Project Summary/Abstract Multimodular polyketide synthases (PKSs) catalyze the biosynthesis of many structurally and pharmacologically diverse antibiotics via an assembly line mechanism. Recently, genomic sequencing has revealed large numbers ? far surpassing known PKS biosynthetic gene clusters (BGCs) ? of cryptic or ?orphan? PKS BGCs associated with no known polyketide products. Therefore, the analysis of orphan PKSs should reveal intriguing novel biosynthetic strategies and new structures with new therapeutic properties as well as reveal paths to pathogenicity in microorganisms. In particular, the NOCAP (NOCardiosis-Associated Polyketide) synthase is an orphan PKS expressed in nine clinical strains of the Nocardia isolated from nocardiosis-affected patients. The NOCAP synthase has been partially reconstituted in vitro. However, because the remainder of the BGC encoding NOCAP and its set of tailoring enzymes was not reconstituted, this approach precludes identification and elucidation of the natural product(s). This proposed research training plan seeks to understand the biosynthetic repertoire of the NOCAP synthase, and also seeks to understand its role in the pathogenicity of Nocardia. Accordingly, the plan is divided into three specific aims: 1) direct cloning, refactoring and heterologous expression of the complete NOCAP BGC in the model expression host Streptomyces coelicolor for the efficient production, discovery and characterization of this synthase?s natural product(s), 2) deletion analysis of the tailoring enzymes accompanying the NOCAP synthase and characterization of their biosynthesis and transfer of different deoxysugars onto the aglycone, and 3) using co-cultures of wild-type and mutant Nocardia and human lung cell lines, investigation of how the NOCAP BGC natural product(s) provides a selective advantage during Nocardia infections. The recent surge in genomic sequencing has exposed many orphan PKSs and may provide a vast resource for pharmaceutical and agrochemical natural products research. Thus, this proposed research training plan represents an important next step: analysis of orphan PKSs and their roles in organism fitness. The results of the proposed study will contribute important tools and strategies for discovering and rationally engineering not only novel polyketides but also products derived from NRPSs. Further, the proposed plan will elucidate how these remarkable enzymes contributes to the biological fitness of microorganisms.