重组酿酒酵母合成谷胱甘肽的研究

Glutathione Biosynthesis by Engineered Saccharomyces cerevisiae

利用PCR方法以酿酒酵母Saccharomyces cerevisiaeS288c基因组DNA为模板,PCR扩增γ-谷氨酰半胱氨酸合成酶(GSH1)及谷胱甘肽合成酶(GSH2)基因,以乙醇脱氢酶(ADH1)启动子进行表达,并以Kanr基因和Hygr基因作为筛选标记,构建了两个重组表达质粒YEplace181AK-GSH1和YEplace181AH-GSH2。采用电击法将这两个质粒分别和同时转入工业酿酒酵母菌株Saccharomyces cerevisiaeTS013,在含有G418或(和)Hygromycin的平板上筛选阳性克隆S.TS013/GSH1、S.TS013/GSH2和S.TS013/GSH1+GSH2。重组菌进行摇瓶发酵,采用四氧嘧啶法测定培养细胞合成谷胱甘肽含量。结果显示,不同转化子重组菌的胞内谷胱甘肽产量比宿主菌分别提高了44.11%、29.79%和56.47%。

The GSH1 and GSH2 genes were amplified from the extracted total DNA of Saccharomyces cerevisiae S288c by PCR and expressed under the control of alcohol dehydrogenase（ADH1）promoter.With Kanr or Hygr as the selective markers,two expression vectors YEplace181AK-GSH1 and YEplace181AH-GSH2 were constructed.The recombinant plasmids were transformed into an industry strain Saccharomyces cerevisiae TS013 respectively and simultaneously by electroporation,and three recombinant strains S.TS013/GSH1,S.TS013/GSH2 and S.TS013/GSH1＋GSH2 were generated by screening on YPD plates supplemented with G418 or（and）Hygromycin.The transformants were cultured in fermentation media,afterwards intracellular GSH content was detected by the method of ALLOXAN.Compared with the content of control strain,the glutathione content of recombinant strains increased by 44.11%,29.79% 和 56.47%,respectively.