| Ethyl carbamate(EC), known as a potential carcinogenic compound, is widely found in Chinese rice wine. Due to its toxicity and high content, EC has been one of the biggest challenges in the Chinese rice wine industry. Urea accumulated due to the effect of nitrogen catabolite repression(NCR) in yeast during Chinese rice wine fermentation process is the major precursor of EC. Therefore, the generation of EC could be depressed by reducing urea content. In the present work, metabolic engineered strains were constructed for urea and EC elimination from an industrial yeast Saccharomyces cerevisiae N85 which was particularly used in Chinese rice wine production. In the engineered strain N85DUR1,2-c, the generation of urea was blocked by disruption of CAR1(encodes arginase), meanwhile, the degradation of urea was enhanced by over-expression of DUR1,2(encodes urea amidolyase). The growth traits and fermentation characteristics of engineered strains during industrial production was investigated in the Chinese rice wine brewery. In addition, the effect of yeast citrulline metabolism on the formation of EC in Chinese rice wine was studied.(1) Haploids with opposite mating type(a and α) were isolated from an industrial Chinese rice wine strain S. cerevisiae N85 by disruption of the HO gene. The resistant gene Kan MX from HO disruption cassette was successfully excised from the genome of the haploids using the Cre/lox P system to get the haploids with no exogenous genes or antibiotic genes. Finally, the haploid strains with uracil-defected, which could be used as selective marker, were constructed for further metabolic engineering manipulation.(2) In the present study, the “self-cloning” engineered strains N85-c1 with a heterozygous deletion(CAR1/car1) and N85-c2 with a homozygous defect(car1/car1) were constructed with CAR1 disruption cassette. After small-scale Chinese rice wine fermentation, there was no significant difference between samples fermented with N85-c1 and the parental strain N85 in the concentrations of urea and EC, which were 35.8±1.5 mg·L-1 and 21.2±0.6 μg·L-1, respectively. However, in the Chinese rice sample fermented with N85-c2, the contents of urea and EC were reduced by 86.9% and 50.5%, respectively, compared to that of N85. The results suggested that urea in the Chinese rice wine fermentation were mainly originated from yeast arginine metabolism. In addition, it indicated that the arginase expressed by one wild-type allele CAR1 was sufficient for cellular arginine degradation, which generated urea and therefore EC.(3) The homozygous engineered strains, N85DUR1,2 and N85DUR1,2-c, were constructed through over-expression of DUR1,2 in the parental strain N85 and N85-c2, respectively. Compared with the parental strain N85, the concentrations of urea in Chinese rice wine samples fermented with N85-c2, N85DUR1,2, and N85DUR1,2-c were reduced by 86.9%, 75.6% and 89.1%, respectively, while the EC concentrations were decreased by 50.5%, 40.0% and 55.3%, respectively. The results indicated that blocking of urea generation pathway by deletion of CAR1 was more effective than enhancement of urea degradaion pathway by over-expression of DUR1,2 for urea elimination in Chinese rice wine. The engineered strain N85DUR1,2-c containing over-expressed DUR1,2 and deleted CAR1 could intensely reduce the contents of urea and EC, which were 4.7±0.5 mg·L-1 and 19.6±2.0 μg·L-1, respectively, in the final fermentation liquor.(4) In the engineered strain N85DUR1,2-c, urea generation pathway was blocked by disruption of CAR1, at the same time, the urea metabolism pathway was enhanced by constitutively expressing DUR1,2 even when preferred nitrogen sources(e.g. glutamine and glutamate) were available. As expected, the expression level of DUR1,2 in N85DUR1,2-c increased significantly during Chinese rice wine fermentation process, resulting in the reduction of urea content. Consequently, the concentration of EC in the Chinese rice wine fermented with N85DUR1,2-c was decreased due to the slowdown of reaction rate between urea and ethanol.(5) The effects of citrulline metabolism in yeast on the formation of citrulline and EC in Chinese rice wine fermentation were studied by disruption of ARG3 and over-expression of ARG1 and ARG4. By small-scale fermentation, there was no significant difference in the concentration of citrulline between Chinese rice wine samples fermented with N85-arg3 and the parental strain N85. The results suggested that citrulline in the Chinese rice wine was not mainly derived from yeast metabolism. In Chinese rice wine samples fermented with engineered strains N85ARG1,4 and N85ARG1,4-arg3, in which the ARG1 and ARG4 genes were over-expressed, the contents of citrulline were reduced by 24.1% and 20.4%, respectively, compared to that of N85. Unexpectedly, the concentrations of EC in these Chinese rice wine samples were higher than that of N85. The results indicated that it was unfeasible to reduce EC contents only by the metabolic engineering of citrulline metabolism pathway in yeast strain.(6) Single factor experiments were carried out to investigate the effects of fermentation conditions on urea and EC elimination property of engineered strain N85DUR1,2-c. The results showed that the parameters including inoculation concentration, fermentation temperature, material-water ratio and wheat Qu amount had no significant influences on urea and EC elimination property of N85DUR1,2-c. To investigate the fermentation characteristics of N85DUR1,2-c during Chinese rice wine production process, pilot-scale fermentation was carried out in a 500 liters tank with N85 and N85DUR1,2-c, respectively. It was found that the variation trends of ethanol, total sugar, amino acid nitrogen and total acid during fermentation processes of N85DUR1,2-c and N85 were similar. And the concentrations of main metabolites in both final Chinese rice wine met the national standard. The results indicated that the metabolic manipulation did not affect the fermentation performances of N85DUR1,2-c under the fermentation conditions of brewery. The concentrations of urea and EC in the Chinese rice wine fermented by N85DUR1,2-c were 3.0 mg·L-1 and 16.6 μg·L-1, which were 91.5% and 55.7% less than that in N85, respectively.(7) An industrial production experiment was carried out in a Chinese rice wine brewery by engineered strain N85DUR1,2-c in 50 kiloliters tank. The contents of ethanol, total sugar, amino acid nitrogen, total acid and main flavour compounds in the fermentation liquor of N85DUR1,2-c and N85 were similar except isoamylalcohol and isobutanol, suggesting that there was no significant difference in fermentation characteristics between N85DUR1,2-c and the parental strain N85. In addition, the concentrations of urea and EC were 2.4±0.2 mg·L-1 and 14.9±0.6 μg·L-1 in the liquor fermented by N85DUR1,2-c, which were 90.7% and 54.6% less than that in N85, respectively. Moreover, it was also found that EC increased in a much lower rate during storage owing to the lower urea concentration in the Chinese rice wine sample fermented with N85DUR1,2-c. In conclusion, the engineered strain N85DUR1,2-c, constructed by food-safty grade genetic manipulation, could efficiently reduce the contents of urea and EC in the Chinese rice wine. Consequently, the safty of the Chinese rice wine was improved. |
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