ABSTRACT/PROJECT SUMMARY Lasso peptides are a class of ribosomally synthesized and post-translationally modified peptide (RiPP) natural products categorized by their unique threaded rotaxane structure. Genome mining studies from my group and others have revealed that these peptides are found in a wide variety of different prokaryotic organisms in environments as varied as the human gut and the deep sea. This proposal will leverage the latest generation of our genome mining software to catalog all known lasso peptides in sequenced genomes. We will continuously update this list and make it available to the natural products community. Leveraging our unique insights into lasso peptide activity, we will prioritize several peptides for further structural and functional studies. In particular, we plan to study lasso peptides predicted to have novel antimicrobial activities against pathogens. Finally, we will also leverage our genome mining work to carry out an in depth biochemical and structural study of lasso peptide biosynthesis.