Heterologous expression of enterocin A, a bacteriocin from Enterococcus faecium, fused to a cellulose-binding domain in Escherichia coli results in a functional protein with inhibitory activity against Listeria

Abstract

The genes for the bacteriocins enterocin A and B were isolated from Enterococcus faecium ATB 197a. Using the pET37b(+) vector, the enterocin genes were fused to an Escherichia coli specific export signal sequence, a cellulose-binding domain (CBD(cenA)) and a S-tag under the control of a T7lac promotor. The constructs were subsequently cloned into E. coli host cells. The expression of the recombinant enterocins had different effects on both the host cells and other Gram-positive bacteria. The expression of entA in Esc. coli led to the synthesis and secretion of functional active enterocin A fusion proteins, which were active against some Gram-positive indicator bacteria, but did not influence the viability of the host cells. In contrast, the expression of enterocin B fusion proteins led to a reduced viability of the host cells, indicating a misfolding of the protein or interference with the cellular metabolism of Esc. coli. Indicator strains of Gram-positive bacteria were not inhibited by purified enterocin B fusion proteins. However, recombinant enterocin B displayed inhibitory activity after the proteolytic cleavage of the fused peptides.

pMPES: A Modular Peptide Expression System for the Delivery of Antimicrobial Peptides to the Site of Gastrointestinal Infections Using Probiotics

“…For example, the Enterococcus -derived AMPs Enterocin A and Hiracin JM79 (HirJM79) have been successfully secreted by the Lactococcus lactis and Lactobacillus native general secretion machinery, using the Lactococcus -derived Usp45 signal peptide [23,24,25]. …”

Enhanced Production, Purification, Characterization and Mechanism of Action of Salivaricin 9 Lantibiotic Produced by Streptococcus salivarius NU10

“…Different genera of lactic acid bacteria (LAB) can produce different kinds of antimicrobial peptides and bacteriocins such as plantaricin produced by Lactobacillus plantarum [1], enterococcin produced by Enterococcus faecium [2], leucocin produced by Leuconostoc carnosum [3], pediocin produced by Pediococcus acidilactici [4] and others. Interest in bacteriocins has increased recently due to their antimicrobial activity towards Gram-positive pathogens [5-7].…”

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

“…The presence of Listeria monocytogenes in food can be reduced by in situ EntA production (23,29), by the addition of semipurified or purified preparations of EntA (1,24), or by the incorporation of EntA into biodegradable packaging films (31). The heterologous production of EntA by bacterial hosts has been attained through the expression of its native biosynthetic genes (29), by exchange or replacement of the EntA leader peptide and/or dedicated processing and secretion systems (33), or by fusion of mature EntA to signal peptides that act as secretion signals (10,28,32). Recently, several yeast platforms have been developed for the large-scale expression of proteins (5,7,13,19).…”