Deletion analysis of the Ure2p in Saccharomyces cerevisiae and effect of NCR on the production of ethyl carbamate during wine fermentations

[摘要] ENGLISH ABSTRACT:The wine yeast Saccharomyces cerevisiae has the ability to utilize several differentnitrogenous compounds to fulfill its metabolic requirements. Based upon differentgrowth rates of the yeast in a particular nitrogen source, nitrogen compounds havebeen classified as either good or poor nitrogen sources. In an environment whichcontains different quality nitrogen sources, such as grape must, the yeast first utilizesgood and then the poor nitrogen sources. This discrimination between good andpoor nitrogen sources is referred to as nitrogen catabolite repression (NCR).Examples of good nitrogen sources are ammonia, glutamine and asparagine.Nitrogen sources such as allantoin, y-aminobutyrate (GABA), arginine and prolineare poor quality nitrogen sources.Several regulatory proteins, Ure2p, Gln3p, Da180p,Gat1pand Deh1p, mediate NCRin S. cerevisiae. These trans-acting factors regulate transcription of NCR sensitivegenes. All these proteins, except Ure2p, bind cis-acting elements in the promotersof genes that are responsible for degradation of poor nitrogen sources. Gln3p is anactivator of NCR sensitive genes in the absence of good nitrogen sources. Thepredominant mechanism by which NCR functions is by using Ure2p to inactivate theactivator Gln3p in the presence of a good nitrogen source.Several research groups have studied the Ure2p, mainly due to its prion-likecharacteristics. The Ure2p has two domains: a prion inducing domain located in theN-terminal region and a NCR regulatory domain located in the C-terminal domain.The aims of this study were (i) to determine the part of the C-terminal domain whichis responsible for NCR, (ii) to establish if ure2 deletion mutants produce less ethylcarbamate during wine fermentations and (iii) if NCR functions in industrial yeaststrains. Nested deletions of the URE2 gene revealed that the NCR regulatorydomain resides in the last ten amino acids of the Ure2p. This was established byNorthern blot analysis on the NCR sensitive genes DAL5, CAN1, and GAP1 genes.Ethyl carbamate in wine is produced by spontaneous chemical reaction betweenurea and ethanol in wine. Urea is produced by S. cerevisiae during the metabolismof arginine. Arginine is degraded to ornithine and urea by arginase, the product ofthe CAR1 gene. Degradation of urea by S. cerevisiae is accomplished by ureaamidolyase, a bi-functional enzyme and product of the DUR1,2 gene which is subjectto NCR. This study investigated if a ure2 mutant strain produced less ethylcarbamate during wine fermentations.Wine fermentations were conducted with diploid laboratory strains: a ure2 mutantstrain and its isogenic wild type strain. GC/MS analysis of the wine revealed that theure2 mutant produced less ethyl carbamate but more ethanol than the wild typestrain when arginine, di-ammoniumphosphate, asparagine or glutamine were addedas nitrogen sources, in combinations and separately. There was no significantdifference between the wild type fermentation and the ure2 mutant fermentationwhen no nitrogen was added. It was found that a combination between the deletionof URE2 and the addition of a good nitrogen source resulted in lower levels of ethylcarbamate.High density micro array analysis done on an industrial strain wine yeast inChardonnay grape must revealed that the GAP1, CAN1, CAR1 and DUR1,2 genes,responsible for transport and metabolism of arginine and degradation of urea, areNCR sensitive. These data strongly suggest that NCR functions in industrial yeaststrains.

[发布机构] Stellenbosch University