Heterologous Expression Facilitates the Production and Characterization of a Class III Lanthipeptide with Coupled Labionin Cross-Links in Sponge-Associated <i>Streptomyces rochei</i> MB037

Cyclic peptides, with remarkable stability, cellular permeability, and proteolysis resistance, display promising potential in pharmaceutical applications. Labionin (Lab), a unique bicyclic cross-link containing both C-C and C-S bonds, provides high rigidity and better control of conformation compared to monocyclic cross-links. To discover more Lab-containing scaffolds with highly rigid conformation for cyclic peptide drug development, herein, a cryptic class III lanthipeptide biosynthetic gene cluster (BGC) (i.e., <i>rcs</i>) was identified in the sponge-associated <i>Streptomyces rochei</i> MB037 and expressed in <i>Escherichia coli</i>, incorporating an N-terminal SUMO-tag on the RcsA precursor peptide to prevent proteolysis. Subsequently, a novel class III lanthipeptide, i.e., rochsin A, exhibiting a highly rigid conformation with coupled Lab cross-links crowded by bulky aromatic amino acids, was produced. Three AplP-like proteases outside the <i>rcs</i> BGC were proven to remove the leader peptide of rochsin A through their dual endo- and aminopeptidase activities, resulting in mature rochsin A <i>in vitro</i>. Ala mutation experiments revealed the C to N cyclization direction, like most class III lanthipeptides. However, RcsKC displays a high substrate breadth, enabling various ring topologies that are rarely observed in other class III lanthipeptides. Overall, the established expression system broadens the chemical diversity of cyclic peptides with unique Lab cross-links and offers a highly rigid scaffold for cyclic peptide drug development.