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Volume 142, Issue 1

A gene replacement strategy for engineering nisin Free

Abstract

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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Keyword(s): antimicrobial peptide , gene replacement , Lactococcus lactis , nisin and protein engineering
© Society for General Microbiology 1996
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1996-01-01
2024-04-23
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References

  1. Biswas I, Gruss A, Ehrlich S. D., Maguin E. 1993; High-efficiency gene inactivation and replacement system for gram-positive bacteria. J Bacteriol 175:3628–3635
     [Google Scholar]
  2. CaSacIaban M. J., Cohen S. N. 1980; Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J MolBiol 138:179–207
     [Google Scholar]
  3. Chambers S. P., Prior S. E., Barstow D. A., Minton N. P. 1988; The pMTL nic-cloning vectors. I. Improved pUC polylinker regions to facilitate the use of sonicated DNA for nucleotide sequencing. Gene 68:139–149
     [Google Scholar]
  4. Chan W. C., Bycroft B. W., Lian L.-Y., Roberts G. C. K. 1989; Isolation and characterization of two degradation products derived from the peptide antibiotic nisin. FEBS Eett 252:29–36
     [Google Scholar]
  5. Chopin M.-C., Chopin A., Rouault A., Galleron N. 1989; Insertion and amplification of foreign genes in the Eactococcus lactis subsp lactis chromosome. Appl Environ Microbiol 55:1769–1774
     [Google Scholar]
  6. Dodd H. M., Gasson M. J. 1994; Bacteriocins of lactic acid bacteria. Genetics and Biotechnology of Eactic Acid Bacteria211–251 Edited by Gasson M. J., de Vos W. M. Glasgow: Blackie Academic & Professional;
     [Google Scholar]
  7. Dodd H. M., Horn N., Gasson M. J. 1990; Analysis of the genetic determinant for the peptide antibiotic nisin. J Gen Microbiol 136:555–566
     [Google Scholar]
  8. Dodd H. M., Horn N., Zhang H., Gasson M. J. 1992; A lactococcal expression system for engineered nisins. Appl Environ Microbiol 58:3683–3693
     [Google Scholar]
  9. Dodd H. M., Horn N., Gasson M. J. 1995; A cassette vector for protein engineering the lantibiotic nisin. Gene 162:163–164
     [Google Scholar]
  10. Engelke G., Gutowski-Eckel Z, Kiesau P, Siegers K., Hammelmann M., Entian K.-D. 1994; Regulation of nisin biosynthesis and immunity in Eactococcus lactis 6F3. Appl Environ Microbiol 60:814–825
     [Google Scholar]
  11. Gasson M. J. 1983; Plasmid components of Streptococcus lactis NCDO 712 and other lactic streptococci after protoplast-induced curing. J Bacteriol 154:1–9
     [Google Scholar]
  12. Holo H., Nes I. F. 1989; High-frequency transformation by electroporation of Eactococcus lactis subsp. cremoris grown with glycine in osmotically stabilized media. Appl Environ Microbiol 55:3119–3123
     [Google Scholar]
  13. Horn N., Swindell S. R., Dodd H. M., Gasson M. J. 1991; Nisin biosynthesis genes are encoded by a novel conjugative transposon. Mol & Gen Genet 228:129–135
     [Google Scholar]
  14. Klein C., Entian K.-D. 1994; Genes involved in self-protection against the lantibiotic subtilin produced by Bacillus subtilis ATCC 6633. Appl Environ Microbiol 60:2793–2801
     [Google Scholar]
  15. Klein C., Kaletta C., Schnell N., Entian K.-D. 1992; Analysis of genes involved in biosynthesis of the lantibiotic subtilin. Appl Environ Microbiol 58:132–142
     [Google Scholar]
  16. Klein C, Kaletta C., Entian K.-D. 1993; Biosynthesis of the lantibiotic subtilin is regulated by a histidine kinase/response regulator system. Appl Environ Microbiol 59:296–303
     [Google Scholar]
  17. Kuipers O. P., Rollema H. S., Yap W. M. G. J., Boot H. J., Siezen R. J., de Vos W. M. 1992; Engineering dehydrated amino acid residues in the antimicrobial peptide nisin. J Biol Chem 267:24340–24346
     [Google Scholar]
  18. Kuipers O. P., Beerthuyzen M. M., Siezen R. J., de Vos W. M. 1993; Characterization of the nisin gene cluster nisABTCIPR of Eactococcus lactis. Eur J Biochem 216:281–291
     [Google Scholar]
  19. Kuipers O. P., Beerthuyzen M. M., De Ruyter P. G. G. A., Luesink E. J., de Vos W. M. 1995; Autoregulation of nisin biosynthesis in Eactococcus lactis by signal transduction. J Biol Chem in press
    [Google Scholar]
  20. Leenhouts K. J., Kok J., Venema G. 1989; Campbell-like integration of heterologous plasmid DNA into the chromosome of Eactococcus lactis subsp. lactis. Appl Environ Microbiol 55:394–400
     [Google Scholar]
  21. Leenhouts K. J., Kok J., Venema G. 1990; Stability of integrated plasmids in the chromosome of Eactococcus lactis. Appl Environ Microbiol 56:2726–2735
     [Google Scholar]
  22. Lennox E. S. 1955; Transduction of linked genetic characters of the host bacteriophage PI. Virology 1:190–206
     [Google Scholar]
  23. Liu W., Hansen J. N. 1992; Enhancement of the chemical and antimicrobial properties of subtilin by site-directed mutagenesis. J Biol Chem 267:25078–25085
     [Google Scholar]
  24. Liu W., Hansen J. N. 1993; The antimicrobial effect of a structural variant of subtilin against outgrowing Bacillus cereus T spores and vegetative cells occurs by different mechanisms. Appl Environ Microbiol 59:648–651
     [Google Scholar]
  25. Mulders J. W. M., Boerrigter I. J., Rollema H. S., Siezen R. J., de Vos W. M. 1991; Identification and characterization of the lantibiotic nisin Z, a natural nisin variant. Eur J Biochem 201:581–584
     [Google Scholar]
  26. Rauch P. J. G., Kuipers O. P., Siezen R. J., de Vos W. M. 1994; Genetics and protein engineering of nisin. Bacteriocins of Eactic Acid Bacteria223–250 Edited by De Vuyst L., Vandamme E. J. Glasgow: Blackie Academic & Professional;
     [Google Scholar]
  27. Rollema H. S., Kuipers O. P., Both P., de Vos W. M., Siezen R. J. 1995; Improvement of solubility and stability of the antimicrobial peptide nisin by protein engineering. Appl Environ Microbiol 61:2873–2878
     [Google Scholar]
  28. Siegers K., Entian K.-D. 1995; Genes involved in immunity to the lantibiotic nisin produced by Eactococcus lactis 6F3. Appl Environ Microbiol 61:1082–1089
     [Google Scholar]
  29. Terzaghi B. E., Sandine W. E. 1975; Improved medium for lactic streptococci and their bacteriophages. Appl Microbiol 29:807–813
     [Google Scholar]
  30. de Vos W. M., Jung G., Sahl H.-G. 1991; Definitions and nomenclature of lantibiotics. Nisin and Novel Eantibiotics457–463 Edited by Jung G., Sahl H.-G. Leiden: ESCOM;
     [Google Scholar]
  31. de Vos W. M., Beerthuyzen M. M., De Ruyter P., Luesink E., Kuipers O. P. 1994; Control of nisin gene expression system. Proceedings of the 2nd International Workshop on LantibioticsPapendal, The Netherlands
     [Google Scholar]
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A gene replacement strategy for engineering nisin
Microbiology 142, 47 (1996); https://doi.org/10.1099/13500872-142-1-47
/content/journal/micro/10.1099/13500872-142-1-47
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