Expanded sequence space of radical S-adenosylmethionine-dependent enzyme involved in post-translational macrocyclization

Abstract Ribosomally synthesized and post-translationally modified peptide (RiPP) represents one of the largest yet largely underexplored natural product families in bacteria. Although varied precursors and maturases lead to remarkable structural diversity, genetically-encoded nature of RiPP results in a residue-defined modification landscape of macrocyclic peptides, which simplifies the prediction and prioritization of their biosynthetic gene clusters (BGCs). Here, we report a small peptide and enzyme co-occurrence analysis workflow (SPECO), which enabled us to perform a large-scale analysis of radical S-adenosylmethionine (rSAM) enzyme-catalyzed RiPPs BGCs and disclosed uncharted enzyme sequence space for unprecedented post-translational modification of macrocyclic peptides. Applying this approach to 161,954 bacterial genomes, we identified 32,220 BGCs of rSAM-catalyzed RiPPs and prioritized 25 new families with unseen biosynthetic architectures or precursor peptide patterns. We further discovered three new enzymes for macrocyclic peptides construction, which catalyzed crosslinks between cysteine to glycine, histidine to aliphatic side-chain, and tyrosine/histidine to arginine, respectively. Together, these results demonstrate the strength of our SPECO approach in discovering new RiPP BGCs and diverse enzymes for macrocyclic peptides biosynthesis.