Applied Microbiology and Biotechnology

, Volume 72, Issue 1, pp 41–51 | Cite as

High-level heterologous production and functional expression of the sec-dependent enterocin P from Enterococcus faecium P13 in Lactococcus lactis

  • Jorge Gutiérrez
  • Rasmus Larsen
  • Luis M. Cintas
  • Jan Kok
  • Pablo E. Hernández
Biotechnological Products and Process Engineering

Abstract

Enterocin P (EntP), a sec-dependent bacteriocin from Enterococcus faecium P13, was produced by Lactococcus lactis. The EntP structural gene (entP) with or without the EntP immunity gene (entiP) was cloned in (1), plasmid pMG36c under control of the lactococcal constitutive promoter P32, (2) in plasmid pNG8048e under control of the inducible PnisA promoter, and (3) in the integration vector pINT29. Introduction of the recombinant vectors in L. lactis resulted in production of biologically active EntP in the supernatants of L. lactis subsp. lactis IL1403 and L. lactis subsp. cremoris NZ9000, and the coproduction of nisin A and EntP in L. lactis subsp. lactis DPC5598. The level of production of EntP, detected and quantified by specific anti-EntP antibodies and a noncompetitive indirect enzyme-linked immunosorbent assay, by the recombinant L. lactis strains depended on the host strain, the expression vector, and the presence of the entiP gene in the constructs of the recombinant L. lactis strains. The highest amount of EntP was produced with derivatives containing entP and entiP, for both L. lactis IL1403 and L. lactis NZ9000. These derivatives produced up to five- to six-fold more EntP than E. faecium P13. Mass spectrometry analysis revealed that EntP purified from L. lactis IL1403 (pJP214) has a molecular mass identical to that purified from E. faecium P13, suggesting that the synthesis, processing, and secretion of EntP progresses efficiently in recombinant L. lactis hosts.

Keywords

Lactis Lactic Acid Bacterium Heterologous Production General Secretory Pathway Lactis IL1403 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Notes

Acknowledgements

The authors wish to express their gratitude to Prof. R. P. Ross (Teagasc Dairy Products Research Centre, Moorepark, Fermoy, Co. Cork, Ireland) for providing the strain L. lactis DPC5598. This work was partially supported by grants 07G/0026/2000 and S-0505/AGR/0265 from the Comunidad de Madrid, and AGL2000-0707 and AGL2003-01508 from the Ministerio de Educación y Cultura, Spain. J. Gutiérrez is the recipient of a fellowship from the Ministerio de Ciencia y Tecnología (MCYT), Spain.

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Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • Jorge Gutiérrez
    • 1
  • Rasmus Larsen
    • 2
  • Luis M. Cintas
    • 1
  • Jan Kok
    • 2
  • Pablo E. Hernández
    • 1
  1. 1.Departamento de Nutrición, Bromatología y Tecnología de los Alimentos, Facultad de VeterinariaUniversidad Complutense de MadridMadridSpain
  2. 2.Department of Genetics, Groningen Biomolecular Sciences and Biotechnology InstituteUniversity of GroningenAA HarenThe Netherlands

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