产甘油假丝酵母产甘油关键酶基因在酿酒酵母中的表达

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（glycerol 3-phosphate dehydrogenase, GPD）和3-磷酸甘油磷酸酶（glycerol 3-phosphate phosphatase, GPP）。利用T-Vector在Escherichia coli JM109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 cerevisiae W303-1A。初步实验结果表明：发酵过程中pYX212-GPD/S. cerevisiae W303-1A的生物量高于pYX212-GPP/S. cerevisiae W303-1A和野生型S. cerevisiae W303-1A；发酵72h后，pYX212 GPD/S. cerevisiae W303-1A发酵液中甘油含量大约为12mmol/L，明显高于野生型S. cerevisiae W303-1A的甘油含量，而pYX212-GPP/S. cerevisiae W303-1A与野生型S. cerevisiae W303-1A在甘油含量上相差不大，均只有4mmol/L 左右。

Expression of Key Enzymes in Glycerol Synthesis of Candida Glycerolgenesis in Saccharomyces Cerevisiae W303-1A

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.