UBC Theses and Dissertations
Metabolic engineering of industrial yeast strains to minimize the production of ethyl carbamate in grape and Sake wine Dahabieh, Matthew Solomon
During alcoholic fermentation Saccharomyces cerevisiae metabolizes L-arginine to ornithine and urea. S. cerevisiae can metabolize urea through the action of urea amidolyase, encoded by the DUR1,2 gene; however, DUR1,2 is subject to nitrogen catabolite repression (NCR) in the presence of high quality nitrogen sources during fermentation. Being cytotoxic at high concentrations, urea is exported into wine where it spontaneously reacts with ethanol, and forms the carcinogen ethyl carbamate (EC). Urea degrading yeast strains were created by integrating a linear cassette containing the DUR1,2 gene under the control of the S. cerevisiae PGK1 promoter and terminator signals into the URA3 locus of the Sake yeast strains K7 and K9. The ‘self-cloned’ strains K7EC- and K9EC- produced Sake wine with 68% less EC. The Sake strains K7EC- and K9EC- did not efficiently reduce EC in Chardonnay wine due to the evolutionary adaptation of said strains to the unique nutrients of rice mash; therefore, the functionality of engineered yeasts must be tested in their niche environments as to correctly characterize new strains. S. cerevisiae possesses an NCR controlled high affinity urea permease (DUR3). Urea importing yeast strains were created by integrating a linear cassette containing the DUR3 gene under the control of the PGK1 promoter and terminator signals into the TRP1 locus of the yeast strains K7 (Sake) and 522 (wine). In Chardonnay wine, the urea importing strains K7D3 and 522D3 reduced EC by 7% and 81%, respectively; reduction by these strains was equal to reduction by the urea degrading strains K7EC- and 522EC-. In Sake wine, the urea degrading strains K7EC- and 522EC- reduced EC by 87% and 84% respectively, while the urea importing strains K7D3 and 522D3 were significantly less capable of reducing EC (15% and 12% respectively). In Chardonnay and Sake wine, engineered strains that constitutively co-expressed DUR1,2 and DUR3 did not reduce EC more effectively than strains in which either gene was expressed solely. Uptake of 14C-urea under non-inducing conditions was enhanced in urea importing strains; parental strains failed to incorporate any 14C-urea thus confirming the functionality of the urea permease derived from the integrated DUR3 cassette.
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