Use of the usp45 lactococcal secretion signal sequence to drive the secretion and functional expression of enterococcal bacteriocins in Lactococcus lactis

Abstract

Replacement of the signal peptide (SP) of the bacteriocins enterocin P (EntP) and hiracin JM79 (HirJM79), produced by Enterococcus faecium P13 and Enterococcus hirae DCH5, respectively, by the signal peptide of Usp45 (SP(usp45)), the major Sec-dependent protein secreted by Lactococcus lactis, permits the production, secretion, and functional expression of EntP and HirJM79 by L. lactis. Chimeric genes encoding the SP(usp45) fused to either mature EntP (entP), with or without the immunity gene (entiP) or to mature HirJM79 (hirJM79), with or without the immunity gene (hiriJM79), were cloned into the expression vector pMG36c, carrying the P(32) constitutive promoter, and into pNZ8048 under control of the inducible PnisA promoter. The production of EntP and HirJM79 by most of the L. lactis recombinant strains was 1.5- to 3.7-fold higher and up to 3.6-fold higher than by the E. faecium P13 and E. hirae DCH5 control strains, respectively. However, the specific antimicrobial activity of the recombinant EntP was 1.1- to 6.2-fold higher than that produced by E. faecium P13, while that of the HirJM79 was a 40% to an 89% of that produced by E. hirae DCH5. Chimeras of SP(usp45) fused to mature EntP or HirJM79 drive the production and secretion of these bacteriocins in L. lactis in the absence of specific immunity and secretion proteins. The supernatants of the recombinant L. lactis NZ9000 strains, producers of EntP, showed a much higher antimicrobial activity against Listeria spp. than that of the recombinant L. lactis NZ9000 derivatives, producers of HirJM79.

Cloning, Production, and Functional Expression of the Bacteriocin Enterocin A, Produced by Enterococcus faecium T136, by the Yeasts Pichia pastoris, Kluyveromyces lactis, Hansenula polymorpha, and Arxula adeninivorans

“…Recombinant LAB, heterologous producers of EntA, also show higher EntA production (1.5-to 18.5-fold) than E. faecium T136, and the EntA they produce shows higher antimicrobial activity (1.5-to 6.6-fold) than that of the EntA produced by E. faecium T136, although the specific antimicrobial activity of the EntA produced was lower than that deduced from its production (10,32). Recombinant LAB, heterologous producers of enterocin P (EntP) and hiracin JM79, also showed higher production and antimicrobial activity of both bacteriocins, whereas their specific antimicrobial activity differed from that of the enterocins produced by the natural enterococcal producers (9,22,36). It has been speculated that the lower specific antimicrobial activity of heterologous bacteriocins produced by LAB may be due to, among many other factors, deficiencies in disulfide bond (DSB) formation, a conserved mechanism for stabilizing extracytoplasmic proteins carried out by thiol-disulfide oxidoreductases (9,10).…”

“…Recombinant LAB, heterologous producers of enterocin P (EntP) and hiracin JM79, also showed higher production and antimicrobial activity of both bacteriocins, whereas their specific antimicrobial activity differed from that of the enterocins produced by the natural enterococcal producers (9,22,36). It has been speculated that the lower specific antimicrobial activity of heterologous bacteriocins produced by LAB may be due to, among many other factors, deficiencies in disulfide bond (DSB) formation, a conserved mechanism for stabilizing extracytoplasmic proteins carried out by thiol-disulfide oxidoreductases (9,10). EntA has two DSBs that seems to improve its antimicrobial activity (15), whereas DSB formation is a prime reason why proteins containing DSBs are difficult to produce in biologically active form by bacterial cell factories (18).…”

“…However, since enterocins may be produced by enterococcal species carrying antibiotic resistance genes and/or genes that code for potential virulence factors, for hygienic, safety, and biotechnological reasons, the production of enterocins in other microbial hosts is being evaluated (9,20,21,22). The enterocin A (EntA) produced by Enterococcus faecium T136 (11) is a class IIa bacteriocin with high antilisterial activity (14,16).…”

Target recognition, resistance, immunity and genome mining of class II bacteriocins from Gram-positive bacteria

“…For example, a larger production, antimicrobial activity and specific antimicrobial activity of the class IIa bacteriocin enterocin P was obtained after heterologous expression in Lc. lactis (Borrero et al, 2011a).…”

Genetic characterization and expression of leucocin B, a class IId bacteriocin from Leuconostoc carnosum 4010

“…The signal peptide of Usp45 has been used previously and shown to be an effective signal peptide for heterologous production of bacteriocins. For instance, class IIa bacteriocins enterocins A and P, pediocin, sakacin A and class IId bacteriocin hiracin JM79 have been functionally cloned as SSusp45 fusions in Lactococcus and other lactic acid bacteria [21,32,33]. Here, leucocins A and B were cloned and expressed in L. lactis and their antimicrobial activities were compared with previously cloned LecC (Figs.…”