PROJECT SUMMARY Nonribosomal peptides are an important class of natural products which display great structural and functional diversity. As many nonribosomal peptides act as antimicrobials, the identification of novel bioactive NRPs can lead to the development of new therapeutics for the treatment of antimicrobial resistant microorganisms. Dehydrated nonribosomal peptides, peptides derived from or containing one or more α,β-dehydroamino acids (dhAAs), are key targets for further therapeutic development due to their structural rigidity, resistance to proteolytic degradation, and enhanced chemical reactivity. The Li Lab has focused on a specific class of dehydrated nonribosomal peptides whose biosynthesis stems from biosynthetic gene clusters (BGCs) containing nonribosomal-peptide synthetases (NRPSs) with C domains which modify amino acids (CmodAA). We have recently characterized a class of these CmodAA domains which dehydrates β-hydroxy amino acids to dhAAs in nonribosomal peptide biosynthesis, and work from our lab and others has demonstrated that these CmodAAderived nonribosomal peptides display a diverse array of bioactivities. However, the identity of many CmodAA derived nonribosomal peptides is undiscovered, and their biosynthetic pathways and biological roles remain uncharacterized. Through genome mining we have identified nearly 4,500 nonidentical CmodAA domains, many of which are distributed among soil bacteria including Streptomyces. The research described herein is aimed to elucidate the unknown products of several of these BGCs from Streptomyces and characterize the role of the CmodAA domain in their biosynthesis. I will use a combination of in silico analysis of the biosynthetic gene clusters, comparative metabolomics, genetic manipulation, and NMR analysis to elucidate the cryptic products of these BGCs. Once the nonribosomal peptide structure has been determined, I will perform a battery of in vitro enzyme assays to elucidate the core enzymes involved in nonribosomal peptide biosynthesis, and the biosynthetic role of the CmodAA domains within each BGC. Completion of the proposed research will lead to the discovery of novel CmodAA derived dhAA nonribosomal peptides with potential therapeutic applications. Furthermore, the findings of this research will provide a base of knowledge that will extend to the discovery of other CmodAA containing BGCs and characterization of their dhAA containing products.