Ribosomally synthesized and posttranslationally modified peptides (RiPPs) are a major group of natural products (NPs) produced in all domains of life. The number of RiPP families continues to expand rapidly based on genome sequencing. RiPPs a·re well suited for genome mining for new natural products by heterologous expression of biosynthetic gene clusters (BGCs) and for library generation of cyclic peptides to recognize diverse targets. Furthermore, RiPPs appear to be widely encoded in the human microbiome and have recently been implicated in causation and prevention of human disease. For the RiPP field to advance, a better understanding of the post-translational modification processes is important. Similarly, tools need to be developed to take advantage of the substrate tolerance of the biosynthetic enzymes. In the current application we describe our progress towards these goals and our proposed studies for continued advances. This application focuses mostly on the lanthipeptides, a group of polycyclic peptides with macrocyclic thioether crosslinks. Individual lanthipeptides have a variety of biological activities including antimicrobial, antiviral, antifungal and anti-allodynic. At least four different routes to lanthipeptides have evolved and lanthipeptide BGCs are ubiquitous amongst the RiPP family in sequenced genomes. For one of these four pathways, substrate recognition is reasonably understood but for the other three this information is lacking and will be a focus of continued research . Another poorly understood aspect is the factors that control the ring topology of the multiple thioether rings that are formed and this question will be a focus of investigation. Furthermore, previous work suggests that lanthipeptide biosynthesis takes place in enzyme complexes that may require its substrate. A method for catalytically competent covalent attachment of the substrate to the key enzyme will be used to try and structurally characterize such enzyme complexes. In addition, the mechanisms of a family of enzymes that make amino acid-derived NPs in a novel pathway will be investigated and the pathways in which they operate will be determined. Improved engineering technology to disrupt protein-protein interactions with cyclic peptides will be developed. RELEVANCE (See instructions): Based on historical precedent, the most likely group of compounds to deliver new antibiotics to fight antibiotic resistance are natural products. Genome sequences offer unprecedented access to new natural products that could be future antibiotics but for this information to be used, biosynthetic enzymes that make the natural products need to be better understood. This application aims to obtain this information.