TMcin RiPP biosynthesis in the cellular membrane

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a large category of natural products and a promising source for new medicines and applications in biotechnology. Their extraordinary diversity stems from the sequences of ribosomally synthesized precursor peptides and the wide repertoire of biosynthetic proteins that post-translationally modify and process the peptides. However, known precursor peptides and biosynthetic events have been characterized as soluble and occurring within aqueous environments in which RiPPs encounter the membrane primarily for secretion via transporters. Here, we report that the cell membrane is the central setting for the biosynthesis of TMcins, a recently discovered RiPP class with antimicrobial activity that contains a transmembrane helix (TMH) in the precursor peptide and mature product. We show that the ribosomally synthesized precursor TmcA is integrated in the producing cell membrane. We then uncovered the biosynthetic roles of gene products encoded on the TMcin biosynthetic gene cluster by integrating structure-prediction with an inducible TMcin biosynthesis platform. All TMcin post-translational modifications occurred in the membrane, for which three of four events were performed by intramembranous biosynthetic proteins. Finally, we assign an escort function to a previously uncharacterized membrane protein and provide insights into its evolution. Thus, our characterization of TMcin expands the setting for RiPP biosynthesis and provides a model for the biosynthesis of membrane-localized peptide natural products. Significance StatementCells modify peptides to make natural products called RiPPs that exhibit a wide range of activities. Many RiPPs are antimicrobial and are important sources for discovering new drugs. Known RiPPs are produced by biosynthetic enzymes in aqueous environments. We characterized the recently discovered TMcin class of RiPPs that possess a transmembrane helix and are found across Gram-positive bacteria. Using a Staphylococcus aureus strain that naturally produces TMcin, we show that TMcin biosynthesis occurs in the membrane and elucidate the steps of TMcin production. This work establishes a model for understanding RiPP biosynthesis within a lipid environment that broadens our understanding of RiPPs, enables discoveries of new RiPPs, and provides a basis for the rational engineering of TMcins.