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Copyright by Charles Evans Melançon, III 2006 The Dissertation Committee for Charles Evans Melançon, III certifies that this is the approved version of the following dissertation: Investigation and Engineering of Macrolide Antibiotic Sugar Biosynthesis and Glycosylation Pathways of Actinomycetes Committee: Hung-wen Liu, Supervisor Walter Fast David E. Graham Stephen F. Martin Christian P. Whitman Investigation and Engineering of Macrolide Antibiotic Sugar Biosynthesis and Glycosylation Pathways of Actinomycetes by Charles Evans Melançon, III, B.S.; B.A. Dissertation Presented to the Faculty of the Graduate School of The University of Texas at Austin in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy The University of Texas at Austin December 2006 Dedication This dissertation is dedicated to my loving and supportive wife Dr. Lin Hong, who has been with me throughout this work, as a coworker, a friend, girlfriend, spouse, and as the mother of our daughter. I also dedicate this work to my beautiful daughter, Samantha Mei-wen Melançon, who was conceived, was born, and has lived her first few months while this dissertation was taking shape. Lastly, but certainly not least, this work is dedicated to my parents, Charles E. Melançon Jr. and Patricia S. Knight, who never stopped believing that I could achieve anything I put my mind to. Acknowledgements The time leading up to writing this dissertation and even more so, the time spent writing it, has been a time of intense reflection for me. Where have I come from? Where am I now? Where am I going? Who and what have been influential in my life during graduate school? My adolescent and early adult years were a time of rapid change for me, and a trying time for my parents. Looking back, I have my parents to thank primarily for the fact that I made it to graduate school in the first place. They never doubted my capabilities even when I doubted them, and always encouraged me to pursue my dreams. They provided me with a stable environment to grow up in, and encouraged my curiosity from a young age. I am eternally grateful to them for all they have done for me. Since my arrival in Austin in July, 2001, my life has changed immeasurably. Joining the Liu Research Group has been a large contributing factor to those changes. I feel like The Liu Group is to science what the Marines are to the military. The training is rigorous, the expectations are high, and the change is permanent. When I started graduate school, I was very enthusiastic about doing research. Dr. Liu’s dedication to his work has inspired me to maintain that enthusiasm. It was also in the Liu Group where I met my wonderful wife, Lin, who has changed my life for the better in innumerable ways. In the Liu Group, I have learned a great deal about natural product biosynthesis and genetic engineering and have gained an appreciation for mechanistic enzymology. The degree to which we are immersed in scientific research in the Liu Group, from the intense three-and-a-half hour literature reviews to the strong encouragement we regularly v receive from Dr. Liu to “namely, work hard, and be serious,” has created an environment conducive to my maximal growth and development as a scientist. Dr. Liu has, from the beginning, shown that he believed in me and has encouraged me along the way. He has also given me the latitude to develop my own ideas and to work independently. This freedom has been instrumental in my development as a scientist, and has also given me a tremendous sense of satisfaction in my work. Some of the most important things I’ve learned have been non-scientific. I have learned the importance of being persistent. I have learned that attention to detail that might be considered unnecessary and even inhibitory in other fields can be a tremendous asset in scientific research. I have identified my strengths and my weaknesses and have learned how to maximize the use of my strengths. I have learned how to be assertive. I have learned how to learn as well as how to teach. I have learned how to work effectively and harmoniously in a group setting. All of these skills will no doubt serve me well for the remainder of my life. Some of my most influential teachers have been the other students and postdocs in the Liu Group. I have learned something from each person with whom I have interacted in the group. I have Sveta to thank for most of my initial training. During the first two years in the lab, she graciously answered hundreds of questions I had, and was instrumental in helping me to develop my laboratory skills. Haruko also taught me a lot about natural product characterization, and gave me a crash course in NMR interpretation. Lin, in addition to being a great wife and mother to our child, gave me a lot of helpful instruction on protein purification, TDP-sugar preparation, and enzyme assay. Yasushi (the all-knowledgeable) has also been extremely helpful in teaching me everything from computer skills and bioinformatics to molecular biology and chemistry. vi But everyone in the group, both past and present, has played a role in helping me to get the most out of my graduate experience. I am forever grateful to everyone in the group. During my time in the Liu Group, many of my coworkers have become my friends. The bond shared among coworkers in the Liu Group is unique. We have spent thousands of hours together, shared meals, stories, embarrassing moments, joys, and disappointments. We have grown up together. We have learned each others’ languages. We have influenced each other in ways we may not even realize. Many of the memories we have shared will remain with me for the rest of my life. I hope both the personal and professional bonds we have forged here will remain after we go our separate ways, and that life will bring us together many more times. In the broadest sense, I am also grateful to Nature itself for the existence of fascinating biochemical systems worthy of detailed study. Without these beautifully intricate phenomena, I would not have nearly as interesting a job as I have now, and would need to find another passion. I feel privileged to live in this time in history when so many interesting questions at the chemistry/biology interface remain unanswered, yet many of the tools to answer them are readily available. I am also thankful to the many scientists who made the discoveries on which our work today is based. As I look to the future, I take comfort in the fact that there will always be new frontiers to explore, and feel confident that I can contribute in a meaningful way to the collective efforts to explore them. I wish everyone in the Liu Group much success and happiness in the future. vii Investigation and Engineering of Macrolide Antibiotic Sugar Biosynthesis and Glycosylation Pathways of Actinomycetes Publication No._____________ Charles Evans Melançon, III, Ph.D. The University of Texas at Austin, 2006 Supervisor: Hung-wen Liu Natural products are an important source of bioactive lead compounds used in drug development. The diverse sugar moieties found in natural product structures are often critical to their bioactivity. Therefore, advances in our understanding of natural product sugar biosynthesis and glycosyltransfer and in our ability to synthesize natural product derivatives through manipulation of the biosynthetic machinery are important and can impact the treatment of human diseases. The work described in this dissertation focuses on the functional elucidation of enzymes involved in biosynthesis and glycosyltransfer of the deoxysugar D-mycaminose, which is a structural component of the macrolide antibiotic tylosin, and on the use of genes encoding the biosynthesis and attachment of D-mycaminose, D-desosamine, and other deoxysugars for the engineered production of macrolide derivatives with altered sugar structures. First, the functions of TylM3 as the activator protein for the glycosyltransferase TylM2 in tylosin biosynthesis in Streptomyces fradiae, and the function of the homologous protein MydC as the activator protein for the glycosyltransferase MycB in viii mycinamicin biosynthesis in Micromonospora griseorubida were elucidated by expression of combinations of their encoding genes in engineered Streptomyces venezuelae hosts. These studies also showed that these glycosyltransferases have relaxed substrate specificity. During this work, a failed attempt to reconstitute the mycaminose biosynthetic pathway in an S. venezuelae mutant resulted in the discovery of a novel hexose 3,4-ketoisomerase, Tyl1a, which is involved in formation of TDP-D-mycaminose. Discovery of Tyl1a allowed reconstitution of the mycaminose pathway in S. venezuelae, and demonstration that Tyl1a alone could convert the desosamine pathway to a mycaminose biosynthesizing pathway. This work resulted in synthesis of several glycosylated macrolide derivatives. The enzymatic activity of purified recombinant Tyl1a was characterized in vitro by 1H NMR product analysis and steady state kinetics, and the substrate specificity of Tyl1a was found to be relaxed. Finally, three S. venezuelae mutants expressing hybrid deoxysugar biosynthetic pathways were constructed, one of which resulted in formation of non-natural deoxysugar-bearing macrolides. This work has provided important functional information on the sugar biosynthesis enzymes and glycosyltransferases studied, and has illustrated the feasibility of constructing complex engineered deoxysugar biosynthesis pathways in the macrolide producer S. venezuelae. ix Table of Contents List of Tables xv List of Figures xvi List of Abbreviations xxii Chapter 1. Background and Significance 1 1. Introduction 1 2. Deoxysugar Biosynthesis in Natural Product Pathways 7 3. Natural Product Glycosyltransferases 21 4. Natural Product Bioengineering using Glycosyltransferases and Sugar Biosynthesis Enzymes 32 5.

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