Biosynthesis of Antimicrobial Ornithine-Containing Lacticin 481 Analogues by Use of a Combinatorial Biosynthetic Pathway in

Lacticin 481, a ribosomally synthesized and post-translationally modified peptide (RiPP), exhibits antimicrobial activity, for which its characteristic lanthionine and methyllanthionine ring structures are essential. The post-translational introduction of (methyl)lanthionines in lacticin 481 is catalyzed by the enzyme LctM. In addition to macrocycle formation, various other post-translational modifications can enhance and modulate the chemical and functional diversity of antimicrobial peptides. The incorporation of noncanonical amino acids, occurring in many nonribosomal peptides (NRPs), is a valuable strategy to improve the properties of antimicrobial peptides. Ornithine, a noncanonical amino acid, can be integrated into RiPPs through the conversion of arginine residues by the newly characterized peptide arginase OspR. Recently, a flexible expression system was described for engineering lanthipeptides using the post-translational modification enzyme SyncM, which has a relaxed substrate specificity. This study demonstrates that SyncM is able to catalyze the production of active lacticin 481 by recognition of a designed hybrid leader peptide, which enables the incorporation of both ornithine and (methyl)lanthionine. Utilizing this hybrid leader peptide, the functional order was established for the production of active ornithine-containing lacticin 481 analogues at positions 8 and 12