Proposal Summary The megasynthases that mediate construction of a vast array of natural products represent some of the most complex molecular machines in Nature. In the Sherman group, polyketide synthases (PKSs) are of interest from a multi-disciplinary perspective. PKSs are responsible for the biosynthesis of diverse secondary metabolites of economic and therapeutic importance including antibiotics, anticancer agents and immune-modulators. Antibiotic resistance is one of the biggest threats of global health according to the World Health Organization (WHO). The Centers for Disease Control and Prevention (CDC) showed that in the US alone, it causes more than 2 million infections and 23,000 deaths a year. These alarming numbers are estimated to continue incrementally every year, with 10 million estimated deaths worldwide in 2050. For these reasons, we are motivated to utilize PKSs to facilitate the design and generation of novel antibiotics from the macrolides class to improve the development of new, effective therapeutics. A diverse subset of PKSs generate macrocyclic ring systems that are essential for macrolide production, include pathways from the Pikromycin (Pik), Erythromycin (DEBS) and Tylosin (Tyl) producing microorganisms. In this project, I will be focusing on the use of synthetic approaches to facilitate assembly of these compounds and their analogs using biocatalysis and enzyme engineering. The synthesis of diverse polyketide chain elongation intermediates in conjunction with late-stage biosynthetic machinery (e.g. glycosyltransferases, P450 monooxygenases) facilitates efficient access to a repertoire of novel molecules, which are challenging to generate using synthetic methods alone. PKS enzymes provide a powerful method to selectively catalyze key transformations on polyketide chains to generate macrolactones, which can be subsequently converted to novel macrolide antibiotics. Previous work in the Sherman lab has revealed that the primary hurdle to applying PKS modules for the production of diverse macrolactones hinges on the selectivity of the Pik thioesterase (TE) domain. These findings suggested that the TE functions as a gatekeeper in the processing of unnatural substrates to generate novel macrocycles. In the proposed research, I plan to (1) Design and synthesize unnatural substrates to explore PKS selectivity and tolerance toward substrate loading, elongation, and cyclization for the generation of odd- membered ring macrolactones, (2) Pursue a TE directed evolution approach for improved total turnover, and expansion of substrate scope to generate new macrolactone products, (3) Apply chemoenzymatic synthesis for diverse macrolides and determine their bioactivity profile against human bacterial pathogens. These efforts will be crucial to developing new macrolide antibiotics to control and overcome emerging resistance in human bacterial pathogens and to improve therapeutic parameters in this important class of anti-infective agents...