NOVEL STRATEGY FOR HETEROLOGUS EXPRESSION OF ENTEROCIN CRL35 IN Saccharomyces cerevisiae

MONICA MECHOUD; CARLOS J. MINAHK; SOFIA CURIA; LUCILA SAAVEDRA; JUAN V. FARIZANO; CLAUDIA VICKERS

San Miguel de Tucumán

Simposio; International Symposium on Lactic Acid Bacteria; 2016

CERELA_CONICET

Bacteriocins are proteinaceous antimicrobial compounds produced by prokaryotes. Mostbacteria produce these peptides but those synthesized by the lactic acid bacteria (LAB) generateparticular interest. Enterocin CRL35 is a bacteriocin produced by Enterococcus mundtiiCRL35. It is active against phylogenetically related bacteria and is a potent bactericidal agentagainst the foodborne pathogen Listeria monocytogenes. The aim of the present work is tofind a novel strategy for heterologous production of enterocin CRL35 in commercial yeastsfor industrial applications. Previously, enterocin CRL35-producing yeasts were constructedusing plasmid pYEULS, which bears the Ura3 gene (auxotrophic yeast). Here enterocin CRL35was associated with intracellular fraction of cells demonstrating that the signal peptide usedwas not efficient in directing enterocin to the culture medium. As novel strategy, we nowcloned the structural gene of enterocin CRL35, munA, as a fusion with a secretion signalencoding sequence in the plasmid pCEV-G4-Km. Importantly, the construction was clonedunder the strong and constitutive promoter TEF1. The clones were selected in Escherichiacoli DH5α and the resulting plasmid pCEV-G4-Km-ENT35 was purified. S. cerevisiae was transformedwith pCEV-G4-Km-ENT35 by two different methods i.e. lithium acetate protocol andelectroporation. Transformed cells were plated on YPD agar supplemented with 100 μg/mlG418 antibiotic as selective marker. Clones were confirmed by colony PCR by amplificationof the complete gene encoding the fused protein (lider peptide + ENTCRL35). As negativecontrol, specific primers for lider peptide and enterocin CRL35 structural gene were used.Bacteriocinogenic yeasts displayed a significant production of enterocin CRL35, almost exclusivelyin the culture medium with negligible cytoplasmic expression. These results open thepossibility of future application of these cells in controlling unwanted LAB in beer and wineindustry, reducing the use of sulfur dioxide and sulfites as preservatives