Metabolic Engineering ofSaccharomyces cerevisiaeto Minimize the Production of Ethyl Carbamate in Wine

Abstract

<i>Saccharomyces cerevisiae</i> metabolizes arginine, one of the major amino acids in grape musts, to ornithine and urea during wine fermentations. Wine yeast strains of <i>S. cerevisiae</i> 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 <i>DUR1,2</i> gene by good nitrogen sources present in the grape must. We expressed the <i>DUR1,2</i> gene under control of the <i>S. cerevisiae PGK1</i> promoter and terminator signals and integrated this <i>DUR1,2</i> expression cassette, flanked by ura3 sequences, into the <i>URA3</i>-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.