Metabolic Engineering Of Saccharomyces Cerevisiae To Reduce Ethyl Carbamate In Rice Wine

In this dissertation, based on the research on the preferred nitrogen sources for a diploidSaccharomyces cerevisiae strain N85, the inhibitory mechanism of normal nitrogen sourceson urea utilization could be proposed. With the guidance of urea metabolism regulation,several metabolic engineering strategies were applied in a model haploid S. cerevisiae strainCEN.PK2-1C. The results showed that the concentration of urea could be significantlyreduced by combining metabolic engineering strategies during the fermentation tests.Furthermore, the optimal strategy used in model strain also had better effect on the haploid S.cerevisiae strain N85. The main results were described as follows:(1) Although diploid S. cerevisiae strain N85is able to use many nitrogen sources for growth,the utilization rates of these components are different. In this study, seven nitrogen sourceswere considered as preferred nitrogen sources for S. cerevisiae strain N85. In addition, it wasfound that there were mainly two kinds of inhibitory effects on urea metabolism by preferrednitrogen sources. Furthermore, regulators of nitrogen catabolite repression (NCR) and targetof rapamycin (TOR) pathway were identified as being involved in urea accumulation byreal-time quantitative PCR. Based on these results, preferred nitrogen sources were found torepress urea utilization by converting them to glutamine or glutamate. Gln3p can be retainedin the cytoplasm by glutamine, while Gat1p can be retained by glutamine and glutamate.(2) Based on the nuclear localization signal (NLS) and nuclear localization regulation signal(NLRS) in Gln3p, the localization of Gat1p, Dal80p and Gzf3p were studied. The residues348–375and366–510were identified as the NLS and NLRS of Gat1p, respectively, and theresidues at positions360(serine) and361(serine) are likely to be the phosphorylation sites inGat1p. Dal80p and Gzf3p are not regulated by phosphorylation, although Gzf3p has an NLSat its C-terminus.(3) In order to increase the nuclear localization of Gln3p and Gat1p, the phosphorylation siteson NLS were mutated and the NLRS was truncated. By combining these strategies, the genes(DUR1,2and DUR3) involved in urea utilization could be significantly activated in thepresence of glutamine. During shake-flask fermentations of the genetically modified strains,little urea accumulated in the media. Furthermore, the disruption of URE2provided anadditional method of reducing urea accumulation. However, this method could not be appliedin S. cerevisiae strain N85because the disruption of URE2would repress the growth of strain.(4) In order to further enhance the expression of DUR1,2and DUR3, several metabolicengineering strategies were attempted on the dephosphorylation regulation, ubiquitylationregulation and the activation of Dal81p and Dal82p. The results showed that the strengtheningeffect of dephosphorylation were limited; whereas overexpression of Dal81p and Dal82pcould enhance the expression of DUR1,2and DUR3. Furthermore, based on the ubiquitylationdetection of regulators, the effect of combining metabolic engineering strategies ondeubiquitylated Dal81p and Dal82p was the best.(5) Based on the former metabolic engineering results, the best strategy used in model strainwas selected. The concentration of urea and EC in a model rice wine system were examined to confirm the effect of metabolic engineering. The results showed that the concentration ofurea and EC could be reduced by63%and72%, respectively. In addition, the examination ofthe normal nutrients and flavour compounds in rice wine indicated that there were fewdifferences in fermentation characteristics between the wild-type S. cerevisiae N85strain andthe genetically modified strain. Therefore, the metabolic engineering strategies attempted inthis study have great potential as the methods for eliminating EC during rice wine production.