A lactococcal expression system was developed which allows the exclusive production of novel nisins encoded by mutated pre-nisin () genes. This system is based on a combination of a specifically constructed host strain and vectors which facilitate the genetic manipulation of the gene. The wild-type chromosomal gene is effectively replaced with a variant gene, by the technique of gene replacement. The recovery of full nisin immunity was employed as a means of directly selecting strains that had acquired an intact gene by the gene replacement process. With this approach the other genes required for pre-nisin maturation are not affected and any alterations to DNA sequences are restricted to only those specific mutations introduced in the gene. The effectiveness of the system was demonstrated by the expression of a number of variant genes leading to the successful production and characterization of nisins containing the substitutions Dha5A, Dha33A, Dha5,33A, H27K, I30W and K12L. The enhanced yields of these engineered nisin molecules, when compared to their production in a plasmid-complementation system, underlines the improvement offered by this gene replacement strategy.


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