The Discovery and Study of Fluvirucin B1 Polyketide Synthase

DEDICATION To my Mother Su-Chu Huang, who had a near-death experience for giving birth of me, for supporting and taking care of me in every aspect of my life and make me a better person than I thought I could be. To my Father Fang-Yen Lin who taught me about Nature and science, cultivated my curiosity since I was a little kid. To my sister Tsu-Ying Lin for standing by my side all the time. And to my love and friends, who support me in my selfish pursuit of PhD. Without you none of this would have been possible. v ACKNOWLEDGMENTS I would like to thank my advisor, Nathan Schnarr for his knowledge, guidance, very strong support to this thesis and my career development. I would also like to thank my committee for their valuable insight and contributions, and the University of Massachusetts for supporting me and my work, and to all of my teachers. I would also like to thank my group members who volunteered their support on these projects. Especially to S. Lawrence Borketey and Gitanjali Prasad for all of the hard work and wisdom they contributed, and to Jon Amoroso and Adam Gann for all the intelligent conversation about organic chemistry. Rapidly decreasing numbers of viable therapeutic leads in the pharmaceutical pipeline demand new, sustainable methods for improved drug discovery and development. Despite vast improvements in de novo drug design and target recognition, Nature remains the richest source of small molecule therapeutics. Among many natural products, polyketides are not only the most promising ones for developing new antibiotic leads, but also exhibit unusually high therapeutic value ranging from clinical use as anticancer, antiviral, and immunosuppressant drugs. Modular polyketide synthases (PKSs) are dedicated nano-machinery that can be manipulated to produce a structurally diverse library for drug discovery programs. The ability to manipulate these natural systems to produce novel metabolites rests largely on increased mechanistic understanding of how these molecules are generated and how these processes can be manipulated. As impressive as their pharmaceutical properties are, the biosynthetic engineering potential of these compounds continues to draw widespread attention from the research community. Although some success has been realized in terms of polyketide structure diversification, severe limitations in engineered product output continue to impede efforts toward practical combinatorial biosynthesis. This thesis is focused on understanding and exploiting a new biosynthetic enzyme assembly and overcoming the engineering hurdles for making novel …