Characterization of a LanC-free pathway for the formation of an ll-MeLan residue and an AviMeCys residue in the newly identified class V lanthipeptide triantimycins.

The thioether-connected bis-amino acid lanthionine (Lan) residues are class-defining residues of lanthipeptides. Typically, the cyclization step of lanthionine formation, which relies on the addition of a cysteine to an unsaturated dehydroamino acid, is directed either by a standalone cyclase LanC (class I) or by a cyclase domain (class II-IV). However, the pathways of characterized class V members often lack a known cyclase (domain), raising a question on the mechanism by which their multi-macrocycle systems are formed. Herein, we report a new RiPP gene cluster in TN 58, where it encodes the biosynthesis of 3 distinct class V lanthipeptides-termed triantimycins (TAMs). TAM A1∼A3 share an N-terminal ll-MeLan residue, and only TAM A1 contains an additional internal ll-Lan residue. TAM A1 also has a C-terminal (2, 3)--(()-2-aminovinyl)-3-methyl-d-cysteine (AviMeCys) residue, which is distinct from the previously reported (2, 3)-AviMeCys residue in other RiPPs. Gene deletion, heterologous coexpression, and structural elucidation demonstrated that the cyclization for an ll-MeLan formation occurs spontaneously and is independent of any known lanthionine cyclase. This study provides a new paradigm for lanthionine formation and facilitates genome mining and engineering efforts on RiPPs containing (Me)Lan and ()Avi(Me)Cys residues.