title: High-level heterologous production and functional expression of the sec-dependent enterocin P from Enterococcus faecium P13 in Lactococcus lactis.
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.
authors.affiliation: Departamento de Nutrición, Bromatología y Tecnología de los Alimentos, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040, Madrid, Spain.
authors.forename: Jorge
authors.initials: J
authors.lastname: Gutiérrez
authors.affiliation: Department of Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, 9751, AA Haren, The Netherlands.
authors.forename: Rasmus
authors.initials: R
authors.lastname: Larsen
authors.affiliation: Departamento de Nutrición, Bromatología y Tecnología de los Alimentos, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040, Madrid, Spain.
authors.forename: Luis M
authors.initials: LM
authors.lastname: Cintas
authors.affiliation: Department of Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, 9751, AA Haren, The Netherlands.
authors.forename: Jan
authors.initials: J
authors.lastname: Kok
authors.affiliation: Departamento de Nutrición, Bromatología y Tecnología de los Alimentos, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040, Madrid, Spain. ehernan@vet.ucm.es.
authors.forename: Pablo E
authors.initials: PE
authors.lastname: Hernández
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001430:Bacteriocins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D003001:Cloning, Molecular; D003470:Culture Media / Q000737:chemistry; D016984:Enterococcus faecium / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D005822:Genetic Vectors; D013294:Lactococcus lactis / Q000235:genetics* / Q000378:metabolism; D013058:Mass Spectrometry; D008970:Molecular Weight; D009561:Nisin / Q000096:biosynthesis; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D001430:Bacteriocins; D003470:Culture Media; D011994:Recombinant Proteins; C417717:enterocin P; D009561:Nisin; C102526:nisin A