产甘油假丝酵母产甘油关键酶基因在酿酒酵母中的表达  

Expression of Key Enzymes in Glycerol Synthesis of Candida glycerolgenesis in Saccharomyces cerevisiae W303-1A

作　　者：赵有玺[1,2] 饶志明[1] 沈微[1] 方慧英[1] 诸葛健[1] 

机构地区：[1]江南大学工业生物技术教育部重点实验室江南大学生物工程学院工业微生物研究中心 [2]北京联合大学生物化学工程学院北京100023

出　　处：《中国生物工程杂志》2006年第1期38-41,共4页China Biotechnology

基　　金：国家自然科学基金资助项目(30300027);江南大学工业生物技术教育部重点实验室开放基金资助项目(KLIB-KF200507);江南大学预研基金资助项目(0004402)

摘　　要：甘油是一种极其理想的耐高渗透压介质。利用PCR方法,从产甘油假丝酵母WL2002-5中扩增出了2个产甘油的关键酶基因GPD和GPP,分别编码3-磷酸甘油脱氢酶(glycerol3-phosphatedehydrogenase,GPD)和3-磷酸甘油磷酸酶(glycerol3-phosphate phosphatase,GPP)。利用T-Vector在Escherichia coliJM109中克隆得到大量的GPD和GPP基因,并成功构建了重组质粒pYX212-GPD和pYX212-GPP;通过LiAc转化法将重组质粒导入酿酒酵母Saccharomyces cerevisiaeW303-1A。初步实验结果表明发酵过程中pYX212-GPD/S.cerevisiaeW303-1A的生物量高于pYX212-GPP/S.cerevisiaeW303-1A和野生型S.cerevisiaeW303-1A;发酵72h后,pYX212-GPD/S.cerevisiaeW303-1A发酵液中甘油含量大约为12mmol/L,明显高于野生型S.cerevisiaeW303-1A的甘油含量,而pYX212-GPP/S.cerevisiaeW303-1A与野生型S.cerevisiaeW303-1A在甘油含量上相差不大,均只有4mmol/L左右。Glycerol is an ideal osmotolerant medium. Two genes of GPD and GPP encoding glycerol 3- phosphate dehydrogenase and glycerol 3-phosphate phosphatase, key enzymes in glycerol synthesis, were cloned from Candida glycerolgenesis WL2002-5 using PCR method. In Escherichia coli JM109, enough GPD and GPP genes were amplified using T-vector, furthermore, expression plasmids pYX212-GPD containing GPD gene and pYX212-GPP containing GPP gene were constructed, which were introduced into Saccharomyces cerevisiae W303- 1A using LiAc transformation method successfully. Primary results showed that the biomass of pYX212-GPD/S. cerevisiae W303-1A was higher than those of pYX212-GPP/S, cerevisiae W303-1A and the wild type during the fermentation. After 72h fermentation, introduction of GPD gene could increase glycerol production to about 12mmoL/L, while GPP gene had little effect on glycerol production （only 4mmoL/L） in comparison with the wild type.

关 键 词：甘油 3-磷酸甘油脱氢酶 3-磷酸甘油磷酸酶 酵母工程菌 

分 类 号：Q786[生物学—分子生物学]