Heterologous\nExpression Facilitates the Production\nand Characterization of a Class III Lanthipeptide with Coupled Labionin\nCross-Links in Sponge-Associated <i>Streptomyces rochei</i> MB037

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