A gene replacement strategy for engineering nisin

• Published in: Microbiology (Society for General Microbiology) 1996-01, Vol.142 (1), p.47-55
• Main Authors: Dodd, Helen M, Horn, Nikki, Giffard, Catriona J, Gasson, Michael J
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Anti-Bacterial Agents; Bacteriological methods and techniques used in bacteriology; Bacteriology; Base Sequence; Biological and medical sciences; Biotechnology; Chromosomes, Bacterial; Fundamental and applied biological sciences. Psychology; Genes, Bacterial; Genetic engineering; Genetic technics; Genetic Variation; Genetic Vectors; Lactococcus - genetics; Methods. Procedures. Technologies; Microbial Sensitivity Tests; Microbiology; Molecular Sequence Data; Mutagenesis, Site-Directed; Nisin - genetics; Nisin - isolation & purification; Protein Engineering - methods; Selection, Genetic; Vectors (cloning, transfer, expression). Insertion sequences and transposons
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/13500872-142-1-47

1 Department of Genetics and Microbiology, Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, UK 2 Department of Biology, University of York, York, YO1 5DD, UK 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, 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. Author for correspondence: Helen M. Dodd. Tel: [phone]. Fax: [phone]. e-mail: [e-mail] Keywords: nisin, protein engineering, gene replacement, antimicrobial peptide, Lactococcus lactis