重组酿酒酵母合成谷胱甘肽的研究

生物技术通报 ›› 2013, Vol. 0 ›› Issue (8): 160-165.

重组酿酒酵母合成谷胱甘肽的研究

陈加利, 吴量, 王娟, 段学辉

（南昌大学食品科学与技术国家重点实验室生命科学与食品工程学院，南昌 330047）

收稿日期:2013-02-12 修回日期:2013-08-11 出版日期:2013-08-11 发布日期:2013-09-02
作者简介:陈加利，女，硕士，研究方向：功能食品发酵；E-mail：ahhblscjl@163.com
基金资助:
南昌大学食品科学与技术国家重点实验室自由探索课题（201113）

Glutathione Biosynthesis by Engineered Saccharomyces cerevisiae

Chen Jiali, Wu Liang, Wang Juan, Duan Xuehui

（State Key Laboratory of Food Science and Technology，School of Life Science and Food Engineering，Nanchang University，Nanchang 330047）

Received:2013-02-12 Revised:2013-08-11 Published:2013-08-11 Online:2013-09-02

摘要/Abstract

摘要： 利用PCR方法以酿酒酵母Saccharomyces cerevisiae S288c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Kan^r基因和Hyg^r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 cerevisiae TS013，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%。

关键词: γ, -谷氨酰半胱氨酸合成酶, 谷胱甘肽合成酶, 谷胱甘肽, 酿酒酵母

Abstract: 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 Kan^r or Hyg^r 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% and 56.47%, respectively.

Key words: γ-Glutamylcysteine synthetase, GSH synthetase, Glutathione, Saccharomyces cerevisiae

陈加利, 吴量, 王娟, 段学辉. 重组酿酒酵母合成谷胱甘肽的研究[J]. 生物技术通报, 2013, 0(8): 160-165.

Chen Jiali, Wu Liang, Wang Juan, Duan Xuehui. Glutathione Biosynthesis by Engineered Saccharomyces cerevisiae[J]. Biotechnology Bulletin, 2013, 0(8): 160-165.

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