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Each original is also photographed in one exposure and is included in reduced form at the back of the book. Photographs included in the original manuscript have been reproduced xerographically in this copy. Hgher quality 6” x 9” black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. UMI A Bell & Howell Infonnation Company 300 North Zed) Road, Ann Arbor MI 48106-1346 USA 313/761-4700 800/521-0600 ANALYSIS OF THE POLYKETIDE SYNTHASE GENES OF THE DAUNORUBICIN PRODUCER, Streptomyces sp. strain C5: GENERATION OF PKS MUTANTS, AND ANALYSIS OF THE UNUSUAL ANTHRACYCLINE PRODUCTS MADE BY THESE PKS MUTANTS DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Vineet B. Rajgarhia, B.S. ***** The Ohio State University 1998 Dissertation Committee: Dr. Tyrrell Conway, Adviser Approved by Dr. William Strohl Dr. Tina Henkin Adviser y Dr. Joe Krvzcki Department of Microbiology UMI Number: 9834051 UMI Microform 9834051 Copyright 1998, by UMI Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. UMI 300 North Zeeb Road Ann Arbor, MI 48103 ABSTRACT Streptomyces sp. strain C5 and Streptomyces peucetius produce the anticancer antibiotics daunorubicin and doxorubicin, which are effectively used for the treatment of several types of cancer. The production of daunorubicin and doxorubicin begins with the sequential decarboxylative condensation of a propionyi-CoA starter moiety with nine malonyl-CoA extender moieties by the anthracycline specific Type II polyketide synthase (PKS) gene products. The PKS gene cluster of Streptomyces sp. strain C5 has two unique open reading frames. dpsC and dpsD, encoding a fatty acid KASHI homologue lacking an active site and an acyltransferase of unproven function, respectively. These two genes are directly downstream of the genes dpsA (ketoacylsynthase a), and dpsB (ketoacylsynthase P), a position normally occupied by the gene encoding the acyl carrier protein. This unique arrangement is speculated to be responsible for Streptomyces sp. strain C5 and Streptomyces peucetius utilizing propionate as the starter unit instead of an acetate, which is commonly used by most other Type II polyketide synthases. Several deletion mutants of Streptomyces sp. strain C5 were generated in which either dpsD alone, dpsCD, or dpsABCD were replaced with a neomycin resistance gene cassette. The dpsCD mutant strain produced daunorubicin, but significantly enough, also produced unique anthracyclines known as feudomycins. These unique anthracyclines are derived from the PKS catalyzed decarboxylative condensation of an acetyl-CoA starter moiety with nine malonyl-CoA extender units. Complementing the mutant strain with dpsC restored normal (propionate initiated) antfiracycline production, while complementation with dpsD failed to restore normal starter unit selection. Similarly, a combination of the PKS genes from Streptomyces sp. strain C5, excluding the dpsC gene, expressed in S. lividans TK24. produced a mixture of both propionate-derived polyketide intermediate, aklanonic acid, as well as an acetate-derived polyketide intermediate, desmethylaklanonic acid. The production of these early intermediates of the daunorubicin and feudomycin pathways, respectively, confirmed our earlier findings. These results suggest that the dpsC gene product is responsible for maintaining natural starter unit selection in Streptomyces sp. strain C5. In the absence of which, the PKS in the mutant strain can select both acetyl-CoA as well as propionyl-CoA to prime anthracycline production. Ill Dedicated to my family for eternal love, support, and understanding. IV ACKNOWLEDGMENTS I am grateful to my adviser. Dr. William Strohl, for encouragement, and perpetual intellectual support, which made this dissertation possible. 1 also thank Drs. Tyrrell Conway, Tina Henkin and Joe Kryzcki for their intuitive ideas, and for their time and efforts spent on guiding my research. 1 am indebted to Nigel Priestley for his unceasing support in my research efforts. 1 wish to thank Don Ordaz for his help with fermentations and with the slides 1 used for several of my seminar presentations. 1 also wish to thank the many friends I made in Baltimore, Maryland and Columbus, Ohio for the wonderful experiences and everlasting moments 1 shared with them. I am grateful to my family members whose constant support and love helped achieve all. 1 thank the past members of the Strohl Lab, Drs. Richard Plater, Yun Li, Gary Kleman, and Mike Dickens who passed on their experiences as well as words of wisdom. 1 am beholden to the current members of the laboratory Anton Woo and Robbie Walczak for their kinship, their friendship, and their support specially during the final year of my graduate school career. 1 am eternally indebted to Balakrishna Shetty and Jignesh Patel for their kinship and their support, which made this process so much smoother. VITA January 4. 1968 ............................................. Bom - Calcutta, India 1990............................................................... B.S. Pharmacy, Bombay University. 1990 - 1991....................................................Graduate Teaching Associate. The University of Maryland at Baltimore 1991 - present................................................ Graduate Teaching and Research Associate. The Ohio State University PUBLICATIONS Research Publications 1. Rajgarhia, V., Priestley, N. D.. and W. R. Strohl. 1995. Efficient synthesis of radiolabeled propionyl-CoA. Anal. Biochem. 224: 159-162. 2. Rajgarhia, V., and W. R. Strohl. 1997. Minimal Polyketide synthase genes of Streptomyces sp. strain C5. J. Bacteriol. 179:2690-2696. 3. Strohl. W. R.. Dickens, M. L.. Rajgarhia, V.. Woo. A., and N. Priestley. Anthracyclines. In: Strohl WR, ed. Biotechnology of Industrial Antibiotics. 2nd ed. New York: Marcel Dekker, Inc, 1997:577 - 657. 4. Strohl, W. R., Dickens, M. L., Rajgarhia. V., Walczak, R., Woo, A., and N. D. Priestley. 1998. Biochemistry, molecular biology and protein - protein interactions in doxorubicin biosynthesis: Proceedings of the Biotechnology of microbial products (BMP 97). C. R. Hutchinson and J. McAlpine. eds. Dev. Indust. Microbiol. 35:15-22. FIELDS OF STUDY Major Field: Microbiology VI TABLE OF CONTENTS Page Abstract....................................................................................................................... ii Dedication ................................................................................................................... iv Acknowledgments ...................................................................................................... v Vita............................................................................................................................... vi Table of Contents ......................................................................................................... vii List of Tables ............................................................................................................... xiv List of Figures .............................................................................................................. xvi Chapters: I. Introduction ........................................................................................................... 1 A. Anthracyclines and the discovery of daunorubicin and doxorubicin ........................................................................................ 4 1. Daunorubicin and doxorubicin producing Streptomyces spp 6 2. Mechanism of action of daunorubicin and doxorubicin ............ 9 3. Quantitative structure/activity relationships .............................. 10 4. Toxicity associated with doxorubicin and daunorubicin ........... 12 5. Uses of daunorubicin and doxorubicin ....................................... 14 VII B. Biosynthetic pathway for daunorubicin and doxorubicin production 15 1. Condensation of acetates and propionates to produce aklanonic acid............................................................................................... 16 a. Architecture of the Streptomyces sp. strain C5 polyketide synthase genes .............................................................................. 22 b. Minimal PKS/role of the dpsC and dpsD gene products.. 26 2. Early pathway gene products that convert aklanonic acid to e- rhodomycinone