NOVEL STRATEGY FOR HETEROLOGUS EXPRESSION OF ENTEROCIN CRL35 IN Saccharomyces cerevisiae

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

Tucumán

Simposio; V International Symposium on Lactic Acid Bacteria; 2016

CERELA (CONICET)

Bacteriocins are proteinaceous antimicrobial compounds produced by prokaryotes. Most bacteria produce these peptides but those synthesized by the lactic acid bacteria (LAB) generate particular interest. Enterocin CRL35 is a bacteriocin produced by Enterococcus mundtii CRL35. It is active against phylogenetically related bacteria and is a potent bactericidal agent against the foodborne pathogen Listeria monocytogenes. The aim of the present work is to find a novel strategy for heterologous production of enterocin CRL35 in commercial yeasts for industrial applications. Previously, enterocin CRL35-producing yeasts were constructed using plasmid pYEULS, which bears the Ura3 gene (auxotrophic yeast). Here enterocin CRL35 was associated with intracellular fraction of cells demonstrating that the signal peptide used was not efficient in directing enterocin to the culture medium. As novel strategy, we now cloned the structural gene of enterocin CRL35, munA, as a fusion with a secretion signal encoding sequence in the plasmid pCEV-G4-Km. Importantly, the construction was cloned under the strong and constitutive promoter TEF1. The clones were selected in Escherichia coli DH5α and the resulting plasmid pCEV-G4-Km-ENT35 was purified. S. cerevisiae was transformed with pCEV-G4-Km-ENT35 by two different methods i.e. lithium acetate protocol and electroporation. Transformed cells were plated on YPD agar supplemented with 100 µg/ml G418 antibiotic as selective marker. Clones were confirmed by colony PCR by amplification of the complete gene encoding the fused protein (lider peptide + ENTCRL35). As negative control, specific primers for lider peptide and enterocin CRL35 structural gene were used. Bacteriocinogenic yeasts displayed a significant production of enterocin CRL35, almost exclusively in the culture medium with negligible cytoplasmic expression. These results open the possibility of future application of these cells in controlling unwanted LAB in beer and wine industry, reducing the use of sulfur dioxide and sulfites as preservatives.