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

Am. J. Enol. Vitic. 57:2 (2006) 113 Many potentially toxic substances can be present in food and beverages. Advances in detection methods have lead to the discovery of many compounds in wines, such as ethyl carbamate, that could be harmful to humans. This naturally occurring compound is considered potentially dangerous to humans, as it exhibits carcinogenic activity in a variety of laboratory animals; degradation products of this compound bind covalently to DNA and subsequently induce mutations (Bartsch et al. 1994, Hübner et al. 1997, Leithauser et al. 1990, Schlatter and Lutz 1990). Ethyl carbamate can be found in many fermented foods and beverages in the μg/L or μg/kg range (Canas et al. 1989, Ough 1976, Wittkowski 1997), and consumption of alcoholic beverages can substantially increase daily exposure to ethyl carbamate (Zimmerli and Schlatter 1991). As a consequence, regulatory authorities in several countries have imposed or suggested restrictions on the maximum concentration of this compound in wine and other alcoholic beverages. Ethyl carbamate (EC) forms spontaneously during wine storage because of the reaction of ethanol with carbamyl compounds released during alcoholic and malolactic fermentation. Citrulline, released by lactic acid bacteria, or carbamyl phosphate can react with ethanol to form EC. However, urea released by yeast cells during alcoholic fermentation is the major precursor for EC in wine (Monteiro et al. 1989, Ough et al. 1988a). The most significant source of urea in wine is arginine (Monteiro and Bisson 1991), one of the major amino acids found in grape must and an important nitrogen source for yeast. Arginine is cleaved by arginase (encoded by the CAR1 gene) into ornithine and urea and both of these compounds can be used as a nitrogen source by Saccharomyces cerevisiae (Figure 1). During wine fermentation, urea degradation does not always immediately follow arginine metabolism (Bisson 1996). Consequently, urea is progressively excreted by yeast cells and can be re-absorbed later to be utilized as a nitrogen source. Urea excretion and re-absorption both affect the amount of urea in the wine at the end of the fermentation (An and Ough 1993, Ough et al. 1988b, 1990, 1991). In musts containing higher amounts of assimilable nitrogen (especially arginine), urea can remain in the wine (An and Ough 1993, Ough et al. 1990). Saccharomyces cerevisiae degrades urea in a two-step reaction yielding ammonia, which can be used to synthesize new complex nitrogenous molecules, and CO2. Urea is first carboxylated into allophanate by urea carboxylase in Metabolic Engineering of Saccharomyces cerevisiae to Minimize the Production of Ethyl Carbamate in Wine