Co-opting the bacterial lipoprotein pathway in the biosynthesis of a lipidated macrocyclic peptide

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are structurally diverse natural products that possess a range of bioactivities, often acting as antibiotics, antifungals, or metallophores. In RiPP biosynthesis, different modifying enzymes install an array of chemical motifs onto a precursor peptide. A recently described RiPP-modifying enzyme, ChrH, catalyzes a remarkably complex reaction on its precursor peptide that results in a macrocycle, heterocycle, and S-methyl group. By leveraging comparative genomics, we demonstrate that the products from a subfamily of enzymes related to ChrH display unexpected structural diversity, including the production of unmethylated macrocyclic congeners and C-terminally modified proteins over 30 kDa in size. Several of these precursors contain a signal peptide, sending them for downstream maturation by the bacterial lipoprotein biosynthetic pathway. Like bacterial lipoproteins, such peptides are modified by addition of a diacylglycerol (DAG) group to the N-terminal cysteine residue along with acylation of the N-terminal amine. Genome mining reveals that these RiPP-lipoprotein hybrids, which we term DAG-RiPPs, are widespread across bacterial phyla and are likely involved in different biological roles. Together, these results highlight a novel maturation paradigm for membrane-bound RiPPs and lay the foundation for the discovery and bioengineering of other RiPP-lipoprotein hybrids.