A GENE REPLACEMENT STRATEGY FOR ENGINEERING NISIN

A lactococcal expression system was developed which allows the exclusi ve production of novel nisins encoded by mutated pre-nisin (nisA) gene s. This system is based on a combination of a specifically constructed host strain and vectors which facilitate the genetic manipulation of the nisA gene. The wildtype chromosomal gene is effectively replaced w ith a variant nisA gene, by the technique of gene replacement. The rec overy of full nisin immunity was employed as a means of directly selec ting strains that had acquired an intact nisA gene by the gene replace ment process. With this approach the other genes required for pre-nisi n maturation are not affected and any alterations to DNA sequences are restricted to only those specific mutations introduced in the nisA ge ne. The effectiveness of the system was demonstrated by the expression of a number of variant nisA genes leading to the successful productio n and characterization of nisins containing the substitutions Dha5A, D ha33A, Dha5,33A, H27K, 130W and K12L. The enhanced yields of these eng ineered nisin molecules, when compared to their production in a plasmi d-complementation system, underlines the improvement offered by this g ene replacement strategy.