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Metabolic Engineering of Saccharomyces cerevisiae to Minimize the Production of Ethyl Carbamate in Wine

¹ Ph.D. student, ⁵ Professor, Faculté d’Oenologie, Unité Associée INRA, Université Victor Segalen Bordeaux 2, Talence, France; ^{2, 6} Ph.D. students, ⁷ Professor and Eagles Chair, Wine Research Centre, The University of British Columbia, Vancouver, BC, Canada; ³ Associate professor, Cool Climate Oenology and Viticulture Institute, Brock University, St. Catharines, ON, Canada; and ⁴ Assistant professor, Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada.

^* Corresponding author [fax: 604-822-5143; email: hjjvv{at}interchange.ubc.ca]

Saccharomyces cerevisiae metabolizes arginine, one of the major amino acids in grape musts, to ornithine and urea during wine fermentations. Wine yeast strains of S. cerevisiae do not fully metabolize urea during grape must fermentation. Urea is secreted by yeast cells and it reacts spontaneously with ethanol in wine to form ethyl carbamate, a potential carcinogenic agent for humans. The lack of urea catabolism by yeast in wine may be ascribed to the transcriptional repression of the DUR1,2 gene by good nitrogen sources present in the grape must. We expressed the DUR1,2 gene under control of the S. cerevisiae PGK1 promoter and terminator signals and integrated this DUR1,2 expression cassette, flanked by ura3 sequences, into the URA3-locus of the industrial wine yeast UC Davis 522. In vivo assays showed that the metabolically engineered industrial strain reduced ethyl carbamate in Chardonnay wine by 89.1%. Analyses of the genotype, phenotype, and transcriptome revealed that the engineered yeast 522^EC– is substantially equivalent to the parental 522 strain.

Key words: wine, ethyl carbamate, DUR1,2, carcinogen

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