Protein Engineering and Biosynthesis of Nisin and Regulation of Transcription of the Structural nisA Gene

The lantibiotic nisin, produced by Lactococcus lactis, is an antimicrobiaI peptide characterized by the presence of three unsaturated amino acid side chains (two dehydroalanines and one dehydrobutyrine) and five (/3methyl)lanthionine rings, which are formed post-translationally. Nisin is widely used in the food industry as a preservative, since it inhibits the growth of unwanted gram-positive bacteria. One of the objectives of our research is to get insight in the complex biosynthesis and regulation of production of nisin. The structure andfunction of several biosynthetic genes were studied by making gene disruptions and by subsequently investigating their effects on nisin gene regulation, biosynthesis, secretion and immunity. An exciting finding is that nisin itself, when added to the culture medium. can induce the transcription of its own structural gene. Another goal is to design and produce altered nisin molecules with desirable properties by protein engineering. In addition to previously reported mutant nisins with improved stability, solubility or activity, recent results on the protein engineering of residues Ilel, Dhb2, AlaS3, Lysl2, AbuS13, Metl7, Asn20 and Met21 indicate that (i) residue I can be replaced without dramatic loss of activity; (ii) the presence of a Thr residue at position 2 significantly lowers the antimicrobial potency, whereas the presence of a Dha residue at position 2 improves activity; (iii) the replacement of AlaS3 by AbuS leads to a dramatic loss of activity, probably due to a conformational change in the first lanthionine ring; (iv) the integrity and hydrophobicity of ring 3 are important for antimicrobial activitv; and (v) the hinge region between rings 3 and 4 is important but not essentialfor antimicrobial activity.