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

Abstract

It is becoming increasingly apparent that innovations from the “golden age” of antibiotics are becoming ineffective, resulting in a pressing need for novel therapeutics. The bacteriocin family of antimicrobial peptides has attracted much attention in recent years as a source of potential alternatives. The most intensively studied bacteriocin is nisin, a broad spectrum lantibiotic that inhibits Gram-positive bacteria including important food pathogens and clinically relevant antibiotic resistant bacteria. Nisin is gene-encoded and, as such, is amenable to peptide bioengineering, facilitating the generation of novel derivatives that can be screened for desirable properties. It was to this end that we used a site-saturation mutagenesis approach to create a bank of producers of nisin A derivatives that differ with respect to the identity of residue 12 (normally lysine; K12). A number of these producers exhibited enhanced bioactivity and the nisin A K12A producer was deemed of greatest interest. Subsequent investigations with the purified antimicrobial highlighted the enhanced specific activity of this modified nisin against representative target strains from the genera Streptococcus, Bacillus, Lactococcus, Enterococcus and Staphylococcus.

Engineering deamidation‐susceptible asparagines leads to improved stability to thermal cycling in a lipase

“…Site saturation mutagenesis (SSM) approach has been used successfully in the recent past to understand the structure-function relationship and also to improve stability of proteins under a variety of in vitro evolution context. [27][28][29][30] Thus, a thorough analysis by SSM would seek optimal substitution for residue susceptible to deamidation to improve the thermotolerance of the protein.…”

Multipronged approach for engineering novel peptide analogues of existing lantibiotics

“…A saturation mutagenesis study on nisin led to the discovery of additional variants with enhanced bioactivity. In the hinge region of nisin, between rings B and C, the substitution K12A had shown an increase in activity [53]. Single amino acid substitutions in another nisin hinge region between rings C and D also enhanced activity against multiple strains tested ( Figure 2A) [54][55][56].…”

“…However, some of the reports on the bioactivity of novel lantibiotic analogues were limited. For instance, several studies performed on nisin only tested the activity against a few strains of pathogenic and non-pathogenic bacteria [53][54][55]57]. Rescreening the activity of these structural variants against a full spectrum of medically relevant Gram-positive strains may elucidate the analogue's medical application.…”

“…Class I lantibiotics; mutacin 1140 and nisin[40,47,48,[53][54][55][56], B. Class II lantibiotics; nukacin-ISK1, mersacidin and actagardine[39,[60][61][62], C. Two-component lantibiotics, lacticin 3147 --Ltna and Lacticin 3147 --Ltnb[64,65].…”

Antimicrobial antagonists against food pathogens: a bacteriocin perspective

“…Indeed, this is the first instance upon which bioengineering of a bacteriocin has led to enhanced activity of this kind [32]. Saturation mutagenesis at another location in nisin, lysine 12, resulted in the finding that a K12A derivative displays increased specific activity against food pathogens such as B. cereus, S. aureus and S. agalactieae but not against L. monocytogenes [33]. 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].…”