Development of Bacteriocinogenic Strains of Saccharomyces cerevisiae Heterologously Expressing and Secreting the Leaderless Enterocin L50 Peptides L50A and L50B from Enterococcus faecium L50

Abstract

A segregationally stable expression and secretion vector for Saccharomyces cerevisiae, named pYABD01, was constructed by cloning the yeast gene region encoding the mating pheromone ␣-factor 1 secretion signal (MF␣1 s ) into the S. cerevisiae high-copy-number expression vector pYES2. The structural genes of the two leaderless peptides of enterocin L50 (EntL50A and EntL50B) from Enterococcus faecium L50 were cloned, separately (entL50A or entL50B) and together (entL50AB), into pYABD01 under the control of the galactoseinducible promoter P GAL1 . The generation of recombinant S. cerevisiae strains heterologously expressing and secreting biologically active EntL50A and EntL50B demonstrates the suitability of the MF␣1 s -containing vector pYABD01 to direct processing and secretion of these antimicrobial peptides through the S. cerevisiae Sec system.

Use of the Yeast Pichia pastoris as an Expression Host for Secretion of Enterocin L50, a Leaderless Two-Peptide (L50A and L50B) Bacteriocin from Enterococcus faecium L50

“…The enterocin L50-producing strain E. faecium L50 (8) Molecular techniques and enzymes. Established protocols were employed for all DNA manipulations, including PCR amplifications, restriction endonuclease digestions, ligations, and transformations, as previously described (3,24). Oligonucleotide primers (Table 2) were obtained from Sigma-Genosys Ltd. (Cambridge, United Kingdom).…”

“…While enterocin P is secreted by the Sec translocase (30), it has been suggested that the enterocin L50 peptides are secreted by a dedicated ABC transporter, as demonstrated for enterocin Q and other leaderless bacteriocins (12,20,43). Although individual EntL50A and EntL50B possess some antimicrobial activity on their own, with EntL50A being the most active, a clearly synergistic effect is observed when both peptides are combined (2,3,8).…”

“…To overcome the concerns related to the safety of enterococci, enterocin production by alternative and safer hosts, including industrially interesting food-grade LAB and yeast strains, has emerged as a suitable strategy, which may also lead to a strict control of enterocin gene expression at the transcriptional and/or translational level and/or optimization of enterocin production and purification. Furthermore, eukaryotic genetic tools developed for gene expression in yeasts have been shown to be adaptable to bacteriocin genes (3,25,26). Despite the high versatility and efficiency of Saccharomyces cerevisiae and Pichia pastoris for the large-scale heterologous production of a variety of functional proteins (4,6,27,33,44,45), the heterologous production of enterocins by these yeast hosts has been addressed in only a few works.…”

“…Despite the high versatility and efficiency of Saccharomyces cerevisiae and Pichia pastoris for the large-scale heterologous production of a variety of functional proteins (4,6,27,33,44,45), the heterologous production of enterocins by these yeast hosts has been addressed in only a few works. With regard to this, the heterologous production of biologically active enterocin L50 (L50A and L50B) by S. cerevisiae and enterocin P and hiracin JM79 by P. pastoris has demonstrated the possibility of developing bacteriocinproducing yeast strains (3,23,42).…”

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

“…cloned into recombinant S. cerevisiae (2,37,41) and P. pastoris (3,4,21,36) hosts have been produced with variable success regarding its secretion and functional expression. 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).…”