Supporting data for 'Genome Mining of Uncharted Ribosomally Synthesized and Post-translationally Modified Peptides'

Ribosomally synthesized and post-translationally modified peptides (RiPPs) represent one of the largest but primarily underexplored natural product families in bacteria. The genetically encoded nature of RiPPs simplifies the prediction and prioritization of their biosynthetic gene clusters (BGCs). We report a small peptide and enzyme co-occurrence analysis workflow (SPECO), which allowed us to identify 32220 prospective rSAM-catalyzed RiPP BGCs from 161954 bacterial genomes and prioritize 25 families with new biosynthetic architectures or precursor patterns. We characterized three new enzymes that respectively catalyze cysteineglycine (BlaB), histidine-aliphatic side chain (ScaB), and tyrosine/histidine-arginine (VguB) cross-links. The cyclophane-forming enzyme ScaB exhibits broad substrate selectivity, allowing it to catalyze diverse triceptide formation. These results demonstrate the strength of the SPECO workflow in discovering new enzymes for peptide macrocyclization.P450-catalyzed RiPPs stand out as a unique but underexplored RiPP family. Here, we introduce a rule-based genome mining strategy that harnesses the intrinsic biosynthetic principles of RiPPs, including the co-occurrence, co-conservation, and interactions between precursors and P450s, successfully facilitating the identification of diverse P450 catalyzed RiPPs. Intensive BGC characterization revealed four new P450s, KstB, ScnB, MciB, and SgrB, that can respectively catalyze Trp-Trp-Tyr (one C-C and two C-N bonds), Tyr-Trp (C-C bond), Trp-Trp (C-N bond), and His-His (ether bond) crosslinks within three or four residues. KstB, ScnB, and MciB could accept non-native precursors, suggesting they could be promising starting templates for bioengineering to construct macrocycles. Our study highlights the potential of P450s in expanding the chemical diversity of strained macrocyclic peptides and enriching biocatalytic tools for peptide macrocyclization.