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. Significance Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a superfamily of natural products that display antibiotic, antifungal, anticancer, and metal-binding activities. Their biosynthesis typically follows a common logic in which modifying enzymes install chemical motifs onto a precursor peptide, followed by proteolytic processing and export from the cell. Herein, we describe the discovery and biochemical characterization of a new class of lipid-RiPP hybrid products. These RiPPs contain a signal peptide that exploits the endogenous bacterial lipoprotein biosynthesis pathway for lipidation, membrane localization, and potential secretion. Genome mining shows that these lipid-peptide hybrids are widespread across bacterial phyla.