title: A gene replacement strategy for engineering nisin.
abstract: A lactococcal expression system was developed which allows the exclusive production of novel nisins encoded by mutated pre-nisin (nisA) genes. 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 wild-type chromosomal gene is effectively replaced with a variant nisA 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 nisA 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 nisA gene. The effectiveness of the system was demonstrated by the expression of a number of variant nisA genes leading to the successful production and characterization of nisins containing the substitutions Dha5A, Dha33A, Dha5, 33A, H27K, 130W 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.
authors.affiliation: Department of Genetics and Microbiology, Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, UK.
authors.forename: Helen M
authors.initials: HM
authors.lastname: Dodd
authors.affiliation: Department of Genetics and Microbiology, Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, UK.
authors.forename: Nikki
authors.initials: N
authors.lastname: Horn
authors.affiliation: Department of Biology, University of York, York, YO1 5DD, UK.
authors.forename: Catriona J
authors.initials: CJ
authors.lastname: Giffard
authors.affiliation: Department of Genetics and Microbiology, Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, UK.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Gasson
mesh_terms: D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents; D001483:Base Sequence; D002876:Chromosomes, Bacterial; D005798:Genes, Bacterial; D014644:Genetic Variation; D005822:Genetic Vectors; D017045:Lactococcus / Q000235:genetics*; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009561:Nisin / Q000235:genetics* / Q000302:isolation & purification; D015202:Protein Engineering / Q000379:methods*; D012641:Selection, Genetic
chemical_list: D000900:Anti-Bacterial Agents; D009561:Nisin