Project Summary / Abstract The emergence of antibiotic resistance has created a global dilemma for the need to discover new antibacterial lead agents. Realizing this critical need for new antibacterial agents with new structure types and targets, we focus here on the biosynthetic interrogation and development of structurally distinct marine bacterial natural products. An underlying theme associated with many marine microbial antibiotics involves the use of aromatic polyketide frameworks that have undergone extensive oxidative tailoring reactions catalyzed by halogenase and oxygenase biosynthetic enzymes. In this application, we propose a multidisciplinary project involving heterologous biosynthesis, mechanistic enzymology, atomic resolution protein X-ray crystallography, chemoenzymatic synthesis, and genetic engineering to understand and control the molecular basis of polyketide diversification in a series of marine bacterial compounds with promising antimicrobial properties. To accomplish the broad goals outlined in this application, we propose four specific aims. First, we plan to functionally and structurally characterize diverse meroterpenoid V-dependent chloroperoxidases and their catalytic properties in promoting antimicrobial chemical diversity. Second, we will discover, characterize, and engineer biosynthetic pathways for structural diversification of halogenated pyrrole containing bioactive natural products. Third, we aim to functionally characterize the unprecedented biosynthesis of thiotetronic acid polyketide antibiotics and apply new biosynthetic reactions to extend the synthesis and bioengineering of novel molecules. And fourth, we will interrogate the antimicrobial activity and mechanism of new meroterpenoid, bipyrrole, and thiotetronate compounds.