PROTEIN ENGINEERING AND BIOSYNTHESIS OF NISIN AND REGULATION OF TRANSCRIPTION OF THE STRUCTURAL NISA GENE

The lantibiotic nisin, produced by Lactococcus lactis, is an antimicro bial peptide characterized by presence of three unsaturated amino acid side chains (two dehydroalanines and one dehydrobutyrine) and five (b eta-methyl)lanthionine rings, which are formed post-translationally. N isin is widely used in the food industry as a preservative, since it i nhibits the growth of unwanted gram-positive bacteria. One of the obje ctive of our research is to get insight in the complex biosynthesis an d regulation of production of nisin. The structure and function of sev eral biosynthetic genes were studied by making gene disruptions and by subsequently investigating their effects on nisin gene regulation, bi osynthesis, 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 alt ered nisin molecules with desirable properties by protein engineering. In addition to previously reported mutant nisins with improved stabil ity, solubility or activity, recent results on the protein engineering of residues Ile1, Dhb2, AlaS3, Lys12, AbuS13, Met17, Asn20 and Met21 indicate that (i) residue 1 can be replaced without dramatic loss of a ctivity; (ii) the presence of a Thr residue at position 2 significantl y lowers the antimicrobial potency, whereas the presence of a Dha resi due at position 2 improves activity; (iii) the replacement of AlaS3 by AbuS leads to a dramatic loss of activity; probably due to a conforma tional change in the first lanthionine ring; (iv) the integrity and hy drophobicity of ring 3 are important for antimicrobial activity; and ( v) the hinge region between rings 3 and 4 is important but not essenti al for antimicrobial activity.