Construction Of Wine Yeast With Low Urea Production

Ethyl carbamate(EC),a pluripotent carcinogen,originates mainly from urea that is produced by the arginine metabolism of Saccharomyces cerevisae in wine.To achieve minimum production of EC,deletion of CAR1 to decrease the enzymatic activities of arginase,overexpression of DUR1,2 to decrease the enzymatic activities of urea amidolyase and overexpression of DUR3 to increase the levels of urea transporter Dur3p were performed.The main research content was as follows:(1)We constructed a recombinant strain YZ11 by successive deletion of one copy of CAR1 gene and a recombinant strain YZ22 by successive deletion of two CAR1 alleles from a diploid wine yeast WY1,respectively.The RT-qPCR results indicated that the CAR1 expression level of YZ11 was reduced by 63%.The fermentation results showed that the content of urea and EC in wine fermented by YZ11 decreased by 14.56%and 23.71%,respectively.Moreover,the recombinant strain YZ11 was substantially equivalent to parental strain in terms of growth and fermentation characteristics.The recombinant strain YZ22 almost didn't express CAR1 gene,and reduced urea and EC in wine by 77.89%and 73.78%,respectively.The fermentation rate of YZ22 was little slower than that of WY1,the content of arginine in wine fermented by YZ22 was higher than that of WY-1,the content of proline in wine fermented by YZ22 was lower than that of WY-1,and the other fermentation characteristics was equivalent to parental strain.(2)Then DUR1,2-overexpression strain WY-DUR1,2 and DUR3-overexoression strain WY-DUR3 were constructed.In wine fermentation,the recombinant strain WY-DUR1,2 reduced urea and EC in wine by 51.88%and 45.81%,respectively.The recombinant strain WY-DUR3 reduced urea and EC in wine by 54.54%and 48.16%,respectively.Moreover,both the recombinant strains WY-DUR1,2 and WY-DUR3 were substantially equivalent to parental strain in terms of growth and fermentation characteristics.(3)We constructed a recombinant strain CD1,2 by overexpressing DUR1,2,in which CAR1 was deleted.The RT-qPCR results indicated that the CAR1 expression level of CD1,2 was reduced by 63%,and the DUR1,2 expression level of CD1,2 was 3.64-fold higher than that of WY-1.The fermentation results showed that recombinant strain CD1,2 reduced urea and EC in wine by 40.35%and 34.44%,respectively.Moreover,the recombinant strain CD1,2 was substantially equivalent to parental strain in terms of growth and fermentation characteristics.(4)We constructed a recombinant strain CD3 by overexpressing DUR3,in which CAR1 was deleted.The RT-PCR results showed that the CAR1 expression level of CD3 was reduced by 63%,and the DUR3 expression level of CD3 was 3.21-fold higher than that of WY-1.The fermentation results showed that recombinant strain CD3 reduced urea and EC in wine by 45.68%and 36.89%,respectively.Moreover,the recombinant strain CD3 was substantially equivalent to parental strain in terms of growth and fermentation characteristics.