The Effect Of Transcriptome Repressor Da180p On Ethyl Carbamate Formation During Chinese Rice Wine Fermentation

Chinese rice wine, one of the most famous traditional fermented beverages, is widely consumed and highly appreciated all around China for its considerable benefits to human nutrition. Ethyl carbamate (EC) commonly found in fermented beverages has been verified to be a multisite carcinogen in experimental animals. EC was upgraded to Group 2A by the Intl. Agency for Research on Cancer (IARC) in 2007, which indicates that EC is a probable carcinogen to humans. Because of its threat to human health, the presence of EC may be a big challenge for the development of Chinese rice wine. The major contributor to EC accumulation is arginine degradation, which is in turn regulated by nitrogen catabolite repression (NCR). To gain a better insight into EC mitigation strategies, we focused on the nitrogen catabolite repression mechanism in this research. The main contents and conclusions were as follows:(1) The effect of ammonium sulfate towards EC formation in Chinese rice wine is investigated, and the results showed that the addition of ammonium sulfate after 20 days fermentation decreased the arginine usage by Saccharomyces cerevisiae, the urea and EC concentration decreased by 24% and 16% in the fermentation product. Fermentation with BY4741 and SC instead of jiuyao, revealed that arginine concentration in fermentation broth is lower than that in jiuyao fermentation broth. While the urea, EC, cthanol content, volatile components, as well as free amino acids in each fermentation products seemed to be similar to each other. As a result, in the following research, BY4741 can be used for further research on EC formation mechanism.(2) The transcriptome of Saccharomyces cerevisiae using arginine as a sole nitrogen source in three different groups (group 2, YNB medium with 2mM ammonium sulfate and 20mM arginine; group 40, YNB medium with 40mM ammonium sulfate and 20mM arginine; group R, YNB medium with 40mM ammonium sulfate,20mM arginine, and 20nM rapamycin). These transcriptome data were compared with the ones obtained under different culture conditions, transcriptome data of group 40 was using as a control. The results revealed that significantly enriched pathways (corrected P-value<0.05) included carbohydrate metabolism and amino acid metabolism when treated with both low nitrogen and rapamycin. Most NCR-dependent genes expression was highly induced with arginine as a sole nitrogen source, explaining a possible mechanism behind the EC accumulation. Dal80p was regarded as a likely regulator of EC production for its supress regulation on DUR1,2 expression. Moreover, expression of these NCR-regulated genes was determined to be independent of the target-of-rapamycin (TOR) pathway.(3) The deduced primary structure of Dal80p contains a zinc finger motif with extensive homology to the metazoan GATA-binding family of transcriptional activators, Gln3p. Gln3p exhibited a broader sequence specificity than Dal80p, which means Gln3p can bind to the DNA fragments that were independent to Dal80p. In this research, Dal80p was identified to bind to DUR1,2, while not CAR], which was previously reported to bind to Gln3p.(4) The effect of DAL80 mutation towards nitrogen metabolism in Saccharomyces cerevisiae and EC formation in Chinese rice wine fermentation was investigated. The results showed that BY4741 and DAL80△ were characterized with similar growth curves. Under lower nitrogen condition, DAL80△ showed higher arginase activity as BY4741 did. While the urease activity kept in a high level regardless of the nitrogen conditions. The RT-PCR results showed that the deletion DAL80 enhanced the expression of GAP1、DUR1,2、DUR3, while not including CAR1. Fermentation with DAL80△ instead of jiuyao revealed that the urea and EC concentration in fermentation broth were lower. The EC content in 30 days’fermentation broth showed 38.5% and 19.6% decrease when jiuyao was total replaced or half replaced with DAL80△ separately. The ethanol content, volatile components, as well as free amino acids in each fermentation products kept in a similar level. Above all, the DAL80A can be applied to the Chinese rice wine fermentation without affecting its quality.