Annual Reports on Fermentation Processes VOLUME 1 EDITED BY D. PERLMAN School of Pharmacy University of Wisconsin Madison, Wisconsin ASSOCIATE EDITOR GEORGE T. TSAO School of Chemical Engineering Purdue University West Lafayette, Indiana ACADEMIC PRESS New York San Francisco London 1977 A Subsidiary of Harcourt Brace Jovanovich, Publishers COPYRIGHT © 1977, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER. ACADEMIC PRESS, INC. Ill Fifth Avenue, New York, New York 10003 United Kingdom Edition published by ACADEMIC PRESS, INC. (LONDON) LTD. 24/28 Oval Road. London NW1 ISBN 0-12-040301-3 PRINTED IN THE UNITED STATES OF AMERICA List of Contributors Numbers in parentheses indicate the pages on which authors' contributions begin. BERNARD J. ABBOTT (205), Microbiological and Fermentation Products Research, Lilly Research Laboratories, Indianapolis, Indiana K. AUNSTRUP (181), Microbiological Research and Development, Novo Industri, DK-2880 Bagsvaerd, Denmark F. R. BERNATH (235), Department of Chemical and Biochemical Engineering, Rut- gers University, New Brunswick, New Jersey L. T. CHANG (1), Industrial Division, Bristol-Myers Company, Syracuse, New York M. CHARLES (365), Department of Chemical Engineering, Lehigh University, Bethlehem, Pennsylvania P. S. S. DAWSON (73), Prairie Regional Laboratory, National Research Council, Saskatoon, Saskatchewan, Canada D. D. DOBRY (95), The Upjohn Company, Kalamazoo, Michigan R. P. ELANDER (1 ), Industrial Division, Bristol-Myers Company, Syracuse, New York M. GORMAN (327), Lilly Research Laboratories, Indianapolis, Indiana F. HUBER (327), Lilly Research Laboratories, Indianapolis, Indiana J. L. JOST (95), The Upjohn Company, Kalamazoo, Michigan MASAKAZU KIKUCHI (41), School of Pharmacy, University of Wisconsin, Madison, Wisconsin (Present Address: Central Research Laboratories, Takeda Chemical Industries, Ltd., Osaka, Japan) K. KIESLICH (267), Department of Mikrobiologische Chemie, Schering AG, D-1000 Berlin, West Germany ALLEN I. LASKIN (151), Corporate Research Laboratories, Exxon Research and En- gineering Company, Linden, New Jersey YOUNG HIE LEE (115), School of Chemical Engineering, Purdue University, West Lafayette, Indiana JAROSLAV MAJER (347), Department of Biochemistry, Medical and Dental Schools, Northwestern University, Chicago, Illinois TAKASHI Ν ARA (299), Tokyo Research Laboratory, Kyowa Hakko Kogyo Co., Ltd., Tokyo, Japan L. K. NYIRI (365), Department of Chemical Engineering, Lehigh University, Bethlehem, Pennsylvania D. PERLMAN (41), School of Pharmacy, University of Wisconsin, Madison, Wiscon- sin v/7 viii LIST OF CONTRIBUTORS COLIN RATLEDGE (49), Department of Biochemistry, The University of Hull, Hull, England O. K. SEBEK (267), The Upjohn Company, Kalamazoo, Michigan GEORGE T. TSAO (115), School of Chemical Engineering, Purdue University, West Lafayette, Indiana R. W. VAUGHN (1), Industrial Division, Bristol-Myers Company, Syracuse, New York K. VEN KATASU Β RAMAN I AN (235), Department of Chemical and Biochemical En- gineering, Rutgers University, New Brunswick, New Jersey W. R. VIETH (235), Department of Chemical and Biochemical Engineering, Rutgers University, New Brunswick, New Jersey Preface ANNUAL REPORTS ON FERMENTATION PROCESSES is designed to furnish readers with a critical account of significant developments published during the past two to three years concerning fermentation processes. Only published material is included, and the main value of the volumes in this series is to assist the reader to keep abreast of developments in areas of fermentation research and developments where he has only peripheral or limited interest. The contributors of chapters to this volume were asked to answer the question "What are the major developments in the field published recently?" and have done so very admirably. Many persons are involved in decisions in launching a new series and we are indebted to them for assisting in this process. The officers past and present of the Division of Microbial and Biochemical Technology of the American Chemical Soci- ety have been very helpful in getting this project started, as has the publisher, Academic Press. We hope that the first volume will meet readers' needs and we will appreciate suggestions on modifications for future volumes. May 20, 1977 D. Perlman School of Pharmacy University of Wisconsin Madison, Wisconsin Introduction The Division of Microbial and Biochemical Technology is extremely pleased to sponsor the publication of "Annual Reports of Fermentation Processes" under the capable editorship of Professor David Perlman. For the first time, a broad cross section of subjects of importance and interest to our members will be reviewed by experts in the field on a regular basis. With this first volume printed in 1977, the Divisional Executive Committee wishes all the success in this new and hopefully lasting activity. We would also thank Academic Press, Inc., for its support and cooperation. George T. Tsao, Chairman Division of Microbial and Biochemical Technology American Chemical Society xi CHAPTER 1 GENETICS OF INDUSTRIAL MICROORGANISMS R. P. BLANDER, L. T. CHANG, AND R. W. VAUGHAN Bristol-Myers Company Industrial Division Syracuse, New York Although basic studies on the genetics and molecular biology of microorganisms have dramatically expanded our understanding of microbial regulation of primary metabolism in a few well studied organisms, relatively little attention has been focused on the molecular and genetic aspects of industrially important microorgan- isms. During the past two decades, basic genetics and the applied genetics of industrial organisms have gone in somewhat diverging directions. Although mutation and selection programs were highly successful, basic new genetic methodology was not easily adopted in industry for a variety of reasons including a lack of apprecia- tion for industrial problems by academic geneticists, a lack of basic understanding of the genetic and recombination mechanisms in industrial strains, and a lack of competent geneticists in the fermentation industry. In order to help bridge this gap, a series of important genetic conferences—Genetics of Industrial Micro- organisms (GIM)—were held in Prague (1970), Sheffield (1974), Orlando (1976) and Madison (1978). These conferences were orga- nized to review recent approaches to tailor microorganisms for practical uses, to expand and include industrially important mi- croorganisms for which genetic studies have been conducted, and to review recent applications of molecular biology and genetics in strain improvement. The proceedings of these highly successful conferences are now published and offer a comprehensive review of topics related to microbial genetics (1-3) Other important genetic topics are covered in other publications and reviews (4,5) % 1 2 R. P. ELANDER ET AL. I. MUTATION, SELECTION AND OPTIMIZATION OF MUTAGENESIS Mutation in microorganisms is thought to arise through two major classes of mutagenic mechanisms: errors directly induced through base mispairing or errors introduced by the various repair mechanisms. Direct induction of mutations can result from the alkylation of guanine and thymine by agents such as ethyl methane- sulphonate (6-9). However, the majority of mutations are deter- mined largely by excision, postreplication, and error-prone repair systems acting on single-strand gaps formed directly or indirectly in DNA. The excision repair process excises a lesion in one strand of the DNA duplex and the resulting gap is filled by a DNA polymerase enzyme using the undamaged strand as a template. Strains that are difficient in excision repair are more apt to give rise to muta- tions than excision repair-proficient strains. A more practical, but less efficient procedure for the industrial microbiologist is to use compounds such as caffeine (11-12) and acriflavine (13,14) which inhibit the excision repair process as part of the mutagen regimen. Induction of mutations by the postreplication repair process involves DNA replication and recombination. Gaps (^10^ bases) are formed in daughter strands opposite induced lesions in the parental strands (9,15,16). The majority of gaps are filled by a recombination process (17) between the two daughter chromo- somes C18). The process is complex since photo-induced dimers are not inherited lineally but are randomly distributed among the pro- geny strands (19). An error-prone repair system, which probably does not involve recombination, may operate as an alternative path- way simultaneously with the excision and postreplication repair processes. The postulated mechanism is incorrect insertion of bases into gaps in the progeny strand DNA but the process is not fully understood (9,20-24). Bridges (25) reported the enzyme DNA polymerase III is necessary for error-prone repair but is not re- quired for postreplication or excision repair. Mutations induced through the above mechanisms are generally base-pair substitutions. Frameshift mutations result from the addition or removal of base pairs in multiples not equal to three which causes the translation process to lose the proper frame of reference (9,26). A converse of mutation induction, the detection of mutagenic compounds, has recently emerged as an area of importance to the industrial microbiologist. Government regulations are requiring increased testing of industrially important compounds for mutagenic and carcinogenic activity. Ames et al. (27-30) have developed sensitive bacterial testor strains which detect both base substi- tution and frameshift mutagens. A few of the common mutagens are cited here and the reader is referred to Fishbein et al. (31) for a more comprehensive treatment. Ultraviolet light, ionizing radi- ations (9,24,32-34) and heat (9) are probably the safest agents to use but alkylating agents (33-38), 4-nitroquinoline-l-oxide (39,40) and sporalens (41,42) in the presence of 360 nm light are sometimes preferred. 1 GENETICS OF INDUSTRIAL MICROORGANISMS 3 Optimization of mutation induction involves the interaction of pH. (35,36) concentration (37) period of treatment, phase of cellular growth, and the interactions are usually organism-depen- dent. Incorporation or elimination of DNA precursor bases can also affect mutation rates (43,44), Addition of compounds which inhibit the excision repair mechanism increase the effectiveness of ultra- violet light (11,12,14). Analysis of the interactions requires histograms or biométrie considerations (45) when random isolation of mutants are used. A simpler and more precise analysis can be obtained with dose-effect plots (48) when specific selection meth- ods are employed (e.g. mutation to resistance or reversion of auxo- trophs to prototrophs). However, the optimum procedure determined for resistance or reversion mutants does not always coincide with the optimum procedure required for increased product formation of compounds of interest to the industrial microbiologist. Selective isolation of biochemical mutants from a mutagenized population is perhaps the most difficult task in a mutant isolation process. Methods are now available which offer distinct advantages over the total isolation process. Replica plating (46) works well with bacteria and fungi which produce spores or fragmented hyphae. Filamentous organisms can be successfully manipulated by the addi- tion of compounds to agar which induce microconidia, spores, frag- mented cells or restrict colony size. The addition of 0.08% sodium deoxycholate to agar induces Aspergillus nidulans to produce re- stricted colonies (34) while sorbose restricts the colonial growth of Neurospora (47). Dense velveteen pads of closely set steel needles or filter paper discs can be used as successful transfer vehicles. Layering techniques (48) are useful with yeasts. An appropriately diluted population of mutagenized cells is plated on minimal agar. Prototroph cells give rise to small colonies after a period of incubation and a layer of complete medium is then poured over the original medium. Auxotrophe begin to grow and can be differentiated by their size difference or the prototrophs can be marked before addition of the overlay medium. Resistance mutants can be isolated by plating large concen- trations of mutagenized populations on solid media containing a toxic substance. Isolation of prototrophic reversions from auxo- trophs requires plating only on a minimal medium. Enrichment techniques based on selective elimination of growing cells before plating on isolation media increase the probability of isolating biochemical mutants. The procedure can be made specific if com- pounds are added to the media which allow all but the desired mu- tants to grow. Filtration procedures, first reported by Fries (49) are primarily applied to filamentous organisms (50-52). Penicillin enrichment, originally developed for bacteria (53) is being widely used with modifications (54-58). The principle of penicillin enrichment can be extended to the isolation of auxo- trophe for many organisms by the use of compounds which have se- lective toxicity. Sodium pentachlorophenate is harmless to spores of Pénicillium chrysogenum, Streptomyces aureofaciens, S. olivaceus and Bacillus subtilis but is lethal to their germinated spores. 4 R. P. ELANDER ET AL. (59,60) Nystatin is effective with P. chrysogenum (61,62) yeasts (63) and Cephalosporium acremonium; 2-deoxyglucose with Schizo- saccharomyces pombe (64) and netropsin with Saccharomyces cere- visiae (65). Certain mutations confer reduced viability to organ- isms when starved in a minimal medium and induction of a second mutation restores survival. This technique has been used to ad- vantage, for example, with inositol (66) and thymine (67,68) mu- tants for the isolation of additional auxotrophic markers. There are instances where specific compounds select specific mutations. Trimethoprim selects for thymine-requiring mutants (69,70) in bac- teria. The specific action of 4-nitropyridine-l-oxide effectively selects proline-requiring mutants in E. coli (71). A chemostat offers great promise if selective pressure can be applied which gives even a minimal growth advantage to the desired mutant. Monitoring the steady-state population metabolite concen- trations in the effluent permits continuous control of the environ- ment and development of a mutant population. Mutants of Ε. coli constitutive for 3-galactosidase (72) P. putida constitutive for mandelate enzymes (73) K. aerogenes with altered xylitol to ribi- tol activity ratio of ribitol dehydrogenase (74) and 5. cerevisiae with an acid phosphatase with an altered pH optimum (75) have been successfully isolated from a chemostat culture. The method has application to more than selection of cultures which overproduce enzymes or have altered enzyme specificities. Mutants resistant to metabolic analogues can be isolated which are derepressed, resistant to feedback inhibition, or altered in regulation of branched or secondary metabolic pathways. More detail can be found elsewhere in this chapter or in review arti- cles (76-78). II. MUTATION AND IMPROVED PRODUCT YIELD IN ANTIBIOTIC-PRODUCING MICROORGANISMS Mutation and selection to increased product formation is pro- bably the most important factor in improving the yield of an anti- biotic C79). Mutation programs continue to be vital to the fer- mentation industry in that mutation to higher productivity in per- iods of increasing labor and raw material costs is the most impor- tant factor in maintaining the industry in an economically healthy state. Intensive strain improvement and concurrent genetic pro- grams are being expanded in both industrial and applied laborator- ies throughout the world. A. Mutagenic Treatment, Morphological and Biochemical Variants, and Antibiotic Productivity The most effective mutagen for improving tetracycline produc- tivity in strains of Streptomyces aureofaciens is UV radiation. Of the mutagens tested, N-methyl-N'-nitro-N-nitrosoguanidine (NG)