THE DISCOVERY AND STUDY OF FLUVIRUCIN B1 POLYKETIDE SYNTHASE

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 polyketide metabolites. Fluvirucin B1, produced by Actinomadura vulgaris, is a 14-membered macrolactam active against a variety of infectious fungi as well as influenza A. Despite considerable interest from the synthetic community, very little information is available regarding the biosynthetic origins of the fluvirucins. Herein, we report the identification and initial characterization of the fluvirucin B1 polyketide synthase and related enzymes. The cluster consists of five extender modules flanked by an N-terminal acyl carrier protein and C-terminal thioesterases domain. All but one of the synthase modules contain the full complement of tailoring domains (ketoreductase, dehydratase, and enoyl reductase) as determined by sequence homology with known polyketide synthases. Active site analyses of several key components of the cluster are performed to further verify that this gene cluster is associated with production of fluvirucin B1. This work will both open doors toward a better understanding of macrolactam formation and provide an avenue to genetics based diversification of fluvirucin structure