Evolution‐Guided Discovery of Antimycobacterial Triculamin‐Like Lasso Peptides

Abstract Triculamin is a ribosomally synthesized and post‐translationally modified peptide (RiPP) lasso peptide with potent antimycobacterial activity, produced by an unusual, non‐canonical biosynthetic gene cluster (BGC). In this study, we elucidate the biosynthetic pathway of triculamin through heterologous expression and show that the biosynthesis proceeds in the presence of a precursor (triA), macrocyclase (triC), and N‐acetyltransferase (triT). Through in vitro triT acetylation and bioactivity assays, we show that acetylation functions as a resistance mechanism. Genomic searches of triculamin BGC genes across bacteria show that triculamin is more widely distributed than previously anticipated, as triculamin‐like core peptides are found in at least three phyla in contrast to previously described lasso peptides that are typically restricted to one phylum. Triculamin BGCs with both canonical and non‐canonical RiPP biosynthetic genes were identified. Two strains containing canonical triculamin‐like BGCs were chemically characterized and shown to produce the novel triculamin‐like lasso peptides palmamin and gelatinamin, the latter of which appears to have an unprecedented additional ring formation. Detailed phylogenetic investigation of the macrocyclases from triculamin‐like BGCs suggests that these molecules are products of convergent evolution. These findings broaden the evolutionary and functional landscape of lasso peptides, revealing their unexpected diversification and cross‐phylum distribution.