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 alpha-factor 1 secretion signal (MFalpha1(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 galactose-inducible promoter P(GAL1). The generation of recombinant S. cerevisiae strains heterologously expressing and secreting biologically active EntL50A and EntL50B demonstrates the suitability of the MFalpha1(s)-containing vector pYABD01 to direct processing and secretion of these antimicrobial peptides through the S. cerevisiae Sec system.

FIG. 1.

(A) Construction of the S. cerevisiae protein expression and secretion vector pYABD01, derived from pPICZαA and pYES2, containing the yeast gene region encoding the mating pheromone α-factor 1 secretion signal (MFα1_s), including the nucleotides encoding the Kex2 signal cleavage site under the control of the galactose-inducible promoter P_GAL1. (B) Construction of the recombinant plasmids pYABD02, pYABD03, and pYABD04, derived from pYABD01, containing MFα1_s fused in frame to entL50A and/or entL50B. Plasmid sizes are given in base pairs. Only relevant restriction enzyme sites are shown. 5′AOX1, promoter region; AOX1 TT, transcription termination; P_TEF1, transcription elongation factor 1, driving expression of the Sh ble gene in Pichia; P_EM7, constitutive promoter driving expression of the Sh ble gene in E. coli; Zeocin gene, zeocin resistance (Sh ble gene); CYC1 TT, transcription terminator; pUC ori, maintenance and high-copy-number replication in E. coli; Amp, ampicillin resistance gene; Ura3, gene for selection of yeast transformants in uracil-deficient medium; 2μ ori, maintenance and high-copy-number replication in yeast; f1 ori, rescue of single-stranded DNA; entL50A, structural gene of EntL50A; entL50B, structural gene of EntL50B; P_T7, T7 promoter driving expression of heterologous gene expression; Koz, Kozak translation initiation sequence of yeasts.

FIG. 2.

Antimicrobial activity of supernatants from S. cerevisiae L50A-20(pYABD02) (EntL50A) and S. cerevisiae L50B-4(pYABD03) (EntL50B) cultures grown in SCGR broth at 30°C, independently and combined to achieve a 1:1 bacteriocin peptide ratio (EntL50A+EntL50B), against P. damnosus CECT4797 as determined by a SPAT.

FIG. 3.

Mass spectrometry analysis of recombinant EntL50A (A) and EntL50B (B) purified from S. cerevisiae L50A-20(pYABD02) and S. cerevisiae L50B-4(pYABD03) cultures, respectively, grown in SCGR broth at 30°C.

FIG. 4.

(A) Tricine-SDS-PAGE of purified recombinant EntL50A and EntL50B after silver staining. (B) Western blotting using rabbit polyclonal antibodies with specificity for EntL50A (anti-LR1-KLH) and EntL50B (anti-LR2-KLH). (C) Antimicrobial activity after gel overlay with the indicator strain P. damnosus CECT4797. Lane 1, purified EntL50A; lane 2, purified EntL50B. SeeBlue Pre-Stained Standard molecular mass markers band sizes (M) (Invitrogen) are indicated on the left.