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Genetic and Biochemical Studies of Biosynthesis Biosynthesis and Regulation of Production of the Antibiotic Myxovirescin A in Myxococcus xanthus DK1622 Dissertation zur Erlangung des Grades des Doktors der Naturwissenschaften der Naturwissenschaftlich-Technischen Fakultät III (Chemie, Pharmazie, Bio- und Werkstoffwissenschaften) der Universität des Saarlandes von Vesna Simunovic Saarbrücken, Germany, Mai 2007 © 2007 Vesna Simunovic All Rights Reserved Tag des Kolloquiums: den 27 June 2007 Dekan: Uni Müller Berichterstatter: Prof. Dr. Rolf Müller Prof. Dr. Manfred Schmitt List of publications featured in this dissertation: Simunovic V., Zapp J., Rashid S., Krug D., Meiser, P., Müller R. Myxovirescin A Biosynthesis is Directed by Hybrid Polyketide Synthases/Nonribosomal Peptide Synthetase, 3-Hydroxy-3-Methylglutaryl-CoA Synthases, and trans-Acting Acyltransferases. ChemBioChem. 2006 Aug; 7(8):1206-20. DOI: 10.1002/cbic.200600075 Simunovic V., Müller R. 3-Hydroxy-3-Methylglutaryl-CoA-Like Synthases Direct the Formation of Methyl and Ethyl Side Groups in the Biosynthesis of the Antibiotic Myxovirescin A. ChemBioChem. 2007 Mar; 8(5):497-500. DOI: 10.1002/cbic.200700017 Simunovic V., Müller R. Mutational Analysis of the Myxovirescin Biosynthetic Gene Cluster Reveals Novel Insights into the Functional Elaboration of Polyketide Backbones. ChemBioChem. 2007 July, 8, DOI: 10.1002/cbic.200700153 Meetings The 32nd International Conference on the Biology of the Myxobacteria Harrison Hot Springs, British Columbia, Canada, July 10-13, 2005. Oral presentation. Myxovirescin Biosynthesis: An intriguing megasynthetase consisting of polyketide synthases, nonribosomal peptide synthetase, 3-hydroxy-3-methylglutaryl-CoA synthases and trans-acting acyltranferases. Page 42. iv Abstract Myxobacteria produce a variety of secondary metabolites displaying important biological activities. Recent sequencing of the Myxococcus xanthus DK1622 genome revealed its high potential for the production of secondary metabolites and led to the identification of the myxovirescin biosynthetic gene cluster. In silico analysis of myxovirescin megasynthetase resulted in the proposal that a number of discrete enzymes work together with the multimodular PKS to build myxovirescin scaffold, unique for the presence of two different β- alkyl groups. To test the myxovirescin biosynthetic model, fourteen in-frame deletion mutants in the myxovirescin biosynthetic gene cluster were created, and their effects on the production of myxovirescin antibiotics evaluated by HPLC-MS analysis of the resulting mutant extracts. Novel myxovirescin analogues arising from certain mutant backgrounds were structurally elucidated to identify the specific positions of these modifications. In silico analysis of an additional 11 kb region encoded upstream from the myxovirescin gene clusters were proposed to be involved in the regulation of its production. Genetic disruption of a gene encoding for a serine/threonine kinase, and two additional genes encoding for proteins of unknown functions, were shown to positively regulate myxovirescin production. v Zusammenfassung Myxobakterien haben sich in den letzten drei Jahrzehnten als vielseitige Produzenten unterschiedlichster Sekundärmetaboliten (SM) mit zum Teil starker biologischer Wirkung erwiesen. Unter diesen Bakterien sind diverse Multiproduzenten bekannt, zu denen auch das Bakterium Myxococcus xanthus DK1622 zählt. Die erst vor kurzem abgeschlossene Sequenzierung des Gesamtgenoms von Myxococcus xanthus DK1622 zeigt das enorme Potential für die Produktion verschiedenster SMs. Auf diesem Weg konnte ebenfalls das Myxoverescin-Biosynthesegencluster identifiziert werden. Die annotierte Genomsequenz lieferte erste Möglichkeiten für eine in silico Analyse der Myxovirescin Megasynthase und führte zum Postulat eines möglichen Biosynthesewegs. In diesem bildet eine multimodulare PKS das Myxovirescin-Grundgerüst, welches nachträglich durch verschiedene separate Enzyme modifiziert wird. Diese Enzyme katalysieren den Einbau zweier ungewöhnlicher β-Alkylgruppen. Um die Beteiligung des identifizierten Genclusters an der Myxovirescin- Biosynthese zu beweisen, wurden vierzehn "in-frame" Deletionsmutanten erzeugt. Die Auswirkung der jeweiligen Mutation auf die Produktion des Antibiotikums wurde mittels HPLC/MS Analyse der erhaltenen Kulturextrakte untersucht. Um in den neuen Myxovirescin- Derivaten die spezifische Veränderung innerhalb des Moleküls zu identifizieren, wurde deren Struktur aufgeklärt. Stromaufwärts des Biosynthesegenclusters konnte eine ca. 11 kb große genomische Region identifiziert werden, in der Gene kodiert sind, die möglicherweise regulatorische Auswirkungen auf die Myxovirescin-Produktion haben. Durch Geninaktivierungen, sowohl eines Serin/Threonin Kinase kodierenden Gens, als auch zweier Gene mit unbekannter Funktion, konnte eindeutig gezeigt werden, dass die jeweiligen Enzyme an der Produktionsregulation beteiligt sind. vi Acknowledgments I would like to thank my mentor, Prof. Dr. Rolf Müller, for his guidance during my PhD work, as well as numerous comments and critical readings in preparation of my manuscripts and this dissertation. I would further like to thank Prof. Dr. Manfred Schmitt for reading this work and being in my doctoral Committee. I would also like to acknowledge Dr. Axel Sandman for translation of the introductory and concluding parts of this work. Drs. Helge Bode and Silke Wenzel are thanked for the critical reading of my papers. Special thanks to Dr. Kira Weissman for significant help in the preparation of the third paper and for editing parts of my dissertation. A big "thank you" goes to Dr. Josef Zapp for teaching me all the secrets of nuclear magnetic resonance (NMR) spectroscopy and natural product structure elucidation. I would also like to acknowledge Prof. Dr. Giffhorn for allowing me the use of fermentors and Drs. Gert-Wieland Kohring and Christian Zimmer for their outstanding assistance with the fermentors. Daniel Krug is thanked for performing the high resolution mass spectrometry (HRMS) measurements and help with the quantitative MS measurements. Michael Ring is thanked for the fabulous help in the domain of computers. It has also been an exceptional pleasure to work with Nora Luniak, Dr. Shwan Rachid, Irene Kochems and Brigitte Lelarge- thank you for your kindness and expertise. I would also acknowledge the past and present members of Prof. Müller’s laboratory. Great thanks go to my close friends and family for their emotional support in the past years. Finally, this work would not have been possible without the financial support provided by the Deutsche Forschungsgemeinschaft (DFG) and the Bundesministerium für Bildung und Forschung (BMBF). vii Table of Contents Page List of Publications and meetings iv Abstract (English version) v Abstract (German version) vi Acknowledgements vii Table of Contents viii Chapter 1 Introduction 1 2 Myxovirescin A Biosynthesis is Directed by Hybrid Polyketide Synthases/Nonribosomal Peptide Synthetase, 3-Hydroxy-3- Methylglutaryl–CoA Synthases and trans-Acting Acyltranferases 28 3 3-Hydroxy-3-Methylglutaryl-CoA-like Synthases Direct the Formation of Methyl and Ethyl Side Groups in the Biosynthesis of the Antibiotic Myxovirescin A 70 4 Mutational Analysis of the Myxovirescin Biosynthetic Gene Cluster Reveals Novel Insights into the Functional Elaboration of Polyketide Backbones 85 5 Regulation of myxovirescin production in M. xanthus DK1622 109 6 Discussion 118 Summary (English version) 138 Summary (German version) 140 References 142 Curriculum vitae 148 viii Chapter 1 Introduction Introduction Natural products-importance, applications, and impacts The serendipitous discovery of penicillin-producing fungus Penicillium notatum by Alexander Fleming, coupled to the later success of Florey and Chain in the development of the large scale fermentative process for the production of penicillin, has marked the beginnings of the modern era in natural product research.[1] This discovery has triggered the golden fever in the discovery of new antibiotics (1940-1960) that were largely based on screenings of soil samples for the presence of microorganisms capable to produce biologically active compounds. Attempts to cultivate these microorganisms, optimize the production of these agents, elucidate their structures, and discover ways of their biosyntheses has made the field of natural product research evolve into an interdisciplinary science which combines multiple aspects of microbiology, molecular genetics, chemistry, biochemistry and recently genomics. In addition to the development of numerous classes of antibiotics in human clinical use, such as penicillins (β-lactams), streptomycin (aminoglycosides), erythromycin (polyketide macrolactones) and vancomycin (glycopeptides), the major impact of natural product research on the history of medicine have had discoveries of immunosuppressive drugs cyclosporine and FK-506 (Figure 1). The discovery, followed by the later success in the proper administration of these drugs has overcome the major limitation in the field of organ transplantation-rejection of the newly acquired organs by the host immune system.[2] Effects of the available antibiotic therapies are evident in the greatly decreased death rates caused by infectious diseases ranging from 797 cases per 100 000 in 1900 to 36 cases per 100 000 in 1980, as reported in the United States.[3] However, increased longevity of humans has caused an inevitable increase in the incidence of cancers. Consequently, development of new and effective agents
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