Bioengineered nisin derivatives with enhanced activity in complex matrices

Abstract

SummaryNisin A is the best known and most extensively characterized lantibiotic. As it is ribosomally synthesized, bioengineering‐based strategies can be used to generate variants. We have previously demonstrated that bioengineering of the hinge region of nisin A can result in the generation of variants with enhanced anti‐microbial activity against Gram‐positive pathogens. Here we created a larger bank of hinge variant producers and screened for producers that exhibit enhanced bioactivity as assessed by agar‐based assays against a selection of target strains. Further analysis of 12 ‘lead’ variants reveals that in many cases enhanced bioactivity is not attributable to enhanced specific activity but is instead as a consequence of an enhanced ability to diffuse through complex polymers. In the case of two variants, which contain the residues SVA and NAK, respectively, within the hinge region, we demonstrate that this enhanced trait enables the peptides to dramatically outperform nisin A with respect to controlling Listeria monocytogenes in commercially produced chocolate milk that contains carrageenan as a stabilizer.

A Bioengineered Nisin Derivative, M21A, in Combination with Food Grade Additives Eradicates Biofilms of Listeria monocytogenes

“…L. lactis strains, L. lactis NZ9700, L. lactis NZ9800-pCI372 Nisin A, L. lactis NZ9800-pCI372 M21A (Field et al, 2008), L. lactis NZ9800–pCI372 AAA (Healy et al, 2013) and L. lactis NZ9800-pCI372 Nisin V (Rouse et al, 2012), were grown on M17 supplemented with 5% glucose (GM17) broth (Oxoid, England) or GM17 agar (GM17 broth substituted with 1.5% w/v agar) at 30°C. Strains were stocked in 40% glycerol and stored at -20°C.…”

“…Those derived from lactic acid bacteria (LAB) have gained particular attention with respect to their use in the food industry. One of the most extensively studied bacteriocins is nisin A, a bactericidal lantibiotic produced by some strains of Lactococcus lactis (Rouse et al, 2012; Campion et al, 2013; Healy et al, 2013). It is a 3.5 kDa peptide consisting of 34 amino acids and is a flexible and elongated amphipathic peptide that has the ability to form pores as well as inhibit cell wall biosynthesis (Breukink et al, 1999; Wiedemann et al, 2001; Prudêncio et al, 2015).…”

Saturation Mutagenesis of Lysine 12 Leads to the Identification of Derivatives of Nisin A with Enhanced Antimicrobial Activity

“…Nisin has been the focus of bioengineering in order to elucidate the relationship between the structure and function of the molecule, as well as with a view to improving functional properties of the peptide [30]. Bioengineering has been fruitful with respect to the introduction of mutations that have a positive impact on the physico-chemical properties of nisin, including better solubility [31], [32], improved stability [32] and an enhanced ability to diffuse through complex polymers [33]. Initial success with regard to increasing the potency of nisin came with the creation of bioengineered nisin derivatives with superior antimicrobial activity against some non-pathogenic targets [13], [34], [35], [36].…”

Antimicrobial antagonists against food pathogens: a bacteriocin perspective

“…Another region of the nisin peptide, the three amino acid 'hinge' region, is particularly amenable to change and bioengineering of this region has had beneficial consequences [34]. Indeed, Rouse et al [35] created a bank of hinge mutants and found that nisin peptides containing hinges consisting of SVA or NAK (rather than the original NMK) displayed an enhanced ability to diffuse through complex polymers, a trait which enabled the variants to outcompete nisin A controlling L. monocyto genes in commercially produced chocolate milk containing the stabiliser carrageenan. Furthermore, Healy et al [36] used site directed mutagenesis of the hinge region to create a novel bank of nisin derivatives and found that AAK, NAI and SLS had enhanced activity towards some microorganisms.…”