Designing and Producing Modified, New-to-Nature Peptides with Antimicrobial Activity by Use of a Combination of Various Lantibiotic Modification Enzymes

Abstract

Lanthipeptides are peptides that contain several post-translationally modified amino acid residues and commonly show considerable antimicrobial activity. After translation, the amino acid residues of these peptides are modified by a distinct set of modification enzymes. This process results in peptides containing one or more lanthionine rings and dehydrated Ser and Thr residues. Previously, an in vivo lanthipeptide production system based on the modification machinery of the model lantibiotic nisin was reported. Here, we present the addition of the modification enzymes LtnJ and GdmD to this production system. With these enzymes we can now produce lanthipeptides that contain d-alanines or a C-terminal aminovinyl-cysteine. We show experimentally that the decarboxylase GdmD is responsible for the C-terminal decarboxylation. Our results demonstrate that different lanthipeptide modification enzymes can work together in an in vivo production system. This yields a plug-and-play system that can be used to select different sets of modification enzymes to work on diverse, specifically designed substrates.

Lantibiotic Reductase LtnJ Substrate Selectivity Assessed with a Collection of Nisin Derivatives as Substrates

“…These resulting pNZ-nisA derivatives were digested by HindIII and XhoI and ligated with a fragment containing the transcriptional attenuator and ltnJ to generate 19 different derivatives of pNZ-nisA-T-ltnJ (Table 1). This fragment was amplified with the primers P-for-HindIII-T-ltnJ and P-ltnJ-rev-XhoI (Table 2) using pNZ-nisA-T-ltnJ (18). By applying two sets of round PCR on pNZ-nisA with two pairs of primers, P-for-K34L/P-rev-K34L and P-for-S29insKIH/P-rev-S29insKIH (Table 2), pNZ-nisin(⌬30 -34)-KI-HIHVSL was created (Table 1).…”

“…Another class II lantibiotic, deoxyactagardine B (NVB302; Novacta Biosystems Limited), is undergoing a phase I clinical trial as a drug candidate for the treatment of Clostridium difficile infections (14). Furthermore, recent research has shown that some class III lanthipeptides have unexpected bioactivity to relieve neuropathic pain (15) and as antiviral compounds (16).Besides the common (methyl)lanthionine, more than 15 extra structures have been unveiled in lanthipeptides playing a significant role in antimicrobial activity, resistance against proteases, and/or physicochemical resistance (12,(17)(18)(19)(20). For instance, D-alanine was found within two lantibiotics: lactocin S and the twocomponent lantibiotic lacticin 3147 (Fig.…”

“…Besides the common (methyl)lanthionine, more than 15 extra structures have been unveiled in lanthipeptides playing a significant role in antimicrobial activity, resistance against proteases, and/or physicochemical resistance (12,(17)(18)(19)(20). For instance, D-alanine was found within two lantibiotics: lactocin S and the twocomponent lantibiotic lacticin 3147 ( Fig.…”

“…It has been shown that the nisin biosynthetic machinery possesses high substrate tolerance and can modify diverse peptides fused to the nisin leader peptide, not only those related to lanthipeptides but also unrelated peptide sequences (26)(27)(28)(29)(30). Previously, we successfully introduced D-alanine into nisin by expressing LtnJ together with the nisin biosynthetic machinery in a dual-plasmid system in L. lactis (18). The Dha at position 5 of nisin has been demonstrated to be modified by LtnJ, while the other Dha, at position 33, is most likely not (18).…”

“…Previously, we successfully introduced D-alanine into nisin by expressing LtnJ together with the nisin biosynthetic machinery in a dual-plasmid system in L. lactis (18). The Dha at position 5 of nisin has been demonstrated to be modified by LtnJ, while the other Dha, at position 33, is most likely not (18).…”