'-- H 0
H"'" H"'" _ _ =0
HO
HO OH
(a)
Fig. 1. Chemical constitution of L-ascorbic acid.
3.2 Properties
L-Ascorbic acid is white, crystalline solid melting at 192°C. Specific rotation in

water at the sodium D line is +21·5°C. In solution, L-ascorbic acid has: pKl of
4·17, pK2 of 11·57. The most acidic proton is that of the 3C-hydroxyl group.
Crystals of sodium and calcium L-ascorbate have the metals associated with
0-3. Reversible oxidation takes place at + 127 m V.
Infrared, ultraviolet, IH-nuclear magnetic resonance (nmr) and 13C-nmr
spectra have all been reported (see: Schauenstein et al., 1948; Billman et al.,
1972; The Merck Index, 1983). Ogawa et al. (1977), studied the conformation
of L-ascorbic acid in deuterium oxide by 13C-nmr spectroscopy. They con-
cluded that in aqueous solution the most favoured rotamer of L-ascorbic acid is
in the form (b) (Fig. 1). The rotamer is not present in the crystalline state of
L-ascorbic acid. The approximate conformation in the crystal is shown by
structure form (a) (Fig. 1).
Elucidation of L-ascorbic acid stereochemistry played an important role in
the determination of its stereomolecular structure and in the development of
chemical synthesis for industrial production. The configuration of L-ascorbic
acid was assignment to the L-series and confirmed by synthesis from o-glucose
and L-xylose. Chiral centers at C-2 and C-3 of these sugars were in the correct
position to become C-5 and C-4 of L-ascorbic acid, respectively (Reichstein &
Griissner, 1934). Other carbohydrates, e.g. L-gulose, L-idose, L-galactose and
L-talose have the correct chirality at C-4 and C-5 to be potential raw materials
for the synthesis, but their high cost makes them impractical.
4 BIOSYNTHESIS IN PLANTS AND ANIMALS
Since fruit and vegetables are the main natural sources of L-ascorbic acid,
biosynthetic pathways in plants were subjected to extensive studies. It has been
shown that L-ascorbic acid is a product of hexose phosphate metabolism
including conversion of D-glucose or o-galactose to L-ascorbic acid (Fig. 2).
o-Glucose o-Galactose o-Glucose
I
UDP-o-Glucose
I
1
UDP-Glucuronic acid
I
o-Galacturonic acid
1
o-Glucuronic acid
D-Glucuronic acid
1
L-Gulonic acid
1
L-Galactonic acid
1
L-Gulonic acid
1
L-Gulono-y-Iactone
~
j j
L-Gulono-y-Iactone L-Gulono-y-lactone
1
L-Galactono-y-Iactone
1
L-2-Keto-gulono- y-lactone
1
L-2-Keto-galactono-y-lactone
1
j
L-Ascorbic acid L-Ascorbic acid
(a) (b)
Fig. 2. Proposed routes of L-ascorbic acid biosynthesis in plants (a) and animals (b),
involving inversion of configuration; UDP-uridine diphosphate.
Studies by using radioactive o-glucose C4 C-1 of 14C_6, respectively) have

shown that the main six-carbon chain is conserved and radioactivity found at
C-1 or C-6 atoms of L-ascorbic acid. In this retention of configuration,
oxidation occurs at C-1, followed by lactonization, then again oxidation at C-2
and C-3, and finally epimerization at C-5 to give L-ascorbic acid (Loewus,
1980). Alternative biosynthetic pathways involve configurational inversion in
which the main precursor is turned to L-galactono-y-Iactone, more active than
L-gulono-y-Iactone, which is oxidized at C-2 to give L-ascorbic acid (in
o-glucose series epimerization at C-3 occurred prior to the last oxidation).
Biosynthesis of L-ascorbic acid in animals is somewhat simpler (Fig. 2). In
mammals, L-ascorbic acid is synthesized in the liver via the glucuronic acid
304 V. Delif, D. Sunif & D. Vlalic
pathway, likewise in plant biosynthesis. Studies with radioactive labelled

D-glucose have shown that 14C_1 (or 14C_6) became the 14C_6 (or 14C_1) atom of
L-ascorbic acid until the D-glucose chain remained intact (Bums, 1967).
5 BIOSYNTHESIS WITH MICRO-ORGANISMS
After 1896, when Bertrand for the first time described microbial oxidation of
sorbitol to sorbose by the micro-organism Bacterium xylinum (subsequently
classified as Acetobacter xylinum) (Bertrand, 1896), more than 30 years passed
until this microbial reaction was used as a biooxidation step in L-ascorbic acid
synthesis. Afterwards, research in the field of microbial biosynthesis of
L-ascorbic acid intermediates became more intensive and further knowledge
accumulated quickly.
During the past 50 years there have been a lot of attempts to find ways of
microbial conversion of intermediates in L-ascorbic acid synthesis which could
compete with the Reichstein-Griissner synthesis from an economical point of
view. Different micro-organisms were screened for their ability to carry out
reactions on carbohydrates as well as methods for new biosynthetic steps in
those routes. These reactions, micro-organisms and media are listed in Table
2. L-Ascorbic acid intermediates biosynthetic routes, including micro-organisms,
are shown in Fig. 3.
In this review we present names of micro-organisms as reported in the
original articles. Since their first presentation in the literature, the taxonomy of
these micro-organisms has undergone many changes due to their morphologi-
cal, cultural and biochemical properties, e.g. Acetobacter suboxydans has been
renamed Gluconobacter oxydans subsp. suboxydans. t For actual names of
micro-organisms see Bergey's Manual of Systematic Bacteriology (1984). Most
of the micro-organisms mentioned here are mutants with blocked side
reactions in 2-keto-L-gulonic acid biosynthetic routes from carbohydrates,
which could utilize alternative carbon sources.
5.1 Biooxidation of L-sorbose to 2-keto-L-gulonic acid
In the classical Reichstein-Griissner synthesis (see p.323), L-sorbose is con-

verted to 2-keto-L-gulonic acid (2-KLG) by chemical reactions. In this part we
are describing the possibilities of L-sorbose conversion to 2-KLG by micro-
organisms (Fig. 3, routes 2, 3).
The first biooxidation of L-sorbose to 2-KLG by micro-organisms was
described by Tengerdy (1961a, b) who used UV-irradiated mutant strains of
Pseudomonas. After 4 days of cultivation in the medium containing 20 g/liter
t 'Names included in the Approved List of Bacterial Names are the only names which
are nomenclaturally valid as at the 1st January, 1980. All other names which have
appeared in the literature prior to 1st January, 1980 are nomenclaturally invalid'
(Skerman et af., 1980).
Table 2
Micro-organisms and Media Used in Biosynthesis of L-Ascorbic Acid Intermediates
Micro-organisms Conversion Media, conditions References

(yield)
Acetobacter D-Sorbitol ~ L-sorbose
suboxydans (>95%)
Acetobacter Sorbitol ~ 2-KLG 30 liters of the medium containing sorbitol 5; glucose 0·5; Obata et aI., 1975
IFO 3243 (4%) yeast ext. 0·5 and CaC0 3 2%. 28-29°C, 150 h,
aeration 15-24 liters/min
Pseudomonas sp. L-Sorbose~ 2-KLG Glycerol 1; L-sorbose 20; com steep liquor 10; NZ amine Tengerdy, 1961a, b ~
(15%) B 5; K2HP04 X 3H20 0·7; KH2P04 0·3; NaCl 0·5; 1';'
MgS04 x 7H20 0·1 and FeS04 x 7H20 0·1 g/liter. 10
a
0-
liters of medium in 14-liter glass fermentors was a
inoculated with 2% cell suspension from inoculum ~
medium. 28°C; pH 7 ·0-7·2; dow silicone oil s::...
I::
<"I
(0· 25 g/liter) was used as antifoam; 96 h; aeration 1 v/v g.
per min; stirred 350 rpm ::s
Pseudomonas L-Sorbose~ 2-KLG KH2P04 0·04; MgS04 x 7H 20 0·02; NZ-amine B 0·25; Huang, 1962 ~
viridiflava yeast ext. 0·5 and sorbose 2%, 28°C, aeration 1 v/v per ~
NRRLB-94 min; pH 8,1-4 days iii
::I
Gluconobacter L-Sorbose ~ 2-KLG L-Sorbose 7; glycerol 0·5; yeast ext. 0·5; MgS04 x 7H2O Tsukada & s·
melanogenus (2%) 0·5 and CaC0 3 1%. 25°C, pH6·5, 220 rpm, 96h Perlman, (")
IFO 3293 19720

Gluconobacter L-Sorbose~ 2-KLG L-Sorbose 7; urea 0·5-0·8; K2HP04 0·007; KH2P0 4 Yin et al., 1980
oxydans (40%) 0·003; glycerol 0·2; MgS04 x 7H20 0·01; CaC03 0·5
N1197 A and com steep liquor 0·5%. pH 6·0-6·5. Addition of Yan et al., 1981
urea or (NH4)zHP04 during fermentation
Gluconobacter L-Sorbose~ 2-KLG L-Sorbose 100; glycerol 0·5; yeast ext. 15·0 g/liter; salts. Fujiwara et ai.,
oxydans U-13 (64%) Cultivation on rotary shaker, 30°C, 4 days 1987
Pseudoglucono- L-Sorbose~ 2-KLG 1000 liters of the ferm. medium containing L-sorbose 15; Nogami et ai., 1987
bacter saccharo- (76%) CaC0 3 5; com steep liquor 2; dried yeast 0·3; Actcol
ketogenes 0·03; (NH4)2S04 0·03; Na2S20 3 x 5H20 0·5 and Vl
IFO 14464 FeS04 x 7H20 0·1 %. 0
U\
(continued)
Table 2-contd. w
~
(yield)
Bacillus 30°C, 110 rpm, aeration 900 liters/min, pressure
megaterium 0·5 kg/cm2, 4 days, 123·1 mg/mI2-KLG formed
IFO 1210S
Pseudomonas Sorbose~ L-idonate Sorbose 5; com steep liquor 2·5; MgS04 x 7H20 0·01; Mochizuki et al. ,
aeruginosa KH2P04 0·03; K2HP04 0·07; NaCl 0·05 and glycerol 1969a
0·05%. Aeration, 2SoC, 120h
Gluconobacter L-Sorbose ~ L-sorbosone L-Sorbose 7; glycerol 0·5; yeast ext. 0·5; MgS04 x 7H2O Kitamura &
melanogenus (6-8%) 0·5; CaC03 1%. pH 6·8, 30°C, 250 rpm, 7 days Perlman, 1975 :0::::
IFO 3293 .t::;)
Pseudomonas L-Sorbosone ~ 2-KLG Glycerol 2·5; Na-citrate 5·0 g/liter; mineral salts; 2SoC, Makover & Pruess, ~
pulida (20%) overnight; fresh medium (9·5 ml) containing. L- 1976 .:>'
sorbosone 10 g/liter was inoculated with 0·5 ml of the ~
old medium, 28°C '-l.
I::
;:
~.
Pseudomonas Ca-L-idonate~ Ca-2-KLG Ca-L-idonate; maltose (or glucose) 3; com steep liquor 3; Gray, 1947a ,""
mildenbergii KH2P04 0·3 and MgS04 x 7H20 0·1 part; 50 parts of R-
P. mildenbergii (grown on 5% glucose and 0·5% ~
yeast). pH 5·5-6·0 ;S
Pseudomonas Ca-L-idonate 2-keto- Idonic acid 250; gluconic acid 250; NH4 0Ac 12; K3P04 Yamazaki, 1955
Ca-n-gluconate ~ hexonic
~
,""
fluorescens 5; MgS04 1 g; 10 liters H 20. pH 6·5; 2-3 days; AcOH
(1: 1) acids added to pH 5·5, kept 7-S days
(40%)
Pseudomonas Ca-idonate~ 2-KLG Ca-idonate and Ca-gluconate (7: 3) Fujisawa et al.,
fluorescens (77%) 50; ~OAc 1·2; KH 2P0 4 0·5; MgS04 x 7H20 0·3; 1963a, b
FeS04 x 7H20 O·S g/liter. Aeration, 6 days
Pseudomonas Ca-L-idonate~ 2-KLG Ca-L-idonate 7; Ca-n-gluconate 3; yeast ext. 2·5; Takeda et al. ,
aeruginosa T-S1 (SO%) (NH4)2HP04 0·2; KH2P0 4 0·1 and MgS04 0·025%. 1964
28°C, 225 rpm, 3 days
Acetobacter Ca-L-idonate~ 2-KLG Glucose 0·5; corn steep liquor 1·0; KHZP0 4 0·1; Mochizuki et al.,
melanogen us (27%) MgS04 x 7H20 0·05; Ca-5-keto-gluconate 1969b
hydrogenation product 34% (containing idonic acid
14·7 and gluconic acid 7·2%); 1 liter. 2SoC, shake
cultured, 76 h, 40 g 2-KLG formed
Acetobacter Ca-L-gulonate- Ca-2-KLG Ca-L-gulonate 120; com steep liquor 5; octadecyl ale. Gray, 1947b
suboxydans (75%) 0·3; maltose or sorbitol 5 and H 20 1000 parts; pH
6·0-6·1. 50 parts of inoculum; 25°C; aerated; 8 days;
90 parts Ca-2-KLG formed
Xanthomonas L-gulonate- 2-keto-L- 4 Liter stirred fermenter containing 2 liters of production Kita, 1979
translucens gulonate medium containing corn steep liquor 8; meat digest 6;
ATCC 10768 (85%) (NH4)zHP0 4 1; glucose 10; CaC0 3 3 and Ca-L-
gulonate 80 g/liter. After ferm. at 28-30°C for 48 h
with stirring and aeration, 40 g/liter Na-L-gulonate
ferm. broth was added followed by an addnl. 30 g/liter
at -72h
Brevibacterium 5-KDG- L-idonic acid Cells of log phase were suspended in 400 ml of 0·1 M Sonoyama et at. , ~
ketosoreductum phosphate buffer (pH 6·86) mixed with 200 ml of 6% 1974 1=;'
FERM-P 1905 K-salt of 5-KDG and held at 30°C for 48 h a
(J-
(ATCC 21914) ~
Acetobacter Glucose- 5-ketogluconic Glucose 10%, 33 h Stubbs et al.,
suboxydans acid 1940
~
s::..
I:
(90%) g.
Acetobacter sp. Glucose- 5-KDG Glucose 10-15; yeast 0·75-1·0%; Teramoto et al., ;:s
(89%) 26°C, 7 days 1946 ~
Acetobacter o-Glucose- 2,5-DKG Glucose 10; yeast ext. 0·5; glycerol 0·5; MgS04 x 7H2O Stroshane &
meianogenus (93%) 0·5; CaC03 1%; pH7·0. 25-26°C, 300 rpm, 3 days Perlman, 1977 ~::!
ATCC9937 s·
Acetobacter o-Glucose-2,5-DKG 2 Liters of medium (pH 6·0) containing glucose 110; corn Kita & Hall, (j
cerinus (95%) steep liquor 0·5; (NH 4)zHP0 4 0·58; KH 2P0 4 1·5; 1981b
IFO 3263 MgS04 x 7H 20 0·05; urea 0·5 g/liter; CuS0 4 x 5H2O
1; nicotinic acid 0·3 mg/liter. 1; nicotinic acid
o· 3 mg/liter. 28°C, 36 h, 1700 rpm, aeration o· 75 v/v x
min. An additional 55 g glucose/liter was added at 20 h
and the pH was maintained at 5·5
Erwinia sp. o-glucose- Ca-2,5-DKG o-Glucose 5·8; com steep liquor 1-13; (NH4)zHP04 O'56; Sonoyama et al.,
SHS 2629001 (94·5%) CaC03 18·4; p-2000 antifoam 0·02%; pH 6·8. 1982
der. from 1·86 m3 /10 m3 ; 160 rpm; 3·6 m3 air/min; 28°C; 26 h.
SHS2006 During fermentation, 2304 kg 50% (wt/wt) o-glucose ...,
(ATCC 31626) was added 0
-..,J
(continued)
~
Table 2-contd.
(yield)
Corynebacterium Ca-5-DKG- Ca-2-KLG o-Glucose 2; com steep liquor 3; NaN0 3 0·345; KH2P04 Sonoyama et ai,
sp. (92·5) 0·067; p-2000 antifoam 0·00167%; ZnS04 X 7H20 4·9; 1982
SHS 752001 MnCl2 x 4H20 0·8; thiamine hydrochloride 0·22; Ca-
der. from o-pantothenate 0·17 mg/liter; pH 6·9; 28°C; 160 rpm;
SHS 0007 1·18 air/min. NaN03 , o-glucose and Ca-2,5-DKG
(ATCC 31090) added during fermentation
Citrobacter 2,5-DKG-2-KLG 100 ml medium contg. cerelose 2; (~)2HP04 1; Kita & Hall, :"'
freundii (30%) KH2P04 1; MgS04 X 7H20 0·5; beet molasses 2 and 1981a t::::I
~
ATCC6750 glycine 0·2 g/liter; pH 6·7. After 22 h 15 ml of an ~
Acetobacter cerinus ferm. broth contg. 15-20% 2,5- !='
DKG was added 28°C; addnl. 52 h; pH 6·5
Erwinia o-glucose- 2-KLG Glucose 3; glycerol 20; yeast ext. 5; peptone 5; CaC03 Anderson et al. , §
7·5 g/liter; pH 7·0. 1 g/liter 2-KLG formed 1985 ~
herbicola (33%)
Estell et al., 1985 Ro
Erwinia o-glucose- 2-KLG 500 ml of medium contg. glucose 10; o-mannitol 20; com Hardy et al. , !='
citreus (50%) steep liquor 10; yeast ext. 1·5; casamino acids 10; 1987 ~
ERl116 K2HP04 4; KH2P04 1; ~Cl1; CaCI2 0·01 and ~
<"I,
K2S04 2·6 g/liter with three further additions of
10 g/liter o-glucose. 28°C; 800 rpm; over 60 h
Candida Galactonic- L-ascorbic Galactonic acid or galactono-y-Iactone 0·5; com steep Roland et al. ,
norvegensis acid acid liquor 0·25; ~CI 0·1; glycine 0·7; MgS04 x 7H2O 1985
KCC MF 42 (8-9%) 0·05; Na-glutamate 0·2; EtOH 1·5%; trace metals;
pH 4.2. 300C, stirring, aeration, 48 h. 0·43 g/liter L-
ascorbic acid formed
PseudogluconOb~cter Si!cch.ilroketog~
~y~
CHO
G. m.,.nog'''' fO HO}COOH
HO OH
1
~P.ut;d' ~X.
r
~p'Ut;d.
tran"u"ns :: H
HO
~ CH,OH CHZOH
L - Sorbosone l-Gulonic aCid
CH20H CH20H
HO HO ~g.nus HO~C~H_ HOfC~O=-~~~C01

mHO
HO
f f _HO
OH
HO
OH
~~
HO
OH
HO
OH
HO
0
CHO CHZOH CH20H CH20H CH,DH

O-Glucose D-Sorbltol L-Sorbose L-Sorbose 2-Keto- L- 2-Keto-L- Methyl- L-Ascorbic
l H O ~C:O~H
diacetone gulonlc .. cid gulonlc acid 2-Keto-L- Clcid
diace-tone gulonate
~glnosa
Po mildenber9li
~~HO~OH
COOH
p tluorescens
p.dlerug~
~g~
® G mel.ilnogenus
OH
HO
CH20H CH20H
L - Idose L - Idonic
acid
A SUbOXy~ HOfC:~H B '.tosocedu'tum

1
OH
o
<;Qrynebacterlum
5-Keto-D- Brevibacterium
ILao_'d_-=:=:'n='~=~'=S~=-~= ;'='~=:__
{
Arthrobact~r
Micrococcus
~P...b):'lococcus
glueo,;, Bacillus
Erwmia
Gluconobact~r
~
Acetobacter cerlnus freundir
Crtrobact~r
AcetomonCiis albosesamae
CHZOH
2,5-0rKeto-O-
gluconrc acid
Erwima cltreus
Erwinla herbrcola
Fig. 3. Biosynthesis of intermediates in L-ascorbic acid routes by different micro-

organisms. 1, Reichstein-Griissner route; 2, L-idose route; 3, L-sorbosone route; 4,
5-keto-o-gluconic and L-idonic acid route; 5, 2,5-diketo-o-gluconic acid route.
L-sorbose, 13 g/liter of substrate was utilized and 3 g/liter of 2-KLG formed.

Tengerdy (1961a, b) proposed the one-step reaction involving oxidation of the
C-1 of L-sorbose to 2-KLG. Another Pseudomonas mutant (P. viridiflava
NRRL B-94), which oxidized L-sorbose to 2-KLG with 50% yield, was isolated
by Huang (1962).
Different genera of eubacteria were screened for their ability to oxidize
sorbitol and L-sorbose. Isono et al. (1968) found that micro-organisms
producing keto-sugar acid from sorbitol were restricted to the genera
Acetobacter, Gluconobacter and Pseudomonas whereas those producing acids
310 V. Delic, D. Sunic & D. VlaIic
from L-sorbose were distri1?uted widely in the 12 genera: Acetobacter,

Alcaligenes, Aerobacter, Azotobacter, Bacillus, Escherichia, Gluconobacter,
Klebsiella, Micrococcus, Pseudomonas, Serratia and Xanthomonas. As shown
by Mochizuki et al. (1969a), P. aeruginosa was able to oxidize sorbose only to
L-idonic acid. Kanzaki & Okazaki (1970) suggested a multi-step conversion of
L-sorbose to 2-KLG by Pseudomonas aeruginosa in which L-idose and L-idonic
acid were intermediates. Pseudomonas aeruginosa IFO 3898 converted L-
sorbose via L-idose and L-idonic acid to 2-KLG efficiently (Fig. 3, route 2).
There are only a few genera of micro-organisms capable of oxidizing sorbitol
to 2-KLG. Some unidentified Acetobacter and Pseudomonas strains were able
to produce 2-KLG from sorbitol (Takeda Chemical Industries, 1964). It was
reported that these strains, after 150 h of fermentation in the medium
containing 5% sorbitol, produced 2-KLG with an overall yield of 8-9%.
Acetobacter sp. IFO 3243 cultivated in 30 liters of fermentation medium
converted 4% sorbitol to 2-KLG (Obata et al., 1975).
Okazaki et al. (1968, 1969) studied the conversion of sorbitol to 2-KLG by
means of G. melanogenus IFO 3292. After 7 days of cultivation in a medium
containing 10% sorbitol, the fermentation broth was found to contain neutral
and acidic compounds which were identified' as: L-sorbose, 5-keto-o-mannonic
acid, 2-keto-o-gluconic acid, 2-KLG, L-idonic acid, o-mannonic acid, 0-
fructose and 5-keto-o-fructose. As a result of the accumulation of these
compounds the authors proposed the following metabolic pathway in G.
melanogenus: sorbitol--+ L-sorbose--+ L-idose--+ L-idonic acid--+ 2-KLG (Fig. 3,
route 2).
In the early 1970s, a number of publications appeared describing the
biooxidation of L-sorbose to 2-KLG by Gluconobacter meianogenus IFO 3293.
Tsukada & Perlman (1972a, b, c) found conditions for the conversion of 2% of
metabolized L-sorbose to 2-KLG and proposed the direct conversion of
L-sorbose to 2-KLG. Later on, the 'sorbosone pathway' was found to be
involved. Employing this route, L-sorbose was converted to 2-KLG via
L-sorbosone as an intermediate (Fig. 3, route 3). The first step of this reaction
was reversible and mediated, in both directions, by an enzyme(s) fraction of
Pseudomonas putida ATCC 21812 (Makover et al., 1975).
Growing cultures or cell-free extracts of G. melanogenus IFO 3293 were also
able to carry out this reaction (Kitamura & Perlman, 1975). The limiting factor
of this route was the reversibility of the first step as well as the poor absorption
of L-sorbose into the cells (Makover et al., 1975). Oxidation of L-sorbose to
L-sorbosone was efficiently improved 2-3 times by the addition of organic
solvents or detergents, due to the increased cell membrane permeability
(Martin & Perlman, 1975).
Yin et al. (1980) and Yan et al. (1981) established the optimal fermentation
medium for producing 2-KLG from L-sorbose by the bacterial strain
Gluconobacter oxydans N 1197 A. In the medium containing 7 or 10% of
L-sorbose and 0·5-0·7% of urea concentration, the yield of 2-KLG was about
30 or 37 g/liter, respectively.
It seems likely that researchers in the People's Republic of China are able to
produce 2-KLG from L-sorbose (10 g/liter) over L-sorbosone on a commercial
scale with a 90% yield using bacteria of the genera Gluconobacter and Bacillus
(Kieslich, 1984; Lu et al., 1985; Ning et al., 1988).
Fujiwara et al. (1987) have used the G. oxydans strain, which has a high
activity of L-sorbose dehydrogenase, to produce 2-KLG. After a 4-day
cultivation of G. oxydans U-13 in the L-sorbose medium 2-KLG was formed
with a 64% yield. Also, G. oxydans U-13 was able to produce 2-KLG during
cultivation in o-sorbitol medium.
Recently, Nogami et al. (1987) reported the capability of Pseudo-
gluconobacter saccharoketogenes IFO 14464 (novel genus, isolated from soil
origin) and a concomitant bacterium Bacillus megaterium IFO 12108 to
produce 2-KLG in a high yield (76%) during cultivation in a 2 m 3 fermenter
containing 1000 liters of L-sorbose medium. Bacillus megaterium, which was
not directly involved in the oxidation process, promoted the growth of the
oxidative strain (P. saccharoketogenes) and increased the yield of 2-KLG. (Fig.
3). Compared with a pure culture of the oxidative strain the mixed culture was
able to oxidize higher concentrations of L-sorbose to 2-KLG in a shorter period
of time. The bacteria of some other genera could be successfully used instead
of B. megaterium, e.g. Pseudomonas, Proteus, Citrobacter, Enterobacter,
Erwinia, Xanthomonas and Flavobacterium.
5.2 Biosynthesis of 2-keto-L-gulonic acid from 5-keto-D-gluconic and L-

idonic acid
Chemical reduction of 5-keto-o-gluconic acid (5-KDG) resulted in a mixture of

L-idonic and o-gluconic acid that could be converted to 2-KLG by various
micro-organisms. The first microbial biooxidation of L-idonic acid to 2-KLG
was reported by Gray (1947a), who found that Pseudomonas mildenbergii was
capable of performing this reaction (Fig. 3, route 4). In this process L-idonic
acid or its salts were prepared along with o-gluconic acid by a chemical
reduction of the corresponding salts of 5-keto-gluconic acid. L-Idonic acid was
subsequently biooxidized by P. mildenbergii to 2-KLG, while o-gluconic acid
was oxidized back to 5-keto-gluconic acid by A. suboxydans. 5-Keto-gluconic
acid was reused in the process.
Pseudomonas fluorescens oxidized well both calcium-L-idonate and calcium-
o-gluconate (Yamazaki & Miki, 1953; Yamazaki, 1953a, b, c). After 7-8 days
of cultivation in the medium containing the mixture of L-idonic and o-gluconic
acid (ratio 1: 1) the producing strains of genus Pseudomonas were able to
convert both of the added compounds to 2-keto-hexonic acids with a 40-50%
yield. The main problem in this process was the isolation of 2-KLG from the
mixture of 2-keto-hexonic acids obtained at the end of fermentation. 2-Keto-
guion ate was isolated in a 50% yield (Yamazaki, 1953c, 1955). 5-Keto-o-
gluconate, used in this procedure, was obtained by the biooxidation of
o-glucose with A. suboxydans (Yamazaki, 1953a).
With a different starting mixture of Ca-idonate and Ca-gluconate (7: 3,

respectively) and after a 6-day cultivation of P. f/uorescens, Fujisawa et al.
(1963a, b) obtained a 77% yield of 2-KLG based on L-idonic acid. Similar
results were obtained by Alieva (1966) using the same micro-organism and by
Takeda et al. (1964) using P. aeruginosa T-81 strain.
It seems likely that the ability for oxidation of L-idonic acid to 2-KLG is
widely spread in genus Pseudomonas, e.g. P. aeruginosa, P. mildenbergii, P.
chlororaphis and P. pseudomallei (Nakanishi et al., 1964a, b). Apart from
genus Pseudomonas, some other micro-organisms have also been reported to
oxidize L-idonic acid to 2-KLG (Shoemaker, 1956; Chas. Pfizer & Co., 1958:
unidentified bacteria ATCC 11867 and ATCC 11868; Mochizuki et al., 1969b:
Acetobacter melanogenus and A. suboxydans).
A new approach to L-ascorbic acid intermediate synthesis was applied by
Sonoyama et al. (1974b). Instead of chemical agents, they used the cells or a
crude enzyme preparation of Brevibacterium ketosoreductum sp. nov. FERM-
P 1905 derived from ATCC 21914 to reduce 5-KDG to L-idonic acid.
5.3 Biooxidation of L-gulonic to 2-keto-L-gulonic acid
A combination of chemical and biochemical reactions for the preparation of

2-KLG from L-idonic acid was described by Gray (1947b). In this combination
the first step was the chemical conversion of L-idonic to L-gulonic acid, while
the second step was the biooxidation of L-gulonic acid to 2-KLG by A.
suboxydans. After 8 days of cultivation 75% of L-gulonic acid was oxidized to
2-KLG. As shown by Perlman (1959), P. aeruginosa was able to oxidize even
L-gulono-Iactone to 2-KLG in a 44% yield. Some strains of genus
Xanthomonas were also able to oxidize L-gulonic acid to 2-KLG. For example,
Xanthomonas translucens A TCC 10768 was cultivated in the medium contain-
ing 80 g/liter calcium-L-gulonate (added twice during cultivation). In this
instance the overall yield of 2-KLG was 85% (Kita, 1979).
5.4 Biosynthesis of 2-keto-L-gulonic acid from o-glucose
Studies on o-glucose biooxidation to keto-gluconic acids began 50 years ago.

Bernhauer & Knobloch (1938, 1940) reported biosynthesis of 2-keto-gluconic
and 5-keto-gluconic acids from glucose or gluconate by micro-organisms of
genus Acetobacter (Fig. 3, route 4, 5). Stubbs et al. (1940, 1943) obtained a
90% yield of 5-keto-gluconic acid from 10% glucose in the medium with A.
suboxydans and 82% with an unidentified strain. Teramoto et al. (1946)
obtained identical yield of 5-keto-gluconic acid using an unidentified
Acetobacter strain. As shown by Razumovskaya & Vasil'eva (1956), A.
suboxydans was able to oxidize o-glucose not only to 5-keto-gluconic but also
to diketo-gluconic acid.
Advancement along this route was achieved when Katznelson et al. (1953)
established oxidation of o-glucose to 2,5-diketo-o-gluconic acid (2,5-DKG) by
Acetobacter melanogenus MA 6.2 without prior splitting of the o-glucose

molecule. The route they proposed was glucose- gluconic acid- 2-keto-
gluconic acid-2,5-DKG. The same route was studied by Wakisaka (1964a)
who reported the microbial oxidation of glucose to 2,5-DKG by Pseudomonas
sesami. Soon afterwards a new species of Pseudomonas genus (P.
albosesamae) capable of a high production of 2,5-DKG from o-glucose was
isolated (Wakisaka, 1964b, c). Acetobacter fragum (Oga et al., 1972) and A.
cerinus (Kita & Hall, 1981b) were also able to produce 2,5-DKG under
aerobic conditions in the medium containing o-glucose. As much as 95% of the
starting o-glucose was converted to 2,5-DKG by A. cerinus. Stroshane &
Perlman (1977) obtained 2,5-DKG in a 93% yield from o-glucose by A.
melanogenus ATCC 9937.
The authors proposed a metabolic pathway which included o-gluconic acid
and 5-KDG as intermediates. In this biosynthesis the main problem was the
instability of 2,5-DKG and decomposition of up to 80% during the isolation
process.
The procedure for 2-KLG biosynthesis from o-glucose was improved by
Sonoyama et al. (1982, 1983b) who used a two-stage fermentation system (Fig.
4). They reported a process which included biosynthesis in 10 m 3 conventional
fermenters for the production of calcium-2-KLG from o-glucose over calcium-
2,5-DKG by two different micro-organisms. The first step in this process was
o-glucose oxidation to Ca-2,5-DKG by the mutant strain SHS 2629001 of
Erwinia sp. derived from ATCC 31626 and, subsequently, the stereospecific
reduction (to eliminate 2-keto-o-gluconic acid formation) of Ca-2,5-DKG to
Ca-2-KLG by the mutant strain SHS 752001 of Corynebacterium sp. derived
from ATCC 31090. At the end of cultivation, Erwinia sp. was treated with
sodium dodecyl sulfate to decrease the number of viable cells so the broth with
accumulated Ca-2,5-DKG could be used directly for the next reduction,
mediated by Corynebacterium sp. Thus, in this process, the isolation of
Ca-2,5-DKG was avoided. The total fermentation time for this two-stage
process was about 100 hand Ca-2-KLG accumulated up to 106·3 mg/ml
(84·6% yield from o-glucose). Media used in this process are listed in Table 3.
Compared to some other processes previously described in this review, a
two-stage fermentation procedure for 2-KLG production has some advantages
that make it a possible candidate for industrial application. These are: (a) high
producing strains; (b) elimination of 2,5-DKG isolation from the broth; (c)
commercially feasible media for both biosyntheses; (d) reliability and re-
producibility and (e) a simplified process for 2-KLG isolation (absence of
compounds like o-glucose, Ca-L-idonate, Ca-o-gluconate, Ca-5-KDG, Ca-2-
KDG).
Results obtained by Sonoyama and his co-workers on this subject have been
very well documented in a number of publications on the mixed cultures of
Acetomonas albosesamae ATCC 21998 and Brevibacterium sp. ATCC 31083,
as well as on the screening, isolation and description of micro-organisms
capable of the oxidation of glucose to 2,5-DKG (Erwinia species) and
11~ ~~';7ri."IP'
(ATCC 31(90)
l28"'C. .ah
[j 10.600 ml 0/ pn:-
seed medium in a
!
2·ntrc=fluk
~ 28'C. lOh. 275 rpm

28"C. 2' h. 215 rpm
~
:.:.:: :::.::::
.....:.. :
.20 ,••<> of the
seed tncdium in
l ~ml rermentor
l 28"C. 10 h. z.w rpm;

0,(2 ml .ir/min.
1860 tilfa of 1M
ferment. medium in
100mJ {ermen.or
inoculated with the
cnlirc conterU of 4DO I:llrCl oIlhc:
the seed fcrmc:nlc:r fennent. medium in
IO-m 3 fennenlor
inoculated with the
entire content of
28"C' 26 h. ,60 rpm; tl'M: JCCd fennenu~:t
1..
3.6 m3 Iit/m.!n. (&Ilk
prcuure 1.5 .... /an 2;
tdd.ition of D1lucosc
28"C. 18h . '60 rpm;
1-18 m1 lir/min:
4200 Ijll'd ollhc: D·""""""dd«I

..
NINO) odQ"j
as a hydrogen
fermen ted broth:
328 m, Ca·2.5-DKGlmL do"".
Add.itJon of SOS. after 6 I'll
the IQlal viabk; cell
populldon docrc.&$Cd
!Torn 5 • 109 t. 9. ,03
c:cUsiml.
7430 litra of tbe

fermented broth;
U16 m& Ca-2-KLGlmi
Fig. 4. Flow-sheet for the production of 2-keto-L-gulonic acid by two-stage fermenta-

tion; SDS, sodium dodecylsulfate; Ca-2,5-DKG, calcium-2,5-diketo-o-gluconate; Ca-2-
KLG, calcium-2-keto-L-gulonate.
Table 3
Media Used in the Biosynthesis of Calcium 2,S-Diketo-o-Gluconate and
Calcium 2-Keto-L-Gulonate by Erwinia sp. SHS 2629001 and
Corynebacterium sp. SHS 725001
Media Concentration
(g/liter)
Erwinia sp. Corynebacterium sp.
Agar slant
o-glucose 5·0
glycerol 5·0
yeast extract 5·0 5·0
peptone 3·0 5·0
KH2 P04 1·0 1·0
MgS04 x 7H2 O 0·2 0·2
agar 20·0 20·0
Preseed medium
o-glucose 10·0
glycerol 5·0
yeast extract 5·0
peptone 5·0
com steep liquor 30·0
NaN0 3 1·0
KH2 P0 4 1·0 1·0
MgS04 x7H2 O 0·2
p-200 antifoam 0·1
Seed medium
o-glucose 10·0
glycerol 5·0
com steep liquor 30·0 20·0
NaN0 3 2·0
KH2 P04 1·0 1·0
MgS04 x7H 2 O 0·2
p-2000 antifoam 0·1 0·05
Fermentation medium
o-glucose 58·0 20·0
com steep liquor 11·3 30·0
NaN0 3 3·45
KH2 P0 4 0·67
(NH4)2HP04 5·6
CaC03 184·0
p-2000 antifoam 0·2 0·0167
ZnS04x 7H2 O 4·9 mg/liter
MnCl2 x 4H2 O 0·8 mg/liter
thiamine hydrochloride 0·22 mg/liter
Ca-0- Pantothenate 0·17 mg/liter
316 V. De/if, D. Sunil & D. Vlalif
reduction of 2,5-DKG to 2-KLG, like Brevibacterium ketosoreductum, Bacillus

megaterium, Arthrobacter simplex, Staphylococcus aureus, Micrococcus,
Pseudomonas, etc. (Sonoyama et al., 1974a, 1976, 1983a, 1986).
Mutants of Corynebacterium sp. ATCC 31088, 31089 and 31090 with
improved bioconversion abilities have been selected after N-methyl-N'-nitro-
N-nitrosoguanidine mutagenic treatment. These mutants were blocked in
degradative metabolic pathways of 2,5-DKG, reducing 2,5-DKG only to
2-KLG in a high molar yield with the addition of glucose as a hydrogen donor
(Sonoyama et al., 1987a, b).
2-KLG is the end product of all described biosynthetic routes starting from
D-glucose, L-sorbose or L-gulonic acid, respectively. This is the ultimate
intermediate in L-ascorbic acid biosynthesis by micro-organisms (or chemicals).
Subsequently, 2-KLG could easily be converted by chemical methylation and
cyclization to L-ascorbic acid.
5.5 Direct Biosynthesis of L-Ascorbic Add
Direct biosynthesis by micro-organisms is the most challenging means of

L-ascorbic acid synthesis. Until now, there have been only few articles
describing micro-organisms and their ability for direct biosynthesis of L-
ascorbic acid from different carbohydrates. Various groups of organisms were
found to be able to produce L-ascorbic acid from different sugar substrates but
concentrations of substrates and yields of L-ascorbic acid are still very low.
Geiger-Huber & Galli (1945) found that Aspergillus niger was able to
synthesize L-ascorbic acid (1·5 mg/lOO ml) after 6-8 days of cultivation in the
medium containing 15% sucrose. Sucrose was also a suitable substrate for
L-ascorbic acid biosynthesis when Aspergillus flavus was used (Ramakrishnan
& Desai, 1956). Sastry & Sarma (1957) cultivated A. niger in glucose medium.
They proposed the following metabolic pathway as follows: D-glucose-
D-glucuronic acid- L-gulono-Iactone- 2-keto-L-gulonolactone- L-ascorbic
acid.
Recently, Roland et al. (1985, 1986) reported that strains of the yeast
Candida (e.g. C. norvegensis KCC MF 42) were able to produce L-ascorbic
acid during cultivation in the medium containing L-galactonic substrates, such
as L-galactonic acid, L-galactonic acid esters or L-galactono-y-Iactone. Accord-
ing to these results it seems likely that biosynthetic pathways in some yeasts
and moulds are similar to those proposed for plants (see Fig. 2).
Some green algae also displayed the ability to produce L-ascorbic acid.
Chlorella pyrenoidosa cultivated on a glucose medium for 101 h produced
1·5 g/liter of L-ascorbic acid (Skatrud & Huss, 1987).
5.6 Biocatalysts Involved in Biosynthesis of L-Ascorbic Add Intennediates
Recently, new methods applying biological catalysts-biocatalysts have been

developed for conversion of different raw materials to desired end-products.
These new technologies and processes significantly reduce the cost of bio-
synthetic production (Aiba et al., 1973; Rehm & Reed, 1981; Karube et al.,
1984; Kulbe & Knopki, 1986).
These methods rely on preparation of biocatalysts such as immobilized
enzyme(s), parts of cells or whole cells (Klein & Wagner, 1978; Scott, 1987;
Brodelius and Vandamme, 1987). The biocatalysts can be used for the
production of various substances in a single-step enzyme reaction (6-
aminopenicillanic acid from penicillin G; fructose from glucose, etc.). The
application of biocatalysts for multi-step enzyme biosynthesis (for instance,
antibiotics) is rudimentally described in the literature (Morikawa et al., 1979,
1980; Vandamme, 1984). This interesting area represents a new type of
biosynthesis, and as such this approach to L-ascorbic acid synthesis should not
be neglected in the future.
Martin & Perlman (1976) described conversion of L-sorbose to L-sorbosone
by biocatalysts. They used immobilized cells of native Gluconobacter melano-
genus IFO 3293 entrapped in polyacrylamide gel. Authors concluded that
conversion depended on the concentration of monomer used. The higher the
monomer concentration, the more L-sorbosone accumulated. Different results
were found with immobilized lyophilized cells. In this case a very active
preparation of biocatalysts was obtained with low monomer concentration due
to the impaired permeability of lyophilized cells, allowing diffusion of the toxic
monomers into the cytoplasm. Fujiwara et al. (1987a, b) described isolation,
purification and characterization of two enzymes (L-sorbose dehydrogenase
and L-sorbosone dehydrogenase) from Gluconobacter oxydans UV-10 involved
in conversion of L-sorbose to 2-KLG via L-sorbosone.
The cells of Gluconobacter oxydans strain No.6 VNIVI capable of oxidizing
o-sorbitol to L-sorbose were entrapped in polyacrylamide gel. The highest
oxidative activity of such a biocatalyst, reused five times, was 3·3 g of L-sorbose
per liter per h (Pomortseva & Krasil'nikova, 1983).
The production of L-sorbose by cells of Gluconobacter suboxydans (ATCC
621) immobilized in polyacrylamide gel was carried out in a continuous
process. The entrapped cells of this strain were capable of almost completely
converting o-sorbitol into L-sorbose at a rate of about 7 kg/m3 per h during a
long period of time (Stefanova et al., 1987).
Bailey et al. (1985) developed a mathematical model on K-carrageenan-
immobilized cells of Acetobacter suboxydans oxidizing ethyl alcohol to acetic
acid. This model, which predicted the cells, substrate and product concentra-
tion in reaction with the biocatalyst, could possibly be used for biooxidative
studies in the L-ascorbic acid pathway.
Studies on enzyme participation in the pathways of o-sorbitol oxidation were
done by Shinaga-,a et al. (1982). They described the solubilization, purification
and characteristics of o-sorbitol dehydrogenase from the membrane fraction of
Gluconobacter suboxydans var. a IFO 3254.
Recently, Sonoyama & Kobayashi (1987) reported two 2,5-DKG reductases
(reductase I and II) isolated from Corynebacterium sp. SHS 752001 mutant
318 V. De/ie, D. Sunie & D. V/aIiC
derived from ATCC 31090. Reductase I differed from reductase II in its

molecular weight, its higher specific activity and its affinity for substrate
2,5-DKG. Reductase II is essentially the same enzyme described by Anderson
et aZ. (1985) and Miller et aZ. (1987). Both reductases had the ability for the
stereospecific reduction of the keto group of 2,5-DKG at the C-5 position and
were NADPH-dependent.
6 CLONING OF GENES RELATED TO L-ASCORBIC ACID

BIOSYNTHESIS
Molecular biology techniques developed in the last 15 years and the possibility
of gene manipulation in vitro (recombinant DNA technologies-rDNA),
particularly in microbial cells, resulted in the creation of hybrid features and
the non-conventional production of several substances in bioreactors. Gene
cloning was extended to many different micro-organisms, showing diversity of
application in a wide range of products like hormones, vaccines, enzymes,
amino acids, vitamins, antibiotics, etc. (Old & Primrose, 1985; Primrose,
1987). The classical example is cloning of genes for human insulin in the
bacterium Escherichia coli (Goeddel et aZ., 1979) and the production of this
hormone by fermentation (Frank & Chance, 1983). These techniques were
also usefully applied in the construction of improved microbial strains for
producing penicillin G-acylase, an enzyme involved in the hydrolysis of
penicillin G to 6-aminopenicillanic acid (Mayer et al., 1979; Vandamme, 1984;
Garcia & Buesa, 1986).
6.1 Cloning of 2,S-Diketo-D-Gluconic Acid Reductase Gene in Genus

Erw;n;a
As described in previous parts of this review, many micro-organisms have the

ability to catalyse a reaction on a sugar molecule and to carry out a conversion
on the L-ascorbic acid pathway. The shortest route for o-glucose conversion to
2-keto-L-gulonic acid (2-KLG) is the two-step reaction via 2,5-diketo-o-
gluconic acid (2,5-DKG) (Fig. 3, route 5). In this route, o-glucose is oxidized
by microbial dehydrogenases to give 2,5-DKG. On the other hand, a group
of-for example-coryneforming bacteria was able to reduce enzymatically
(2,5-DKG reductase) 2,5-DKG to 2-KLG, the last intermediate in L-ascorbic
acid synthesis that could be easily chemically converted into L-ascorbic acid.
Sonoyama et al. (1982) demonstrated a two-stage fermentation using mutants
of bacteria Erwinia sp. and Corynebacterium sp. to convert o-glucose to
2-KLG.
Based on this data, the idea of Anderson et al. (1985) was to combine the
traits of these two micro-organisms in a single cell by gene manipulation
techniques and to simplify the conversion of o-glucose to 2-KLG. The strategy
was to clone the 2,5-DKG reductase gene from Corynebacterium sp. ATCC
31090 in Erwinia herbicola ATCC 21988 (also known as Acetomonas

albosesamae) which was able to oxidize D-glucose into 2,S-DKG. Anderson et
al. (198S) isolated and characterized 2,S-DKG reductase from
Corynebacterium sp. and through Edman degradation determined a sequence
of 40 amino acids from the NH2 -terminus of the enzyme. Two synthetic DNA
probes, 43 nucleotides long and corresponding to the NH2 -terminal amino acid
sequence were used to detect by hybridization the 2,S-DKG reductase gene
between fragments obtained by Bam HI digestion of total Corynebacterium sp.
DNA. Fragments ranging in size from 2·0 to 2·S kb were isolated and cloned in
a Bam HI site of plasmid pBR 322. A partial genomic library was obtained in
E. coli and the transformed colonies were checked for the presence of
2,S-DKG reductase gene by hybridization. A fragment was subcloned in
bacteriophage M13mp9 cut by a combination of two restriction enzymes Pst I
and Sma I for further vector construction. A simplified route for suitable
vector construction is given in Fig. S. For good 2,S-DKG reductase gene
expression in bacterium Erwinia herbicola, deletion was constructed upstream
of the ATG start codon. A 'strong' tryptophan (trp) promoter linked to the
Shine-Dalgarno sequence (Shine & Dalgarno, 1974) from plasmid pHGH207-
1ptrpL\RIS' (De Boer et al., 1983) was attached to the 2,S-DKG reductase
coding region instead of the deleted seque~ce. This was accomplished by using
deletion primer and Alu I fragments of M13mp9 treated with Klenow fragment
of DNA polymerase I, T4 DNA ligase and deoxynucleotide triphosphates
(dNTPs) to give heteroduplex mit12 RF (replicative form) molecule used to
transform E. coli JM101. Deletion of the upstream region between Nco I
restriction site and A TG starting codon was selected by plaque hybridization
(A). A replicative form of one isolate mit12L\ having desired deletion was used
to excise 2,S-DKG reductase gene by Eco RI and Hind III digestion. A new
hybrid vector was constructed from three different parts (B). 2,S-DKG
reductase gene (l·S kb) from mit12L\ was mixed with a small fragment (1·0 kb)
of plasmid pHGH207-1ptrpL\R1S' digested with Pst I and Eco RI. This small
fragment contained tryptophan promoter region essential for 2,S-DKG reduc-
tase gene expression. The third part of the hybrid vector was a larger fragment
(3·6 kb) of plasmid pBR322 obtained after digestion with Hind III and Pst I.
Three parts were mixed and ligated and this mixture was used to transform E.
coli MM294. Transformants were selected for ampicillin resistance. Hybrid
plasmid pmit12L\ trp1 AmpR could not be used for the selection of transformed
cells of Erwinia herbicola since this micro-organism is naturally resistant to
ampicillin. Thus, a functional gene for tetracycline resistance was re-
constructed, since after pBR322 digestion with Hind III-Pst I loss of tetracy-
cline resistance occurred. For this purpose plasmid pBR322 was digested with
Eco RI and Sph I to generate the fragment of 0·S6 kb containing the
tetracycline promoter region. This fragment was ligated with a S·4 kb fragment
from plasmid pmit 12L\/trp1 AmpR isolated from E. coli MM294 and digested
with Hind III and Sph I (C). The resulting 2,S-DKG reductase expression
vector ptrpl-3S was used to transform Erwinia herbicola and transformants
320 V. Delic, D. Sunic & D. VlaIic
-
2.5- DKG red. gene
Annealing of deletion primer

and Alul f ra9m~nt$, DNA
pOlymerase I (Klenow), dNTPs.
T4 DNA ligase
HindW
S
Transfectron ot E. colt JMIOI
w ith heterodupr;;-;;~ ' 2RF
(A)
ECOR!
mil 12 6 f Screening for phage
containing deletion
e)
m
I
Hind
E. CORl e
PS~ H;nd m
pBR 322
(B)
--
Handm
15kb 1·0kb 3 ·6kb
-----Ligation
Transformation J,I ~
------
MM294.
select ion for AmpR (olonies
I .
(C)
5 .6kb~
8
/ o.56kb
SR322 lE~:tR!
SphI
L1gation
I
Transfo,.mat ion of "",,!!,!Ill>1!....!lS=~. selection for Tet R cOlOnies
Erw ini. herblcola w ith

2, 5- DKG reductase gene on
pl.smid ptrpl- 35
Fig. 5. Construction of 2,5-diketo-D-gluconic acid reductase cloning vector and

transformation of Erwinia herbicola. Only relevant restrictipn sites are given; AmpR,
ampicillin resistance; Tet, R tetracycline resistance; trp, tryptophan promoter; kb, kilo
base; dNTPs, deoxynucleotide tryphosphates.
were selected on tetracycline. Isolated transformants, checked for enzyme

activity, showed 54 times higher 2,5-DKG reductase activity in crude extracts
in comparison with E. herbicola containing plasmid pBR322. Activity was
50-100 times higher in E. herbicola than in E. coli MM294 when transformed
with the same ptrpl-35 plasmid.
For the production of 2-KLG, E. herbicola with plasmid ptrp1-35 was

cultivated in a o-glucose-containing medium and subsequently on the medium
containing 2% glycerol. After additional incubation, 1 g/liter 2-KLG was
found and analysed by HPLC. Identity of 2-KLG was confirmed by the usual
analytical methods (Lazarus & Seymour, 1986).
A similar approach has recently been applied by Hardy et al. (1987) and
Grindley et al. (1988) for the construction of 2,5-DKG reductase expression
vector. Fragments containing 2,5-DKG reductase sequence were taken from
Corynebacterium sp. SHS 7520001 after Sau 3A digestion and inserted into
plasmid pUCS digested by Bam HI restriction enzyme. Recombinant plasmid
was used for the transformation of Escherichia coli JM83 and colonies of the
resulting library were screened by hybridization with the 17-mer DNA
synthetic probe to locate the 2,5-DKG reductase gene. Two recombinant
plasmids, pCBR10 and pCBR13, were found to contain 2,5-DKG reductase
gene approximately 3·0 kb long. These plasmids were used for further vector
construction using different promoters (lac, trp, tac, the operator and
promoter regions of phage A, the control region of fd coat protein, the
promoters of yeast, etc.). Plasmid pPLred332 containing APL promoter region
upstream of 2,5-DKG reductase region from pCBR13 was used to transform
the mutant strain Erwinia citreus ER 1026 obtained from E. citreus SHS 2003
(ATCC 31623). The newly-formed E. citreus ER 1116 strain was able to
convert o-glucose in a single biosynthetic step to 2-KLG after 65 h of
cultivation. o-Glucose was added three times during biosynthesis and at the
end 19·8 g/liter of 2-KLG was found (overall yield was 49·4%).
6.2 Molecular Biology of Other Micro-organisms Involved in 2-Keto-L-Gulonic

Add Biosynthesis
Members of the genus Acetobacter and Gluconobacter are industrially impor-

tant because of their ability for high yield conversion of carbohydrates to
various oxidation products. In recent years more data on the molecular biology
and genetics of these micro-organisms have been accumulated.
Fukaya et al. (1985a) showed that as much as 81·8% of Acetobacter strains
harboured mainly multicopy plasmids of low molecular weight while 63·8% of
Gluconobacter strains harboured plasmids ranging from 1·0 to 17 Md.
The transformation method for Acetobacter strains was improved by
polyethylene glycol and treatment of cells by divalent cations (Ca2 +, S~+,
Ba2+, Mn2 +). Efficiency of transformation was HP transformants per I-'g DNA
(Fukaya et al., 1985b).
Construction of shuttle vectors for Gluconobacter suboxydans and
Escherichia coli was a further step in the application of recombinant DNA
technology for this group of micro-organisms, although efficiency of transfor-
mation was low (1ij2 per I-'g DNA) (Fukaya et al., 1985c).
Two phages isolated from 54 different strains of Gluconobacter (Robakis et
al., 1985a) were characterized and discovered to be unusually large, containing
322 V. Delit, D. Sunil & D. Vlaiil
double stranded DNA. Construction of a restriction map of bacteriophage A-I

genome revealed circular DNA containing cohesive ends (Robakis et al.,
1985b). Palleroni (1986) showed that Gluconobacter oxydans IFO 3293 could
accept plasmid RSF 1010 both by transformation and conjugation but the
yields of transformants were low, and obviously could be improved.
A new approach was used by Kahn and Manning (1988) to create
Gluconobacter oxydans strains capable of higher production of 2-KLG from
L-sorbose. They carried out additional mutation in G. oxydans UV-lO mutant
strain by using insertional transposon mutagenesis (PI:: Tn5). Mutant strain
M23-15 produced more 2-KLG (33·3 g/liter) than the parent strain
(19·6 g/liter) due to reduced 2-KLG-reductase activity. As a consequence of
this reduction, decreased flux of 2-KLG to L-idonic acid occurred. Besides this,
new recombinant strains of G. oxydans IFO 3293 were obtained by using
plasmid vectors constructed of broad host range plasmid RSF 1010 and a part
of chromosomal DNA of the wild type strain containing L-sorbose de-
hydrogenase DNA sequence. One of these recombinant strains, obtained by
conjugative transfer with constructed plasmid pURU010 into the parent strain
(1·8 glliter of 2-KLG) showed much higher conversion of L-sorbose to 2-KLG
(28 g/liter).
Some species of the genus Pseudomonas, particularly P. aeruginosa and P.
putida, are well characterized genetically and biochemically. This includes the
development of host-vector systems for gene cloning by using restriction-
negative host strains of P. aeruginosa and P. putida, and the construction of
suitable plasmids or bacteriophage DNA as vectors (Bagdasarian & Timmis,
1982; Holloway, 1986).
Broad host range plasmid RSF 1010 is one of the possible candidates for a
cloning vector in Pseudomonas cells. Some derivatives of this plasmid (pKT
230; pKT 231) with stable maintenance and propagation in host strains, have
several unique cleavage sites, and insertional inactivation inside antibiotic
resistance determinants is possible. Recently, new cloning vectors were
constructed by inserting the entire sequence of plasmid pUC 13 into
derivatives of the RSF 1010 plasmid. These new vectors, named pWS, were
stable both in E. coli and Pseudomonas putida (Werneke et al., 1985). With
regard to the cloning genes of Pseudomonas, P. putida possesses a potent gene
engineering system, even more promising than E. coli.
These findings in those industrially important micro-organisms, which have
the capacity for oxidation products from carbohydrates and polyalcohols, have
opened up further possibilities for the creation of new routes for L-ascorbic
acid biosynthesis by recombinant DNA technology.
7 INDUSTRIAL PROCESS
L-Ascorbic acid was the first vitamin to be produced in commercial quantities

(Table 1). It is produced in a very large, integrated and automated process
involving both batch and continuous operations. Modern industrial production

of L-ascorbic acid is based on Reichstein-Griissner synthesis established more
than 50 years ago. According to available information, it seems that most
L-ascorbic acid producers use this procedure for industrial production.
7.1 The Reichstein-Griissner synthesis
The first chemical synthesis was reported by Reichstein et al. (1933; 1934).
L-Ascorbic acid was synthetized from L-xylose over L-xylosone but this route
was never commercialized. It is interesting that when Reichstein and co-
workers published the first synthesis of L-ascorbic acid, the correct structure
was not yet known.
The most important synthesis of L-ascorbic acid for the manufacturing
processes is that reported by Reichstein & Griissner (1934), well known in
literature as Reichstein's second synthesis (Fig. 3, route 1).
This synthesis from o-glucose included the inversion of the glucose chain,
which means the rearrangement of the molecule where C-1 of o-glucose
became C-6 of L-ascorbic acid. The main steps of the Reichstein-Griissner
synthesis were: reduction at C-1 (o-glucose- o-sorbitol) followed by microbial
biooxidation at C-5 (o-sorbitol- L-sorbose), acetone treatment (L-sorbose-
L-sorbose diacetone), oxidation at C-6 (L-sorbose diacetone- 2-keto-L-gulonic
acid (2-KLG) diacetone), hydrolysis (2-KLG diacetone- 2-KLG), esterifica-
tion (2-KLG- 2-KLG methyl ester) and lactonization (2-KLG methyl
ester- L-ascorbic acid). o-Sorbitol was oxidized to L-sorbose with Acetobacter
xylinum in a 60% yield. Later on A. suboxydans became the micro-organism
of choice due to its high effective oxidative ability. The modified Reichstein-
Griissner synthesis is a very efficient process for L-ascorbic acid synthesis
and served as the basis for modern industrial production.
From an industrial point of view, the Reichstein-Griissner synthesis has two
crucial features. The first is low cost of raw material, e.g. o-glucose which is a
starting compound, and second, fully protected intermediate L-sorbose diace-
tone for the second oxidation which precludes other side reactions.
Many chemical and technological modifications have improved the
efficiencies of each step. This multi-step chemical synthesis, which includes
micro-organisms for oxidation in one step, remains till now the principal and
the most economical process for industrial production of L-ascorbic acid. The
process steps including microbial oxydation are outlined in Fig. 6.
o-Sorbitol obtained by the hydrogenation of o-glucose was oxidized by
Acetobacter suboxydans into the key intermediate L-sorbose. L-Sorbose is then
condensed with acetone to form sorbose diacetone which is oxidized to a
diacetone derivative of 2-keto-L-gulonic acid (2-KLG). After hydrolysis and
esterification 2-KLG diacetone gives 2-KLG methyl ester which is converted to
L-ascorbic acid by cyclization. The procedure requires about 1·7 kg of
L-sorbose per kg of L-ascorbic acid with an overall yield of 66% (Jaffe, 1984).
324 V. Delil, D. Sunil & D. V[alil
D-GLUCOSE
!
D- SORBITOL
~
r-------------------------,
FERMENTATION
Acetob~cter Suboxy'd~ns
Filtered air
L-SORBOSE
~
L - SORBOSE DIACETONE
~
2 - KETO - L- GULONIC ACID
l
L- ASCORBIC ACID
Fig. 6. Synthesis of L-ascorbic acid including biooxidation of D-sorbitol to L-sorbose by
Acetobacter suboxydans.
7.2 Product recovery and purification
Some steps of industrial processing include generally sophisticated product

purification and recovery.
L-Sorbose isolation from cultivation broth is related with demands for the
separation of A. suboxydans biomass accumulated in the course of n-sorbitol
fermentation and elimination of other accompanying impurities. The product
purification and recovery of L-sorbose as a solid is a continuous process which
includes not only filtration, evaporation and crystallization steps but also
purification in anion and cation ion exchange columns.
The acetone treatment of L-sorbose results in a mixture of mono- and
diacetone-L-sorbose. Because benzene readily extracts diacetone-L-sorbose,
diacetone-L-sorbose can be separated in a benzene/water system, while the
monoacetone-L-sorbose in the aqueous layer is recovered and recycled.
2-Keto-L-gulonic methyl ester is recovered as a crystalline product. After the
formation of 2-KLG methyl ester crystals, mother liquors and methanol

washes are accumulated for a second recovery of ester crystals.
At the end of the Reichstein-Griissner process crude L-ascorbic acid is
filtered and purified by recrystallization. The pure product is isolated and
dried. Mother liquors are accumulated and recycled for L-ascorbic acid
recovery (Jaffe, 1984).
8 ASSAY METHODS FOR L-ASCORBIC ACID
Since L-ascorbic acid is widely distributed in different plant and animal tissues,
as well as in crystalline form added to a variety of products (e.g. in food,
drinks, etc.), the determination and quantification of its content deserve a
special analytical approach.
Apart from these difficulties L-ascorbic acid is easily subjected to oxidation
by moderate oxidants, thus yielding dehydroascorbic acid which frequently
interferes with L-ascorbic acid determination.
As analytical details on L-ascorbic acid determination are not within the scope
of this review, readers interested in this field of L-ascorbic acid are recommended
to a very pertinent review by Pelletier (1985). The main analytical methods will
be mentioned here only briefly.
Bioassay as a method was not frequently used for L-ascorbic acid determina-
tion. This method based on guinea-pig protection against scurvy, was further
applied to determine if certain L-ascorbic acid derivatives or other compounds
exhibited anti-scurvy activity.
Most determinations are based on colorimetric methods using blue dye
2,6-dichloroindophenol (Hughes, 1983). In the presence of L-ascorbic acid, dye
is reduced, resulting in colourless or pink colour solution. Direct titration of
L-ascorbic acid with 2,6-dichloroindophenol is at present the most frequently
used oxido-reduction method applicable to diverse types of samples yielding
reproducible results.
Murty & Rao (1979) used iodine salts for the determination of L-ascorbic
acid. In this case different dye indicators could be used (naphthol blue black,
Amaranth, Brilliant Ponceau 5R) in titration mixture, liberating iodine which
in the presence of starch yielded a blue colour. Reduction of ferric, cupric and
mercuric ions by L-ascorbic acid has been the basis of simple and sensitive
methods for measuring L-ascorbic acid. Corresponding ferrous, cuprous or
mercurous coloured complexes with different substances were used as a
measure of L-ascorbic acid.
Pelletier & Brassard (1977) developed a method for the determination of the
total content of L-ascorbic acid, dehydroascorbic acid and diketogluconic acid
in the samples on the basis of coupling with 2,4-dinitrophenyl hydrazine.
Gas-liquid chromatography of trimethylsilyl L-ascorbic acid derivatives was
also adapted for quantitative determination of L-ascorbic acid in different
samples (Schlack, 1974).
326 V. De/ie, D. Sunie & D. V/aIie
High performance liquid chromatography (HPLC) seems to be the most

precise method for the determination of L-ascorbic acid in samples of different
origins (Geigert et al., 1981; Rose & Nahrwold, 1981) although it remains to
be established which method is the most accurate and precise for general
application.
Recently developed peroxidase-based colorimetric determination of L-
ascorbic acid has certain advantages over previously mentioned analytical
methods (Thompson, 1987). Determination is based on the interference of
L-ascorbic acid with the horseradish enzyme peroxidase which causes a
decrease in the rate of oxidative coupling of chromogenic reagents like
4-aminoantipyrine or 3,5-dichloro-2-hydroxy-phenylsulphonate. The resulting
change in absorbance at 510 nm is related to the concentration of L-ascorbic
acid.
Pharmaceutical applications represent the largest segment of the L-ascorbic

acid market. L-Ascorbic acid is not used only for the prevention and treatment
of scurvy, which is of course its primary application, but also for the
prevention and treatment of other pathological conditions and maintenance of
good health in general. It can be used for the prevention and treatment of
certain types of anaemia, some cardiovascular diseases, wound repair and
normal healing processes. Due to its stimulative role toward the immune
system, L-ascorbic acid is used in the prevention and treatment of various
infections such as common colds and others.
L-Ascorbic acid has gained extensive application in the food industry due to
Table 4
Main World Producers of L-Ascorbic Acida
Producers City, country Nominal

capacity
(t/year)
Hoffmann-La Roche Grenzach, FRG 11000

Belvedere, New Jersey, USA 13500
DaIry, UK 13500
Takeda Chemicals Ltd Osaka, Japan 7000-8000
Merck Darmstadt, FRG 6000
BASF Grenaa, Denmark 3000
Pfizer' Croton, Connecticut, USA 2000
PLIVA Zagreb, Yugoslavia 1200
a Total world production (including Eastern countries, China, India and
Brasil) is assumed to be 65 000-70 000 t/year.
b According to available information Pfizer's plant in Croton has been
closed.
its antioxidant properties. It is used as an antioxidant in the commercial

preparation of oils, fats, meats, beer, soft .drinks, milk products and canned
and frozen food. It is also used as a dough and flour conditioner in the flour
industry. L-Ascorbic acid is applicable in photography as a developing agent,
and in metallurgy as a reducing agent (Jaffe, 1984).
At the present time, L-ascorbic acid is one of the most used and the most
widely produced vitamins of importance in the pharmaceutical, food, cosmetic
and other industries. The largest world producers are listed in Table 4
(Ullmans, 1983; Kieslich, 1984).
10 CONCLUSION
Although no alternative chemical route has reached the economical impor-

tance of Reichstein's second synthesis, current developments of chemical
synthesis and biosynthesis by micro-organisms may provide the important basis
in the future of L-ascorbic acid production. Many future views are focused on
the direct biosynthesis of 2-KLG from o-sorbitol or o-glucose by genetically-
manipulated organisms.
Nevertheless, the main effort in the future could be designated as the
substitution of the chemical pathway for L-ascorbic acid synthesis with a
stereospecific bioconversion in one single step and continuous operation.
ACKNOWLEDGEMENTS
The authors gratefully acknowledge the expert secretarial assistance of Miss

Jasenka Korajlija during the preparation of the manuscript, and thank Dr
Ivanka Pavusek for suggestions and reading of the manuscript.
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OTHER GROWTH FACTORS
Chapter 18
MICROBIAL PRODUCTION OF ATP

Y. TANI
1 INTRODUCnON
A TP plays a central role in energy metabolism as a carrier of chemical energy

from catabolic reactions to anabolic reactions. It has two high-energy
phosphate bonds which give a free energy of 14-16 kcal/mol on hydrolysis.
Since ATP was first isolated in 1929 from a tissue (muscle) having high
glycolysis activity, by Lohmann, and Fiske and Subbarow, the mechanism of
its formation has been studied through elucidation of the mechanisms of the
glycolytic pathway and the respiratory chain. Thus, A TP is an important
chemical in biochemical research and medicine. Recently, ATP has also
attracted much attention as the energy donor in bioreactors for the produc-
tion of useful metabolites.
There are various methods for the preparation of A TP, which include
extraction from rabbit muscle, as originated by Lohmann, inhibiting the
activity of ATPase with magnesium ion, chemical synthesis using AMP as a
starting material, and fermentation. Regarding ATP formation by fermen-
tation methods, Lutwak-Mann & Mann in 1935 and Ostern et al. in 1938 found
that added AMP or adenosine was phosphorylated to A TP in the alcohol
fermentation of yeast.
During the past two decades, several biochemical processes of A TP
production were developed in Japan; (1) phosphorylation of adenosine and
AMP through glycolysis by yeasts, using glucose as the energy donor; (2)
phosphorylation of AMP by Escherichia coli with a hybrid-plasmid containing
genes for phosphofructokinase and triose phosphate isomerase, (3) direct
ribotidation of adenine through salvage synthesis and phosphorylation by
Brevibacterium ammoniagenes, and (4) phosphorylation with or without
ribotidation of AMP, adenosine and adenine using the oxidative phosphoryla-
tion system of methylotrophic yeast and methanol as the energy donor. These
methods will be described in this chapter.
337
338 Y. Tani
2 CHEMISTRY AND ASSAY OF ATP
ATP has the chemical structure shown in Fig. 1, and a molecular weight of
507·2. The absorption maxima are 257nm at pH2 and 259nm at pH7, with
the molecular extinction coefficients of 14·7 and 15·4, respeCtively. It is rather
stable in neutral solution but unstable in acidic solution; 60% of phosphorus is
liberated by heating at 100°C for 10 min in 1 M HCl. The breakage of P-O
bonds at y and fJ positions produce free energy of 7-8 kcal (30-40 kJ).
ATP and its related compounds can be determined quantitatively by several
methods.
A convenient paper chromatographic method is as follows. The sample on
filter paper is developed in a solvent system containing 5 vol. of iso-butyric
acid and 3 vol. of 0·5 N ammonium hydroxide. Each compound on paper is
detected under UV irradiation. Absorbing parts are cut off and eluted with
5 ml of 0·1 M hydrochloric acid for about 15 h at room temperature. Absor-
bancy at 260 nm is measured by a spectrophotometer and the amounts of
compounds are calculated from standard curves. Since inosine and AMP are
developed on the same position with this solvent system, these compounds
were calculated by measuring absorbancy at 250 and 260 nm.
High voltage paper electrophoresis using two kinds of buffers, 0·1 M borate,
pH 9·4, and 0·2 M acetate, pH 3·5, is carried out at 3 kV for 30-60 min.
Detection and determination of nucleic acid-related compounds are the same
as the paper chromatography.
High performance liquid chromatography is carried out with a Hitachi
liquid chromatograph system 655 under the following conditions: column,
M&S PACK C18 (4·6 mm i.d. x 150mm); mobile phase, 0·2M potassium
phosphate buffer, pH 6·0 (solvent A), and methanol (solvent B); gradient, 2%
of solvent B (0-6 min) and 10% of solvent B (6-20 min); flow rate,
1·0 ml/min; injection volume, 5 Ill; detection, absorbancy at 260 nm. Figure 2
demonstrates the elution pattern of a reaction mixture to phosphorylate
adenosine by a methylotrophic yeast, which will be described below.
ATP and ADP are determined by enzymatic methods using
o 0 0
II II II
HO-PY-O-Pf3-O-p a -O-H2 CS' 0
I I I
OH OH OH
OH OH
Fig. 1. Chemical structure of ATP .
Microbial Production of A TP 339
::
o II 10 111 20
min
Fig. 2. High performance liquid chromatography of A TP and its related compounds in
the reaction mixture.
hexokinase/glucose 6-phosphate dehydrogenase and pyruvate kinase/lactate

dehydrogenase systems, respectively, in measuring the absorbance of NADH
at 340nm.
Isolation of A TP and its related compounds can be carried out by a
purification process with column chromatography employing Dowex 1 x 2 as
ion exchanger.
3 ATP PRODUCTION THROUGH GLYCOLYTIC PATHWAY

(SUBSTRATE-LEVEL PHOSPHORYLATION)
3.1 Yeast Cell Reaction Method
It was found by Tochikura et al. (1967) that AMP or adenosine, added to a

fermentation system with ground or acetone-dried baker's yeast cells, was
effectively phosphorylated to A TP and ADP. In this system, 72% of AMP
added as substrate was converted to ATP. The phosphorylation of AMP
scarcely occurred with air-dried or fresh yeast cells, while the phosphorylation
of adenosine to A TP proceeded only with acetone-dried yeast. The concentra-
tion of inorganic phosphate as buffer in the reaction mixture is a special
requirement of this method: the phosphorylation of AMP with acetone-dried
yeast proceeds at a concentration of 1/3 M but not at 1/9 M or 2/3 M, and that
with ground yeast proceeds at 1/4M. The mechanism of ATP formation is
thought to be the substrate-level phosphorylation of AMP or adenosine via
ADP, using the energy of glycolysis of glucose. The essential features of this
method are presumably the inhibition of phosphatase by a high concentration
340 Y. Tani
of inorganic phosphorus and the release of the inhibited glycolysis in high salt
concentrations by AMP.
This method was improved to permit easier cell preparation, and by adding
higher concentrations of the substrate (Tanaka & Hironaka, 1972). They
obtained 15·7 g of ATP·Na2 from 10 g of adenosine in 1·2 liters of the reaction
mixture after treatment of the yeast cells with a surfactant, Cation S, and the
addition of manganese chloride.
Further improvement of the productivity was studied in detail by
Tochikura's group. High amounts of ATP (202 mM, 102·5 g/liter) and ADP
(37 mM, 15·8 g/liter) were obtained at 35-h reaction at 28°C in the reaction
mixture consisting of 300 g dried yeast cells per liter, 0·4 M adenosine, 1 M
inorganic phosphate and 1·5 M glucose initially and with feeding of 0·6 M
inorganic phosphate and 0·5 M glucose at 3-h incubation periods.
The mechanism of the A TP production by this method is as follows:
adenosine + ATP adenosine kinase) AMP + ADP (1)
AMP + ATP adenylate kinase) 2ADP (2)
glucose + 2Pi + 2ADP glycolysis) 2 ethanol + 2C02 + 2ATP (3)
By adding one-and-a-half times equation (3) to the sum of equations (1) and
(2), equation (4) is derived:
adenosine + 1· 5 glucose + 3Pi ~ A TP + 3ethanol + 3C02 (4)
Screening to find an enzyme source other than baker's yeast has also been
carried out. The AMP-phosphorylating activity was searched for among 59
strains of 17 genera of yeasts, and Debaryomyces nilssonii and Hansenula
jadinii were found to be good ATP producers. These yeasts could phosphory-
late 80-100% of AMP (lOmM) to ATP. It was also found that a hydrocarbon-
assimilating yeast, Candida sp., possessed the ability to form A TP from AMP
in high yield; the activity was markedly affected by the culture conditions for
cell production.
The mechanism of phosphorylation was further studied with respect to
the hexokinase isozyme (Kimura et al., 1980). Dried cells of Hansenula jadinii,
that had been cultured aerobically with acriflavine, contained three hexokinase
isozymes and metabolized glucose to produce A TP in the presence of a high
concentration of inorganic phosphorus. The cells cultured aerobically without
acriflavine contained two hexokinase isozymes and could not metabolize
glucose under the same conditions. Two of the isozymes of the yeast cultured
with acriflavine were similar to isozymes of the yeast cultured without
acriflavine. The third isozyme was resistant to a high phosphorous concentra-
tion. Therefore, the isozyme was considered to be responsible for regeneration
of ATP through glycolysis and phosphorylation of nucleotides in the A TP-
producing system.
Recently, genes of hexokinase and glucokinase of Saccharomyces cerevisiae
were cloned and the yeast cells harbouring the hybrid plasmid could
phosphorylate glucose about three times as effectively as the original strain
(Fukuda et al., 1984). These cells completely converted adenosine (130 mM)
into A TP, consuming glucose rapidly by the enhanced glycolytic pathway.
The yeast cell method could also be applied to the production of CfP, UTP
and GTP from the corresponding mononucleotides in high yield.
3.2 Escherichia coli-CeU Reaction Method
The production of A TP from AMP by the glycolytic pathway in dried cells of

E. coli was achieved by applying recombinant DNA techniques.
A plasmid pLC16-4 which contained an E. coli phosphofructokinase
structural gene was introduced into E. coli C600 (Shimosaka et al., 1981). It
was designated as C600 (pLC16-4). By gene dosage effect the C600 (pLC16-4)
showed a sevenfold higher level of phosphofructokinase than the original strain
E. coli C-600. By the use of this plasmid-carrying E. coli C600 (pLC16-4), a
high level of A TP was produced in the A TP formation system consisting of
AMP and glucose 6-phosphate as the substrate: about 75% of 20 mM AMP was
phosphorylated to ATP in a 3-b reaction, while only 25% of AMP was
converted to ATP by the original strain C600. However, this method was
difficult to operate practically since glucose 6-phosphate is very expensive.
It could also be prepared from sucrose and inorganic phosphate with
Leuconestoc mesenteroides sucrose phosphorylase (Vandamme et al., 1987)
which yields fructose and glucose-1-phosphate; subsequent action of a phos-
phoglucose mutase then results in glucose-6-phosphate formation.
Methods were therefore sought to produce ATP more economically from
AMP and glucose, and E. coli B was found to be a suitable strain for this
purpose. An ATP production method from AMP and glucose by dried E. coli
B-cells dosed with the glucokinase gene was then developed (Yamaguchi et al. ,
1984). The glucokinase activity of E. coli B cells was increased about 2·2-fold
by culturing the cells in a medium containing D-fructose and ammonium
phosphate. After drying, these cells converted AMP to ATP, consuming
glucose by the glycolytic pathway. Under the optimal conditions, AMP
(309 mM) was almost completely converted into ATP within 3 h. The dried E.
coli B cells harbouring a hybrid plasmid (pGK100) carrying the glucokinase
gene showed higher A TP-producing activity than cells without hybrid plasmid.
3.3 Continuous ATP Regeneration by Yeast Cells
There has been extensive work on bioreactors which use immobilized enzymes,
resting cells and living cells as catalysts (Kennedy, 1987).
A TP regeneration systems so far studied are classified into four categories;
chemical synthesis, whole cells (mainly yeasts), organelles, and cell-free
enzymes. Chemical synthesis was eliminated because it uses an excess of
342 Y. Tani
expensive reactants and non-aqueous media which must be separated and

recovered before and after ATP regeneration. Although organelles such as
mitochondria, chloroplasts and chromatophores are efficient in A TP regenera-
tion, with very cheap energy sources, they are too unstable. It was considered
that cell-free enzyme systems were the most feasible routes for large-scale A TP
regeneration processes, in which acetyl phosphate as phosphoryl donor and
acetate kinase were employed, considering the stability and cost of the
phosphoryl donor, and the stability of the enzyme. On the other hand, the
yeast cell system for A TP production was also considered to be suitable for the
continuous A TP regeneration in the following respects: (1) the energy source
for ATP formation, glucose, is easily supplied; (2) enzyme preparation is easy,
and (3) the most abundant reaction intermediate formed as a by-product is
energy-rich fructose 1,6-bisphosphate, which can be used as an energy source
for further A TP regeneration.
Continuous A TP regeneration from adenosine using enzymes of the
glycolytic pathway was demonstrated using a reactor equipped with a
semi-permeable membrane by Asada et al. (1981). Firstly, ATP regeneration
from adenosine in batch systems with acetone-dried yeast cells, acetone-dried
yeast cells immobilized in photo-crosslinked resin gel, and an extracted crude
enzyme preparation was carried out. The substrate solution contained 600 mM
glucose, 600 mM potassium phosphate buffer, pH 7·3, 20 mM magnesium
sulfate and 20 mM adenosine. A crude enzyme preparation was extracted from
150 mg of acetone dried yeast cells by soaking the cells in 1 ml of a medium
containing 600 mM potassium phosphate buffer, pH 7· 3, 1 M glucose and 25 mM
magnesium sulfate for 3·5 h at 28°C with stirring.
The phosphorylation of adenosine proceeded similarly in all three cases, in
which A TP is accumulated abruptly several hours after the beginning of
incubation, concomitant with fructose 1,6-bisphosphate accumulation, and
reaches more than 75% yield. However, accumulated ATP and fructose
1,6-bisphosphate decreased rapidly with further incubation in the case of whole
cells and immobilized cells, while they remained at a high level for a fairly long
time in the case of the extracted enzymes, suggesting a decrease in ATP-
degrading activity. From this result, the extracted crude enzyme preparation
was chosen for use in a continuous ATP regeneration system.
Since the immobilization of the 13 enzymes participating in A TP regenera-
tion without loss of activity was difficult, and carbon dioxide gas generated
during the reaction must be removed from the system, a membrane-type
reactor was chosen. A plug-flow type continuous reaction apparatus consisting
of eight chambers, each separated into two compartments by a collodion
membrane of 40-50 m thick, was successfully constructed.
The addition of dithiothreitol, a protector of the enzyme-thiol group,
increased the duration of the period of ATP formation from 42 to 100 h. With
the addition of a yeast extract containing intermediates of the glycolysis, a
stable steady state was attained, in which a yield of more than 75% ATP
continued for 2 weeks. It was considered that the long-term continuous ATP
formation obtained might be due to the protection and stabilization of enzymes
Microbial Production of ATP 343
by the yeast extract which was added to prevent inactivation. Another role of
the yeast extract was to act as an energy-rich substance which is necessary to
initiate the reaction.
4 ATP PRODUCTION THROUGH SALVAGE SYNTHESIS BY

BREVIBACTERIUM AMMONIA GENES
4.1 Culture Method
A culture method to ribosylate and phosphorylate adenine, a cheaper starting

material than AMP and adenosine, by B. ammoniagenes for the production of
A TP was shown to be useful for the industrial production of A TP by Tanaka et
al. (1968).
Brevibacterium ammoniagenes was cultivated for 4 days at 30°C in the
medium consisting of 100 g glucose, 6 g urea, 10 g monopotassium phosphate,
10 g dipotassium phosphate, 10 g magnesium sulfate, 10 g yeast extract, 0·1 g
calcium chloride and 30 g biotin in 1 liter , pH 7 ·4, with the addition of adenine
(2 g/liter) after 3 days. It was found that A TP, AD P and AMP accumulated in
amounts of 1·57, 1·59 and 2·16 g/liter of the culture filtrate, respectively. GTP,
GDP and GMP similarly accumulated on cultivation with the addition of
guanine. The mechanism of phosphorylation is considered to be the further
phosphorylation of AMP from adenine, since the accumulation of AMP has
been demonstrated with adenine-supplemented cultures of the same organism.
Although adenine can be used as the precursor with this method, a drawback
appeared for practical production. Brevibacterium ammoniagenes cells grown
with a strictly limited supply of manganese ion showed a high ability to convert
adenine to A TP. However, excessive addition of the metal ion (above
30 g/liter) to the culture media severely inhibited ATP production. It became
clear that limitation of manganese ions caused large morphological changes in
the cells which result in the removal from the cell membrane of a permeability
barrier to the excretion of A TP. The optimal concentration of the metal ion
was very low, around 10 g/liter, and the optimal range was very narrow.
4.2 CeU Reaction Method
A cell reaction system for the production of A TP from adenine with

manganese ion-unlimited cells of B. ammoniagenes was developed by Fujio &
Furuya (1983). They first screened agents which allow A TP to permeate
through the cell membrane and accumulate in the reaction mixture. Polyoxy-
ethylene stearylamine, a cationic detergent, was obtained as the most effective
additive. With resting cells of B. ammoniagenes grown in manganese-ion-rich
medium, the reaction conditions for A TP production from adenine were
optimized at 33°C and pH 7·4. Magnesium ions inhibited the ATP production.
For efficient ATP production, it was necessary to maintain the concentration of
soluble phosphate between 35 and 150 mM. A practical method fulfilling this
344 Y. Tani
requirement was developed, in which disodium phosphate was added to the

reaction mixture intermittently every hour, starting at the third hour of the
reaction. Under these optimum conditions, 13 g of ATP·Na2·3H20 per liter
was produced from 3·5 g of adenine per liter. The molar ratio of the
transformation of adenine to A TP was approximately 83%.
Further improvement of this method was performed by Fujio & Furuya,
(1985). The ATP formation in this system ceased within 6-8 h. Simultaneous
addition of magnesium ion and phytic acid, a chelator of divalent cations, was
found to allow ATP formation to continue longer, and 24·2 g of
ATP·Na2·3H20 per liter was accumulated in lOh. However, ATP formation
ceased thereafter.
This second cessation was proved to be caused by the lack of magnesium ion
active as a co-factor. To provide the active magnesium ion, sufficient phytic
acid was added at the beginning of the reaction and magnesium ion was also
added intermittently. Under these conditions ATP production continued
further, and the rate of ATP production was increased; 37·0 g of
ATP·Na2·3H20 per liter was accumulated in a 13-h reaction.
5 ATP PRODUCTION THROUGH OXIDATIVE

PHOSPHORYLATION BY METHYLOTROPHIC YEASTS
Industrially applicable methods for biochemical production of A TP have been

developed and established based on substrate-level phosphorylation of yeasts
and on salvage synthesis in B. ammoniagenes as described above. However,
there has been no report on A TP production from AMP or adenosine by use
of the oxidative phosphorylation system. Moreover, the energy source
substrate was exclusively glucose.
Methylotrophic yeasts reduce NAD+ in the oxidation pathway to supply
energy for the assimilation of C 1 compounds through glyceraldehyde 3-
phosphate by the xylulose monophosphate pathway. The NADH formed is
oxidized in the respiratory chain, and ADP is phosphorylated by the
proton-driving force in the oxidative phosphorylation system.
C 1 compounds are currently attracting much attention as convenient raw
material for the chemical industry and for biotechnology. Methanol could be
expected to be one of the most useful of these compounds in the very near
future because it can be easily produced from natural gas. Methanol is
advantageous as a carbon and energy source of microbial growth because of its
high purity and miscibility with water in all proportions. Therefore, attempts
have been made to establish new processes for fermentative production in
which both unique and conventional aspects of metabolism of methylotrophs
are utilized.
Recently, an A TP production system from AMP, adenosine and adenine
with Zymolyase® and sorbitol-treated cells of a methylotrophic yeast, Candida
boidinii (Kloeckera sp.) No. 2201 was developed from the viewpoint described
above. The system was based on conversion of free energy of methanol to

ATP through oxidative phosphorylation in respiration.
5.1 Construction of the ATP-producing System
A reaction system producing A TP from AMP and reduced C 1 compounds was

constructed with cells of C. boidinii No. 2201 (Tani et al., 1982). Yeast cells
were treated with Zymolyase, a cell wall-lytic bacterial enzyme. The treated
cells release protoplasts on transfer to a hypotonic reaction mixture from a
hypertonic suspension, and then burst to yield intact mitochondria and
cytoplasmic enzymes. A reaction mixture containing AMP, methanol (or
formate), NAD+, inorganic phosphate and the treated cells was used for ATP
production. Production of A TP could be inhibited by replacement of air in the
reaction tube by nitrogen gas, and by addition of an uncoupler, an electron-
transport inhibitor or an ATPase inhibitor. Involvement of substrate-level
phosphorylation in the presence of xylulose 5-phosphate, an initiator of the
xylulose monophosphate pathway, was negligible.
Thus, Zymolyase-treated cells of C. boidinii No. 2201 produce ATP from
AMP by adenyl ate kinase, oxidation of methanol by formaldehyde and
formate dehydrogenases to reduce NAD+ and oxidative phosphorylation of
ADP to ATP in the respiratory chain as shown Fig. 3.
The phosphorylation of AMP proceeds by the following scheme:
AMP + ATP .Q4 2ADP ~ 2ATP
ATP for reaction (1), adenylate kinase, is initially supplied from the
endogenous pool in the protoplast and then by reaction (2), oxidative
CH 30HT'""'\,HCHO ~ CHo-SG- HCOO~ cO 2
o H;o2 If. \ (. \
2 ~ NAD+ NADH NAD+ NADH
Microbody
H20
ATP
Fig. 3. ATP-producing system from AMP by methylotrophic yeast.
346 Y. Tani
phosphorylation. Thus, oxidative phosphorylation is required twice for the

formation of one molecule of A TP in this system.
Reaction conditions for A TP production were optimized in respect of
substrate and coenzyme concentrations, pH and temperature, osmotic pres-
sure, and oxygen supply. Under optimal conditions, 13 and 4 g/liter of ATP
were produced with methanol and formate as C 1 substrate, respectively (Tani
et a/., 1984a).
Subsequently, cell treatment was simplified to obtain yeast cells with stable
ATP production ability, by incubation with added sorbitol (Tani et a/., 1984b).
The sorbitol-treated cells did not lose this activity even in the presence of 3 M
methanol or after incubation for 36 h. Under optimal reaction conditions, the
amount of ATP in the reaction mixture reached 30 g/liter (Tani et a/., 1984c).
The effect of sorbitol treatment on the cell structure of C. boidinii No. 2201
was demonstrated by analysis with transmission electron microscopy (Yone-
hara & Tani, 1988). The high ATP-producing activity of the cells was proved
to be due to the plasmolysis without destruction of essential organelles for the
A TP-producing system.
To prepare stable active cells, the essential elements of the yeast extract in
the culture medium were identified: biotin was an essential growth factor;
ferric ion and thiamin stimulated growth more or less and zinc ions strongly
stimulated growth. Limitation of zinc ions in the culture medium (0·5 mg/liter
of zinc chloride) improved the A TP yield from AMP, the conversion rate
being 92%, possibly due to repression of AMP deaminase, for which the zinc
ion was a cofactor and which affected the A TP-producing activity. A zinc
ion-supplemented culture provided fully-active cells as to ATP production.
Transmission electron microscopy revealed that the zinc ion was an essential
factor for the formation of peroxisomes in which alcohol oxidase and catalase
are localized.
S.2 Energy Efficiency of the ATP·produdng System
The AMP phosphorylation system can be characterized as energy conver-

sion from methanol to high-energy phosphate bonds in nucleotides as well
as the biotransformation of adenine nucleotide. As described below, AMP can
be replaced with adenosine or adenine as a substrate for A TP formation.
The sequential conversion system can be illustrated as shown in Fig. 4.
NADH produced by the methanol-dissimilation system is located in the cytosol
of this yeast cell and cannot permeate through the inner mitochondrial
membrane. The electron transport system coupled to the oxidative phos-
phorylation is located in the inner mitochondrial membrane. Subsequently,
electrons from cytoplasmic NADH may enter mitochondria by the glycerol
phosphate shuttle mechanism and reduce FAD bound to the inner membranes.
The reduced flavin inside the mitochondria transfers its electrons to the
respiratory chain at the level of coenzyme Q. Consequently, the P / 0 ratio in
this system is not 3·0 but 2·0.
Adenine CH 3 0H
Adenosine
1
J\
HCHO
1
AMP
\ NATyto'~H
ADP
H' cO 2
ATP ADP O2
H'"
Fig. 4. ATP-producing system from adenosine or adenine by methylotrophic yeast.
The material balance of A TP production from adenosine can be summarized

in the equation below:
adenosine + 3Pi + 3/4 methanol + 9/8 O 2 - - ATP + 3/4C02 + 9/2 H 2 0
Table 1 shows the free energy changes of respective reactions involved in the
system. The theoretical value of the total energy efficiency, Ke(t), from
methanol to A TP is,
Ke(t) = 18·0/(167·91 x 3/4) x 100 = 14·3%
The practical energy efficiency, Ke (p ), was calculated using a result of
Table 1
Free Energy Changes of Reactions Involved in A TP
Production from Adenosine
Reaction - Ll.G(kcallmol)
CH3 0H + 3/202~ CO 2 + 2H20 167·91

NADH + H+ + 1/202~ NAD+ + H 20 52·6
Adenosine + 3Pi ~ ATP + 3H20 -18·0
3ADP + 3Pi ~ 3ATP + 3H20 -7·3 x 3
AMP+ATP~2ADP o
Adenosine + ATP~ AMP + ADP 3·9
348 Y. Tani
adenosine phosphorylation and methanol consumption in a typical reaction

(Yonehara & Tani, 1986). The amount of not only adenine nucleotides but
also that of IMP, a by-product, as energy stored compounds in the reaction
mixture was also estimated. Furthermore, the evaporation of methanol from
the reaction mixture was prevented by an air-sealed system.
The practical energy efficiency against the methanol consumption is ex-
pressed as follows,
Ke(p) = (2181/51302) X 100 = 4·25%
The ratio of the practical efficiency against the theoretical efficiency is
expressed as follows,
Ke = (Ke(p)/Ke(t» X 100 = (4·25/14·3) X 100 = 30%
Thus, the energy efficiency of the ATP-producing system is quite high as in
a biochemical energy conversion system.
5.3 ATP Production from Adenosine or Adenine
The use of adenosine or adenine instead of AMP, which is chemically rather

unstable and commercially expensive as a starting material for A TP produc-
tion, was investigated to construct a more profitable process with sorbitol-
treated cells of C. boidinii No. 2201 (Tani & Yonehara, 1985).
The reaction conditions for A TP production from adenosine or adenine
were optimized as to initial concentrations of substrates and coenzymes, pH of
potassium phosphate buffer, temperature, osmotic pressure and other addi-
tives. Efficient A TP production from adenosine was achieved by the addition
of pyrophosphate together with magnesium ions. On the other hand, significant
A TP production from adenine was obtained but not in high yield under the
conditions tested.
Orthophosphate, pyrophosphate and divalent metal ions inhibited the
deamination of AMP to IMP, a major by-product. No hypoxanthine nucleo-
tide compound was accumulated by addition of an adenosine deaminase
inhibitor, coformycin. By successive feeding of potassium phosphate to
maintain the phosphorous concentration at over 100 mM, the conversion rate
from adenosine to A TP was improved to 70%. Simultaneous feeding of
potassium phosphate and adenosine resulted in the accumulation of 100 mM
A TP (50·7 g/liter) after 28 h of incubation and the increase of IMP without
decrease of A TP for 48 h.
A TP production was further prolonged up to 60 h of incubation and a high
amount, 198mM ATP (100 g/liter) , was accumulated (conversion rate of
77-4%) from adenosine by control of pH in the range of 6·5-6-8 during the
reaction (Yonehara & Tani, 1987).
Biochemical systems for A TP production so far studied are summarized in
Table 2, in which raw material, ATP yield, efficiency in grams of ATP
Table 2
ATP Production by Biochemical System
System Raw material A TP(g/liter) Efficiency * Molar yield (%)
Culture of Adenine, 1·57 20
B. ammoniagenes glucose
Cell of Adenine, 10·9 0·10 83
B. ammoniagenes glucose
Cell of yeasts Adenine, 102·5 0·34 50
glucose
Cell of E. coli AMP, 14·0 0·14 92
glucose
Chromatophore of ADP, 0·4 2·0 80
R. rubrum light
Chromatophore of ADP, 200·0
R. capsulata light
Cell of blue- ADP, 0·17 56·0 17
green alga light
Acetate kinase ADP, 10·0 100
acetyl-P
Adenylate kinase ADP 0·24 1·3 60
Cell of AMP, 30·0 1·5 60
C. boidinii methanol
Adenosine, 100·0 4·0 77
methanol
Adenine, 4·1 0·20 40
methanol
* ATP produced (g) by 1 g of dry cell weight, protein or chlorophyl.
produced by 1 g of dry cell weight, protein or chlorophyll, and molar yield are
compared. The highest amount is obtained by yeast cell methods using
glycolysis (substrate level phosphorylation) and oxidative phosphorylation.
The efficiency of A TP productivity for cells is more than 10 times higher in the
latter than that in the former. The efficiency is quite high in the system using
photophosphorylation, but the amount of A TP produced by these methods is
quite low and the substrate needed is ADP.
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Tochikura, T., Kuwahara, M., Yagi, S., Okamoto, H., Tominaga, Y., Kano, T. &
Ogata, K. (1967). Fermentation and metabolism of nucleic acid-related compounds
in yeasts. J. Ferment. Technol., 45, 511-29.
Vandamme, E. J., Vanloo, J., Machtelinckx, L. & De Laporte, E. (1987). Microbial
sucrose phosphorylase: fermentation process, properties and biotechnical applica-
tions. Adv. Appl. Microbiol., 32.
Yamaguchi, S., Fukuda, Y., Shimosaka, M. & Kimura, A. (1984). Phosphorylation of
AMP to ATP by dried Escherichia coli B cells, J. Ferment. Technol., 62,29-33.
Yonehara, T. & Tani, Y. (1986). Comparative studies on ATP production from
adenosine by a methanol yeast. Agric. BioI. Chem., 50,899-905.
Yonehara, T. & Tani, Y. (1987). Highly efficient production of ATP by a methanol
yeast, Candida boidinii (Kloeckera sp.) No. 2201. J. Ferment. Technol., 65,255-60.
Yonehara, T. & Tani, Y. (1988). ATP production by a methanol yeast, Candida
boidinii (Kloeckera sp.) No. 2201: effects of sorbitol treatment and zinc on cell
structure as to ATP production. Agric. BioI. Chem., 52,909-14.
Chapter 19
ADENOSYLMETHIONINE, ADENOSYLHOMOCYSTEINE
AND RELATED NUCLEOSIDES
1 INTRODUCTION
The function of L-methionine as a methyl group donor in biological trans-

methylation reactions requires ATP to form so-called active methionine. This
compound was identified as S-adenosyl-L-methionine (AdoMet) by Can toni
(1952, 1953), who showed, with a liver preparation, that it was synthesized by
the enzyme, methionine adenosyltransferase (EC 2.5.1.6) through the follow-
ing reaction:
L-methionine + ATP ~ AdoMet + PPi + Pi
Ado Met is the most versatile methyl group donor for many biological
transmethylation systems in both prokaryotic and eukaryotic organisms,
whereas 5-methyltetrahydrofolic acid, methylcobalamine and betaine are
involved in relatively few methylation reactions. Many low molecular weight
metabolic compounds receive the methyl group from AdoMet through the
reactions catalyzed by AdoMet-dependent transmethylases. These include
catechols, norepinephrine, histamine, serotonine, tryptamine, and so on.
Much attention has recently been paid to the methylation of membrane
phospholipids in relation to regulation of membrane structure and function.
The methylation of proteins, RNAs and DNAs has also been suggested to play
important roles in regulation of cell differentiation, protein synthesis, car-
cinogenesis and so on. Such versatility of AdoMet-dependent transmethylation
reactions in biological systems suggests that they play very important roles in
cellular regulations. In this respect, transmethylation reactions can be com-
parable to phosphorylation reactions.
S-Adenosyl-L-homocysteine (AdoHcy), the product of AdoMet-dependent
transmethylation reactions, is known to be a potent inhibitor of a number of
AdoMet-dependent transmethylases. The inhibition of AdoMet-dependent
reactions by AdoHcy is usually relieved by the enzymatic hydrolysis of
351
AdoHcy to adenosine and L-homocysteine. The reaction is catalyzed by

AdoHcy hydrolase (EC 3.3.1.1), which was first demonstrated in rat liver by
de la Haba & Cantoni (1959). The activity of this enzyme is thought to playa
critical role in the control of cellular levels of AdoHcy and thereby influence a
variety of methyltransfer reactions.
Since the discovery that AdoHcy hydrolase is an adenosine binding protein
(Hershfield & Kredich, 1978), this area of research has expanded very rapidly.
One of the most important findings in the last 10 years was that an antiviral
agent, arabinosyladenine, attacks this enzyme as a suicide inactivator (Hersh-
field, 1979). This observation soon led to further studies which showed that
AdoHcy hydrolase is a target for dozens of naturally occurring and artificial
adenosine analogs exhibiting antibiotic, antiviral and/or antitumor activities.
In connection with these findings, studies on the methylation of DNAs and
RNAs in relation to cell growth, differentiation and carcinogenesis also
indicated the potential of these nucleosides as pharmacologically active agents.
Another attractive and widely expanding line of investigation on trans-
methylation reactions is their relation to the central nervous system. The
metabolic relationships between AdoMet, AdoHcy and related nucleosides,
and biogenic amines suggests their importance in this field. For example,
AdoMet has been suggested as an effective treatment for depression, sleep-
lessness and a variety of neuroses. AdoHcy is expected to have unique sedative
and anticonvulsive properties.
In addition, AdoMet, after decarboxylation by AdoMet decarboxylase (EC
4.1.1.50), provides the aminopropyl group for the synthesis of polyamines
which also play important roles in control of cell growth, differentiation and
protein synthesis.
The involvement of methyl and aminopropyl group transfer reactions in such
a wide variety of biological phenomena suggests the potential of these
nucleosides in the pharmaceutical and chemotherapeutic fields. However, no
process for the practical production of these nucleosides had been established
before the current work performed by Yamada and co-workers (Shimizu &
Yamada, 1984; Yamada et aI., 1984; Yamada & Shimizu, 1985, 1988). In this
chapter, we summarize current progress in the biotechnological production of
these nucleosides. Several reviews summarize other various aspects of these
nucleosides and provide excellent background information (Cantoni, 1975;
Salvatore et ai., 1977; Usdin et ai., 1979, 1982; Zappia et ai., 1979; Ueland,
1982; Schlenck, 1983, 1984; Shimizu & Yamada, 1984; Tabor & Tabor, 1984;
Borchardt et ai., 1986).
2 CHEMISTRY
The methyl group of the methionine unit in the molecule of Ado Met is
activated by the positive charge on the adjacent sulfur atom, which makes it
very reactive. Thus, AdoMet is used as a methyl donor in versatile biological
Adenosylmethionine, Adenosylhomocysteine and Related Nucleosides 353
transmethylation reactions. Four stereoisomers are possible for AdoMet due to

two asymmetric centers at the sulfonium center and at the a-amino carbon
atom in the methionine moiety. Only the natural form, i.e. ( - )-S-adenosyl-L-
methionine, is active as the methyl donor (de la Haba et al., 1959). The
conformation of AdoMet was studied by Stolowitz & Minch (1981). AdoMet is
unstable at room temperature, both as a dry solid and in aqueous solution,
being easily decomposed to mainly 5'-deoxymethylthioadenosine (MeSAdo).
Several AdoMet analogs, i.e. S-adenosyl-D-methionine, S-adenosyl-2-
methylmethionine and S-adenosylethionine have been reported to be synthe-
sized enzymatically (see Table 1).
AdoHcy is the demethylated product of Ado Met. The structure contains a
single asymmetric center at the a-amino carbon atom in the homocysteine
moiety, so that it is optically active. In most cases, only the L-isomer shows
potent inhibitor activity toward biological transmethylation reactions which
utilize Ado Met as the methyl donor. The conformation of AdoHcy has been
reported (Ishida et al., 1982). Many analogs of AdoHcy, modified in the sugar
moiety, amino acid portion or purine ring, have been synthesized to obtain
information on the structural requirements for binding of AdoHcy to various
transmethylases and compounds that resist metabolic degradation. Some of
these compounds have been reported to show antiviral and growth inhibitory
activity. Recent reviews (Ueland, 1982; Borchardt et al., 1986) provide further
information.
Table 1 lists several important derivatives of Ado Met and AdoHcy.
3 BIOSYNTHESIS
3.1 Activated Methyl Cycle
Ado Met is synthesized by the transfer of an adenosyl group from A TP to the

sulfur atom of L-methionine. The reaction is unusual in that there is a complete
dephosphorylation of A TP, and the energy of ATP is used to activate the
methyl group of the methionine unit. AdoHcy is formed when the methyl
group of AdoMet is transferred to an acceptor compound such as catechol. In
eukaryotic and most prokaryotic organisms, the thioether of AdoHcy is then
hydrolyzed by AdoHcy hydrolase to form adenosine and L-homocysteine. This
reaction had long been thought to be characteristic in eukaryotic organisms
(Duerre, 1962; Miller & Duerre, 1968; Walker & Duerre, 1975). An
alternative reaction that yields adenine and S-ribosyl-L-homocysteine for the
metabolism of AdoHcy had been suggested to be involved in prokaryotic
organisms. However, it was recently demonstrated that most prokaryotic
organisms also metabolize AdoHcy through the thioether hydrolysis as well as
eukaryotic organisms (Shimizu et al., 1984a, 1988). Although the reaction
favors synthesis of AdoHcy in vitro, the metabolic flow is in the hydrolytic
direction because adenosine and L-homocysteine are rapidly metabolized in the
<.H
Table 1 ~
Some Derivatives of AdoMet and AdoHcy
Amino acid modifications Chemical structures of each amino acid moiety (R) are as shown below left
NH2
~JyN~
':~ ~
HO OH ~
§.
Accumulated in Candida utilis when grown with D-methionine (Schlenk et al., 1978); I:j.
S-Adenosyl-D-methionine I:
CH 3 shows methyl donor activity in some transmethylation systems (Nakamura & Schlenk, Roo
• I 1976) ;t:
HOOC-CH-(CH 2)2-S-CH2-
I + ~
NH2 ~
~
S-Adenosyl-L-ethionine Accumulated in baker's yeast (Schlenk et al., 1965) and molds (Kusakabe et al., 1974)
CH 2-CH3 when grown with L-ethionine
I
HOOC-CH-(CH 2)2-S-CH2-
I +
NH2
S-Adenosyl-2-methyl-DL-methionine Accumulated in Candida utilis when grown with 2-methyl-DL-methionine (Schlenk et
CH] CH3 al., 1978); shows methyl donor activity in some transmethylation systems (Nakamura
I . I & Schlenk, 1976)
HOOC-C-(CH 2)2-S-CH2-
I +
NH2
S-Adenosyl-o-homocysteine Synthesized chemically and shows inhibitor activity in some transmethylation reactions
• (Borchardt & Wu, 1974)
HOOC-CH-(CH2h-S--CH2-
I
NH2
;...
S-Adenosyl-L-cysteine Synthesized by lupin seed and beef liver AdoHcy hydrolases (Guranowski & ~
::r
Jakubowski, 1983)
HOOC-CH-CH 2-S--CH 2- ~
I
NH2 I§.
Sinefungin Isolated from Streptomyces griseoius as an antifungal antibiotic (Hamill & Hoehn, s·
1973); a potent inhibitor of some transmethylation reactions (see Ueland, 1982) .';..."
HOOC-CH-(CH2h-CH-CH,-
I 1-
NH2 NH2 ~
~
A9145C A metabolite of Sinefungin (Hamill & Hoehn, 1973); a potent inhibitor of some s::
transmethylation reactions (see Ueland, 1982) ~
HOOC-CH-(CH2h-CH-CH= ~
I I '"~
NH2 NH2 s·
5' -Deoxy-5' -S-isobutyl-thioadenosine §'"
Chemically synthesized as an antiviral agent (Hildesheim et ai., 1971); an inhibitor of >:>..
CH,,,, polyamine synthesis and transmethylation reactions (see Ueland, 1982) ~

S"
CH-CH 2-S--CH z- [
CH/,
Nucleoside modifications Chemical structures of each base are as shown. In the case of S-aristeromycinyl-L-
H2N~
homocysteine, the ribose moiety is modified as shown.
,
~
CH-CH 2-CH 2-S--CH z base
HOOC/ ~
U\
""
(continued) U\
HO OH
....
VI
Table l-contd. 0"1
S-Formycinyl-L-homocysteine Synthesized by beef liver, lupin seeds and bacterial AdoHcy hydro lases (Guranowski et
at., 1981; Shimizu etal., 1984a, b; Shimizu & Yamada, 1988)
NH z
N~N\
~NJ--C~
S-Nebralinyl-L-homocysteine Synthesized by beef liver, lupin seeds and bacterial AdoHcy hydro lases (Guranowski et
at., 1981; Shimizu et at., 1984a, b; Shimizu & Yamada, 1988)
H
~
N~N
~
§.
~NJ-) N·
I:
R-
S-Tubercidinyl-L-homocysteine Synthesized by beef liver, lupin seeds and bacterial AdoHcy hydrolases (Guranowski et
aI., 1981; Shimizu et aI., 1984a). A potent inhibitor of various transmethylation ~
NH z reactions (Coward et at., 1974; also see Ueland, 1982) ~
(j) I
N N
S-2-Chloroadenosyl-L-homocysteine Synthesized by bacterial AdoHcy hydrolase (Shimizu et aI., 1984a, b; Shimizu &
Yamada, 1988); 2-Chloroadenosine shows a potent inactivator activity toward
AdoHcy hydrolase (Shimizu et aI., 1984a; see also Ueland, 1982)
NJ:-N
CIAN~~
S -It'-Methyladenosyl-L-homocysteine A potent inhibitor of some transmethylation reactions (Hoffman, 1978); Synthesized by
H-N-CH 3 liver, lupin seeds and bacterial AdoHcy hydrolases (Hoffman, 1978; Guranowski et
at., 1981; Shimizu et at., 1984a)
ex)
S-l-Methyladenosyl-L-homocysteine Synthesized by bacterial AdoHcy hydrolase (Shimizu et al., 1984a)
HC NH z
~
, "~~N~ f}
5
"""NJ-N ~
S-Inosyl-L-homocysteine Synthesized by beef liver, lupin seeds and bacterial AdoHcy hydrolases (Guranowski et i
OH ai., 1981; Shimizu et al., 1984a) ";;.
5·
;:s
s·
-"
7JyN~ ~
"""NJ-N ~
S-3-Deazaadenosyl-L-homocysteine Synthesized by beef liver and lupin seeds AdoHcy hydrolases (Guranowski et al., 1981); ~
s:
3-Deaza-adenosine is a potent competitive inhibitor of AdoHcy hydrolase
(Guranowski et al., 1981) ~
~~ ~
..
~
N~C~~ ~.
S-2-Aza-3-deazaadenosyl-L-homocysteine Synthesized by beef liver and lupin seeds AdoHcy hydrolases (Guranowski et ai., 1981)
~
~
NH z Ei""
~
1:1.
..,~
~:X) ~
N..,C ~
S-8-Azaadenosyl-L-homocysteine Synthesized by beef liver and lupin seeds AdoHcy hydrolases (Guranowski et al., 1981)
.~
NH z
N~\ w
(continued) ~
~NJ...N
~
00
Table l---contd.
S-8-Aminoadenosyl-L-homocysteine Synthesized by beef liver and lupin seeds AdoHcy hydrolases (Guranowski et al., 1981)
NH2
N~N
~~TJ- )-NH2
N N
S-(5' -Deoxyadenosine N'-oxide- 5') Synthesized by beef liver, lupin seeds and bacterial AdoHcy hydrolases (Guranowski et
-L-homocysteine aI., 1981; Shimizu et al., 1984a, b; Shimizu & Yamada, 1988)
~
NH2
~
O'-NJ)-N) §.
N·
I:
~N~ R-
S-Pyrazomycynyl-L-homocysteine Synthesized by beef liver and lupin seeds AdoHcy hydrolases (Guranowski et al., 1981); ~
pyrazomycin is a potent inactivator of AdoHcy hydrolase (see Ueland, 1982) ;;,<
o
H2N~N~ l
T~~'/;
HO C
S-Aristeromycinyl-L-homocysteine Synthesized by beef liver and lupin seeds AdoHcy hydrolases (Guranowski et al., 1981);
aristeromycin is a potent competitive inhibitor of AdoHcy hydrolase (Guranowski et
NH2 al.,1981)
HOCH 2
0:) ~
HO OH
normal cells. Deamination of adenosine by adenosine deaminase (EC 3.5.4.4)

and phosphorylation of adenosine to AMP through the action of adenosine
kinase (EC 2.7.1.20) are the responsible reactions for the further metabolism
of adenosine. The other product, L-homocysteine, is used to regenerate
L-methionine through the transfer of a methyl group from N 5 _
methyltetrahydrofolate. This reaction is catalyzed by 5-methyltetrahydrofolate:
L-homocysteine methyltransferase (EC 2.1.1.13). Alternatively, it can be
methylated to L-methionine by utilizing betaine as a methyl donor with the
enzyme, betaine: L-homocysteine S-methyltransferase (EC 2.1.1.5).
These reactions constitute the activated methyl cycle (Fig. 1). The methyl
groups enter the cycle in the conversion of L-homocysteine into L-methionine
and are then converted to a highly reactive form as Ado Met. The high transfer
potential of the methyl group in AdoMet enables it to be transferred to a wide
variety of acceptor compounds.
When cellular levels of adenosine or L-homocysteine or both are high, the
reaction catalyzed by AdoHcy hydrolase is directed toward synthesis of
':Hz
lY)
....2
¥l>+r%-~-CH~
Hj:. 0
N~N,; C~2./ S-Adenosylmethylthlopropylamlne
~_J--N ~ l
j--J~ 1<H3
I«l OH
ATP
~- -~-CHz-CHr~
~,
HO OH
N:.JLN
S-Adenosylmethionine ~N~I)
HJC-!i-Di2
~
¥,
l?=~
~
HO OH
I
Methylthloadenoslne
HOOC'CH-CHz-CHrS-CH6
L
Adenine lIethylthlor!bO$e-1P
HOOH
,._~ Methionine S-Adenosylhomocysteine L s-Ribosylhomocysteine
5-AdenOSYUJomlysreine hydrolase
CH3- Homocysteine
~
~ Adenosine
,.
\ Ribose
,'
I
...... _----- -- ------ --

-------------- ----- --- --- -----'
Fig. 1. Major routes for the metabolism of AdoMet and AdoHcy and the activated
methyl cycle.
360 S. Shimizu & H. Yammla
AdoHcy and the enzymatic degradation of AdoHcy is inhibited (de la Haba &
Cantoni, 1959). The accumulated AdoHcy blocks some metabolically impor-
tant AdoMet-dependent transmethylation reactions, because AdoHcy is a
potent inhibitor of many AdoMet-dependent transmethylases (Usdin et al.,
1979; Borchardt et al., 1986). Furthermore, AdoHcy hydrolase is an adenosine
binding protein (Hershfield & Kredich, 1978)\ Various adenosine analogs
including adenosine have been shown to inactivate this enzyme irreversibly. As
the result of this inactivation, the cellular level of AdoHcy increases, which
also results in inhibition of transmethylation reactions. Thus, the hydrolysis of
AdoHcy by AdoHcy hydrolase is a key step for the regulation of the activated
methyl cycle and AdoHcy itself is thought to be a key regulator compound for
transmethylation reactions (Cantoni & Chiang, 1980; Ueland, 1982; Borchardt
et al., 1986).
The route which yields adenine and S-ribosyl-L-homocysteine for the
degradation of AdoHcy is present in only few bacteria, especially in Entero-
bacteriaceae (Duerre, 1962; Miller & Duerre, 1968; Shimizu et al., 1984a,
1988). This route involves the following two reactions: cleavage of glycosyl
linkage of AdoHcy by AdoHcy nucleosidase (EC 3.2.2.9), yielding adenine
and S-ribosyl-L-homocysteine, and hydrolysis of S-ribosyl-L-homocysteine to
L-homocysteine and ribose by S-ribosylhomocysteine hydrolase (EC 3.3.1.3)
(see Fig. 1).
3.2. Decarboxylation of AdoMet
Decarboxylation of AdoMet is a key step in the biosynthetic pathway for
spermidine and spermine. This reaction is catalyzed by AdoMet decarboxylase
(EC 4.1.1.50) (Tabor & Tabor, 1984). The decarboxylated AdoMet, S-
adenosylmethylthiopropylamine, donates its aminopropyl group to putrescine
to form spermidine, or spermidine to spermine in the presence of
aminopropyltransferases. On transfer of the aminopropyl group, the decar-
boxylated AdoMet itself is converted to MeSAdo, which has been demonstr-
ated to be a potent inhibitor of the aminopropyltransferases and AdoHcy
hydrolase (Ueland, 1982; Schlenk 1983). The inhibition by MeSAdo is relieved
by the enzymatic cleavage of MeSAdo to adenine and 5-deoxy-
methylthioribose-l~phosphate by MeSAdo phosphorylase (EC 2.4.2.28).
Thus, this enzyme, as well as AdoHcy hydrolase, plays an important role in
regulation of the activated methyl cycle. This enzyme activity has been
detected in the organisms having AdoHcy hydrolase, but not in the bacteria
having AdoHcy nucleosidase instead of AdoHcy hydrolase. In such bacteria,
MeSAdo is hydrolyzed to adenine and ribose by AdoHcy nucleosidase
(Shimizu et al., 1988) (see Fig. 1).
3.3 AdoHcy Hydrolase
As mentioned above, AdoHcy hydrolase is the most important enzyme for the
regulation of the activated methyl cycle. Hydrolysis of AdoHcy by this enzyme
is reversible, the reaction equilibrium in vitro greatly favors the synthetic

direction (de la Haba & Cantoni, 1959), and the specificity of the enzyme for
adenosine analogs is broad (Guranowski et al., 1981; Shimizu et aI., 1984a),
suggesting that it is also important as a practical catalyst for the preparation of
AdoHcy and related nucleosides (Shimizu & Yamada, 1984).
The enzyme activity has been detected in a variety of organisms ranging
from mammalians to micro-organisms. The enzyme has been isolated from
mammalian tissues (Richards et aI., 1978; Palmer & Abeles, 1979; Fujioka &
Takata, 1981), lupin seeds (Guranowski & Pawelkiewicz, 1977) and a
bacterium Alcaligenes faecalis (Shimizu et aI., 1984a) and characterized in
some detail.
Bacterial AdoHcy hydrolase can be easily purified and crystallized from cells
of A. faecalis, in which the cellular content of this enzyme is about 2·5% of the
total extractable protein. This value is about 100 times higher than the enzyme
content of eukaryotic sources. The purified enzyme has six identical subunits,
each of which (mol wt = 48000-50000) binds 1 mol of NAD+, whereas most
mammalian enzymes are tetramer whose subunit molecular mass is about
48000 and the plant enzyme is composed of two subunits identical in molecular
Table 2
Properties of AdoHcy Hydrolase of A. faecalis·
Mol. wt.
Sedimentation equilibrium 280000
Gel filtration 290000
HPLC 290000
SDS-PAGE 48000
Sedimentation velocity (S20.w) 9·79
Number of subunits 6
pI 4·7
Km (mM) AdoHcy 0·014
adenosine 0·050
DL-homocysteine 1·0
Vm (Ilmol/min per mg) Hydrolysis 546·5
Synthesis 7·47
Optimum temperature (0C) 50
Optimum pH Synthesis 8·0-10·5
Heat stability CC) 45
pH stability 6·0-10·0
COOH-teiminal amino acid Tyr
Other substrates Formycin A, neburalin,
adenosine N' -oxide,
tubercidin, inosine, etc.
Inhibitor Adenosine 5' -carboxylate,
5'-deoxyadenosine, etc.
Inactivator Arabinosyladenine, 2'-
deoxyadenosine,5'-deoxy-
adenosine, adenosine, etc .
• See Shimizu et al. (1984a) for details.
mass (55 (00). The function of NAD+ in the reversible reaction was estab-
lished by Palmer & Abeles (1979). The prokaryotic enzyme efficiently
catalyzes thioether formation between L-homocysteine and several novel
nucleosides, most of which are known as antibiotic, antiviral or antitumor
agents (for chemical structures, see Table 1), yielding the corresponding
S-nucleosidyl-L-homocysteines. This fact suggests that the enzyme can be used
as the catalyst for the production of not only AdoHcy but also these novel
nucleoside derivatives. The enzyme is a characteristic adenosine binding
protein as well as mammalian enzymes. Properties of the bacterial enzyme are
summarized in Table 2.
4 PRODUCTION OF ADOMET
4.1 Microbial or enzymatic production
Since AdoMet was first prepared from L-methionine and A TP with a liver
preparation containing methionine adenosyltransferase (Cantoni, 1953), there
have been several reports for the preparation of Ado Met. Schlenk and
co-workers (Schlenk & DePalma, 1957; Schlenk et ai., 1965) pointed out that
several common yeasts, such as Saccharomyces cerevisiae and Candida utiiis,
might be suitable sources of AdoMet. They reported that AdoMet is
accumulated in growing or washed cells in the presence of relatively large
amounts of L-methionine. Several molds were also reported to be suitable
sources of AdoMet (Kusakabe et ai., 1974). However, the cellular contents of
AdoMet were not satisfactory in these micro-organisms.
i o
•U100 0
~
"a 0
QI
"- 0 0
§
:8
c 0
•
~ •
c
g• 50 aa
-;
0
u
•
~
0 /j.
"a
C /j. A
~
i
16 0 0.05 0.10
Methlon"'e adenosyltransferase activity
(pmo1/30 m .../mg)
Fig. 2. Relationship between methionine adenosyltransferase activity and cellular

AdoMet content of various yeasts. See Shiozaki et ai. (1984) for details. 0,
Saccharomyces sake strains; e, other Saccharomyces strains; 6., yeasts of other genera.
Recently, Yamada and co-workers (Shimizu & Yamada, 1984; Shiozaki et

al., 1984; Yamada et al., 1984) assayed a variety of micro-organisms for their
ability to accumulate Ado Met intracellularly using several media containing
L-methionine. The ability to accumulate significant amounts of AdoMet was
found to be widely distributed in yeasts belonging to the genera Saccharomyces
and Candida. A group of Japanese traditional sake yeasts, i.e. Saccharomyces
sake strains, accumulated especially high levels of Ado Met. The AdoMet
contents of most of these strains reached more than 100 mg/g dry cells or
1 mg/ml of culture broth (Fig. 2). They selected S. sake K-6, as the most
promising strain.
In a study on the optimization of the culture conditions for the accumulation
of AdoMet by S. sake K-6, they found that sucrose and urea are very suitable
carbon and nitrogen sources, respectively, for obtaining high density growth
12
'l
i 5 ._ ..\ ' - / / .. _... _., ............. _.,... -..., ............_._.-
r
i 0"",
--
0'1 /
E 10 50 0\ / E
/i\ Y- 0'1
Qj
::.: ~ E
10
;,<~\
0 I '0
'0 c
« I 0
8 I 40 / 0 ..c
~ / W
a a /
::2
OJ
u
l:
6 ~30 /
£/ /
~I
x E /
/
Vl
QI
L.. ..c / 300 ~
u
0 I
Q
3 / A-·A, C
4 ~ 20 A"'/ "'A
U
I
'., .f
0'1
L..
0 0
::2 I /
200
OJ I I '\
u
~
I I ~
2 10 I i A C
C A' 100 0u
0 I OJ
;§ 9
)e _e_e",
L
0
0 0 ~
0 2 3 4 5 6 7
Time (days)
Fig. 3. Typical time course of AdoMet production by S. sake K-6. S. sake K-6 was
cultivated in a lO-liter bench scale fermentor with 5 liters of a medium containing 10%
sucrose, 1% yeast extract, 1·8% urea, 1% L-methionine, 0·2% glycylglycine, 0·4%
KH2P04, 0·01 % MgS04·7H20, biotin (2,Ltg/ml) and 10 ml of a mixture of metal salts
and trace elements (20 g of CaCI2·2H20, 0·5 g of MnS04'5H20, 0·5 g of FeS04·7H20,
1·0 g of ZnS04'7H20, 0·01 g of CuS04·5H20, 0·01 g of CoCI2·6H20, 0·01 g of H 3 B03 ,
0·01 g of Na2Mo04 and 0·01 g of KI in 1 liter H 20), pH 6·3. Cultivation was carried out
at 28°C with aeration at 1 vvm and agitation at 300 rpm. Ethanol (50 ml) was added to
the medium at the time indicated by arrows in the figure. MgS0 4·7H20 (0·25 g) was
further added to the medium on the 3rd day of the cultivation. See Shiozaki et al.
(1986) for details.
364 S. Shimizu & H . Yamada
Fig. 4. Ultraviolet photomicrograph showing accumulation of Ado Met in vacuoles of S.

sake K-6. (0) indicates control cells grown for 3 days without L-methionine . (2) and (5)
indicate cells grown with L-methionine for 2 and 5 days, respectively, under the
conditions given in Fig. 3.
without any decrease in intracellular level of AdoMet. They also pointed out
that addition of glycylglycine, biotin and CaCl2 to the culture medium
increases cellular AdoMet content (Shiozaki et al. , 1986). Under the optimal
culture conditions in a lO-liter bench scale fermentor , S. sake K-6 produced
lO·8 g/liter of AdoMet (Fig. 3). Almost all the AdoMet was accumulated in
vacuoles in the cells (Fig. 4), the extracellular concentration being very low.
The maximum AdoMet content of cells reached 260 mgt g dry cells; this
compares favorably with the commercially available yeast cells rich in AdoMet
which contain only about 20 mg/g solids.
Leakage of AdoMet from vacuoles leads to its rapid decomposition to
MeSAdo and it is important to retain vacuolar integrity and high concentra-
tions of Ado Met in order to produce satisfactorily large amounts of the
nucleosides. Although it is not yet clear why sake yeasts show notably high
ability to accumulate AdoMet, one possible explanation is suggested by the
data shown in Fig. 2, where methionine adenosyltransferase activities are
compared among various kinds of yeasts. The results reveal a positive
correlation between accumulation of Ado Met and the enzyme activity.
Another interesting characteristic of this yeast is that it is able to grow well in
the presence of high concentrations of L-methionine. L-Methionine has been
reported to markedly inhibit the growth of common Saccharomyces yeasts
(Takahashi & Takahashi, 1968). This may also be an important characteristic
for the high accumulation of Ado Met.
Another approach to produce AdoMet was reported by Gross et al. (1983) ,
who synthesized 7·8 mmol of AdoMet on incubation of a reaction mixture
(850 ml) containing 20 mmol of AMP, 0·5 mmol of A TP, 40 mmol of phos-
phoenol pyruvate, lO mmol of MgCI2 , 150 mmol of KCl, 20 mmol of L-
L-Methlonine
AMP ; ; P\ ATP -"'-_...,..-------"

PEP Pyruvate PPi ~ Pi
Fig. 5. Reaction sequences of AdoMet synthesis using immobilized cell-free enzymes.
See Gross et al. (1983) for details. PEP, phosphoenol pyruvate; Met
adenosyltransferase, methionine adenosyltransferase; AK, adenylate kinase; PK,
pyruvate kinase; PPase, inorganic pyrophosphatase.
methionine and 1 mmol of dithiothreitol with methionine adenosyltransferase

purified from baker's yeast, inorganic pyrophosphatase, adenylate kinase and
pyruvate kinase for 6 days at room temperature. In this case, the enzymes
immobilized on gels were used as catalysts and the A TP used for the synthesis
was supplied in situ from AMP using phosphoenol pyruvate as the source of
phosphate, as shown in Fig. 5.
4.2 Methods for Isolation of AdoMet and its Stabilization
Some important problems to be improved for the practical production of

AdoMet are its separation from other low molecular weight cell constituents
and the stabilization of the finally isolated AdoMet (Shimizu & Yamada, 1984,
1986). The conventional large-scale preparation method involves the following
successive steps: extraction of AdoMet from yeast cells with perchloric acid,
decolorization of the extract, separation of AdoMet and salt formation.
Decolorization can be achieved with synthetic adsorbents, and is indispensable
for obtaining a final product of high purity. Separation of Ado Met from other
low molecular weight compounds may be carried out on a cationic ion-
exchange resin due to its high positive electric charge at the sulfonium center.
Adenosine, adenine, MeSAdo and similar compounds having positive electric
charges sometimes interfere with the separation of AdoMet. Shiozaki et al.
(1986) reported a new and efficient isolation procedure involving
freezing/thawing of cells for the extraction of AdoMet and chromatographies
on Amberlite IRA-45, XAD-2 and IRC-50. They obtained AdoMet of 96·1%
purity in a recovery yield of 69·7% from cells of S. sake K-6 (28 g as dry weight)
containing 5·01 g of AdoMet through this procedure.
A variety of salt forms have been proposed for stabilization of AdoMet.
They can be grouped into the following types: salts with haloacids (i.e.
AdoMet+Cl-, AdoMet+Cl-HCI, AdoMet+r, AdoMet+Br-, etc.), salts with
sulfate compounds (i.e. AdoMet+HSO; and AdoMet+HSO;·(H2S04 )1_3, and
double salts with H 2 S04 and inosine-5'-monosulfate, adenosine-5'-
monosulfate, uridine-5'-monosulfate, etc.), salts with sulfonate compounds
(i.e. salts with p-toluenesulfonic acid, p-chlorobenzenesulfonic acid, methane-
sulfonic acid, etc., and a double salt with H 2S04 and p-toluenesulfonic acid),
salts with phosphoric acid (i.e. AdoMet+H3 PO;, etc.), salts with dicarboxylic
acids (i.e. salts with citric acid, tartaric acid, malic acid, succinic acid, etc.),
salt with ascorbic acid, and cyclodextrin complexes of AdoMet+HSOi,
AdoMet+HSOi . (H2 S04 )n , etc. Among these, the p-toluenesulfonate salts and
cyclodextrin complexes seem to be considerably stable. The double salt with
p-toluenesulfonic acid and H 2S04 has been used to treat various liver diseases
in Italy and some other countries.
Another problem recently revealed is that most commercially available
AdoMet products contain 10-20% of the inactive ( + )-enantiomer, the source
of which is unknown (Stolowitz & Minch, 1981). Matos et al. (1987) reported
that the ( - )-enantiomer is the sole product of the enzymatic reaction with E.
coli methionine adenosyltransferase. This suggests that conversion of the
( - )-enantiomer to the ( + )-enantiomer might take place during isolation or
storage.
4.3 Chemical Synthesis of AdoMet
Chemical approaches to prepare AdoMet generally involve the methylation of

AdoHcy with appropriate methylating agents such as methyl iodide. However,
the chemical methylation is not stereoselective. Molar ratios of the (- )- to
( + )-enantiomer are 55: 45 and 65: 35 for methyl iodide and trimethylsul-
fonium iodide, respectively (Matos et al., 1987). In addition, AdoHcy is
required for the preparation. It is considerably expensive when compared with
L-methionine.
5 PRODUCTION OF AdoHcy
5.1 Enzymatic Methods
The in vitro hydrolysis of AdoHcy by AdoHcy hydrolase greatly favors the

synthetic direction and the enzyme is considered to be a useful catalyst for the
synthesis of AdoHcy and S-nucleosidylhomocysteines.
Recently, a simple and efficient method for the production of AdoHcy using
the reverse reaction of AdoHcy hydrolase has been reported (Shimizu et al.,
1984b, 1986a, b; Yamada et al., 1984) based on the finding that bacterial cells
are rich sources of AdoHcy hydrolase (Shimizu et aI., 1984a). Shimizu et al.
(1986a) found that washed cells of several bacteria belonging to the genera
Alcaligenes and Pseudomonas, and some strains of actinomycetes, efficiently
converted the added adenosine and L-homocysteine to AdoHcy. They selected
A. faecalis AKU 101 as the most promising catalyst. With the washed cells of
this bacterium as the catalyst, and adenosine (200 mM) and L-homocysteine
(200 mM) as the substrates, they obtained nearly 100% molar conversion with a
yield of about 80 g/liter after 40 h at 37°C (see Table 3).
A problem with this synthesis was that the D-isomer of homocysteine could
not be utilized as the substrate. To overcome this, they screened for microbial
strains capable of synthesizing AdoHcy from D-homocysteine. Several
Pseudomonas strains were found to have this ability. When washed cells of P.
putida were used as the catalyst, AdoHcy synthesis from adenosine, and each
of the three forms of homocysteine, i.e. L-, D- or DL-homocysteine, proceeded
at the same reaction rate. In each case, the product was the L-form of AdoHcy.
They demonstrated that, although the P. putida AdoHcy hydrolase as well as
the A. faecalis enzyme catalyzes only the condensation of adenosine and
L-homocysteine, the bacterium also produces a racemase which catalyzes the
interconversion of the D- and L-homocysteine isomers but not that of the
AdoHcy produced. For practical purposes, A. faecalis cells are still superior to
P. putida cells in that they show high AdoHcy hydrolase activity and low
adenosine deaminase activity. In the mixed cell system containing the two
strains, the reaction rate greatly decreased when the content of P. putida cells
was increased. Therefore, they used P. putida cells just as a source of the
racemase. With 200 mM adenosine and 200 mM DL-homocysteine, the molar
conversion to AdoHcy was 86%, when a mixture of A. faecalis cells
(36 mg/ml) and P. putida cells (4 mg/ml) was used as the catalyst (Table 3).
The reaction mechanism for this system is considered to be as shown in Fig. 6
(Shimizu & Yamada, 1984; Shimizu et al., 1986b).
Table 3
AdoHcy Synthesis with Varying Concentrations of Adenosine and L- or DL-
Homocysteine with Cells of A. faecalis or Mixed Cells of A. faecalis and P. putida
Cells used as Concentrations AdoHcy

catalysts (I1mol/ml) of produced
(I1mol/ml)
Adenosine Homocysteine
L- DL-
A. faecalis 100 100 100
200 200 178
300 300 199
400 400 153
A. faecalis 50 100 50
100 100 53
100 200 76
200 400 103
300 600 71
A. faecalis: 100 100 100
P. putida 200 200 173
(9: 1) 300 300 173
400 400 155
Reaction mixture (1·0 ml) containing 40 mg of air-dried cells, as indicated,

50 Ilmol of potassium phosphate buffer, pH 8·0, and the indicated amounts of
adenosine and homocysteine were incubated for 36 h at 37°C. See Shimizu et al.
(1986b) for details.
AdoHcy
hydTo/ase
(A.faecalls)
L-Homocysteine + Adenosine ~ " AdoHcy
:
1:R.cemase
l(P.putlda)
D-Homocysteine
Fig. 6. Reaction mechanism for the synthesis of AdoHcy from adenosine and
DL-homocysteine.
5.2 Chemical Methods
Two routes have been used generally for the chemical synthesis of AdoHcy.
One involves (1) protection of the 2'- and 3'-hydroxyl groups of adenosine
using an isopropylidene protecting group; (2) activation of the nucleoside
5'-position by formation of the 5'-tosylate; (3) condensation of the intermedi-
ate 5'-tosylate with S-benzyl-L-homocysteine; and (4) removal of the
isopropylidene protecting group using dilute acid (Baddiley & Jamieson, 1955;
Borchardt & Wu, 1974). The other route involves replacement of the
5'-hydroxyl group of the adenosine by chlorine followed by the condensation
of the resulting 5'-chloro-S'-deoxyadenosine with L-homocysteine (Borchardt et
al., 1976). Condensation of unprotected adenosine with a protected homocy-
steine in the presence of trialkyl phosphine was also reported (Serafinowski,
1985). These standard procedures have been used for the synthesis of a broad
spectrum of AdoHcy analogs.
6 PRODUCTION OF NOVEL S-NUCLEOSIDYLHOMOCYSTEINES
Because A. faecalis AdoHcy hydrolase utilizes a variety of adenosine analogs

as substrates (Shimizu et al., 1984a), the cells containing this enzyme can be
used as a catalyst for the synthesis of a variety of S-nucleosidylhomocysteines.
Shimizu et al. (1984b) reported that neburalin and formycin A, as well as
adenosine, were converted to the corresponding S-nucleosidylhomocysteines in
high yields on incubation with L-homocysteine as the cosubstrate and dried
cells of A. faecalis as the catalyst. They also reported that K-carrageenan-
entrapped cells of the same organism efficiently catalyze the conversion of
adenosine Nl-oxide and 2-chloroadenosine, in addition to neburalin and
formycin A, to the corresponding S-nucleosidylhomocysteine congeners. The
immobilized cells showed high stability on repeated reactions, whereas free
cells rapidly inactivated (Fig. 7) (Shimizu & Yamada, 1988).
These novel S-nucleosidylhomocysteines may also be promising in the
pharmaceutical and chemotherapeutic fields (Ueland, 1982; Borchardt et al.,
1986).
o 1 2 3 4 5 6 7 8 9 10 11
Number of repeat
Fig. 7. Synthesis of S-nucleosidylhomocysteines with K-carrageenan entrapped cells of
A. faecalis. See Shimizu & Yamada (1988) for details. Adenosine (Ado), formycin A
(For), adenosine N1-oxide, (Ado-N-oxide) and 2-chloroadenosine (Cl-Ado) were used
as nucleoside substrates.
7 CONCLUSION
The biotechnological area of research on AdoMet and related nucleosides

has advanced considerably in the past few years. Important progress in this
area can be summarized as follows: (1) screening of S. sake K-6 as a potent
producer of AdoMet; (2) determination of the optimal conditions for
large-scale culture, by which Ado Met content of the yeast reached 200-
300 mg/g dry cells; (3) finding of A. faecalis as a potent AdoHcy hydrolase
producer, which made possible to synthesize AdoHcy and related nucleosides
in high yields; and (4) development of a new synthetic scheme for the
production of AdoHcy, involving AdoHcy hydrolase and homocysteine
racemase as the catalysts. This made possible to use cheap racemic mixture of
homocysteine as the amino acid substrate. Rapid expansion of research and
accumulation of biochemical and pharmacological information on these
nucleosides suggest that their pharmacological values are very high.
ACKNOWLEDGEMENT
Research from the author's laboratory was supported, in part, by a Grant in

Aid of Scientific Research from the Ministry of Education, Science and
Culture of Japan and a grant from the Uehara Memorial Foundation.
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Chapter 20
OTHER VITAMIN-RELATED COENZYMES
1 INTRODUCTION
The name 'coenzymes' has been given to a group of dissociable, low molecular
weight active components of enzymes which catalyze transfer of chemical
groups, hydrogen or electrons. Coenzymes, defined in this way, usually
function as cosubstrates for enzymes to couple two otherwise independent
reactions. Thus, they can be regarded as transmitters of metabolites. In a
wider sense, any catalytically active, low molecular weight, non-protein
components of enzymes are also included in the coenzyme group. This
definition includes coenzymes which are covalently bound to enzymes as
prosthetic groups. In this case, the coenzyme and the apoenzyme together
make up the active holoenzyme.
Many coenzymes are biosynthesized from vitamins and also contain a
nucleoside (or nucleotide) moiety in their molecules. The relationships of some
coenzymes to vitamins and metabolic functions are listed in Table 1. Some
coenzymes are produced on an industrial scale and are used as pharmaceuti-
cals. For example, ATP, COP-choline, FAD, NAO, coenzyme A, S-
adenosylmethionine, coenzyme Q1O, etc., are used for muscular dystrophy,
metabolic diseases, clouding of consciousness, cerebral surgery, hepatic or
renal dysfunction and so on. They are also used for analysis and research in
clinical chemistry, biochemistry, metabolism and pharmacology. Because
several coenzymes are already mentioned in the preceding individual chapters
describing the corresponding parent vitamins, this chapter outlines a few of the
remaining ones. A previous review on coenzymes (Shimizu & Yamada, 1986)
seen from a biotechnological point of view may be useful for further
understanding.
373
w
Table 1 -...I
.;..
Classification, Metabolic Functions and Sources of Coenzymes
Coenzyme Function Vitamin source Production Major use

methodO
I, Oxidoreduction coenzymes
NAD Hydrogen and electron Nicotinic acid EX,E Pharmaceuticals,
transport analytical reagent
NADP Hydrogen and electron Nicotinic acid E Analytical reagent
transport
Flavin mononucleotide Hydrogen and electron Riboflavin C,F
transport
FAD Hydrogen and electron Riboflavin E,C,F Pharmaceuticals ~
transport ~
Pyrroquinoline quinone Hydrogen and electron Ex

'"§'
transport N'
;::
Coenzyme Q Hydrogen and electron Ex Pharmaceuticals R-
transport ::t:
Menaquinone-6 Hydrogen and electron Ex Pharmaceuticals
~
transport
Lipoic acid Hydrogen and acyl C Pharmaceuticals
~
~
transfer
Cytochromes Electron transport Ex Pharmaceuticals
Ferredoxins Electron transport and
hydrogen activation
Thioredoxins Hydrogen transport
2, Group transfer coenzymes

S -Adenosylmethionine Transmethylation Ex Pharmaceuticals
Methylcobalamin Transmethylation Vitamin B12 EX,F
Coenzyme M Methyl transfer
Tetrahydrofolic acid and Transfer of formyl, Folic acid C
conjugates hydroxymethyl and
methyl groups
Coenzyme A Transacylation, etc. Pantothenic acid EX,E Pharmaceuticals,
analytical reagent
4' -Phosphopantetheine Transfer of acyl and Pantothenic acid
(or acyl carrier protein) amino acid groups,
etc.
Nucleoside diphosphate Transfer of sugars and E Pharmaceuticals
coenzymes phosphorylcholine
Pyridoxal 5' -phosphate Transamination, Vitamin B6 C,F Pharmaceuticals,
decarboxylation, etc. analytical reagent
Biotin (or CO 2 -biotin Carboxylation, trans- Biotin E,C,F Feed additive, pharmaceuticals
enzymes) carboxylation, etc.
Thiamine pyrophosphate ~-group transfer Thiamine C Pharmaceuticals 0
Adenosine 3' -phosphate- Sulfate transfer So
...
.,
5' -phosphosulfate
Phosphorylation, pyro- $
ATP E Pharmaceuticals, S"
phosphorylation and analytical reagent 3
transfer of adenosyl
s·
group ~
is"
3. Isomerization coenzymes 1>
~
5' -Deoxyadenosylcobalamin Carboxyl shifts Vitamin B12 EX,F g
UDP-sugars, etc. Sugar isomerization E ~
~
a E, enzymatic method; Ex, extraction from microbial cells or animal tissues; C, chemical synthesis; F, fermentation. :I
~
:j
Ul
2 NICOTINAMIDE COFAcrORS
Various aspects of the studies on these cofactors were reviewed in a recent

book by Dolphin et al. (1987). A review by Chenault & Whitesides (1987)
focussed on the biotechnological aspect of the cofactors.
2.1 Preparation of NAD and NADP

Both extraction from yeast cells and enzymatic synthesis are performed for the
commercial production of NAD. Baker's yeast (Saccharomyces cerevisiae)
and brewer's yeast (S. carlsbergensis or S. uvarum) have been shown to be
favorable sources of NAD. On cultivation in a medium containing adenine and
nicotinamide, values of 12 mg/g dry cells and 42 mg/g dry cells for S. cerevisiae
and S. carlbsergensis, respectively, were reported (Sakai et al., 1973a, b). The
enzymatic process utilizes the salvage route for the biosynthesis of NAD from
nicotinamide (or nicotinic acid) and adenine. Brevibacterium ammoniagenes
was found to convert the added precursors to NAD efficiently. When the
precursors were added to the medium after culturing for 48 h and incubated for
a further 120 h, the amount of NAD in the medium reached 2·3 mg/ml. The
mechanism involved in the NAD production has been suggested to be that
adenine and nicotinamide are first ribosylated to yield A TP and nicotinamide
mononucleotide, respectively, which are then condensed by pyrophosphoryla-
tion to form NAD (Nakayama et al., 1968). Several combined cell-free
enzymatic and chemical syntheses of NAD starting from AMP or ATP have
also been reported. For example, on incubation of 40 mmol of nicotinamide
mononucleotide (synthesized from AMP in three steps) and AMP for 10 days
in a 2000 ml of reaction mixture containing immobilized NAD pyrophos-
phorylase, inorganic pyrophosphatase, acetate kinase and adenylate kinase,
39 mmol of NAD was produced (Walt et al., 1980). These procedures seem to
be inferior to the extraction from yeast cells and to the enzymatic synthesis
with cells of B. ammoniagenes because of their complexity.
NADP is prepared by the phosphorylation of NAD using NAD kinase and
ATP. The conditions for the continuous phosphorylation of NAD have been
studied with immobilized cells of Achromobacter aceris (Uchida et al., 1978)
and B. ammoniagenes (Tanaka et al., 1982). With a column packed with the
immobilized A. aceris, 6-8 Ilmol/ml of NADP was obtained on a continuous
passage of the substrate solution (25·1 mM) at space velocity of 0·1 h- 1 .
Recently, a thermostable NAD kinase of Corynebacterium flaccumfaciens was
shown to be useful for practical preparation of NADP (Matsushita et al.,
1986). Brevibacterium ammoniagenes has been reported to produce a novel
enzyme, polyphosphate kinase, which catalyzes the phosphorylation of NAD
with methaphosphate as a phosphate donor. Continuous phosphorylation of
NAD with immobilized cells of this bacterium was also reported (Murata et
al., 1979).
NADH and NADPH can be prepared from the corresponding oxidized
coenzymes by chemical, enzymatic, or microbial reduction.
Other Vitamin-related Coenzymes 3n
2.2 Regeneration of NADD and NADPD
Many systems for regenerating nicotinamide cofactors are involved in the

complex and regulated metabolism of living cells. For example, the pathway
for glucose oxidation via glucose-6-phosphate involves two important reactions
yielding NADPH, i.e. the oxidation of glucose-6-phosphate by glucose-6-
phosphate dehydrogenase and the oxidation of 6-phosphogluconate by 6-
phosphogluconate dehydrogenase. Endogeneous NADPH or a catalytic
amount of added NADPH can be efficiently regenerated by the simple mixing
of microbial cells with glucose in an appropriate reaction mixture. The
reductions of various carbonyl compounds by yeast cells occasionally per-
formed in synthetic organic chemistry are mainly based on this system.
Similarly, oxidation of ethanol to acetic acid by yeasts and oxidation of
methanol to CO2 by methylotrophic micro-organisms are considered to be
useful for NADH regeneration.
When cell-free enzymes requiring NAD(P)H are to be used, the construc-
tion of a suitable regenerating system is an additional requirement. Many
systems have been tested for this purpose (for a review, see Chenault &
Whitesides, 1987). Among them, the formate/formate dehydrogenase,
glucose/ glucose dehydrogenase and glucose-6-phosphate/ glucose-6-phosphate
dehydrogenase systems are most widely used, because of the availability of
these hydrogen donor substrates and enzymes (Table 2). The formate/formate
dehydrogenase system has been used successfully to regenerate a polymer-
bound NADH in a membrane-reactor synthesis of several optically active
amino acids from the corresponding keto acids (Wandrey et ai., 1984; Hummel
et aI., 1986). Successful application of the same system has also been reported
for the production of L-phenylalanine with phenylalanine dehydrogenase
(Asano & Nakazawa, 1987) and R-( - )-mandelic acid with 2-hydroxyiso-
caproate dehydrogenase (Yamazaki & Maeda, 1986).
3 PYRROLOQUINOLINE QUINONE
Pyrroloquinoline quinone (POO), 2,7 ,9-tricarboxy-H-pyrrolo[2,3-f)quinoline-

4,5-dione, was found as the prosthetic group of a primary alcohol de-
hydrogenase (EC 1.1.99.8) in methylotrophic bacteria. Subsequent work has
demonstrated that in addition to the NAD(P)-dependent and flavoprotein
dehydrogenases, there is a third group of enzymes, 'quinoprotein oxidoreduc-
tases', in which PQQ is involved as the coenzyme. Alcohol, aldehyde and
glucose dehydrogenases of acetic acid bacteria and several other bacteria have
been found to be POQ-dependent dehydrogenases. Evidence has been
provided for the quinoprotein nature of several other microbial and mam-
malian enzymes such as amine dehydrogenase, amine oxidase, nitrile hydra-
tase, and several more (for review, see Duine et ai., 1986, 1987). These results
suggest that PQQ is widely distributed among living organisms, although no
~
00
Table 2
Systems for Regeneration of NAD(P)H from NAD(P)"
System and reaction Advantage Disadvantage

Formate/formate dehydrogenase High reducing potential High expense of enzyme
HCOO- + NAD- CO 2 + NADH Inexpensive reductant Low specific activity of enzyme
Easy removal of by-product (C0 2) Inability to reduce NADP
Glucose/ glucose dehydrogenase High reducing potential High expense of enzyme
Glucose + NAD(P)- gluconate + NADP(H) Inexpensive reductant Difficulty in removal of :->
High stability of enzyme by-product (gluconate) ~
High specific activity of enzyme §.
Ability to reduce NAD or NADP
...
;::
Glucose-6-phosphatel glucose-6-phosphate High reducing potential Expensive reductant R-
dehydrogenase Inexpensive enzyme Difficulty in removal of ;t:
G-6-P + NAD(P)- 6-PG + NAD(P)H High specific activity of enzyme by-product (6-PG) ;;.<
Ability to reduce NAD or NADP
Ethanol/ alcohol dehydrogenase-aldehyde Inexpensive enzyme (yeast alcohol Low reducing potential l
dehydrogenase dehydrogenase) Product inhibition by acetaldehyde
~H50H + NAD- CH 3COO- + NADH Inexpensive reductant Inability to reduce NADP
High specific activity of enzymes
Volatility of ethanol and
acetaldehyde
a Modified from Chenault & Whitesides (1987). G-6-P, glucose-6-phosphate; 6-PG, 6-phosphogluconate.
Other Vitamin-related Coenzymes 379
systematic search has been reported. POO has been shown to have a
growth-promoting effect on several bacteria (Ameyama et al., 1984a; Shimao
et al., 1984).
Several bacteria have been reported to excrete PQO into their culture
medium. Especially, methylotrophic bacteria seem to be favorable sources for
PQQ production. Reported fermentation yields range from 0·01 to 10 ,ug/ml
(Ameyama et al., 1984b). From a culture filtrate (1100 liters) of
Hyphomicrobium X grown with 0·5% methanol, a yield of 1385 mg POO was
obtained through the procedures involving absorption with Amberlyst A21
anion exchanger and elution from it (Duine et al., 1987).
4 COENZYME 0
Coenzyme Q (CoQ, ubiquinone) is a 2,3-dimethyl-5-methyl benzoquinone
with an isoprenoid side chain composed of dihydroisoprene units. There are
various types which are designated according to the number of isoprene units
in the side chain: Co06 , COQ7, CoOs, Co0 9. COQIO, etc. CoQ6 -COQIO are
widely distributed (the name 'ubiquinone' is from its ubiquitous occurrence).
Co0 1 -CoQs were found in several bacteria and yeasts, such as Rhodospirillum
rubrum, Escherichia coli and Saccharomyces cerevisiae (Friis et aI., 1966;
Daves et aI., 1967; Bentley & Meganathan, 1987). The occurrence of CoO u ,
C00 12 and Co0 13 was also reported (Natori & Nagasaki, 1979, 1980). Among
these homologs, CoO IO has been extensively studied as to practical production,
because it is the natural coenzyme in humans and it is used in therapeutic
treatment for diseases of such tissues as heart and muscle.
An ethionine-resistant mutant derived from Agrobacterium sp. has been
reported to be a potent producer of CoO lO . This mutant produced two types of
morphologically distinguishable colonies, rough and white, and smooth and
yellow, on an agar plate. Only the former showed excellent production
(4·1 mg/ g dry cells), but changed easily to the latter with lower productivity
(2·8 mg/g dry cells) during cultivation or on transfer. The concentration of
ammonium ions in the production medium and aeration or agitation during the
cultivation were found to be important factors affecting cellular COQlO content.
Under optimum cultivation conditions, a value of 5·1 mg/g dry cells or
211 ,ug/ml of culture broth was attained with this mutant (Kuratsu et al.,
1984a, b, c). A facultative methylotroph, Protaminobacter ruber, produced
1· 52 mg/ g dry cells on cultivation with methanol as sole carbon source for
growth. In this case, no other CoO homologs than COQlO were detected in the
cells (Natori et aI., 1978). Paracoccus denitrificans (Matsumura et al., 1983)
and tobacco cells (Ikeda et al., 1981) have also been shown to be potent
producers of CoO lO .
Microbial production of several CoO homo logs other than COQlO has also
been studied. An n-alkane-utilizing yeast, Candida tropicalis, produced
4·7 mg COQ9/g dry cells (52,ug/ml of culture broth) on cultivation in a
medium containing n-alkanes with successive addition of p-hydroxybenzoate as
a precursor of the quinone nucleus. n-Alkanes have been suggested to be

superior to other usual growth substrates for the production of CoQ, because
the isoprenoid moiety of the CoQ molecule is derived from acetyl-CoA which
is thought to be sufficiently available through the degradation of n-alkanes
(Shimizu et al., 1970a, b).
REFERENCES
Ameyama, M., Shinagawa, E., Matsushita, K. & Adachi, O. (1984a). Growth

stimulation of micro-organisms by pyrroloquinoline quinone. Agric. BioI. Chem.,
48, 2909-11.
Ameyama, M., Hayashi, M., Matsushita, K., Shinagawa, E. & Adachi, O. (1984b).
Microbial production of pyrroloquinoline quinone. Agric. Bioi. Chem., 48,561-5.
Asano, Y. & Nakazawa, A. (1987). High yield synthesis of L-amino acids by
phenylalanine dehydrogenase from Sporosarcina ureae. Agric. Bioi. Chem., 51,
2035-6.
Bentley, R. & Meganathan, R. (1987). Biosynthesis of the isoprenoid quinones,
ubiquinone and menaquinone. In Escherichia coli and Salmonella typhimurium, ed.
F. C. Neidhardt. American Society for Microbiology, Washington, DC, pp. 512-20.
Chenault, H. K. & Whitesides, G. M. (1987). Regeneration of nicotinamide cofactors
for use in organic synthesis. Appl. Biochem. Biotechnol., 14, 147-97.
Daves, G. D. Jr, Muraca, R. F., Whittick, J. S., Friis, P. & Folkers, K. (1967).
Discovery of ubiquinones-l, -2, -3, and -4 and the nature of biosynthetic
isoprenylation. Biochemistry, 6,2861-6.
Dolphin, D., Avranovic, O. & Poulson, R. (Eds) (1987). Coenzymes and Co/actors,
Vol. 2: Pyridine Nucleotide Coenzymes: Chemical, Biochemical, and Medical
Aspects, Part B. John Wiley and Sons, New York.
Duine, J. A., Frank, Jzn; J. & Jongejan, J. A. (1986). PQQ and quinoprotein enzymes
in microbial oxidations. FEMS Microbiol. Rev., 32, 165-78.
Duine, J. A., Frank, Jzn, J. & Jongejan, J. A. (1987). Enzymology of quinoproteins.
Adv. Enzymol., 59, 169-212.
Friis, P., Daves, G. D. Jr & Folkers, K. (1966). Isolation of ubiquinone-5, new
member of ubiquinone group. Biochem. Biophys. Res. Commun., 24,252-6.
Hummel, W., Schmidt, E., Wandrey, C. & Kula, M. R. (1986). L-Phenylalanine
dehydrogenase from Brevibacterium sp. for production of L-phenylalanine by
reductive amination of phenylpyruvate. Appl. Microbiol. Biotechnol., 25, 175-85.
Ikeda, T., Matsumoto, T., Obi, Y., Kisaki, T. & Noguchi, M. (1981). Characteristics
of cultured tobacco cell strains producing high levels of ubiquinone-lO selected by a
cell cloning technique. Agric. Bioi. Chem., 45, 2259-63.
Kuratsu, Y., Hagino, H. & Inuzuka, K. (1984a). Effect of ammonium ion on coenzyme
QlO fermentation by Agrobacterium species. Agric. BioI. Chem., 48, 1347-8.
Kuratsu, Y., Sakurai, M., Hagino, H. & Inuzuka, K. (1984b). Productivity and colony
morphology associated with coenzyme QlO production by Agrobacterium species.
Agric. Bioi. Chem., 48, 1997-2002.
Kuratsu, Y., Sakurai, M., Hagino, H. & Inuzuka, K. (1984c). Aeration-agitation effect
on coenzyme QlO production by Agrobacterium species. J. Ferment. Technol., 62,
305-8.
Matsumura, M., Kobayashi, T. & Aiba, S. (1983). Anaerobic production of
ubiquinone-lO by Parococcus denitrificans. Eur. J. Appl. Microbiol. Biotechnol., 17,
85-9.
Other Vitamin-related Coenzymes 381
Matsushita, H., Yokoyama, S. & Obayashi, A. (1986). NADP+ production using

thermostable NAD+ kinase of Corynebacterium flaccumfaciens AHU-1622. Can. J.
Microbio!., 32,585-90.
Murata, K., Kato, J. & Chibata, I. (1979). Continuous production of NADP by
immobilized Brevibacterium ammoniagenes cells. Biotechnol. Bioeng., 21,887-95.
Nakayama, K., Sato, Z., Tanaka, H. & Kinoshita, S. (1968). Production of nucleic
acid-related substances by fermentation processes. Part XVII. Production of NAD
and nicotinic acid mononucleotide with Brevibacterium ammoniagenes. Agric. Bio!.
Chem., 32, 1331-6.
Natori, Y. & Nagasaki, T. (1979). Formation of coenzyme Q n by Pseudomonas M16, a
mutant. Agric. Bioi. Chem., 43,797-802.
Natori, Y. & Nagasaki, T. (1980). Occurrence of coenzyme Q 12 and coenzyme Q13 in
facultative methanol-oxidizing bacteria. Agric. Bio!. Chem., 44, 2105-10.
Natori, Y., Nagasaki, T., Kobayashi, A. & Fukawa, H. (1978). Production of
coenzyme Q10 by Pseudomonas N842. Agric. Bioi. Chem., 42, 1799-1800.
Sakai, T., Uchida, T. & Chibata, I. (1973a). Production of nicotinamide adenine
dinucleotide by Saccharomyces carlsbergensis. Agric. Bioi. Chem., 37, 1041-8.
Sakai, T., Uchida, T. & Chibata, I. (1973b). Accumulation of nicotinamide adenine
dinucleotide in baker's yeast by secondary culture. Agric. Bioi. Chem., 37, 1049-56.
Shimao, M., Yamamoto, H., Ninomiya, K., Kato, N., Adachi, 0., Ameyama, M. &
Sakazawa, C. (1984). Agric. Bioi. Chem., 48,2873-76.
Shimizu, S. & Yamada, H. (1986). Coenzymes. In "Biotechnology", Vol. 4, ed. H.-J.
Rehm & G. Reed. VCH Verlagsgesellschaft, Weinheim, pp. 159-84.
Shimizu, S., Tanaka, A. & Fukui, S. (1970a). Studies on the utilization of hydrocar-
bons by micro-organisms (X). Production of coenzyme Q by Candida tropicalis pK
233 in hydrocarbon fermentation. (Part III) Effect of compounds related to the
respiratory system on coenzyme Q production. J. Ferment. Techno!., 48, 542-8.
Shimizu, S., Tanaka, A. & Fukui, S. (1970b). Studies on the utilization of hydrocar-
bons by micro-organisms (XI). Production of coenzyme Q by Candida tropicalis pK
233 in hydrocarbon fermentation. (Part IV) Effect of various medium components
on coenzyme Q production. J. Ferment. Technol., 48,549-55.
Tanaka, Y., Hayashi, T., Kawashima, K., Yokoyama, T. & Watanabe, T. (1982).
Production of NADP by immobilized cells with NAD kinase. Biotechnol. Bioeng.,
24,857-69.
Uchida, T., Watanabe, T., Kato, J. & Chibata, I. (1978). Continuous production of
NADP by immobilized Achromobacter aceris cells. Biotechnol. Bioeng., 20, 255-66.
Walt, D. R., Rios-Mercadillo, V. M., Auge, J. & Whitesides, G. M. (1980). Synthesis
of nicotinamide adenine dinucleotide (NAD) from adenosine monophosphate
(AMP). J. Am. Chem. Soc., 102,7805-6.
Wandrey, c., Wichmann, R., Berke, W., Morr, M. & Kula, M. R. (1984). Continuous
cofactor regeneration in membrane reactors. In Third European Congress on
Biotechnology, Vol. I. Verlag Chemie, Weinheim, pp. 239-44.
Yamazaki, Y. & Maeda, H. (1986). Enzymatic synthesis of optically pure (R)-( -)-
mandelic acid and other 2-hydroxycarboxylic acids: Screening for the enzyme, and
its purification, characterization and use. Agric. Bioi. Chem., SO,2621-31.
Chapter 21
FUNGAL GIBBERELLIN PRODUCTION
B. BRUECKNER, D. BLECHSCHMIDT
Friedrich-Schiller-University lena, Dept. of General Microbiology, DDR-6900
lena, Neugasse 24, GDR
&
G. SEMBDNER, G. SCHNEIDER
Institute of Plant Biochemistry, Academy of Sciences of the GDR, DDR-4050
Halle, Weinberg 3, GDR
1 HISTORICAL SURVEY
Micro-organisms are capable of producing a series of plant growth regulators

such as gibberellins, auxines, abscisic acid, ethylene, fusicoccin, jasmonic acid,
etc., but only the microbiological production of gibberellins is of industrial
importance. The story of the identification of gibberellins as a plant growth
regulator is a classic example of the interactions between soil micro-organisms
and plants. It is well known that gibberellins were first obtained from culture
filtrates of the soil fungus Gibberella fujikuroi (Saw.) Wr. (Fusarium monili-
forme Sheld). In Japan and other Far East countries this plant-pathogenic
fungus is one of the most important pathogens of rice. When it attacks rice
seedlings an early disease symptom is a superelongation of the shoots, which
are more pale in colour than normal shoots. Farmers have called them bakanae
or 'foolish seedlings'. After growing very rapidly, infected plants die. The
disease was a serious cause of rice destruction in Japan during the last century.
The first scientific description of the bakanae disease was published in 1898
by Hori, who detected the causative agent and identified it as an imperfect
fungus. The Japanese scientist Kurosawa (1926), after several failures, showed
that the sterilized filtrate of the culture medium in which Giberella fujikuroi
had grown is capable of causing the same marked growth stimulation in rice
seedlings without infection of the fungus. He reported that the phytopathoge-
nic fungus secretes a toxin which promotes the rice growth. Kurosawa
concluded that the activity of the sterile culture filtrate 'was not due to enzyme
action but rather to some kind of chemical' (Kurosawa, 1926, cit. Phinney,
1983). This observation was confirmed and extended by other Japanese
scientists. The main results established by this work are these:
(a) cultural filtrates of Giberella fujikuroi produced overgrowth of shoots,
growth of roots was unaffected;
383
384 B. Brueckner et al.
(b) other plants, including dicotyledons as well as monocotyledons, re-

sponded by increased shoot growth;
(c) the rice plant does not respond by forming an antibody against the
toxin.
After Kurosawa's report a number of plant pathologists started to isolate the

active principle and to study its biological properties. More than 50 publica-
tions appeared between 1927 and 1940, mainly in Japan. In 1934 Yabuta's
group succeeded in obtaining an active component from the fungus filtrate.
However, they showed rather inhibitory effects than stimulatory activity. The
substance was given the name fusaric acid (Yabuta et ai., 1934). After
changing the composition of the culture medium Yabuta (1935) announced the
isolation of the growth stimulant as a fairly pure non-crystalline solid which
was called 'gibberellin' according to the bakanae fungus. This was the first
appearance of the term in scientific literature. In 1938 Yabuta & Sumiki
isolated two crystalline substances, 'gibberellin A' and 'gibberellin B'. Later
the terms were reversed because the former was shown to be inactive
(Tamura, 1971). After the war the Japanese experiments initiated the studies
in other countries too. In the USA, Stodola and his colleagues (1955)
developed the fermentation techniques for mass production of gibberellins and
showed that the substance they had isolated was composed of two gibberellins,
which they named 'gibberellin A' and 'gibberellin X'. Independently, workers
at the Imperial Chemical Industries Ltd (ICI) in Great Britain in 1954 obtained
a new active substance with similar biological properties but different in
chemical and physical properties called 'gibberellic acid' (Curtis & Cross,
1954).
In the 1950s the Japanese workers at the Tokyo University reinvestigated
their original gibberellin A preparation and could separate it into four
gibberellins and named them gibberellins AI> A 2 , A3 and A4 (Tamura, 1971).
Furthermore, they demonstrated that gibberellin A3 was identical with
gibberellin X and gibberellic acid, and that gibberellin Al was the same as
Stodola's gibberellin A. The successful work with the fungus induced further
studies on higher plants. At once the number of publications on gibberellin
responses in plants increased exponentially. In 1954 Brian reported the
elongation of a pea dwarf cultivar in response to exogenous gibberellin. The
spectacular growth response of Brian's dwarf pea to gibberellic acid led the
workers to speculate that dwarfism might be due to the absence of endogenous
gibberellins and that endogenous gibberellins would be present in non-dwarf
cultivars. The next step in the story of gibberellins was the discovery in 1956
that extracts from higher plants contained gibberellin-like substances that
induced biological responses identical to those elicited by the fungal gib-
berellins (Radley, 1956). The detection of gibberellin-like substances in several
species of plants led to studies that resulted in the isolation and identification
of gibberellins in higher plants. In 1958 MacMillan and his colleagues were
successful in the identification of gibberellin Al from runner bean seeds, and
Fungal Gibberellin Production 385
later on in the identification of gibberellin As from the same material

(Phinney, 1983). Up to now, a total of 72 gibberellins have been isolated and
structurally elucidated, of which 62 have been isolated from plants and 26 from
the fungus.
Gibberellins (GAs) exhibit a bifacial nature, on one hand they are products of
the secondary metabolism in fungi, especially Gibberella fujikuroi, on the
other hand they are biologically active, endogenous hormones in the higher
plants. From the chemical point of view, gibberellins represent a group of
diterpenoids having a typical tetracyclic ring system that, according to the
nomenclature (Rowe, 1968) is called ent-gibberellane (Fig. 1).
The trivial nomenclature attributes gibberellins to a numbered A series
(GAl ... GAx). This system was originally dedicated to the plant hormone
status of gibberellins, because in addition to having the typical structure and
the natural occurrence, the gibberellins have to be biologically active in certain
bioassays (MacMillan & Takahashi, 1968). Currently, the group of gibberellins
(A-series) comprises altogether 72 different GAs; 26 of them were isolated
from fungal sources (Bearder, 1980; Muromtsev & Agnistova, 1984; Takahashi
et al., 1986). Also 3-0-acetyl-GA3 was found to be a genuine product of the
fungus (Schreiber et al., 1966). The structures and some physical data of fungal
gibberellins are compiled in Table 1.
Gibberellins fall into two groups: ~o-gibberellins, which possess the
complete diterpenoid skeleton, and C 19-gibberellins, which have biogenetically
lost the C-20 resulting in a 19,1O-y-lactone structure that is typically for GAs
with high bioactivity. Other inevitable features of physiologically important
GAs are the 6-carboxy function and the 16(17) methylene group.
The most prominent representative GA is GA3 (=gibberellic acid, ent-
3£t', 10, 13-trihydroxy-20-nor-gibberella-1(2), 16(17)dien-7 ,19-dicarboxy-19,1O-
lactone) because of its high abundance in microbial fermentation but also
because of its high bioactivity.
Due to the structural diversity and the manifoldness of functional groups
GAs cover a broad range of polarity, e.g. hydrophilic and lipophilic properties.
Fig. 1. The ent-gibberellane skeleton.

Table 1
Fungal Gibberellins
Structure Formula M mp caC) (a)D
GA, o H C'9 H 2. 0 0 348 255-258 +38 0
HOJ11y[},~~
(dec.)
CH, COOH 2
GA 2 o H
C'9H 26 0 0 350 255 +12 0
HO~OH
(dec.)
CH, COOH CH,
@
GA, C'9H 22 0 0 346 234-236 +920
(dec.)
~o nn OH
HO - H CH 2
CH, COOH
@
GA. C'OH24O, 332 214-216 _3 0
(dec.)
co
HO 2 (H
H ,
CH 3 COOH
GA7 C'9 H 22 0 , 330 169-172 +20 0
HO· @~
CH,
H
(DOH
-(II
2
202 (dec.)
W
GAo C,oH2.04 316 208-211 -220
,. H CH,
CH, COOH
C,oH 20 O, 334 245-246 +3 0
~O"
GAIO
CH 3 (DOH (H 3
W
GAil C'OH22O, 330 242-244 + 11 0
~O
H CH z
CH, COOH
GA'2 ~H, H C2oH 2X O. 332 245-248
HOOC""\~
CH,
H
COOH
CH
z
GA" C2"H260 7 334 194-196 -48 0
"@
HOOC\""
(H,
H
COOH
CH z
Table l-contd.
Structure Formula M mp (OC) (a)D

GAl. ~fi:J H C ZOH 2,O, 348 242-243 -73 0
HO~
HOO(~ H
(H 3 (DOH
(H,
C ZOH 26O. 330 274-276 +5
~ytL,
GA" 0
(H 3 (DOH
#
GAI6 C I9H z.06 348 157-165
=0
H =
(H 3H DOH (H,
GA2• CHO H C 2oH 26 O, 346 198-203 -88 0
®CH,
HOOC CH3 COOH
GA z5 (DOH H 362 248-252 -69

CZOHZ606 0
HO DC
~CH'
CH 3 COOH
GA36 ~HO H CZOHZ606 362 205-208
HO~CH'
HOOC CH
3
(OOH
@
GA37 C 2oH z6 O, 346 228-232
CO
HO ~ H CH,
CH 3 COOH
GA40 Q H C I9H 2.O, 332 212-213

HO/////%tl
(H 3H COOH (H,
GA' l Cz"HzgOg 396 am.
366 174-182
(continued)
Table l-contd.
Structure Formula M mp t'C) (a)D
348
Ho.@,.H
~o
HO :; H (H 2
(H 3 (OOH
"4P",
GAs. 348 243-246
4htt"
GA" 364 260-263
H H (H
(H, COOH 2
H 364 am.
HO
(O()-j
~ H
364 147-150
H~(H
~IIIOH
(H, COOH 2
3-0-
~
~H
acetyl- ~O
380 226
GA, AcO" ,,,,OH
H (H_
(H, COOH '
glbbe ri c ac Id
""""'''''''"~ )''"'"'' "d'
HO @~" (H,
H
(OOH
(H 2
,~~ ~
HO
~
"~O
H
- H
II... OH
(H 2
~
H(k~H
~gO("· H
lu.. OH
(H 2
rn, (OOH ~ (OO~
iso-GA, d icarboxyllc acid
Fig. 2. Typical reactions of ring A of GA3 type gibberellins.

H H
H; Lewis Ac id
0)
OOH 100 D C n C H3
CH 2 = - 0
H
H+
fLCH' n'CH;
b)
H H
c)
U CH, H+
14CH,
OH
Fig. 3. Typical reactions of ring C/D site of gibberellins.
Accordingly GAs differ considerably in solubility and in their partitioning

coefficients (Durley & Pharis, 1972; Takahashi et ai., 1986). The pK-values of
of monobasic GAs range between 3·8 and 4·3.
The structural elucidation of GAs is based on comprehensive data of mass
spectrometry and of IH_ and 13C-NMR investigations (Takahashi et ai., 1986;
Hedden, 1987). The stereochemistry of GA3 was finally confirmed by X-ray
analysis (Hartsuck & Lipscomb, 1963), where other GAs can be related to.
As far as the chemical reactivity of GAs is concerned the multifunctional
substitution pattern and tensions of the ring system induce a series of typical
degradation processes, which may also play some role in fermentation and/or
processing steps. Thus, already slight alterations of the pH value can lead to
irreversible rearrangements and transformations, especially with GA3-type
gibberellins as shown in Fig. 2. Allogibberic acid, gibberellenic acid and
iso-GA3 are almost concomitants of GA3.
Some general routes of reactions taking place in the C/D-ring site of GAs
are outlined in Fig. 3. These reactions depend on the specific surrounding of
the 16(17) methylene group, which is accessible to both nucleophilic and
electrophilic attack. Quite common is the Wagner-Meerwein rearrangement
of the 13-hydroxy-16(17) methylene structure, which for GA3 leads to the
formation of gibberic acid (Graebe & Ropers, 1978; Takahashi et ai., 1986).
As described above, gibberellins were first isolated from culture filtrates of

Gibberella fujikuroi. Although several micro-organisms have been tested for
their potency in gibberellin formation, the most productive system continues to
be Gibberella fujikuroi (Saw.) Wr. (Fusarium moniliforme Sheld). In the first

publications after the discovery the taxonomic position of the phytopathogenic
fungus responsible for the bakanae disease was poorly defined. This led to
considerable confusion in the literature in the naming of the gibberellin
producer and in relation to the symptoms of the rice disease to a specific
species of the fungus. The reason for the controversy was the lack of a
systematic nomenclature for the genus. Jefferys (1970) listed all names which
have been attributed to producing organisms:
Fusarium heterosporum Nees
Fusarium moniliforme Sheldon
Fusarium oxysporum Schlechtendahl
Gibberella fujikuroi (Sawada) Wollenweber
Gibberella moniliforme (Sheldon) Wineland
In 1931 Wollenweber in his publication on the taxonomy of the genus Fusarium
resolved the problem and named the imperfect stage of the bakanae fungus
Fusarium moniliforme (Sheldon) and the perfect stage Gibberella fujikuroi
(Saw.) Wr. (Phinney, 1983). Borrow et al. (1955) had tested a lot of strains
isolated from several host plants for their capacity to synthesize gibberellins.
The bakanae effect was produced by most of the rice strains but only by one of
the strains from other hosts. He concluded a connection between the ability of
isolates to produce gibberellins and the origin of the strains from infected rice.
In contrast to these results Gordon (1960) showed that gibberellins were
produced by isolates from rice and other host plants such as maize, sugar cane
and cotton. He had acquired a large collection of strains from widely differing
hosts and geographical locations. All high-yielding strains of Gibberella
fujikuroi have their origin from the best wild-types, which were isolated from
infected rice seedlings by an extended screening programme. So in 1966 and
1967 Tamura isolated 1500 strains of Fusarium moniliforme in Japan. The
infected rice seedlings were collected from fields on the islands of Hokkaido,
Honshu, Shikoku and Kyushu (Phinney, 1983). One of the most cited high
producing wild-strains, isolated from infected rice seedlings in Japan is
Gibberella fujikuroi (Saw.) Wr., BRL (ACC) 917. This is the C.B.S., Baarn,
'Sawada' strain, and it is also culture No. NRRL 2633 and Commonwealth
Mycological Institute culture No. 58290 (Borrow et al., 1955, 1961). The BRL
(ACC) 917 was the subject of many physiological, biochemical and genetic
studies in connection with biosynthesis of gibberellins. The highest yields
reported were approximately 1 g GA per liter of culture filtrate obtained in
improved medium with this strain (Rehm, 1980).
Beside Gibberella fujikuroi other fungi were also found to produce GA., but
generally in lower amounts. In 1972 J. C. Lozano at the Centro Internacionale
de Agricultura Tropical (CIAT) in Cali (Columbia) reported a 'superelonga-
tion disease' of cassava plants. The independent work of two research groups
has established the occurrence of GA4 in the Deuteromycete Sphaceloma
manihoticola causing growth promotion in cassava (Rademacher & Graebe,
1979; Zeigler et al., 1980). G~ appears to be an end-product of gibberellin

biosynthesis in Sphaceloma manihoticola, while it is an intermediate in the
biosynthesis of GA7, GA3 and GAl in Gibberella fujikuroi.
In the culture filtrate of Sphaceloma manihoticola some other gibberellins
could be detected: GA13 (5 mg/liter), GA l4 (1 mg/liter), GA24 (100 Ilg/liter),
GA9 (50 Ilg/liter) and GA I5 , GA25 , GA36 , and GA37 in trace amounts. GA4 is
obtained in a very pure state in high amounts of maximum 7 mg/liter (EP
0,024,951). By improving culture conditions and selecting high-producing
strains it might be possible to produce G~ in a very pure form.
From mycelia, but not from culture filtrate, of Neurospora crassa, low
amounts of gibberellic acid could be isolated (Kawanabe et al., 1983). The
authors concluded that gibberellic acid has a regulatory function in the sexual
cycle of this fungus. Coolbaugh et al. (1985) reported the detection of several
gibberellins from the culture filtrate of the producer of abscisic acid,
Cercospora rosicola. Gibberellin-like substances were found to be produced in
trace amounts by several groups of micro-organisms, especially soil bacteria,
actinomycetes, algae, farns and fungi (Muromtsev & Agnistova, 1984; Brueck-
ner & Blechschmidt, 1986). But the structure of these active compounds was
not identified unequivocally.
As diterpenes the gibberellins are formed through the isoprenoid biosynthetic

pathway, starting from mevalonic acid which is first converted via the
isopentenyl, dimethylallyl, geranyl and farnesyl pyrophosphates to geranylger-
anyl pyrophosphate (GGPP), being an important pivotal intermediate. As
shown in Fig. 4, several points of divergence occur in this pathway which lead
to other important groups of compounds.
GGPP is converted further to ent-kaurene, which is the first committed
intermediate in gibberellin biosynthesis. The formation of ent-kaurene is
catalyzed by ent-kaurene synthetase in a two-step sequence. In the first step
(A-activity of the enzyme) GGPP is partially cyclized to form the bicyclic
copalyl pyrophosphate which is cyclized again in the second step (B-activity) to
give ent-kaurene. Both the A- and B-activity of the enzyme appear in the
supernatant after high speed centrifugation. The molecular weight of the
purified ent-kaurene synthetase, which exhibited both activities, was estimated
at 4·3-4·9 x 105 dalton and the pH optimum of the A- and B-activities were 7·5
and 6·9, respectively. The presence of a divalent cation, preferably Mg2+, is
required for both activities. Knowledge about these early stages of gibberellin
biosynthesis in Gibberella fujikuroi has been comprehensively reviewed by
Sembdner et al. (1980) and Coolbaugh (1983); and a more recent review
(Graebe, 1987) provides some supplementary information. It is apparent from
the extensive literature that the early stages of GA biosynthesis are identical in
the fungus and in higher plants. However, the extraordinary rate of GA
~H3
Ho-~-CH2COOH mevalonic acid
CH 2CH2 0H
ATP---1
ATP-i
ATP-i
~OPP 63-isopentenyl pyrophosphate - - - - , cytokinin side chains

(IPP)
J
(opp
~ 3.3'- dimethylallyl pyrophosphate
1PP-i
(Copp geranyl pyrophosphate - - monoterpenes
IPP-1
abscisic acid
farnesyl pyrophosphate ~
- - squalene __ sterols
~I
~
I ~
OPP
geranylgeranyl pyrophosphate
phytoe ne -
,ChlorOPhyuesters
0(. tocopherol
phylloqUInone
plas toquin one
carotenolds
A I fatty acid esters

noncyclic diterpenes
~I ;;
H
,# OPP
copalyl pyrophosphate :::::::::
______ macrocyclic dlterpenes
,H - - ent-isokaurene
!
B
17
ent- kaurene gibberellins
18
Fig. 4. Gibberellin biosynthetic pathway from mevalonic acid to ent-kaurene.

production in Gibberella fujikuroi indicates that the pathway in the fungus

lacks some of the controls working in higher plants.
ent-Kaurene synthesis is a branch point in the pathway which commits the
cell (organelle) to the production of either GA or alternative products, the
variety of which is much wider in the higher plants than in the fungus (cf. Fig.
4). ent-Kaurene is a direct precursor of GAs, and the key position of
ent-kaurene synthetase suggests that its activities A and B might be metabo-
lically controlled. However, regulation of ent-kaurene synthetase in Gibberella
fujikuroi is much less studied than in higher plants. A number of plant growth
inhibitors, like Phosphon D and quarternary ammonium compounds (e.g.
AMO-1618, CCC), are known to affect ent-kaurene synthetase, especially its
activity A.
Further conversion of ent-kaurene is characterized by stepwise oxidation to
form ent-kaurenol, ent-kaurenal, ent-kaurenoic acid and ent-7a-
hydroxykaurenoic acid (Fig. 5). These reactions are catalyzed by microsomal
monooxygenases, requiring NADPH as the only cofactor and probably
containing cytochrome P-450 (Graebe, 1987). The pathway branches, ap-
parently at two points, and leads to either ent-7 a, 18-dihydroxykaurenolide or
ent-6a, 7 a-dihydroxkaurenoic acid. The intermediates and end products of
these branches are not converted to GAs and lost to GA biosynthesis. The first
gibberellin compound formed from ent-7 a-hydroxykaurenoic acid by contrac-
tion of the B ring is GA 12-aldehyde.
The enzymes catalyzing the conversion of ent-kaurenolids into gibberellins
possess a low substrate specificity, as was shown by supplying ent-kaurenes
other than the true intermediates to Gibberella cultures. The various results
obtained by feeding kaurenoids to Gibberella fujikuroi have been comprehen-
sively reviewed (Sembdner et aI., 1980; Bearder, 1983).
The three steps of oxidative metabolism of ent-kaurene to ent-kaurenoic acid
are the sites of action of highly efficient plant growth retardants acting as
inhibitors of GA biosynthesis such as substituted pyrimidines (ancymidol),
norbornenodiazetine derivatives (tetcyclacis) and triazole derivatives (paclo-
butrazol) (see Sembdner et al., 1987).
GA 12-aldehyde being the first compound with ent-gibberellane skeleton is
the starting point for two parallel pathways through which all gibberellins are
formed in Gibberella fujikuroi (Fig. 6). One metabolic route leads from GA 12
via two further non-hydroxylated Czo-stages (GAlS and GA24) to GA9 and
subsequent products like GA lO , GAll and GA40 • A second pathway starts from
GAwaldehyde which is derived from GA12-aldehyde by 3p-hydroxylation.
This route leads via GA14 and two further C 2o-intermediates (GA37 and GA 36)
to a number of Cwgibberellins, the origin of which is GA4. Metabolic
transformation of C 2o-gibberellins is characterized by stepwise oxidation of
carbon 20, starting from -CH3, via -CH20H and -CHO, to the -COOH
stage. These transformation steps are catalyzed by soluble 2-oxoglutarate-
dependent dioxygenases. The conversion of Czo-gibberellins to Cwgibberellins
by elimination of C-20 and formation of the characteristic y-Iactone bridge in
~c~
HJw~'
H C"' ,"Ho,~".
~3
H
e nt- kaurenol
.. H
Hf "'-(H 2 0H
!
~
(H3 -c."'~
H ent- kaurenal
H -
Hf ~(HO
~ l
~~
',~H
H
c."'~
ent-kaurenoic ac id
-
___.~ ~§H
H : t~7oc. ~ H ~
OH - .
18-dih'Jdroxy-
kaurenolide
Hf ~OOH HOH 2 ( --C 0-0
1
~
H ~(:'0, ~_ CH z
a H OH ent-1x..hydroxy - - -... ~ H
OH ent- 60(,70( -
H3(
!
">~~OOH kaurenoic acid H3 C %.~OOH OH dihydroxy-
kaurenoic acid
Hi
~"" "(OOH
GA" - old.hyd.
Fig. 5. Gibberellin biosynthetic pathway from ent-kaurene to GA 12-aldehyde.
ring A occurs when the aldehyde oxidation step is reached; tricarboxylic acids
like GA13 , GA25 , and GA41 are not converted to Cwgibberellins. Feeding
experiments using 14C_ and 13C-Iabelled precursors demonstrated that C 20 is
lost as CO2 (see Graebe, 1987).
The main product of GA biosynthesis in Gibberella fujikuroi is GA3, which
is formed from G~ via GA7 by 1,2-dehydrogenation (G~-+ GA7) and
13-hydroxylation (GA7-+ GA3). The alternative pathway leading through GAl
to GA3 is a minor one and may be due to non-specificity of the dehydrogenat-
ing enzyme. However, in Sphaceloma manihoticola GA4 is the main product
Po thlJay lJithout PathlJay lJith

h yd ro x y la ti 0 n early 3r-hydroxylation
d uri ng (20 - s toges at (20 -level
R= H R = OH
erl,
'" GA 12 GA'4~GA2
.D
(\-\,
GAlS GA 3-
(hydroxy acid) ( hydroxy ac id)
R
'"
1
u
1 1
0\,
GA36
GA 25 II \A~GA13 41
----------------11--- --------- ----

GA~
GA-~GA~GA
,:l'/~~ ~ __ ----~ 3
0/ :«\ ~ GAl ---k -0/.

R GAs
I
GAS4 GA'6 GA4" 130£-OH~
~ 6
GA,s GAS7
Fig. 6. Gibberellin biosynthetic routes after GA 12 -aldehyde (derived from review

articles by Sembdner et aI., 1980; Bearder, 1983; Dathe, 1986; Graebe, 1987).
and GA7, GA3 and GAl are not formed. In Gibberella fujikuroi 13-
hydroxylation is a major reaction in conversion of Cwgibberellins such as GA4
( -+ GAl) and some subsequent products derived from GA4 by hydroxylation
at position 10 (GA I6 ), 1 (GAS4) and 20 (G~7)' respectively. It is interesting to
note that 13-hydroxylation occurs only at the end of the pathway and is
catalyzed by soluble enzymes, whereas 3P-hydroxylation is microsomal and
occurs at the beginning of the pathway (GA12-aldehyde-+ GAI4-aldehyde).
The oxidation of GAwaldehyde to GA l4 also proceeds in fungal microsomes
(Hedden, 1983). Opposite to the fungus in higher plants two further pathways
starting with early 13-hydroxylation (GAS3) or 3p,13-dihydroxylation (GAlS)
at the ~o-level are operating (see Graebe, 1987).
Both the amount and the type of gibberellins formed by the fungus are
dependent on the genetic constitution of the strain used (see Section 5) and the
fermentation conditions applied (see Section 6). Though rapid progress has
been made in the biosynthetic routes and the enzymes catalyzing conversion
steps, only little is known about the metabolic regulation of GA biosynthesis.
This holds true especially for fungal systems, whereas some more insights have
been gained during the last few years into the physiological and biochemical
control of GA biosynthesis in higher plants (see Graebe, 1987; Sembdner et
ai., 1987).
5 STRAIN IMPROVEMENT AND GENETICS
For the first time the genetics of the fungus Gibberella fujikuroi was
investigated by Gordon, who was occasionally successful in crossing some of
the strains of his large collection. He could show that the fungus was
heterothallic (Gordon, 1960). Physiological-genetic studies on gibberellin
production presented evidence for the presence of two genes that control
different steps in the gibberellin biosynthetic pathway. Genetic studies were
difficult at first because production of perithecia was erratic and a rare process.
A number of natural and synthetic media were tested which would support the
development of the sexual stage of the fungus. Spector & Phinney (1966, 1968)
found a medium containing stems of Citrus medica stimulated perithecial
production. When fungal strains of opposite mating type were grown on stems
of this tree, perithecia were regularly produced within 3-6 weeks. The number
of ascospores present in an ascus varied, a maximum number of eight spores
was observed. Mating between a high and low gibberellin-producing strain
gave tetrads that segregated 2: 2 in terms of the total amount of produced
gibberellins. One of the analyzed tetrads was of special interest, since one of
the four strains produced high amounts of G~, GA7 and GA9 but no GAl
and GA3 (Spector & Phinney, 1968). Genetic studies with this strain resulted
in the identification of two genes, gl and g2. Gene gl is responsible for the con-
trol of the overall gibberellin production, and gl-mutants neither produced
gibberellins nor metabolized those added. A second pair of alleles, gene g2,
controls 13-hydroxylation since g2+ strains produced the whole spectrum of
gibberellins, whereas g2 strains did not synthesize GAl or GA3 but accumu-
lated GA4 and GA7.
As indicated above, the best wild strains such as Gibberella fujikuroi ACC
917 produce about 1 g GA3 per liter of culture filtrate in an optimized medium,
and this was the yield in the early years of GA manufacturing. Current yields
are reported to be many times higher (Martin, 1983). Increased yields and
production rates are the result of improvement in strains and in fermentation
processes. Strain development of gibberellin-producing organisms had received
a great deal of attention. Since most of this research has been done at the ICI
Pharmaceutical Division and other industrial laboratories, it is mostly un-
published because of its proprietary nature. Like other natural products the
techniques included a combination of direct selection for high-producing
strains and mutagenesis by several different agents. Conventional mutation
processes have been published by Soviet workers (Imshenetsky & Ulyanova,
1962; Erokhina & Sokolova, 1966; Erokhina & Efremov, 1970). Mutants with
increased GA-titers of up to 60% were found after UV irradiation, fast
neutrons, and gamma treatments from spores or mycelia in the case of
non-sporing organisms (Erokhina & Sokolova, 1966).
A mutant strain resulting from UV and ethylenimine treatments gave yields
of approximately 2 g/liter in a medium with plant oil as the sole carbon source
(USSR Patent 440408). Another way of increasing yields is the screening of
mutants with blocked carotenoid biosynthesis as a competing biosynthetic
pathway with the same precursor mevalonate. Avalos & Cerda-Olmedo (1987)
found that strains with particularly low basal levels of neurosporaxanthin,
which usually represents about 75% of total carotenoids, are good gibberellin
producers.
In general, Gibberella fujikuroi is a highly suitable organism for the
induction and isolation of mutants. The uninucleate microconidia commonly
allow the expression of recessive mutations (Avalos et al., 1985). Besides
gibberellic acid, the last few years' commercial interest has been concentrated
on the production of GA4 and GA7. Gibberellin A3-producing strains can be
switched over to increased production of the precursors of GAl and GA3 by
increasing the pH value to the range of pH 6-7·5 (Jefferys, 1970). In
g2-mutants the hydroxylation at C-13 is blocked and consequently, relatively
large amounts of GA4 and GA7 could be isolated without separation from GA3
and GAl' Bearder (1983) described such a g2-mutant R-9, which did not
produce GAl and GA3. Mutation and selection for increased product
formation are probably the most important factors in improving the yield of
gibberellins. There is only a small number of publications concerning methods
of parasexual recombination to obtain diploid strains in Gibberella fujikuroi.
Crossing both wild strains and mutants as parents Calam et al. (1973) found
different recombinants, some of which gave increased yields of gibberellic acid,
compared with the already high-yielding parents.
6 FERMENTATION PROCESS AND PHYSIOLOGY
Beside strain improvement of wild-type strains, medium development ano

appropriate cultivation techniques are very important prerequisites for suc-
cessful economy of gibberellin production. Jefferys (1970) and Brueckner &
Blechschmidt (1986) made suggestions for maximizing production of GA. The
following section concentrates primarily on the effects of inoculum-
preparation, medium composition and culture conditions on the course of
fermentation.
6.1 Inoculum
Inoculum quality and quantity strikingly affect the production of gibberellins.

Based on experiments with a mutant strain of Fusarium moniliforme Gancheva
& Dimova (1984) established that at the submerged culture fermentation the
growth and biosynthesis of gibberellins depended on the age and quantity of
vegetative inoculum. The highest yield was obtained when a 48-h vegetative
culture at the phase of slowed-up mycelium growth was used as the inoculum.
The productive phase continues for a longer time (250-264 h) than with 72-h
mycelium (216 h). With increasing age of vegetative inoculum the hyphae
broke and autolysis began. The yield of fermentations inoculated with such a
mycelium was very small (Gancheva & Dimova, 1984). The necessary number
of conidia for inoculation of 1 ml inoculum medium is from 4 x 104 to 6 X 104 •
The best results were found when the inoculation of the fermentation medium
was carried out with 10 vol% vegetative mycelium.
Mycelium used as inoculum for large-scale fermentations is prepared in
progressively larger submerged-culture stages until sufficient of it is available.
For still vigorously growing vegetative culture little adaptation is needed and
growth in production fermentors commences quickly (Vass & Jefferys, 1979).
6.2 Nutrient Media and Environmental Factors for GibbereUin Production
The synthesis of secondary metabolites depends principally on biomass.

Therefore, the selection of medium components is based on both the aspects of
growth and product formation. A criterion for medium composition and other
ingredients is a fast enrichment of gibberellins at high concentrations. As
carbon sources, glucose and sucrose have frequently been used. However, if
the initial concentration of glucose was higher than 30% the specific growth
rate and the rate of gibberellin production were decreased (Borrow et al.,
1964). In view of the inhibitory effect of high glucose amounts on productivity,
feed processes were introduced (Brit. Patent 783 611; US Patent 2906 670; US
Patent 2906671). Glucose was added at intervals during the production
phase, and the concentration maintained below 4%. Another way to avoid
the inhibitory effect of glucose is to use carbohydrate polymers, such as starch
and plant meals (Brit. Patent 839652; Fuska et al., 1961; CS Patent 104329) or
combinations of fast and slowly utilized carbon sources (Brit. Patent 919 186;
Darken et al., 1959). Darken et al. (1959) obtained yields of 880 mg gibberellic
acid per liter in a 7-day period using a basal fermentation medium with com
steep liquor, ammonium sulfate, potassium dihydrogen phosphate and a
mixture of glycerol (20 g/liter), glucose (10 g/liter), and lactose (20 g/liter).
Moderate, but economically reasonable yields of gibberellin were produced
on molasses, sulfite liquors, and skimmed milk (FRG Patent 1081402;
Maddox, 1977). The Soviet workers have successfully used plant oils, e.g.
sunflower oil (Muromtsev et al., 1968; Muromtsev & Agnistova, 1984; USSR
Patent 440 408). They compared growth and gibberellin formation on mediums
with adequate amounts of oil and sucrose. The fat was introduced into the
initial nutrient medium as a single additive while sugar was supplemented to
the liquid in fractional doses. In the oil-containing medium significantly more
biomass was formed, and the production phase continued for a long time by
high mycelium productivity (Muromtsev & Agnistova, 1984). Sucrose con-
sumption for the gibberellic acid synthesis (as calculated per carbon) was
2·3-3·6 times higher than oil consumption. The results in Fig. 7 display the
increased yields with plant oil in comparison with sucrose. Besides plant oils,
hydrocarbons (Rehm, 1980) and fatty acids (Muromtsev & Dubovaya, 1964)
have also been tested for their ability to support growth and gibberellin
production.
Very important for the gibberellin fermentation are the quality and quantity
of nitrogen. Favorable nitrogen sources are ammonium sulfate, ammonium
chloride and slowly assimilable sources such as glycine, ammonium tartrate
and natural sources of nitrogen (Jefferys, 1970). The productivity on am-
monium acetate was considerably lower than on the other N-sources. It seems
possible that the stimulation in productivity by natural sources of carbon and
nitrogen such as plant meals, plant oils and com steep liquor could be
attributed to the content of precursors.
In submerged culture significant production of gibberellins starts only at the
time of nitrogen exhaustion. Production was greater the higher the value of
initial nitrogen. Further increases in nitrogen concentrations lead to a decrease
of the mycelium productivity because of the decreasing efficiency of oxygen
transfer (Borrow et al., 1964). Therefore, the selection of both the initial
concentration of nitrogen and of an optimal C/N-ratio is very important.
Besides carbon and nitrogen sources magnesium, potassium, phosphate and
sulphate were all needed. Trace element requirements would be met by
impurities in commercial media (Vass & Jefferys, 1979). For a maximum
production of GA Jefferys (1970) recommended a temperature of 31-32°C for
growth and for product formation 29°C. The airflow-agitation regime should be
as vigorous as possible.
Borrow et al. (1964) showed that the specific growth rate and the gibberellin
yield are fairly constant over the range of pH 3·5-6·5. However, the
composition of the gibberellin mixture produced depends on the pH value. In
a wild-type culture of Gibberella Jujikuroi the normal end-product of the
400 B. Brueckner et aJ.
, I
, a) 0
, <l
0 ~
9
0 I
/ 6 ____6 I
'-...
Ol
2000 <l- 5
.C-
01
5,5 .... ,
,
"\ 6
/~
"en
E
c::;;
55 ~
E
E .- 4 I 5,0
\
\ A/ OJ 45 c::;;
OJ
<{
M
3 I
a.
\
,,/~, ~ /
""
0 0
-'"
l:J 1 000 ;::-3 4,5 V............... \ /,/ -", ./0 35 ~
.--\-~~
Y '---'- -----
u /t:i. c::;;
/ \. / 0----,,0
2 4,0 30 25
.i
0 .y /0 20
-t!;
200 3,5 '0 0 10 15
3 5 7 9 11 13 15 17 19
days
0 .q
"
b) q
,
I
0 I
, '-... '-...
9 2 ODD- <l-5 S,S ""
E 55 E
Ol
->-
.C- "- c::;;
'-...
en Vl
- 4 '.... ./' - . . . x::-:6~::s;:::~:::::::,A_6 OJ
E I 5,0 '--- / ~ 0 0 en 45 c::;;
OJ
). ./A /o~ "-
~ 0
/Y ',
3 I "- 0
<{
M
a. / X
'6 ..... ..- -'"
// /
l:J 1 000 ;::-3 4,5 ./ li '_ . . . . . . . . . . 0 '--- 35 CI
'0
'\ c::;;
2 4,0 A 0 ,--- 3D 25
o 0/ 20
200 3,5 D~o 10 15
3 5 7 9 11 13 15 17 19
day s
Fig. 7. Growth and GA3 production by Fusarium moniliforme (strain F6) with different
carbon sources: (a) medium with glucose (fed batch); (b) medium with plant oil (after
Muromtsev & Agnistova, 1984).
gibberellin biosynthesis is GA3. At higher pH values, e.g. at pH 7, smaller

quantities of GA3, but increased production of the intermediates GA4, GA7,
GA9 , GA 12 , GA l4 and GA 16 could be detected. This pH effect may be ex-
plained in terms of increased dissociation of intermediates expelled into the
brew of the end-product GA3 at high pH. Therefore, these substances cannot
be transported back into the mycelium to be further metabolized (Bearder, 1983).
When the fungus was grown on a complex nitrogen source, relatively larger
amounts of GAl, GAl6 and GA47 were produced compared with the growth on
a synthetic medium (McInnes et aI., 1977). It was suggested by Bearder (1983)
that the dehydrogenation of GA4 to GA7 was readily inhibited by inorganic
nitrogen. The complex medium slowly releases nitrogen, which inhibits the
GA4 1,2-dehydrogenase preferentially and allows the accumulation of GAb

GA47 , GA 16 and G~. Japanese workers (JP 58,152,499) found that alumi-
nium, zinc and copper stimulated the preferable production of gibberellin A 4.
6.3 Fermentation Course and Fermentation Process Development
Production of GA is a classical fermentation in which significant production of

gibberellins and other secondary metabolites starts only after growth has
ceased because of exhaustion of assimilable nitrogenous compounds. Borrow et
al. (1961, 1964) defined producing and non-producing phases of the fermenta-
tion process. Growth during the balanced phase is exponential, and the
uptakes of glucose, nitrogen and the other nutrients remain constant. In the
following storage phase, when nitrogen was exhausted, the dry weight
continued to increase due to accumulation of lipid (maximum 45%) and
storage carbohydrates (maximum 32%). But under production conditions
these maxima are not reached because of controlled feeds of carbon source.
This phase is characterized by the onset of rapid production of gibberellins.
The storage phase is the main producing phase which is associated with the
production of gibberellins used for commercial purposes. With optimum
carbohydrate feeds this phase can be continued for a long time. Other phases
of growth which were described by Borrow et al. (1961) do not occur in
production conditions (Vass & Jefferys, 1979).
Bu'Lock et al. (1974) studied the synthesis of two different secondary
metabolites of Gibberella fujikuroi, namely the polyketide pigment bikaverin
and the diterpenoid gibberellic acid. In batch culture under conditions of
increasing nitrogen limitation the fungus first accumulates bikaverin and then,
under very strong nitrogen limitation, gibberellins are produced. It is very
interesting that this series of events can be reproduced in chemostat cultures at
different growth rates. Bu'Lock et al. (1974) concluded that the gibberellin
pathway is under the same type of overall regulation as bikaverin synthesis,
but that the GA synthesis begins at a level of limited nutrient and a
corresponding lower growth rate.
The conventional lag phase is by-passed in large-scale fermentations if high
initial glucose concentrations (more than 20%) or the use of ammonium
acetate as a nitrogen source is avoided, and if vigorously growing inoculum
is used to commence a fast mycelium development (Vass & Jefferys,
1979).
A critical factor in the gibberellin production is the supply of oxygen. Since
the demand for oxygen of growing mycelium increases with increasing dry
weight, oxygen limitation can be initiated which results in subsequent decrease
of GA production in the thickening broth. The dry weight at the end of the
exponential growth phase was shown to be dependent on the initial nitrogen
concentration (No) and on the rate of agitation of the fermentor (Borrow et
at., 1964). It is well known that the Q~ of the fungus decreases when the cells
switch to storage phase metabolism and the oxygen transfer capacity is no
longer saturated. That is why the initial nitrogen concentration, optimum
medium composition and nutrient feeds must be selected in such a way that
some further proliferation and, therefore, a mixed linear growth and storage
phase occurs after the balanced phase (Jefferys, 1970).
The gibberellin fermentation has a long history of development. Early trials
of the Japanese workers were based on surface cultivation, and low yields
(40-60 mg/liter) after prolonged periods of incubation were reported (Rehm,
1980).
In shake-flask experiments with the same media 200 mg/liter GA3 was
obtained. Optimization of medium composition, especially of the initial
nitrogen and carbon concentrations, led to a further increase of GA yield up to
1 g/liter. The early gibberellin production, prior to 1961, used a purely batch
technique. Later fermentations are batch-fed processes (Vass & Jefferys,
1979). From this time onward the feed regimes have been developed using
natural and synthetic carbohydrates and nitrogenous nutrients in the feedstock.
The carbohydrate feed rate also must be carefully restricted to limit the storage
activity of the fungus without lowering the rate of GA production. In the
patent literature it was said that some precursor feeds, e.g. mevalonic acid,
kaurene and kauranol, increased the yield of gibberellin A3 (Brit. Patent
957634). However, it seems unlikely that the obtained increases have any
economic interest because of the high cost of these compounds.
Holme & Zacharias (1965) and Bu'Lock et at. (1974) described the
production of gibberellin A3 in glycine-limited continuous culture conditions
and found maximum gibberellic acid synthesis at a growth rate of 0·005 h- 1 •
But because of the little specific rates of synthesis and the long period of
fermentation the chemostate culture has a theoretical importance only.
Besides submerged fermentation techniques employed throughout the
world, solid state fermentation is known to produce the metabolites in most of
the cases at high yield, especially if molds are involved (Lindenfelser &
Ciegler, 1975). First information on the cultivation of the gibberellin-producing
fungus on com grains was published by Focke et at. (1967). Indian workers
compared the production of GA3 by solid state fermentation and submerged
fermentation. Based on adequate carbohydrate contents they found that in the
case of solid medium (wheat bran) the accumulation of GA3 was 1·6 times
higher (Kumar & Lonsane, 1987). The degree of biomass formation varied
between 9·7 and 11·9 g/kg commercial wheat bran, the maximum quantity of
GA3 accumulated at the end of a incubation period of 7-8 days was 1·2 g/kg
solid substrate.
Up to now, there is no information on scaling up the extension of the solid
state fermentation technique for technical purposes in GA3 production on an
industrial scale. Probably this is due to the problems with the process control,
especially with oxygen transfer and moisture control on an enlarged scale.
6.4 Fermentation Process Economics
The total cost of a fermentation process comprises many factors, namely the
cost of raw materials, fixed costs, costs for separation and purification of the
product and so on. Process improvements, especially increase of the yield,
include strain improvement, optimization of the medium composition and
extension of the production phase. Conditions which lead to increases in the
overall rate of production, for example, by decreasing the percentage of
by-products, are also very important.
Vass & Jefferys (1979) calculated the changes in production data over a
period of 18 years. The process development obtained resulted in the
introduction of new strains, transition from a purely batch technique to
batch-fed process and modifications in medium compositions (Fig. 6.2). Only
those process changes which have shown a cost reduction or yield improve-
ment in laboratory experiments were implemented.
As can be seen in Fig. 8 the producing strains of Gibberella fujikuroi (A-D)
were developed during this period. For example, the introduction of strain D
shows an improvement in volumetric production rate of 35% in comparison
with strain C. The authors defined the term volumetric production rate as the
Period- Upto1961 11961 1970 11970-11972

1972 -19761
150
100
I
E
i
50 §
§ ~
dsrlilll
~
§
E
§
E I
'1=
Medium
Strain
Fig. 8. Changes in productivity of gibberellic acid up to 1976. Relative values have
been calculated from process data of Imperial Chemicals Industries. Horizontally
striped histograms indicate yield (mol), open histograms production rate (mol liter- 3 ) ,
and vertically striped histograms volume-specific production rate (molliter3 (t + tr )-I),
where tr denotes the time for turn-round (after Vass & Jefferys, 1979).
average rate of change of concentration of product with time, where the time
includes the period for vessel cleaning and preparations for new fermentation.
On the other hand, in the period from the introduction of strain D 1970 to
1976 an improvement in the volumetric production rate of more than 60% was
achieved by variations of the medium components in both the initial batching
and in the feed-liquor and by control of the supply of assimilable nitrogenous
nutrients for the growth period. The feed regime is also controlled during the
course of fermentation to nearly maintain a state of oxygen depletion (Vass &
Jefferys, 1979).
It is interesting that although the total fermentation costs per batch have
increased by 50% during these 18 years, the unit production costs decreased
with the increase of gross yield per batch up to 1700%.
Vass & Jefferys (1979) have mentioned that at the end of fermentation a
greater percentage of available carbohydrates from the feed was metabolized
to cell storage components rather than gibberellins. The decrease of these
losses of expensive components seems to be a target for further improvements.
During the fermentation the gibberellins are secreted into the culture medium.
Therefore, the fermentation of Gibberella fujikuroi normally results in the
production of a mixture of gibberellins among which GA3, GA4 and GA7
predominate. GA3 is the most abundant gibberellin.
Prior to isolation of gibberellins, the mycelium should be separated from the
broth by means of filter press, centrifuge, or drum filter with a filter aid.
Afterwards, the gibberellins can be prepared from the broth by adsorbing on
activated charcoal, by various types of ion exchange processes or by liquid-
liquid extraction. But it is very difficult to describe the processes in use on a
large commercial basis because these processes are kept as company secrets
and are not published. This is why the product recovery and purification of
gibberellins can only be described by a general discussion of the kinds of
technology available and published in literature and in patent specifications.
The adsorption on active carbon (e.g. charcoal) was the first method for the
extraction of GA3 developed by Japanese researchers. Probably it is also used
in commercial processes. After the adsorption the elution of the gibberellins is
carried out by percolation with water-miscible solvents, e.g. methanol or
acetone. The solvents may be used pure or mixed with ammonia (Jefferys,
1970).
However, this method has some disadvantages:
(a) it requires large amounts of solvents;
(b) the complete recovery of the gibberellins from the carbon is not
possible;
(c) the carbon can be used just once;
(d) the purification is poor.
Therefore, subsequent purification processes with buffer-ethyl acetate systems

proved to be necessary.
Another possibility for fermentation recovery is the application of ion-
exchange resins. A patent filed by the Societe d'Etudes et d' Applications
Biochimiques (Brit. Patent 936 548) describes the isolation of the gibberellins
and their purification by means of ion-exchange resins. The pre-treatment of
the culture filtrate is carried out by additive of alkaline-earth metal hydroxides
(e.g. Ba(OHh). This treatment effects the precipitation of impurities such as
organic acids, proteins, pigments and others. Moreover, the volume of the
resins needed for the ion-exchange processes is reduced to a half or to a fifth of
the original amount without pre-treatment (FRG Patent 1288044). After a
further purification on a weak cation-exchange resin (e.g. Amberlite IRC 50,
H. form) the culture filtrate passes a column of a weak anion-exchange resin
(e.g. Amberlite IR 4 B, acetate or formate form). Also the use of strongly
alkaline resins (e.g. Amberlite IRA 401, Dowex 1 x 2, chloride form) is
described.
The gibberellins are eluted from the resins by slow percolation with 1 N
ammonia or with buffered alkaline solutions of ammonia salts (e.g. ammonium
diphosphate). Also the elution is possible with methanol acidified with H 2 S04
or HCl (Jefferys, 1970).
The first fractions of the ammoniacal eluate obtained are sufficiently pure to
make possible the direct production of raw crystals by mere extraction of the
gibberellins from those fractions of the eluate with ethyl acetate and
concentration of the extract, without recourse to other purifying steps (US
Patent 3118909). The obtaining of such fractions represents a characteristic
feature and an important advantage of the extraction of gibberellins from a
culture broth by means of weak anion-exchange resins. Therefore, in commer-
cial processes employing this method it suffices to segregate these fractions to
produce crystallized gibberellins. The raw crystals prepared in such a manner
contained about 77% gibberellins. The extraction efficiency achieved with this
method was near to 100%.
The further purification of these raw products is possible by a second
purification step. It is carried out by the adsorption of the products obtained in
the first stage onto active carbon and the desorption of the gibberellins by
means of a solvent such as ethyl acetate. Evaporation, crystallization of the
gibberellins in pure ethyl acetate, and subsequent washing and drying resulted
in crystals with a purity of the order of 95% (US Patent 3118909).
However, the application of ion-exchange resins for the fermentation
recovery comprises some problems: the procedure requires large amounts of
resins, and numerous impurities and organic acids are adsorbed onto the
ion-exchange resins together with the gibberellins. Furthermore, the lactone
ring of the gibberellin molecule may be destroyed on the anion-exchange resin
or during the desorption under alkaline conditions (see Chapter 10). In this
way the gibberellin would lose its biological activity.
The Polish patent 0 152 578 avoids these disadvantages of the ion-exchange
resins by the use of synthetic sorption resins with non-ionic character and with
unpolar or middle-polar surfaces of the type Amberlite XAD-4, XAD-2 or
XAD-7. Gibberellins are caught quantitatively by resins of this type, whereas
the impurities and by-products are bound only to a negligible extent. The
gibberellins may be eluted by aqueous solutions of acetone, methanol or
ethanol. A further removal of impurities is possible by the choice of selective
elution media. The advantage of this procedure is based on the fact that the
solvent mixtures for the desorption of the gibberellins and of the impurities are
identical. They only differ in their concentrations. For example, the desorption
of the gibberellins is carried out with 35-80% methanol and with acetone
>70% or <45%, respectively; by comparison the desorption of the impurities
is possible by elution with 80-95% methanol and 45-70% acetone.
The third way of gibberellin recovery is the liquid-liquid extraction of the
culture filtrate with water-immiscible solvents. These methods were developed
as the yields in the fermentation broths were increased. The solvents mostly
used are esters such as ethyl acetate or butyl acetate and ketones such as ethyl
methyl ketone or methyl isobutyl ketone. However, n butanol or esters can be
used either alone or in mixtures. Ethyl acetate appears to be used as solvent on
plant-scale processes. Acid conditions (pH 2-4) are needed for the extraction.
The subsequent recovery of the gibberellins from the solvent is effected by
adsorption on solid sodium or potassium bicarbonate or by the buffer-solvent
processes being based on the relative solubilities of the free acid in solvent and
of the sodium or potassium salt in an aqueous phase. The extraction
efficiencies described in the literature varied between 40 and 100% (Jefferys,
1970). An important disadvantage of the liquid-liquid extraction is the large
demand for solvents and their recovery.
Definitely, it can be judged that the cost for each of the three procedures of
the recovery described is much higher than the fermentation cost itself because
the yields of the recovery processes on the large commercial scale are still
relatively low. The recovery cost is the main reason for the expensive price of
the product. The procedures mentioned up to now relate to the separation of
the gibberellins from the culture medium. But the culture broth contains a
mixture of gibberellins amongst which GA3, G~ and GA7 predominate. It is
desirable to separate the gibberellins not only from the culture broth but also
from each other because the gibberellins GA3 and G~/GA7 have different
plant gJ,:owth-regulatory effects, and, thus, the compounds have distinct and
specific practical applications.
The separation of GA3 and a mixture of G~/GA7 is possible by a
liquid-liquid extraction which is based on the different partition coefficients of
GA3 and G~/GA7 between certain organic solvents and water within a
particular pH range. For example, the culture filtrate is extracted with an
organic water-immiscible solvent (preferably ethyl acetate) at a pH value
between 4 and 8·5. The result is an extract rich in G~/GA7 and an aqueous
broth rich in GA3. The precipitation of G~/GA7 from the solvent is difficult
because other substances inhibit this process. The leI patent EP 83 306 682
Microorganism Manufacturing process Product recovery Product
Gibberella fujikuroi
(Saw.) Wr.
~ MY',li.m ~P7'ti., i : Mycelium I
= Fusarium moniliforme
(Sheld.)
Adsorption on active carbon I

~ S"'m'~ fu~"llitio, ~ -I
I
31-32°C for growth
I Elution with methanol or I GA3
acetone 1
29°C for product formation J I
pH 3·5-6·5 ~
4-7 days ;:
~I Purification by ion-ex- ~
Substrates change resins I
I
-~
glucose I Elution with ammonia or [
carbohydrate polymers ammonium salt solutions I ~
(starch, plant meals)
plant oils (sunflower oil) - I
Extraction with ethyl I
ammonia or ammonium salts l l J GA3
corn steep liquor acetate 1
1
oxygen i
~ Liquid-liquid extraction
with ethyl acetate I
pH 2-4
I GA3
1
H Jl I
pH 4-8·5 I GA4n
Y J -I I
Fig. 9. Flow chart of the gibberellin fermentation and recovery. ~
408 B. Brueckner et aJ.
describes the additive of a N-hydrocarbyl-N-arylmethyl amine to an organic

extract of a fermentation broth containing GA4/GA7. The reaction caused a
selective precipitation of the salts of GA4/GA7 with that amine, leaving the
residues of GA3 in solution. The recovery of the precipitated salt is carried out
by filtration. The isolation of G~/GA7 is also practicable by a reaction of the
amine salt with an organic acid (e.g. citric acid) and subsequent filtration.
Preparations obtained by these processes contained the GA4/GA7 mixture in
concentrations of about 95%. Consequently, the GA4/GA7 preparation is
sufficiently pure so that it may be used directly after isolation and no
recrystallization is necessary. Furthermore, the GA4/GA7 mixtures are free
from GA3 and other gibberellins. The application of this method on a large
commercial scale is described in the ICI patent cited above.
The production and the recovery of the gibberellins are summarized in
Fig. 9.
8 ASSAY METHODS
8.1 General
The identification and quantification of endogenous GAs in higher plants is

one of the most important aims for analysts (Crozier & Durley, 1983; Crozier,
1987; Sembdner et al., 1988). In microbial fermentations, where GAs
represent an abundant group of constituents, the analysis does not require such
high sensitivity as in plant hormone analysis, but it has almost to cope with
routine work for a large number of samples. This has to be considered
especially for sample preparation and prepurification steps. Commonly, the
first step of working up of fermentation broths consists of partitioning of the
acidified aqueous phase against ethyl acetate. More recently, solid phase
extraction using various cartridges became more favoured. This easy-to-handle
method, which is mostly based on partitioning at reversed phase material,
ensures both high efficiencies and recoveries (Hedden, 1987; Sembdner et al.,
1988).
8.2 Physical Methods
For some moderate analytical purposes, purified GA samples can be subjected

straight away to spectroscopical analysis. Thus, for the estimation of GA3 its
reaction with Folin-Ciocalteu reagent has been used for colorimetry at 730 nm
(Muromtsev & Agnistova, 1973).
Another possibility consists of inducing GAs to fluoresce by treating them
with sulphuric acid. The obtained fluorescence spectra are characteristic
for structurally related GAs (Table 2) and allow the quantification at the
ng-level (Schneider, 1988a). However, these spectroscopical methods fail if
complex GA mixtures or troublesome admixtures are to be considered.
Table 1
Experimental Conditions for Fluorimetric Analysis of
some Fungal Gibberellins (AE, Exciting Wavelength, AF,
Fluorescence Wavelength)
AE AF Incub. temp. Incub. time

(nm) (nm) ("C) (min)
GAl 410 455 80 120

GA3 410 455 20 30
G~ 390 410 80 90
GA7 455 468 80 60
GA9 385 410 80 180
More reliable analyses, therefore, usually include some chromatographic

steps in order to separate single GAs before quantifying them. In this respect
the combination of TLC and fluorimetric assay in situ turned out to be quite
versatile (Winkler, 1978). Based on the automated measurement on HPTLC
plates this method was reported to be both accurate and effective, especially if
large sample series have to be processed. This way GAs can be estimated at
concentrations of 1-200 Ilg/ml with SDs as low as 3-4% (Sackett, 1984).
Better resolution of complex GA samples can be achieved by HPLC
separation. Especially for the distinction of GA3 in the presence of the
biologically inactive concomitant iso-GA3 (see Section 2) HPLC is the method
of choice. If non-selective detection at 204-210 nm is used, some simple
pre-purification steps, e.g. by cartridge, is mandatory prior to HPLC. Analyses
are then performed on RP 8 or RP 18 materials with methanol/water (acidified
with, e.g. 0·1% H 3P04) (Jensen et al., 1986; Schneider, 1988b). Standard
deviations are as good as for HPTLC (1-4%). Automated HPLC-systems
guarantee high reproducibility and effectiveness in routine work. Considerable
increase in detection sensitivity for GAs can be achieved by the introduction of
a suitable chromophor (precolumn derivatization). Thus, GA-p-
bromophenacyl esters have been used for the detection at 254 nm (Morriss &
Zaerr, 1978). By reaction with 7-methoxy-4-bromomethyl coumarin GAs also
become accessible to fluorometric detection (pg-range) at AE = 320 nm and
AF = 400 nm (Crozier et al., 1982).
The highest level of information, accuracy and reproducibility in analysing
GAs is offered by GC-MS. Free acidic GAs have to be derivatized before. For
this, methyl esters and their trimethylsilyl derivatives are commonly used
(Crozier & Durley, 1983; Takahashi et al., 1986; Schneider, 1988c). The
extraordinary discriminating power of capillary GC sometimes becomes
necessary also for the analysis of fungal GAs, if they are isomers or closely
related otherwise. Subsequent on-line MS detection including isotope-labelled
internal standards, provides highest precision in quantifying GAs at pg-
amounts (Hedden, 1986, 1987).
8.3 Bioassays
Bioassays are based on typical physiological effects of gibberellins (cf. Section

9). Bioassays have been used successfully in gibberellin research since the
original purification and isolation of GAs from fungal cultures, and they are
still significant tools in GA analysis. Bioassays are most helpful in detection
and preliminary quantification of GA-containing fractions and in physiological
characterization of the GAs as well as in studies on structure-activity
relationships. Limitations in bioassaying are given by the fact that the single
GAs possess different physiological potencies. Thus, GAs of low activity like
Czo-GAs and GA9 may give only 1 % or less of the response produced by GA3.
On the other hand the activity of a given GA differs in different bioassays.
Therefore, the biological activity measured by bioassay has always to be
related to the identity of the GA and the specific sensitivity of the assay. For
practical use only those bioassays the responsiveness of which is well known,
should be used. Furthermore, it has to be considered that bioassays normally
result in relative GA quantities only, e.g. measured as {tg GA3 equivalents.
The sensitivity of a bioassay is characterized by the minimal GA concentration
(or GA amount) necessary to give a significant response. Sensitivity of good
GA bioassays is of the same order as the most sensitive physical methods.
Another characteristic is the concentration range within which the increase is
proportional to the dose (Table 3).
Quantification of bioassay results is possible by use of a log dose-response
curve (Fig. 10) obtained by measuring different concentrations of a standard
GA (normally GA3)' Furthermore, Fig. 10 demonstrates that the deviation
ranges are differing in dependence upon the logarithmic dose-responsiveness.
Many physiological effects of gibberellins (d. Section 9) have been put to
Table 3
Sensitivity of GA Bioassays
Assay Minimum GA3 dose Range of linear
concentration log dose-response
giving significant
response
Dwarf rice 0·1 ng/plant 0·1-100 ng/plant
'Tan-ginbozu'
microdrop variant
Root application 10- 8 _10- 7 mol/liter 10- 7 _10- 5 mol/liter
Dwarf maize--d 1 5 ng/plant 5-10,Ltg/plant
Dwarf pea--cv. 'Meteor' O· 5-1·0 ng/ plant 1 ng-lO ,Ltg/plant
Lettuce bypocotyl- 5 x 10- 7 mol/liter 5 x 10- 7 _10- 4 mol/liter
cv. 'Arctic'
a-amylase; barley 10- 10_10- 9 mol/liter 10- 9 _10- 7 mol/liter
endosperm
Response
o
. \1/ 10 I 00 \
logOose
I
1000
-111''-
0,7~X1~1.1
,/ ~
----~I~/-'-__+_I -+1--
I \ 1 ~~~~~tati
Lp 0n
70 100110
70~X2~110
Fig. 10. Schematized log dose-response curve. Deviation ranges (Yl/Xl; Y2/X2) pre-
sented in logarithmic and linear scale.
use as a bioassay. However, only three types of bioassays have become widely
used due to their ease of performance, reliability, sensitivity and range of
response. These are the dwarf mutants, the hypocotyl and the a-amylase
bioassays. The very extensive literature dealing with GA bioassays including
complete descriptions of their performance is covered by a number of reviews
and monographs (see Bailiss & Hill, 1971; Reeve & Crozier, 1975, 1980;
Graebe & Ropers, 1978; Hoad, 1983; Bergner, 1988; Bernhardt, 1988).
Some dwarf mutants of various plant species are known to be deficient in
endogenous gibberellins and, therefore, can be normalized by exogenous GA.
Such GA-sensitive mutants are favoured bioassay objects. Most important are
special mutants of Zea mays L., Oryza sativa L. and Pisum sativum L., the
dwarfism of which is controlled by a single recessive gene. The GA sensitivity
of those mutants is due to a block in GA biosynthesis (see Section 3)
preventing the formation of GAl necessary for normal stem growth. GA
biosynthesis can be blocked either at a very early stage (d 5 mutant of maize, dx
mutant of rice and na mutant of pea) or immediately before GAl formation (d l
mutant of maize, dy mutant of rice and Ie mutant of pea). Mutants belonging to
the first type respond to a broad spectrum of different gibberellins, and the
second type is sensitive only to GAs having a 3f3-0H group, like GAb GA3 ,
G~, GA7 , etc. Dwarf rice bioassays are widely used either with a microdrop
or root application technique. Both dx-mutants (cv. 'Tan-ginbozu') and
dy-mutants (cv. 'Katake-Tamamishikai' and 'Waito-C') are applied. Rice
seedlings are pre-germinated and-after application of the test solution-
further cultivated under artificial light and controlled temperature conditions.

Measurement of seedling length is performed after 4 days (microdrop assay)
and 7 days (root application), respectively. The root application assay is most
easy in performance, rather sensitive and well suited for screening of GA
activities in extract fractions or even non-purified culture liquids of the fungus.
Quantification can be done more precisely by the microdrop variant.
Dwarf maize bioassays can be performed under varying conditions.
Favoured are the mutants d 1 and d s which, however, are not available
commercially. Normally caryopses are pregerminated and seedlings grown
under controlled light and temperature conditions either in soil or nutrient
solution. Test solutions are applied as 50 Jll drops into the unfolding first leaf
and after about 6 days the length of the second leaf is measured.
Dwarf pea bioassays are in most cases performed with varieties the
genotypes of which are not known, like cv. 'Progress Nr. 9' and cv. 'Meteor'.
Seeds are germinated in wet sawdust and selected seedlings grown in nutrient
solution under controlled light and temperature conditions. About 5 days after
application of the test solution (50 Jll to the apex) the length of three
internodes above the first complete leaf is measured. By use of suited
genotypes and appropriate conditions, quantitation of GA amounts in extract
fractions is possible.
Hypocotyl growth in various plant species can be stimulated by low amounts
of GA. Among the different bioassays based on this response the lettuce
hypocotyl assay is rather widely used. Seeds of Lactuca sativa L. cv. 'Arctic'
are germinated and selected seedlings thereafter grown on filter paper wetted
with test solution. The assay is easy in performance, but not very sensitive and
often disturbed because of non-specific inhibition, e.g. by traces of solvent,
acetic acid, etc.
a-Amylase bioassays are based on the GA controlled induction of the
enzyme in the aleurone layer of germinating cereal caryopses. Several variants
of the assay have been described in the test object, measurement and some
other parameters; however, all are rather complicated in performance and
reliability. In any variant embryoless parts of cereal caryopses (wheat, barley,
rice) are used and incubated with the GA test solution. Sensitivity varies highly
depending on the variety, age and provenance of the seed, etc. Enzyme
activity is determined either by iodometric measurement of starch degradation
or by measuring the formation of reducing sugars. Because of the complicated
procedure the assay is only seldom used routinely in screening research, but it
may be helpful in physiological studies.
8.4 Immunoassays
Immunoassays are the most recent tools in quantitative GA analysis. During

the last years both the radioimmunoassay (RIA) and enzyme immunoassay
technique, especially the enzyme-linked immunosorbent assay (ELISA) have
been introduced to GA analysis and have gained considerable importance. The

first group of immunoassays that have been developed comprises radioim-
munoassays which make use of polyclonal antibodies raised in rabbits. Such
assays are available for the fungal gibberellins At. A 3, ~, and A24 (Weiler,
1984; Weiler et al., 1986a, b; Knoefel, 1988). They employ tritium or, less
frequently, iodine-125-labelled tracers which allow the detection of minimal
GA amounts of 0·05-0·1 pmol. Even higher sensitivity could be reached by
solid-phase enzyme immunoassays detecting 1 fmol of GA3 methyl ester,
0·5 fmol of G~ methyl ester and 1 fmol of GA7 methyl ester (Atzorn &
Weiler, 1983). With the introduction of the monoclonal antibody technology
the use of immunoassays in plant hormone analysis has entered a new
dimension (Weiler, 1986). Thus, monoclonal antibodies against GAlJ-imide
recognizing GA4 and related gibberellins have been applied to analyse the GA
spectrum produced by the fungus Sphaceloma manihoticola (Eberle et al.,
1986). A series of monoclonal antibodies allowing recognition of, and the
discrimination between GAl> GA4 and GA9 , have been derived from
immunisations with immunogens in which the proteins were linked to GAl at
carbon-3 and to GA4 and G~ at carbon-17 (Knox et al., 1987). Formerly
immunogen synthesis had been carried out only via carbon-7. Antisera raised
against apolar gibberellins, like G~, proved to be most selective, whereas
antisera raised against more polar gibberellins, like GAl and GA3, showed
cross-reactivity with few related GAs and, thus, gave 'group-selective' assays.
To date the immunoassays available cover only part of the fungal gibberellins;
however, the major ones are among them.
There is no doubt that immunoassays will be used to an increasing extent to
quantify the levels of gibberellins. They complete the traditional methodical
spectrum and can profitably be applied in combination with chromatographic
techniques, especially HPLC (see Section 8.2). Nevertheless, we must be
aware of their limitations resulting mainly from the fact that each material to
be analysed may contain substances that will interfere in the immunoreaction.
Therefore, in any case appropriate purification procedures and validation of
the assay have to be checked (Pengelly, 1986; Wang et al., 1986; Knoefel,
1988).
The bakanae disease syndrome in rice plants, including the typical overgrowth
symptoms, led to the discovery of gibberellins as the active metabolites of the
pathogenic fungus Gibberella fujikuroi (see Section 1). Apparently, the
gibberellins are part of the substantial interactions between the parasite and
the host and, therefore, might have some functional role in disease develop-
ment. However, there is only one piece of evidence that gibberellins have
some physiological significance in Gibberella fujikuroi. Nakamura et al. (1985)
found a promotive effect of GA3 on conidial germination and elongation of
young hyphae in Gibberella fujikuroi, but also in Penicillium notatum and

Neurospora crassa.
The work with the fungus led to the discovery of gibberellins in higher
plants. By use of bioassays (see Section 8.3), originally developed on the base
of the action of fungal GAs on higher plants, both the existence of gibberellins
in higher plants and their various physiological potencies could be demon-
strated. As a result of intensive GA research concerning the natural occur-
rence, structural elucidation and quantification, the biosynthesis, metabolism
and translocation as well as the physiological actions, the gibberellins were
found to represent a group of endogenous plant hormones. Within the
hormonal system the gibberellins interact with auxins, cytokinins, abscisic acid,
ethylene, and possibly other plant growth regulators, like brassinosteroids and
jasmonic acid, in the coordination and regulation of growth and development
of plants. The most obvious function of gibberellins is to control the rate of
growth and final length of stem internodes. This is best seen in certain dwarf
mutants which due to disturbed GA biosynthesis (see Section 8.3 and the
review of Graebe, 1987) have very short internodes but, upon treatment with
GA, grow to normal height. Gibberellins are also active in the reactions
of cold-requiring and long-day plants in response to induction. Furthermore,
gibberellins affect sex expression, the formation of flowers-at least in some
gymnosperms-and the extension of floral organs, the pollination and fertiliza-
tion, the development of seeds and fruits, especially pod growth, the
germination of seeds and vegetative propagation organs as well as the breaking
of dormancy and the abscission processes. Gibberellin functions are mediated
by external factors such as light conditions, temperature, water supply and
nutrition; and gibberellins might also be involved in the response of plants to
different stress factors.
The biological properties of gibberellins, including the multiple structure-
activity relationships and their physiological functions as plant hormones, have
been studied most intensively and the published literature contains numerous
original papers, many reviews and a number of monographs. Within the
Encyclopedia of Plant Physiology, New Series, three volumes deal with the
hormonal regulation of plant development and, thus, contain remarkable
knowledge on gibberellin physiology (MacMillan, 1980; Scott, 1984; Pharis &
Reid, 1985). Besides several early reviews (see Graebe & Ropers, 1978) two
special gibberellin books were published by Krishnamoorthy (1975) and
Muromtsev & Agnistova (1984). Important aspects of gibberellin physiology
were comprehensively summarized into a monograph edited by Crozier (1983).
It contains reviews on gibberellin-induced growth in two extensively studied
objects; these are excised lettuce hypocotyls (Jones & Moll, 1983) and Avena
internodes (Kaufman & Dayanandan, 1983). Many effects of gibberellins on
flowering have been described, although their physiological role in flower
induction is not completely understood (Zeevaart, 1983). Also, the interac-
tions between gibberellins and phytochrome need further investigation (Reid,
1983). However, photoperiodic control of stem growth is clearly mediated by
gibberellins (a recent review is given by Graebe, 1987). Some information on

the physiological role of gibberellins in fertilization and reproductive develop-
ment has been derived from either GA effects obtained by exogenous
application or studies on the endogenous hormonal situation. Present knowl-
edge in these fields has been summarized by Tsao & Linskens (1986)
concerning the fertilization processes and by Pharis & King (1985) with respect
to seed and fruit development. The physiological significance of gibberellins in
seed germination is well established; some more recent aspects have been
reviewed and discussed in view of gibberellin-deficient mutants (Karssen &
Lacka, 1986). A general survey on plant hormones in seed dormancy and
germination is given by Lewak (1985).
Gibberellin effects on conifers, especially with respect to flowering, have
gained increasing interest and it could be shown that there may be profound
differences between angiosperms and gymnosperms as far as GA physiology is
concerned (Dunberg & Oden, 1983). On the other hand, gibberellins are
known to have widespread effects on cryptogams, when exogenously applied.
They promote growth in many species of algae, liverworts, mosses, and ferns,
induce spore germination in ferns and regulate differentiation of reproductive
organs in fern gametophytes. As analogues processes in the life cycle of higher
plants are often similarly influenced by GA treatment, it might be assumed
that the mechanism of GA action in green cryptogams is essentially the same
as in higher plants (Furuya & Takeno, 1983).
The mechanism of gibberellin action is most intensively studied in cereal
aleurone cells known to respond to exogenous gibberellins with the de novo
synthesis and secretion of hydrolytic enzymes, predominantly a-amylase
isoenzymes (see Section 8.3). During germination of caryopses the embryo is
assumed to provide aleurone cells with gibberellins (reviewed by Jacobsen,
1983). The GA action on a-amylase synthesis by affecting gene expression is
intensively studied at present. Considering the results published it can be
concluded that GA controls the synthesis of certain aleurone cell enzymes by
enhancing mRNA transcription. Mechanism of GA action on growth processes
is also studied on the level of gene expression but less intensively than in
aleurone system (see Sembdner et af., 1987). a-Amylase and some other
hydrolytic enzymes are influenced by gibberellins also in systems other than
aleurone cells. Furthermore, it is well known that gibberellins can affect either
the biosynthesis or the activity of various enzymes covering nearly all aspects
of plant metabolism, though the modes of action and physiological roles are
understood in a few cases only (see Barendse, 1986).
With respect to gibberellin primary action, a number of investigations deal
with the binding properties of radioactively labelled gibberellins to high
molecular weight compounds and the characterization of proteinaceous recep-
tor sites. However, only little progress is available in gibberellin receptor
research up to now. The evident knowledge in this field has been summarized
with competency by Stoddart (1986).
According to the data published gibberellins apparently display only low
activities or they are inactive in animal systems. The acute toxicity on animals
is very low. The tolerance limit to GA3 is much higher than to antibiotics; for
mice it is given as 6300 and 25 ()()() mg/kg after intravenous and oral
application, respectively (Schwartz et al., 1983). The acute toxicity for
G~/GA7 on mice is also>500mg/kg (Abdel-Rahman et al., 1977). Neither
in animals nor in tissue cultures were teratogenic or carcinogenic effects
observed over a period of years. Pharmacological studies have been done with
GA3 using predominantly mice, rats and guinea-pigs. Among the results
reviewed by Schwartz et al. (1983), there are stimulating effects on the body
weight in various animals, influences on endocrinologic functions and on the
functions of the macrophaga and changes in mitotic activities as well as
protective effects against pathogenic infections, toxicants and X-rays.
10.1 Total Synthesis
The total synthesis of any natural product is a challenge to chemists even if

commercial interests might be far beyond. In first attempts for total synthesis
of complex GA structures, other totally synthesized compounds, e.g. enmein,
were tried to transform to GAs, e.g. GAlS (Somei & Okamoto, 1970). The
group of 13-dehydroxy-GAs became linked to total synthesis by converting
Fig. 11. Main routes in conversion of gibberellins.

synthetic epigibberic acid via gibberelline C-methyl ester to GA4 or G~ (Mori

et al., 1968). The highly complicated structure of GA3 with eight chiral
C-atoms could finally be synthesized in the course of a 34-step sequence by
Corey et al. (1978). Using another approach, Lombardo et al. (1980) succeeded
in synthesizing GA3 and GAl> too. As many other GAs can be derived from or
related to either GA3 and GAl or GA4 (see Fig. 11) all these GAs are formally
linked to total synthesis.
10.2 Conversion
Most of the endogenous plant GAs occur in traces only. Thus the microbiolog-
ical source of GAs is of high importance in order to get access to substantial
amounts of these substances. Commonly the main fungal GAs like GA3, G~
and GA7 act as starting material for preparing CwGAs, and GAl3 for
Cw-GAs. Figure 11 compiles the main routes in GA conversion, for references
see Takahashi & Yamaguchi (1983). Sometimes a single step like hydrogena-
tion leads easily to another GA (GA3~ GAl> GA7~ G~). Other conver-
sions need a complex sequence of reactions, e.g. GA3~ GA29 •
10.3 Labelling
In principle, many of the reactions used in conversion of GAs (see above) are
suitable for introducing stable or radioactive labels into GAs (see Takahashi &
Yamaguchi, 1983). For example, hydrogenations with deuterium or tritium
lead efficiently to labelled analogues. A widely used method for double
labelling of GAs consists of oxidatively removing the exocyclic methylene
group followed by refixing it with 13C, 3H2 -Wittig reagent.
Sometimes biotransformation by Gibberella fujikuroi or plant enzymes are
included in modifying initial products. Other possibilities for introduction of
3H are the catalytic hydrogen exchange with GA3-type gibberellins (Nadeau &
Rappaport, 1974) or the 6-H exchange on the 6-aldehyde level (Lischewski,
1985).
10.4 Structural Modification
With respect to structure-activity relationships of GAs in plant systems,

structural modifications of natural GAs are of high scientific but also
commercial interest. Thus, a series of papers is dealing with the introduction of
halogen atoms, e.g. fluorine, chlorine, bromine, and iodine, especially at
positions C-l, C-3, C-13 (Banks & Cross, 1977; Cross & Simpson, 1981; Keith
et al., 1979).
Quite promising were experiments toward the blockage of metabolic
oxidation by the alkylation of C-2, e.g. 2,2-dimethyl GAl or GA4 (Beale et al.,
1984). Other facets in modifying GAs are alterations or removal of the
6-carboxy group (Lischewski & Adam, 1980) or the introduction of hetero
418 B. Brueckner et aI.
atoms like N, e.g. as l-azido-, l-amino-GAs (Voigt & Adam, 1982) or amino
acid conjugates (Adam et ai., 1977). Physiologically relevant glucosyl conjug-
ates of GAs have also been prepared. Thus a series of GA-O-glucosides and
GA-glucosyl esters were synthesized (see Schneider, 1983).
11 FORMULATIONS, APPLICATIONS AND ECONOMICS
From the 72 gibberellins only gibberellic acid (GA3) and, to a lesser extent,
mixtures of GA,. and GA7 have found practical use. Sometimes it is difficult to
calculate the financial return to the grower for the application of gibberellins.
For example, climatic effects, environmental factors and differences in varietal
response can effect or veil the efficiency of the hormones applied so that a
reliable calculation of the economic value is connected with many elements of
uncertainty. Moreover, it has been difficult to gather accurate information
separating actual commercial use of gibberellins from wishful thinking or
potential uses.
The gibberellins are the most widely used group of native plant hormones.
In 1980 the world production of gibberellins for commercial use was estimated
to be 12-15 tons (Martin, 1983). Countries with a good tradition in the
production of gibberellins are the USA (Abbott Laboratories, Eli Lilly), the
UK (Imperial Chemical Industries) and Japan (Takeda Chemical Industries).
Further countries producing more or less large amounts of gibberellins are the
USSR, Hungary, Poland and the People's Republic of China. Well-known
trade names are Pro-Gibb, Promalin, Gib-Sol, Gib-Tabs, Activol, Berelex,
Auxilin, Gibersib and Gibreskol. The preparations often contain GA3 and
small quantities of other gibberellins (At, A 4 , A7 and the artefact iso-GA3)
together with spreader activators, effervescent mixtures or desiccants. At
present, practical uses of gibberellins are most extended in the brewing
industry, in increasing both yields and quality of grapes and in stimulating the
growth of sugar cane.
In the routine commerical malting of barley the use of GA3 is a widespread
method because the development of malt is a costly, time-consuming step in
brewing beer. Normally, malt is developed by weighing a certain quantity of
barley seed into a tank where germination occurs. During this process en-
dogenous gibberellins passing from the germinating embryo to the aleurone
layer of the seed induce the synthesis of hydrolytic enzymes, especially
a-amylase and diastase. These enzymes degrade endosperm food reserves such
as cell wall carbohydrates, storage protein and a limited quantity of starch. The
resulting liquor is then drained from the steep for further processing. Small
additions of GA3 accelerate the production and release of the enzymes which
degrade the hemicellulosic-protein-starch complex into essential brewing ma-
terials such as sugar, peptides and amino acids. Today the average steeping
and germinating time amounts to 7-10 days. It can be shortened to 1-3 days
by the addition of GA3. In commerical use GA3 is applied as an aqueous
spray, or in the final steeping water in concentrations ranging from 0·25 to

1·0 ppm (Palmer, 1974) resp. 0·02S-0·5ppm (Martin, 1983) with respect to the
weight of the barley. The economic effect of using GA3 during malting is
difficult to evaluate, nevertheless a net gain of 2% production can be estimated
(Martin, 1983). At present, the US brewing industry does not use GA3 in the
malting process (Martin, 1983), and in the FRG the use of GA3 is also
prohibited (Sommer, 1987).
Besides the brewing industry the gibberellins have found a wide propagation
in horticulture and agriculture caused by their ability to increase the vegetative
growth, to enhance flowering, to increase fruit set, size, development and
quality and to delay senescence in numerous plant species.
One of the most important and traditional fields of application of the
gibberellins is wine-growing. Most of the GA3 used in the world is applied to
grapes. Seedless grape varieties contain only small quantities of endogenous
gibberellins. Therefore, for more than 20 years the growers of seedless grapes
have been using gibberellins as a safe and practical method of reducing the cost
of cultivation by regulating the time of harvesting or by increasing yields and
improving the quality of the crop. In Japan, the seeded grape cultivar
'Delaware' is treated with GA3 prior to bloom and 2 weeks later to induce
parthenocarpic fruit set and to accelerate apparent fruit maturity. The berries
attain their full size, irrespective of seed number. At least 12000 ha are treated
in this way (Rappaport, 1980).
GA3 is also being routinely used to increase the crop yields in sugar cane.
Treatment of sugar cane with about 62 g GA3 per acre can increase the yield to
more than 5 tons and raise the output of sugar from 0·2 to 0·5 tons per acre
(Nickell, 1978). These results were achieved with several types of gibberellins
including broths, semi-purified and crystalline gibberellins· under Hawaiian
plantation conditions (Nickell, 1979).
Besides these main commercial uses the gibberellins have reached a certain
importance by a limited, sometimes only local application in selected fields of
agriculture, horticulture and fruit growing. For example, in the USA and in
the Mediterranean countries the application of GA3 is widespread by reason of
the beneficial effects on citrus species. GA3 is widely used by Californian navel
orange growers to improve fruit quality by delaying rind senescence and thus
reducing surface defects such as 'rind staining', 'puffy rind', 'sticky rind', and
'water spot'. In mandarins GA3 treatment with 100 mg/liter applied in the
period between anthesis and 2 weeks post-anthesis increases fruit set, total
fruit number and the number of seedless fruits. Ten milligrams of GA3 per liter
applied to lemons prior to degreening results in delayed degreening and
prolonged storage life of the fruit up to 3 months (Rappoport, 1980). In the
USA, sweet cherries are treated with 25-50 ppm GA3 up to 4 weeks before
harvest (Martin, 1983). The fruits are larger and firmer, and the GA3
treatment suppresses the development of post-harvest surface pitting and
bruising defects during harvest or transport. GA3 is also being used routinely
in Europe to increase the crop yield in pears, to induce parthenocarpic fruit
set, and to treat frost damage of pear (Martin, 1983). In the UK a mixture of
GA3 and naphthalene acetic acid is registered for use on Cox's Orange Pippin
applies to improve fruit set and yield (Looney, 1979). In the UK, the
Netherlands and Canada GA3 is recommended for use on rhubarb. The
treatment with the hormone breaks the dormancy in some early cultivars and
increases stick yield from plants emerging from dormancy in all cultivars.
GA3 is also effective in breaking dormancy of potatoes. The treatment with
GA3 is particularly useful in climates where two potato crops are grown in one
season (e.g. in India), by breaking the dormancy of seed potatoes saved from
the first crop. Moreover, GA3 hastens shoot emergence and increases the
number of stems produced and daughter tubers formed (Rappaport, 1980).
The ability of GA3 to stimulate flowering and to increase the flower number
in a wide range of vegetable species has been utilized in the production of seed
from poor-bolting lettuce cultivars. GA3 (10 ppm) applied at 4- and 8-leaf stage
increased the seed yield about 4-7-fold compared with the unsprayed and not
beheaded control (Martin, 1983)_
Yields of certain cultivars of hops are improved and the number of cones are
increased up to 35% after spraying with GA3 (Palmer, 1974).
In Mediterranean countries and in South Africa GA3 is used commercially
for the production of globe artichokes. By spraying the hormone onto young
plants it is possible to increase the head number per plant and to advance the
harvest by several weeks, especially at low temperatures which limit bud
growth. In such a way total crop yields can be increased by up to 40% without
affecting earliness (Thomas, 1985).
Besides these uses, on a large commercial scale numerous possibilities of
GA3 application are known in crops with a limited market size: GA3 can be
used for the early harvest of chicory, cabbage, mint, parsley and spinach, for
the increase of the yields of beans, celery, endives, peppers, watercress and
tomatoes under glasshouse conditions, for the seed production of carrots,
celery and onions, for the improvement of the germination of celery and
onions, for a better fruit set of blueberries and plums, for the increase of the
fruit size of lemons and melons and for the induction of parthenocarpic
development of many fruits such as black and white currants, figs, straw-
berries, apples, pears and tomatoes (Martin, 1983).
In addition to GA3, the gibberellins GA4 and GA7 are going to reach a
greater utilization. It is supposed by numerous experts that their importance
will rise further in the near future, because the physiological potencies of GA 4,
GA7 and GA3 differ from each other in some important respects. For example,
GA4/GA7 have the remarkable ability to modify the sex expression of
cucurbits. Treatment of gynoecious cucumbers with GA4/GA7 (e.g. ProGibb)
can enhance male flower formation, and this phenomenon can be used
commercially for producing pollen parents in gynoecious cucumber fields
(Rappaport, 1980).
In celery seeds, thermodormancy can be broken by GA4/GA7 treatment
(Biddington & Thomas, 1978). This procedure has some importance for the
UK with a celery production of about 41000 tons (Thomas, 1985).
In the USA the russet incidence of the apple cultivar 'Golden Delicious' is
reduced by spraying with GA4/GA7 (Meador & Taylor, 1987). In Italy five to
six treatments of GA4 applied 8-15 days after full bloom reduced russeting of
the 'Golden Delicious' apple by 70%.
A recent addition for the growth-regulator treatment of apples is a
combination product of a mixture of GA4+ 7 and the cytokinines N6 _
benzyl adenine (Promalin). This treatment increases the ratio of length to
diameter of 'Golden Delicious' apples, resulting in an apple which better meets
market expectation with respect to shape (Looney et al., 1979). In forestry,
promoting influences of GA4/GA7 on the flowering and the seed production of
conifers were observed in field experiments (Rappaport, 1980). For example,
the strobili production in western hemlock (Tsuga heterophylla) and the
flowering in white spruce (Picea glauca) were promoted (Pharis et al., 1986;
Rottink, 1986). These important economic forest trees have a poor natural
regeneration because of the infrequency of good seed years. Thus there is a
need for a reliable method to increase and stabilize seed production. However,
a true commercial scale application will not be possible before sufficient
amounts of GA4/GA7 are available.
Finally, it seems to be worthwhile to mention some outlooks for new and
unconventional fields of gibberellin application. Japanese patents describe the
addition of gibberellins to a cosmetic cream for the decolorizing of freckles
(Japan Patent 58103307 and Japan Patent 58077 808) and to a hair tonic for
the promotion of the growth of hair (Japan Patent 8059906).
By adding GA3 to the food of fattened bulls, the daily increase in weight of
these animals was increased by 18%. The GA3 stimulated the protein,
carbohydrate and fat metabolism (Shamberev et al., 1985). Furthermore, GA3
stimulated the growth of lactic acid bacteria resulting in an increase in biomass
yield of 4-8-fold. This effect of GA3 will possibly gain importance for the
silage of green fodder (Erzinkyan, 1981). It is to be expected that the
commercial interest in the gibberellins will grow further, especially as this
group of compounds has very low mammalian toxicity and occurs naturally in
many vegetable foodstuffs.
In spite of the applications described above, the scale of gibberellins is
relatively low compared with the market of the herbicides, defoliants and
desiccants. Some important factors limiting use of gibberellins are:
(a) the relatively high cost and the low availability, especially for GA4+ 7;
(b) the action over a long period of time in contrast to the herbicides which
are expected to produce a rapid effect;
(c) potential side-effects, such as inhibition of flowering in fruit trees for a
long period of time after treatment;
(d) the insufficient reproducibility of the results obtained under controlled

conditions in the field, and
(e) the inaccurate knowledge of the ideal time of application and of the
concentration required.
12 CONCLUSIONS
In this chapter we have shown that the production of gibberellins is an almost

classical fermentation, in which typical phases of growth can be discerned.
Gibberella fujikuroi (Saw.) is up to now the only gibberellin-synthesizing
fungus of economic importance. High producing strains of Gibberella fujikuroi
were developed from wild strains. Obviously, process improvement involve a
complex of conditions, which:
lead to increases in the overall rate of production;
extend the production phase;
hasten the onset of production; and
yield fewer by-products (Vass & Jefferys, 1979).
A detailed knowledge of the gibberellin biosynthetic pathway and well-
characterized mutants are required for maximizing production. Further impor-
tant phenomena in the gibberellin production, which for economic reasons is a
fed-batch process, include medium composition, the feed regime and airflow-
agitation regime. The separation and purification process constitutes a famous
part of gibberellin production cost, and from recovery process improvements
there may be a decrease of gibberellin losses. Continuing reductions in the
costs make gibberellins more attractive for existing applications and open
possibilities for further applications.
In the future, the introduction of molecular genetics in order to increase
rate-limiting enzyme levels by means of gene amplification may contribute to
strain selection programs. Fermentation process computer control develop-
ment is also important for the near future.
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for Pesticides and Plant Growth Regulators, Vol. X. New and Updated Methods, ed.
G. Zweig & J. Sherma. Academic Press, New York, San Francisco, London, pp.
454-9.
Yabuta, T. (1935). Biochemistry of the 'bakanae' fungus of rice. Agr. Hort., 10, 17-22.
Yabuta, T., Kambe, K. & Hayashi, T. (1934). Biochemistry of the 'bakanae' fungus of
rice. Part 1. Fusaric acid, a new product of the bakanae fungus. J. Agr. Chem. Soc.
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Yokota, N., Murofushi, N. & Takahashi, N. (1980). Extraction, purification and
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Zeevaart, J. A. D. (1983). Gibberellins and flowering. In The Biochemistry and
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Sphaceloma manihoticola, causal agent of cassava superlongation disease.
Phytopathol., 70,589-93.
INDEX
Acetobacter, 309, 310, 321 ADP, 338, 339

Acetobacter cerinus, 313 Aerobacter, 262, 310
Acetobacter fraqum, 313 Aerobacter aerogenes, 177, 287
Acetobacter melanogenus, 312, 313 Agrobacterium, 262, 264, 379
Acetobacter suboxydans, 168, 199, 209, tJ-Alanine, 206
275, 304, 311, 312, 317, 323, 324 Alcaligenes, 262, 310
Acetobacter xylinum, 168, 304, 323 Alcaligenes faecalis, 361, 366, 367, 368,
Acetomonas albosesamae, 313, 319 369
Acetyl-CoA carboxylase, 232 Algae
Achromobacter aceris, 376 composition of growth medium, 51
Adenine, 348 physical factors, 51
Adenosine, 348, 367 5-Aminoimidazaole ribonucleotide
Adenosylhomocysteine (AdoHcy), 351- (AIR), 140-1
72 AMP, 337-40, 343,344, 346,376
activated methyl cycle, 353-60 Antivitamins, 5, 6
biosynthesis, 353-62 Arachidonic acid, 111-15
chemical synthesis, 368 Arthrobacter globiformis, 177
chemistry, 352-3 Arthrobacter parafJineus, 289
derivatives of, 353 Arthrobacter simplex, 316
enzymatic methods, 366-7 L-Ascorbic acid, 299-334
hydrolase, 360-2 application, 326-7
production of, 366-8 assay methods, 325-6
Adenosylmethionine (AdoMet), 351-72 background history, 299-301
activated methyl cycle, 353-60 biocatalysts involved in biosynthesis of
biosynthesis, 353-62 intermediates, 316-18
chemical synthesis, 366 biosynthesis
chemistry, 352-3 cloning of genes related to, 318-22
decarboxylation, 360 in plants and animals, 302-4
derivatives of, 353 micro-organisms and media used in,
enzymatic production, 362-5 305-8
isolation of, 365-6 with micro-organisms, 304-18
microbial production, 362-5 chemical properties, 302
production of, 362-6 development data, 300
stabilization of, 365-6 direct synthesis of, 316
Adonis vernalis, 169 economics, 326-7
431
432 Index
L-Ascorbic acid-contd. Biotin-contd.

industrial process, 322-5 chemical properties, 232-3
nomenclature, 302 chemical reactions, 233
physical properties, 302 chemical structure, 233
product recovery and purification, chemical synthesis, 248-50
324-5 deficiencies in animals, 243-8
Reichstein-Griissner synthesis, 323 economics, 250
Ashbya gossypii, 151-60, 162, 165 fermentation, 241-2
Aspergillus flavus, 151, 316 genetics, 240-1
Aspergillus giganteus, 28 growth-promoting activity, 248
Aspergillus niger, 151, 235, 316 historical background, 231-2
Astaxanthin, 58-9 physical properties, 232-3
extractability of, 61 producing micro-organisms, 234-5
ATP, 337-50, 376 product recovery and purification,
assay methods, 338-9 242-3
chemical structure, 338 regulation of biosynthesis, 238
chemistry, 338-9 screening, 234-5
construction of reaction system strain improvement, 240-1
producing, 345-6 structure of, 232
energy efficiency of phosphorylation Biotin carboxylase (BC), 232
system, 346-8 Bixa orellana, 27
production Blakeslea trispora, 27, 28, 30-3, 36, 55
adenosine or adenine, from, 348-9 Blood clotting mechanism, 124
biochemical system, by, 349 Brevibacterium, 287, 313
glycolytic pathway, through, 339-43 Brevibacterium ammoniagenes, 212-14,
oxidative phosphorylation by 177,208,215,289,292,337,
methylotrophic yeast, through, 343-4,376
344-9 Brevibacterium divaricatum, 237, 238
salvage synthesis, through, 343-4 Brevibacterium flavum, 250
role in energy metabolism, 337 Brevibacterium ketosoreductum, 312,
Azotobacter, 262,310 316
Brevibacterium KY-4313, 62-4, 71
Brevibacterium thiogenitalis, 177
Bacillus, 72, 168, 171, 174, 175-83, 185, Bupleurum falcatum, 169
262, 289, 310
Bacillus circulans, 61
Bacillus megaterium, 237, 238, 264, 311, Calcium 2,5-diketo-o-gluconate, 315
316 Calcium 2-keto-L-gulonate, 315
Bacillus pumilus, 177, 180 Calcium pantothenate, 216
Bacillus sphaericus, 234-8, 241, 242 Candida, 84
Bacillus subtilis, 177, 243, 258, 289, 291 Candida arborea, 151
Bacterium xylinum, 304 Candida boidinii, 344-6, 348
Bacteroides rumincola, 175 Candida chalmersi, 151
Bicyclic ketocarotenoids, 58-65 Candida flareri, 151
Bifidobacterium, 216 Candida ghoshi, 151
Biotin, 231-56 Candida quilliermondia, 151
absolute configuration, 233 Candida humicola, 101
antimetabolites, 240 Candida krusei, 151
application, 250 Candida lactis, 151
assay methods, 244-5 Candida lipolytica, 151, 289
bioassay methods, 243 Candida meliblosi, 151
biodegradation, 238-40 Candida norvegensis, 316
biological properties, 243-8 Candida olea, 151
biosynthesis, 231,235-6 Candida parapsilosis, 210
Index 433
Candida puicherrima, 151 Carotenoids---contd.

Candida solani, 151 product recovery and purification, 69
Candida tropicalis, 89, 289, 292, 379 scarcity in diet, 27
Candida tropicalis varrhaggi, 151 strain improvement, 53-4
Candida utilis, 362 structural formulae, 45
Cantharellus cinnabarinus, 28 structure of, 28
Canthaxanthin, 58-9, 62 Cell reaction method, 341, 343-4
Capsicum annuum, 27 Cempasuchil, 27
Carboxyl carrier protein (CCP), 232 Cercospora rosicola, 391
Carboxyl transferase (Cf), 232 Ceroplastes, 169
p-Carotene,15-26 Chilomonas paramoecium, 152
applications, 23 Chlamydomonas, 97
biological properties, 22-3 Chlorella, 97
biosynthesis, 18-19 Chlorella pyrenoidosa, 56, 316
chemical properties, 15-16 Chlorella sorokiniana, 56
chemical synthesis, 23 Choanephora cucurbitarum, 28
economics, 23-4 Cholecalciferol, 82
formulations, 23 Citrobacter, 311
HPLC analysis, 16 Citrus medica, 396
industrial purification, 37 Claviceps purpurea, 83
physical properties, 15-16 Clostridium, 262
product recovery and purification, 22 Clostridium acetobutylicum, 151
regulation, 18-19 Clostridium butylicum, 151
syntheses, 27 Clostridium kainantoi, 226
Carotenes, 54-5 Clostridium tetanomorphum, 102, 275
Carotenoids CMN complex, 62
applications, 73 Cobalamins, 259, 260
assay methods, 68-71 biosynthesis of, 264
basic structures, 44 Cobaltcorrinoids, 259, 260
biological properties, 47 Coccus cacti, 6
biosynthesis and regulation, 48-53 Coenzyme A, 199,207-9,212-14
biosynthetic relationships, 50 application, 216
chemical properties, 44-5 biosynthesis, 214
chemical synthesis, 71-3 economic aspects, 216
compounds inhibiting normal structure of, 205
biosynthesis of, 52 Coenzyme Q, 379-80
content variations, 30-2 Coenzymes, 373-81
culture conditions, 32 classification, metabolic functions and
discovery in nature, 43 sources of, 374-5
eady observations in micro-organisms, Cordyceps militaris, 169
43-4 Corynebacterium, 235, 262, 287, 313,
economics, 73 315-19
environmental factors, 50-2 Corynebacterium glutamicum, 250, 286,
fungal,28 287, 289, 292, 293
fungi production, 27-42 Corynebacterium sp. XG, 67
future prospects, 73 Crocus sativus, 27
genetic control, 53 Croton bean, 169
industrial applications, 47 Croton tiglium L., 169
industrial production, of, 28 Culture method, 343
microbial production, 43-79 Cyanobacteria, 59
micro-organisms of interest in Cyanocobalamin, 257
production of, 47-8 Cyclic GMP, 248
nomenclature, 46
physical properties, 44-5 Debaryomyces nilssonii, 340
434 Index
Deinococcus radiodurans, 58 Fermentation--contd.

Deinococcus radiophiLus, 58 production, 54-65
Deinococcus radiopugnans, 58 micro-organisms designed for
Deoxyadenosylcobalamin,271-2 applications other than pigment
I-Deoxy-ketoses, 180 production, 65-9
2-DeoxY-D-ribose, 167 Flavin adenine dinuleotide (FAD), 164
Dethiobiotin, 241-2 FLavobacterium, 57, 73, 127, 129-33,
Dihomo-y-linolenic acid, 115-16 226,262, 311
2,5-Diketo-D-gluconic acid reductase, Flavobacterium meningosepticum, 127-9
318-21 Flavobacterium soLare, 258
Dimethylallypyrophosphate (DPP), 86 Fungal gibberellins. See Gibberellins
DipLoascus, 84 Fungi, 60, 66
DunalieLLa, 15, 119 blue illumination, 31
DunalieLLa salina, 15 carotenoid composition, 28-30
production process requirements, 19- carotenoid production, 27-42
21 chemical stimulation, 32-3
strain improvement, selection and composition of growth medium, 51
maintenance, 16-18 culture conditions, 32
Eicosapentaenoic acid (EPA), 116-19 genetical stimulation, 34-6
EmiLiania huxLeyi, 169 growth medium, 55
Endomyces, 84 industrial production, 36-8
Enterobacter, 311 inhibitors, 55
EntomophthoraLes, 111 photocarotenogenesis,31
Enzyme immunoassay, 412 physical factors, 51
Enzyme-linked immunosorbent assay sexual interaction, 31
(ELISA), 412 sources of xanthophylls, 57
Eremothecium ashbyii, 151-62 Fusarium, 89, 151
Ergocalciferol, 82 Fusarium aquaeductuum, 28
Ergosterol, 81-93 Fusarium heterosporum, 390
chemical properties, 83 Fusarium moniLiforme, 383, 390, 398,
fermentation, 88-9 400
occurrence and producing organisms, Fusarium oxysporum, 390
83
patents for production of, 90
physical properties, 83 Geotrichum candidum, 102
production, 87 GibbereLLa fujikuroi, 28, 30, 36, 383,
Erwinia, 311, 313, 315, 318-21 385, 390, 391, 393, 394, 396, 397,
Erwinia citreus, 321 399,401,403,404,413,414,417,
Erwinia herbicoLa, 319, 320, 321 422
Escherichia, 262, 289, 310 GibbereLLa moniLiforme, 390
Escherichia coLi, 139-41, 143, 174, 176, Gibberellins, 7, 383-429
180, 207, 209, 226, 232, 235, 238, applications, 418-22
241, 243, 261, 274, 276, 287, 289, assay methods, 408-13
291, 318, 320-2, 337, 341, 379 bioassays, 410-12
Escherichia freundii, 227 biological properties, 384, 413-16
Eubacterium saburreum, 169 biosynthesis and regulation, 391-6
EugLena, 97 biosynthetic pathway, 394
EugLena graciLis, 95, 276 chemical properties, 385-9
EugLena graciLis Z, 95, 97-100, 98 chemical synthesis, 416-18
economics, 418-22
Famesylpyrophosphate (FPP)-C15 , 86 fermentation and recovery flow chart,
Fermentation, 54 407
carotenoid-producing micro-organisms fermentation process, 398-404
specifically designed for pigment economics of, 403-4
Index 435
Gibberellins--contd. International units (IV), 4

ftuorimetric analysis, 409 Isopentenyl pyrophosphate (IPP), 86
formulations, 418-22 5-Keto-D-gluconic acid, 311-12
genetics, 396-7 2-Keto-L-gulonic acid, 304-16, 321-2
historical survey, 383-5 Klebsiella, 310
immunoassays,412-13
labelled analogues, 417
main routes in conversion of, 416, 417 Lactobacillus, 143, 151, 174
physical properties, 385-9 Lactobacillus arabinosus. 258
plant growth regulator, 383 Lactobacillus bulgaricus, 199, 216, 286,
producing micro-organisms, 389-91 287
product recovery and purification, Lactobacillus casei, 214, 163, 222, 223
404-8 Lactobacillus helveticus, 209, 214
screening, 389-91 Lactobacillus lactis, 258,272
strain improvement, 396-7 Lactobacillus leichmanii, 272, 276
structural modification, 417-18 Lactobacillus plantarum, 214, 243, 247
structure of, 385 Lactuca sativa L. cv. Arctic, 412
total synthesis, 416-17 Leuconostoc mesenteroides, 163
Gluconobacter, 309, 310, 321 y-Linolenic acid, 109-11
Gluconobacter melanogen us , 310 Linseed oil, 117-19
Gluconobacter oxydans, 310, 311, 317 Lycopene, 54-5
Gluconobacter oxydans subsp.
suboxydans, 304
Gluconobacter roseus, 101 Menaquinones, 123-33
Gluconobacter suboxydans, 317, 321, biosynthesis of, 125
322 chemistry of, 124
D-Glucose, 312-16 fermentative production of, 125
Green algae, 56-7, 59 menaquinone-4, 131-3
Growth factors, 2, 3 menaquinone-5,128-9
basic source of, 7 menaquinone-6,127-8
production and application, 7-10 screening of micro-organism, 125-8
technical function, 6-7 Methanobacillus, 262
L-Gulonic acid, 312 Methionine, 351
Methylotrophic yeast, 344-9
Methyltrophs, 66-7
Hansenula, 84 Micrococcus, 228, 310, 316
Hansenula jadinii, 340 Micrococcus denitrificans, 177
Helminthosporium, 169 Micrococcus glutamicus, 286
Hexuronic acid, 299 Micrococcus roseus, 64-5, 70
High performance liquid Micromucor, 110, 111
chromatography (HPLC), 326, Micromucor ramanniana, 111
338 Micromucor vinacea, 111
High voltage paper electrophoresis, 338 Micro-organisms, 1-11
Hydrocarbon assimilating bacteria, 52 Monascus, 6
Hydrocarbon-utilizing bacteria, 64 Monocyclic ketocarotenoids, 58
Hydroxocobalamin, 271 Mortierella, 110-13, 117, 118
3-hydroxy-3-methylglutaric acid (HMG), Mortierella alpina, 114-117, 119
86 Mortierella elongata, 114
R-tJ-hydroxyisobutyric acid (tJ-HIBA), Mortierella isabellina, 110
101 Mucor ambigus, 111
Hyphomicrobium X, 379 Mucorales, 109, 111
Mutants
blocked,53
L-Idonic acid, 311-12 enhanced pigment levels, with, 53-4
436 Index
Mutation to non-production of Pantothenic acid, 199-219

undesirable carotenoids, 53 assay methods, 214-16
Mutational biosynthesis, 53 biosynthesis, 205-9
Mycobacterium, 52, 262 chemical production, 209-12
Mycobacterium brevicale, 64 chemistry of, 199-205
Mycobacterium lactiola, 64 control mechanisms for biosynthesis,
Mycobacterium phlei, 54, 57 209
Mycobacterium smegmatis, 58, 175, 258 microbial production, 209-12
Mycoderma, 84 Pantothenyl alcohol, 205, 216
Mycoplana, 238, 240 Paper chromatography, 338
Paprika, 27
Paracoccus denitrificans, 379
NAD,376 Penicillium, 28
NADH,377 Penicillium brevi-compactum, 175, 180,
NADP, 376 181
NADPH,377 Penicillium chrysogenum, 151
Nadsonia, 84 Penicillium notatum, 414
Nematospora, 84 Penicillium westlinghi, 84
Neurospora, 286, 287 Phaeolactylium, 119
Neurospora crassa, 28, 38, 207, 391, 414 Phaffia rhodozyma, 28, 30, 38, 60, 61,
Nicotinamide cofactors, 376-7 70,73
Nocardia asteroides, 210 growth medium, 60
Nocardia gardneri, 262 use of cheap media, 61
Nocardia rugosa, 262, 266, 272 Phlebodium dictyocallis, 169
S-Nucleosidylhomocysteines, 368 5'Phosphate (FMN), 164
4' -Phosphopantetheine, 214
Photocarotenogenesis, 31
Ochromonas malhamensis, 274 Photoinduction, 52
Oenothera biennis, 106 Photosynthetic bacteria, 52, 67-9
Oenothera lamarckiana, 106 Phycomyces, 28, 30
Orotic acid, 285-97 Phycomyces blakesleanus, 28, 30, 32-6,
application, 294-5 38, 143
assay, 287 Phylloquinone, 133
biosynthesis and regulation, 289-92 Pigments
production, 54-65
chemical synthesis, 294
technical function, 6-7
chemistry, 286-7
fermentation, 292-3 Pisum sativum L., 411
Plant growth-regulators, 7
historical background, 285-6
Polyunsaturated fatty acids (PUFAs),
isolation, 294
105-21
producing micro-organisms, 287-9,
biosynthesis, 108-9
288
naturally occurring, 105-7
properties of, 287
production, 109-19
purification, 294
sources of, 109
structure of, 286
Propionibacterium, 262
Orotidine
Propionibacterium freudenreichii, 267-
properties of, 287
9,271-2
structure of, 286
Propionibacterium sherman ii, 232, 262,
Oryza sativa L., 411
266-9, 271-2, 275
Protaminobacter ruber, 67, 379
Protective factor X, 231
Pachysolen tannophilus, 175 Proteus, 262, 311
Pantetheine (Pantethine), 199,216 Proteus mirabilis, 171
Pantoic acid, 207 Proteus morgan ii, 207
Index 437
Protomonas extorguens, 65 Rhodoturula minuta, 28, 210

Provitamin A. See p-Carotene Rhodoturula mucilaginosa, 66
Provitamin D 3 , 89 Rhodotorula rubra, 28
Pseudo-gluconobacter Rhodoturula gracilis, 84
saccharoketogenes, 311 Rhodoxanthin, 65
Pseudomonas, 262, 269, 309-11, 316, Riboflavin, 149-66
366 applications, 164
Pseudomonas aeruginosa, 310, 312, 322 bioassay methods, 163-4
Pseudomonas albosesamae, 313 biological properties, 164
Pseudomonas chlororaphis, 312 biosynthesis and regulation, 152-3
Pseudomonas denitrificans, 262, 264, chemical properties, 150
265,267, 269-71 chemical synthesis, 164
Pseudomonas extorquens, 65 economics, 164
Pseudomonas jluorescens, 311, 312 fermentation/bioconversion process,
Pseudomonas graveolens, 238 154-61
Pseudomonas maltophilia, 207, 209 formulae of, 168
Pseudomonas mildenbergii, 311, 312 formulations, 164
Pseudomonas pseudomallei, 312 history, 149
Pseudomonas putida, 310, 322, 367, 101 physical properties, 150
Pseudomonas reptilivora, 175, 180, 181, producing micro-organisms, 151-2
182, 185 product recovery and purification,
Pseudomonas sesami, 313 160-3
Pseudomonas viridiflava, 309 screening, 151-2
Pyridoxal,228 strain improvement, 153-4
Pyridoxal-P, 221, 223, 227-8 structure of, 149
Pyridoxamine 5' -phosphate, 221 D-Ribose, 167-97
Pyridoxamine-P, 221, 223, 225 application, 188
Pyridoxine, 221, 225, 227-8 assay methods, 185
Pyridoxine glucoside, 229 biosynthesis, 172-5
Pyridoxine-P, 223, 225, 227-8 chemical properties, 171-2
Pyrimidine nucleotides, 289-91 chemical synthesis, 185-8
Pyrroloquinoline quinone (PQQ), 377-9 economy, 188
fermentation, 180-4
fermentation time course, 184
Radioimmunoassay (RIA), 412 formation from carbon sources, 183
Red peppers, 27 formation from D-glucose, 178
Red yeasts, 66 formula of, 172
Reichstein-Grussner synthesis, 323 metabolism, 172-5
Retrocarotenoids, 65 nitrogen sources for, 183
Rhizobium, 262 occurrence in nature, 169-71
Rhizobium lequminosarum, 175 physical properties, 171-2
Rhizobium melioti, 171, 174,264 producing micro-organisms, 175-80
Rhizobium tritolii, 264 producing tkt mutants
Rhodobacter capsulatus, 54, 67-9 improvement of, 177-9
Rhodococcus erythropolis, 210, 211 pleiotropic properties, 179-80
Rhodococcus maris, 64 recovery and purification, 184-5
Rhodopseudomonas capsulatus, 67 Rye ergot, 83
Rhodopseudomonas sphaeroides, 275
Rhodospirillum rubrum, 275, 379
Rhodosporidium, 66 Saccharomyces, 84-6
Rhodosporidium diobovatum, ·28 Saccharomyces carlsbergensis, 84, 89,
Rhodotorula, 66 139,376
Rhodotorula aurea, 28 Saccharomyces cerevisiae, 38, 85, 86,
Rhodotorula glutinis, 235 89, 92, 102, 140, 141, 143, 207,
438 Index
Saccharomyces cerevisiae--contd. Thiamine--contd.

223,228,234,243,247,341,362, biological properties, 144
376,379 biosynthesis, 140-2
Saccharomyces fermentati, 89 chemical assays, 143-4
Saccharomyces fragilis, 151 chemical properties, 137-8
Saccharomyces sake, 363-5, 369, 376 chemical synthesis, 145
Saccharomyces uvarum, 89, 214, 376 enzyme assays, 144
Saffron, 27 metabolism, 144
Salmonella typhimurium, 139, 141, 142, microbiological assays, 143
169, 174, 176, 206, 289, 291 physical properties, 137-8
Sarcina, 228 physiological functions, 144-5
Serratia, 262, 310 producing micro-organisms, 138-40
Serratia marinorubra, 287 regulation, 142
Skeletonema, 119 structure of, 137
Sphaceloma manihoticola, 390-1, 394, Thiamine monophosphate (TMP), 137-8
413 Thiamine pyrophosphate (TPP), 137-8
Spongiococcum excentricum, 56 Thiamine triphosphate (TIP), 137-8
Staphylococcus aureus, 143,316 Tocopherol, fermentative production of,
Staphylococcus hyicus subsp. hyicus, 175 97-100
Sterols a-Tocopherol
biosynthesis and regulation, 86-8 isolation of, 98
production, media and environmental production of, 98-100
factors, 85-6 Tocopherols, 95
Streptococcus, 262 producing micro-organisms, 97
Streptococcus faecalis, 162 Transketolase mutants, 175-80
Streptococcus faecalis R, 222, 223 Trichoderma spp., 89
Streptococcus liquefaciens, 162 Trimethylhydroquinone, 103
Streptococcus zymoge, 162
Streptomyces, 216, 246
Streptomyces albidoflavus, 262 UMP, 291-2
Streptomyces alboniger, 169 Ustilago violacea, 28, 30, 36
Streptomyces antibioticus, 258, 262
Streptomyces aureofaciens, 260-1, 262
Streptomyces aureus, 262 Verticillium agaricinum, 28
Streptomyces chrestomyceticus subsp. Vibrio parahaemolyticus, 171
rubescens, 55 Vitamin A, 2
Streptomyces chrestomyceticus var. Vitamin antagonists, 5
aurantioideus, 57 Vitamin B, 2, 137-48
Streptomyces farinosus, 262 Vitamin B), 2
Streptomyces fradiae, 262 Vitamin B 2 , 149-66
Streptomyces griseus, 258, 262 Vitamin B s , 199-219
Streptomyces hygroscopicous, 180 Vitamin B 6 , 221-9
Streptomyces mediolani, 57 assayof,221-3
Streptomyces olivaceus, 262 biodegradation of, 223
Streptomyces roseochromogenes, 258, biosynthesis, 223-6
262 chemistry of, 221-3
Streptomyces showdoensis, 289 de novo synthesis and bioconversion,
Streptomyces vinaceus, 262 226-9
derivatives, 228
fermentation, 226
Tagetes tenuifolia, 27 metabolism, 223-6
Tetrahymena pyriformis, 164 structure of, 222
Thiamine, 137-48 Vitamin B 12 , 66, 67, 257-84
assay methods, 143-4 absorption, 276-7
Index 439
Vitamin B 12-contd. Vitamin O 2 , 5, 81, 92

analogues of, 259 Vitamin 0 3 ,5,81,92
applications, 277-9 Vitamin deficiencies, 1
bioassay methods, 272-5 Vitamin E, 95-104
biochemical mechanisms, 275-6 assayof,96
biological properties, 275-7 bioconversion reactions for synthesis
biosynthesis, 263-5 of,100-2
chemical properties, 259-61 biosynthesis of, 97
chemical structure, 259 chemistry of, 96
deficiency, 277-8 Vitamin H, 231
derivatives, 271-2 Vitamin K, 2,123,133
economics, 277-9 chemical structure of, 124
elimination, 276-7 Vitamin Kb 133
forms and grades of, 278 Vitamin K 2 , 123-33
formulations, 277-9 Vitamin nomenclature, 2-4
historical background, 257-9 Vitamin PP, 2
improvement of producing strains, Vitamins
265-6 basic source of, 7
isolation of, 272 fat-soluble, 5
metabolism, 276-7 history of, 1
nutritional-dependent deficiency, 278 nutritional, clinical and physiological
pharmaceutical use of, 278 aspects of, 6
physical properties, 259-61 physiological functions, 4-6
producing micro-organisms, 261-3 production and application, 7-10
production of, 266-71 research history, 1-2
regulation, 263-5 technical function, 6-7
screening, 261-3
transport, 276-7
Vitamin B12 coenzyme, 260-1, 271-2, Xanthomonas, 262, 310, 311
275 Xanthomonas translucens, 312
Vitamin B 12a, 259 Xanthophylls, 55-8
Vitamin B 13 , 285-97 X-ray crystallography, 259
Vitamin C, 2, 5, 299-334
Vitamin compounds, 2
Vitamin content, 4
Vitamin 0, 2, 81-93 Yeast cell reaction method, 339-41
application and economics, 92 Yeast cell regeneration, 341-3
chemical nature, 81
formation of, 89-91
metabolic transformations, 91-2 Zea mays L., 411

Biotechnology of Vitamins, Pigments and Growth Factors

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BIOTECHNOLOGY OF

VITAMINS, PIGMENTS AND GROWTH FACTORS

ELSEVIER APPLIED SCIENCE

Sole distributor in the USA and Canada

WITH 51 TABLES AND 113 ILLUSTRATIONS

© 1989 ELSEVIER SCIENCE PUBLISHERS LTD

British Library Cataloguing in Publication Data

Biotechnology of vitamins, pigments and

Library of Congress Cataloging-in-Publication Data

Biotechnology of vitamins, pigments, and growth factors/edited by

1. Vitamins-Biotechnology. 2. Growth promoting substances-

Special regulations for readers in the USA

1. Vitamins and Related Compounds via Micro-organisms: a

Fat-Soluble Vitamins and Pigments

14. Microbial Production of Biotin (Y. Izumi & H. Yamada) 231

Other Growth Factors

VITAMINS AND RELATED COMPOUNDS VIA

2 VITAMINS: WHAT'S IN A NAME?

Vitamin nomenclature was initially based on the use of letter symbols,

Vitamin group Important examples Important natural International Recommended adult

Provitamin A group ,B-carotene Vegetables, I IV = 0,6 pg 2·4 mg,B-carotene

Vitamin group Important examples Important natural International Recommended adult

Folic acid Pteroyl-glutamic acid, Cereals, liver, 400 I'g

Vitamins have a catalytic role in the body in enabling optimal synthesis,

As to the function of fat-soluble vitamins and vitamin C, much controversy

Vitamin Physiological functions

Vitamin A Retinol is part of rhodopsin, the light sensitive

thrombosis and leukemia, respectively. Antivitamins present in our daily food

4 TECHNICAL FUNCTION OF VITAMINS, PIGMENTS AND

In addition to their nutritional, physiological and medical importance, vitamins

5 PRODUCTION AND APPLICATION

Vitamin Industrial Application World production

Provitamin A E F 100 450 2, 5, 6, 6a, 15a,

Vitamin Industrial Application World production

Vitamin B13 B (fermenta- M 100 21

(vitamins A, D 3 , E, K, Bh H, etc.), although microbiological methods exist or

combination of chemical and microbiological reactions (Sakaguchi et al., 1971;

Vol. 2. Plenum Press, New York, London.

Algal Biotechnology Laboratory, School of Biological and Environmental

Dunaliella is a unicellular, biflagellate, naked green alga (Chlorophyceae,

2 CHEMICAL AND PHYSICAL PROPERTIES

p-Carotene is accumulated as droplets in the chloroplast stroma of Dunaliella

high temperature, high salinity and deficiency of nitrogen source (Lerche,

3 STRAIN IMPROVEMENT, SELECTION AND MAINTENANCE

Fig. 2. Aerial photographs of Western Biotechnology's Dunaliella production facility at

4 BIOSYNTHESIS AND REGULATION

There is an inverse relationship between growth rate and carotenogensis in D.

Factor Biomass Jj-carotene

Increase in salinity +++

a Rate of decrease is very slow.

Open-pond D. salina production is perhaps more closely related to farming

HIG H SALINITY BRINE •

differently to particular wavelengths of light (Mil'ko, 1963), and high

6 PRODUCT RECOVERY AND PURIFICATION

Commercial D. salina production is a young industry, only 3 years old, and

tJ-Carotene is marketed mainly as an orange colouring, for foods such as

for use of {:1-carotene as a pigment supplement in the commercial feeds for

The first commercial synthesis of f3-carotene was established by Hoffman-La

9 FORMULAnONS AND APPLICA nONS

{:1-Carotene is a lipid- and oil-soluble product. As suspensions or solutions in

Moulton, T. P., Borowitzka, L. J. & Vincent, D. J. (1987a) The mass culture of

PRODUCTION OF CAROTENOIDS WITH FUNGI

into p-carotene. In Phycomyces these reactions are carried out by an enzyme