PROJECT SUMMARY/ABSTRACT Natural products are indisputably the most productive source of chemical matter for antibiotic development. Unfortunately, the pharmacological deployment of new natural products has been outpaced by the waning utility of approved antibiotics, especially those active against Gram-negative bacterial pathogens, Genomic technologies and synthetic biology are uniquely positioned to address this shortfall, and towards that critical goal, the previous project period developed a fully 6utomated, §,calable, and high-Ihroughput (FAST) pipeline for the discovery of Ribosomally synthesized and Post-translationally modified Peptides (RiPPs), RiPPs are a class of natural product covering nearly 50 known structural families. Despite their impressive structural and functional diversity, RiPPs are united by a common biosynthetic strategy. A gene-encoded precursor peptide is comprised of an N-terminal leader region, providing a binding site for the modification enzymes, and a C-terminal core region, which receives all of the post-translational modifications. This renewal project builds on our previous success in unlocking the potential of RiPPs as a source of new antibacterials. For this project, we propose three interrelated but independently achievable specific aims. Aim 1 addresses pitfalls of the FAST-RiPPs procedure uncovered during the previous project period. Here, we will enhance our ability to achieve high titers of any desired RiPP structural class. Among others, solutions to the challenge of obtaining mature products involving radical S-adenosylmethionine-dependent enzymes are proposed. Aim 2 develops the ways and means to prioritize RiPPs with a high probability of displaying antibacterial activity. Lastly, Aim 3 addresses the grand challenge of predicting ring patterns directly from primary sequence for multicyclic RiPPs. With several RiPP classes associated with multimacrocyclic scaffolds and often encoding numerous macrocycle donor and acceptor residues in the core region, determining the structural outcome of the enzymatic pathways has been impossible. However, with advances in bioinformatics, artificial intelligence (Al), and new ring patterns discovered as a consequence of a functional FAST-RiPPs pipeline, a solution to this problem becomes feasible. Given their proven success rates from the previous project period, including the discovery of a potent anti-Klebsiella compound, lanthipeptides serve as our testbed for Al algorithmic development. This project blends cutting-edge Al, synthetic biology, biofoundry, and analytical chemistry techniques with innovative solutions to heterologous expression deficiencies. We further will engineer broad host range plasmid compatibility to overcome the need to reclone pathways when a heterologous host is deemed insufficient. Success on this project will have a profound impact on the synthetic biology community and pharmaceutical industry.