| Urethane (Ethyl carbamate, EC) is a potential carcinogen present in the Chinese rice wine. Studies showed that the EC content of commercial rice wine was generally higher than the standard of FAO. So it is necessary to carry out a research to reduce the content of EC in the rice wine. EC is mainly generated by the reaction between urea and ethanol. Therefore, we can construct a genetic modified Saccharomyces cerevisiae strain or change raw material to avoid the introduction of the EC precursor.In this study, SFH-PCR-mediated gene knockout technique was adopted to delete Saccharomyces cerevisiae arginase gene. One arginase gene deletion strain was obtained and named SAcarl. The fermentation performance, EC content and amino acids content of rice wine fermentated by SAcarl and AH 109 were compared. Moreover, two amylase expression vectors S△car1/Al with ADH1 promoter and SAcar1/A2 with ADH2 promoter were constructed, then transformed into S△car1 strain, respectively. Comparative analysis of amylase expression in the transformants was carried out. Results are as following.1. Deletion of Saccharomyces cerevisiae arginase gene car1In order to knockout the car1 gene of Saccharomyces cerevisiae, car1 gene knockout cassette was constructed by SFH-PCR and transformed into the wild-type AH109. A arginase gene deletion strain S△carl was obtained and the arginase activity of the S△car1 strain cultivated in YNBG (containing 0.1% arginine) was analyzed. The results showed that no arginase activity was dectected in the SAcarl strain while the arginase activity of the wild-type strain AH109 reached the maximum value,5.2 U, at 18 h. These results suggested that the arginase gene had been proved successfully knockouted in S. cerevisiae.2. Analysis of rice wine fermentation with arginase knockout mutant S.cerevisiae S△carlRice wine was fermented by adding SAcarl and AH109 into glutinous rice. The fermenting process was measured by analyzing the alcoholicity, saccharides content, pH and total acidity of broth. The results indicated that the fermentation efficiency was not obviously influenced by the arginase gene disruption. The category and content of amino acids in rice wine fermented by SAcarl and AH 109 were analyzed. AH 109 strain produced 0.44 g/100L, and S△carl strain produced 5.3 g/100L arginine (about 12 times of AH109); S△carl strain produced 0.58 g/100L omithine, AH109 strain produced 1.98 g/100L (about 3 times of SAcarl). Moreover, the content of lusine and cysteine was analyzed, AH109 strain produced 1.46 g/100L lusine and 0.63 g/100L cysteine, SAcarl strain produced 3.16 g/100L lusine and 1.38 g/100L cysteine (approximately 2 times of the wild-type)The fermentation rice wine was heated under 80℃and the content of EC was detected. S△carl produced 5.93μg/L EC while AH 109 produced 40.65μg/L EC (approximately 8 times of the SAcarl). These results suggested that the content of EC can be decreased because the arginase gene knockout in S. cerevisiae, but cannot avoided completely.lt may because of EC precursor containing in the raw material.3. Construction of amylase expression cassette and the analysis of yeast transformantAmylase expression vector pADHl/ALP1 with ADH1 promoter and pADH2/ALP1 with ADH2 promoter were constructed and transformed into SAcarl. Transformants named as S△car1/Al and S△carl/A2 were screened on the sole carbon source YNBS starch plate and verified by PCR. The amylase production capacity under the different concentration of the glucose of the SAcarl/A1 and SAcarl/A2 strains were analyzed. The results showed that the halo circle of S△carl/Al was becoming bigger with the increase content of glucose. Yet, the situation in SAcarl/A2 were opposite to SAcar1/A1. Such results illustrated that ADH2 promoter was negatively regulated by glucose, and ADH1 is not affected by this effect. Even so, ADH2 promoter show more efficient than ADH1 promoter. |
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