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.