Iterative glycosylation on a single residue of a mature lasso peptide

Iterative catalysis is a unique characteristic of the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs) to boost structural and biological diversity. Lasso peptides are an abundant class of RiPPs featuring lariat knot structures, which pose obstacles to iterative post-translational modifications (PTMs) after macrocyclization. Here, we present a unique glycotransferase (GT), IgtG, that iteratively glycosylates a Ser residue up to four times within a mature lasso peptide, as the final biosynthetic step of davasins. Such iterative catalysis diverges from classical iterative modifications on linear peptides. Bioinformatic analysis reveals 24 IgtG-like GTs that are potentially involved in the biosynthesis of graspetides, and IgtG-like GTs are evolutionarily distinct from known RiPP GTs. The discovery of PTM enzymes functioning on mature lasso peptides showcases the divergent biosynthetic strategies of RiPPs and provides valuable enzymatic tools for diversification of lasso peptides through combinatorial biosynthesis.