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Studies on the Biosynthesis and Isolation of the Biosynthetic Gene Cluster for the Salinosporamides of the Marine Bacterium Salinospora Tropica

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Studies on the Biosynthesis and Isolation of the Biosynthetic Gene Cluster for the Salinosporamides of the Marine Bacterium Salinospora Tropica Studies on the Biosynthesis and Isolation of the Biosynthetic Gene Cluster for the Salinosporamides of the Marine Bacterium Salinospora tropica Item Type text; Electronic Dissertation Authors Beer, Laura Lynn Publisher The University of Arizona. Rights Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. Download date 02/10/2021 21:14:20 Link to Item http://hdl.handle.net/10150/194242 STUDIES ON THE BIOSYNTHESIS AND ISOLATION OF THE BIOSYNTHETIC GENE CLUSTER FOR THE SALINOSPORAMIDES OF THE MARINE BACTERIUM SALINISPORA TROPICA by Laura Lynn Beer ___________________________ Copyright © Laura Lynn Beer 2006 A Dissertation Submitted to the Faculty of the DEPARTMENT OF PHARMACOLOGY AND TOXICOLOGY In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY In the Graduate College THE UNIVERSITY OF ARIZONA 2006 2 THE UNIVERSITY OF ARIZONA GRADUATE COLLEGE As members of the Dissertation Committee, we certify that we have read the dissertation prepared by Laura Lynn Beer entitled Studies on the Biosynthesis and Isolation of the Biosynthetic Gene Cluster for the Salinosporamides of the Marine Bacterium Salinispora tropica and recommend that it be accepted as fulfilling the dissertation requirement for the Degree of Doctor of Philosophy ________________________________________________Date: June 27, 2006 Bradley S. Moore ________________________________________________Date: June 27, 2006 Laurence Hurley ________________________________________________Date: June 27, 2006 Elaine Jacobson ________________________________________________Date: June 27, 2006 Matthew Cordes Final approval and acceptance of this dissertation is contingent upon the candidate’s submission of the final copies of the dissertation to the Graduate College. I hereby certify that I have read this dissertation prepared under my direction and recommend that it be accepted as fulfilling the dissertation requirement. ________________________________________________ Date: June 27, 2006 Dissertation Director: Bradley S. Moore 3 STATEMENT BY THE AUTHOR This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at The University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the library. Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgement of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the copyright holder. SIGNED: Laura Lynn Beer 4 FUNDING ACKNOWLEDGEMENTS This research was supported in part by a Washington Sea Grant Industry fellowship (R/B-47) in partnership with Nereus Pharmaceuticals, La Jolla, CA. ACKNOWLEDGEMENTS I would like to acknowledge all of the employees of the College of Pharmacy with whom I have interacted over the past years and who have been very helpful and supportive. I would like to thank all members of my graduate committee for their critical review of this dissertation and guidance throughout my graduate studies and especially Dr. Moore for providing the opportunity to work on this fascinating project. A special thanks to Professor Bill Fenical and Dr. Paul Jensen at SIO, UCSD for providing the Salinispora tropica strains, protocols for growth and isolation of salinosporamide A and for standards of purified salinosporamide A. Thanks to Dr. John Kalaitzis for assistance with NMR analysis at the University of Arizona and Ray Lam, Barbara Potts and Kathy Reed at Nereus Pharmaceutical Company for assistance with the propionate feeding experiments and for providing protocols for purification of salinosporamides A and B and standards. 5 TABLE OF CONTENTS LIST OF FIGURES ..................................................................................................9 LIST OF TABLES ...................................................................................................14 LIST OF ABBREVIATIONS .................................................................................15 ABSTRACT..............................................................................................................17 CHAPTER 1: INTRODUCTION..........................................................................19 An Overview of the History of Natural Products as a Source of Medicine ...........................................................................................................19 Microbes as a Source for Therapeutics .............................................................20 The Rise of Marine Natural Products as Potential Clinical Agents..................23 Marine Natural Products as Anticancer Agents................................................26 Marine Actonomycetes are the Premier Source for Novel Bioactive Molecules from the Sea......................................................................................27 The Marine Actinomycete Salinispora tropica ..................................................28 The Structure and Activity of Salinosporamide A..............................................29 The 20S Proteasome: Structure and Function...................................................32 The Development of Proteasome Inhibitors ......................................................34 Proteasome Inhibitors as Antitumor Agents......................................................36 Salinosporamide A Activity against the 20S Proteasome..................................37 NRPS and PKS Derived Molecules and their Biosynthesis...............................40 β-lactones as Therapeutics and their Biosynthesis............................................41 β-lactam Biosynthesis ........................................................................................43 6 TABLE OF CONTENTS - continued CHAPTER 2: CHARACTERIZATION OF THE BIOSYNTHESIS OF SALINOSPORAMIDES USING 13C-LABELED PRECURSORS.....................45 Introduction and Background ............................................................................45 Proposed Biosynthesis of Salinosporamide A....................................................45 Proposed Biosynthesis of the C-2 Alkyl Group..................................................48 Proposed Biosynthesis of the Amino Acid Moiety .............................................50 Materials and Methods........................................................................................53 Chemicals...........................................................................................................53 Strains ................................................................................................................53 Chromatography................................................................................................54 Nuclear Magnetic Resonance (NMR) ................................................................54 Salinispora tropica Fermentation and Isolation of the Salinosporamides...............................................................................................55 Preparation of Crude Extracts...........................................................................56 Monitoring the Production of Salinosporamides by Analytical HPLC .............56 Purification of Salinosporamides ......................................................................57 Feeding Experiments with 13C-Labeled Precursors ..........................................58 13 Preparation of [U- C10]Chorismic Acid...........................................................59 Optimization of Salinosporamide A Yields and Purification Techniques .........................................................................................................60 Feeding Experiments and NMR Analysis ..........................................................63 7 TABLE OF CONTENTS - continued Results ...................................................................................................................63 Acetate Feeding Experiment..............................................................................64 Feeding Experiment with the General Precursor Glucose................................66 Glucose Feeding Experiment Results: the Alkyl Carbons .................................66 Glucose Feeding Experiment Results: the CHHA Moiety .................................68 The CHHA Moiety: Glycerol Feeding Experiments ..........................................70 Phenylalanine and Shikimate Feeding Experiments..........................................72 Chorismate Feeding Experiment .......................................................................74 Origin of the 4-Carbon Alkyl Moiety.................................................................74 Biosynthesis of Salinosporamide Analgos .........................................................77 Discussion of Results and Conclusions...............................................................80 CHAPTER 3: CLONING AND SEQUENCING THE SALINOSPORAMIDE BIOSYNTHETIC GENE CLUSTER............................83 Introduction and Background ............................................................................83 The Genetic Organization of PKSs and NRPSs.................................................83 Tailoring Enzymes in NRPS and PKS Biosynthesis...........................................87
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