Biosynthesis of Antimicrobial Ornithine-Containing Lacticin 481 Analogues by Use of a Combinatorial Biosynthetic Pathway in <i>Escherichia coli</i>

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 <i>in vivo</i>. Furthermore, this study demonstrates that prior lanthionine (Lan) and methyllanthionine (MeLan) formation may preclude ornithine incorporation at specific sites of lacticin 481. The antibacterial activity of ornithine-containing lacticin 481 analogues was evaluated using <i>Bacillus subtilis</i> as the indicator strain. Overall, the synthetic biology pathway constructed here helped to elucidate aspects of the substrate preferences of OspR and SyncM, offering practical guidance to combine these modifications for further lantibiotic bioengineering.