Bioengineered nisin derivatives with enhanced activity in complex matrices

Abstract

Nisin 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.

Figure 1

Structure of nisin A. Modified residues are in grey. Ala‐S‐Ala, Lanthionine; Abu‐S‐Ala, β‐methyllanthionine; Dha, dehydroalanine; Dhb, dehydrobutyrine. (β‐methyl)lanthionine rings are labelled A–E. The location of the hinge region, consisting of Asn‐Met‐Lys, is also indicated.

Figure 2

Impact of nisin A (grey diamonds) and the nisin variants, SVA (white circle) and NAK (white triangle), relative to a non‐nisin‐containing control (black squares) on the growth/survival of L. monocytogenes UCC 35 (spiked at a level of 10⁵ cfu ml⁻¹ or 10⁴ cfu ml⁻¹) in chocolate milk at 22°C and 4°C. Data points are the average of triplicate experiments. Asterix indicate significant differences (*P < 0.05, **P < 0.005, **P < 0.001; two‐tailed Student t‐test) in UCC 35 numbers between the variant and nisin A‐treated samples (ND, not detected).