Biosynthesis of\nAntimicrobial Ornithine-Containing\nLacticin 481 Analogues by Use of a Combinatorial Biosynthetic Pathway\nin Escherichia coli

Lacticin 481, a ribosomally\nsynthesized and post-translationally\nmodified peptide (RiPP), exhibits antimicrobial activity, for which\nits characteristic lanthionine and methyllanthionine ring structures\nare essential. The post-translational introduction of (methyl)lanthionines\nin lacticin 481 is catalyzed by the enzyme LctM. In addition to macrocycle\nformation, various other post-translational modifications can enhance\nand modulate the chemical and functional diversity of antimicrobial\npeptides. The incorporation of noncanonical amino acids, occurring\nin many nonribosomal peptides (NRPs), is a valuable strategy to improve\nthe properties of antimicrobial peptides. Ornithine, a noncanonical\namino acid, can be integrated into RiPPs through the conversion of\narginine residues by the newly characterized peptide arginase OspR.\nRecently, a flexible expression system was described for engineering\nlanthipeptides using the post-translational modification enzyme SyncM,\nwhich has a relaxed substrate specificity. This study demonstrates\nthat SyncM is able to catalyze the production of active lacticin 481\nby recognition of a designed hybrid leader peptide, which enables\nthe incorporation of both ornithine and (methyl)lanthionine. Utilizing\nthis hybrid leader peptide, the functional order was established for\nthe production of active ornithine-containing lacticin 481 analogues\nat positions 8 and 12 <i>in vivo</i>. Furthermore, this\nstudy demonstrates that prior lanthionine (Lan) and methyllanthionine\n(MeLan) formation may preclude ornithine incorporation at specific\nsites of lacticin 481. The antibacterial activity of ornithine-containing\nlacticin 481 analogues was evaluated using Bacillus\nsubtilis as the indicator strain. Overall, the synthetic\nbiology pathway constructed here helped to elucidate aspects of the\nsubstrate preferences of OspR and SyncM, offering practical guidance\nto combine these modifications for further lantibiotic bioengineering.