产甘油假丝酵母胞浆3-磷酸甘油脱氢酶基因(CgGPD)功能分析

Characterization of glycerol-3-phosphate dehydrogenase gene (CgGPD) from the glycerol producing Candida glycerinogenes

【目的】从高产甘油生产菌株产甘油假丝酵母(Candida glycerinogenes)基因组中克隆了NAD^+依赖3-磷酸甘油脱氢酶编码基因(CgGPD),但是该基因及其上游调控序列具体的功能还是未知的。本文研究了CgGPD基因及其上游调控序列的功能。【方法】本文以酿酒酵母(Saccharomyces cere- visiae)及其渗透压敏感型突变株为宿主,构建3种不同的酵母表达载体导人酵母细胞,研究了不同酵母转化子在渗透压胁迫条件下CgGPD基因表达对细胞的耐高渗透压胁迫应答及其细胞的甘油合成能力的影响。【结果】实验结果表明无论是以来源于S.cerevisiae的TPI启动子还是来源于CgGPD基因的启动子,过量表达CgGPD基因的转化子均能够显著加速葡萄糖消耗速度和提高甘油合成能力,在gpd1/gpd2突变株中表达CgGPD基因能够消除细胞对外界高渗透压的敏感性,同时转化子胞内甘油大量积累。【结论】CgGPD基因在野生型酵母S.cerevisiae W303-1A表达显著提高细胞的甘油合成能力,在gpd/1gpd2突变株中能够互补GPD1基因的功能,CgGPD基因表达受渗透压诱导调控。

[Objective] Candida glycerinogenes, an excellent glycerol producer, has been used for commercial scale glycerol production. Recently, we cloned and sequenced the gene encoding NAD＋-dependent glycerol 3-phosphate dehydrogenase （GPD） from C. glycerinogenes and this gene was named CgGPD, which plays an important role in glycerol production. However, compared with GPD1 and GPD2 from S. cerevisiae, the function of CgGPD was unclear to date. [Methods] In this study, a functional charaterization of CgGPD was undertaken, using S. cerevisiae and its isogenic gpd1/gpd2 mutant as expression host under high osmotic stress. [Results] Expression of CgGPD in wide type S. cerevisiae, using either TPI promoter from S. cerevisiae or upstream regulatory sequence of CgGPD accelerated glucose consumption rate and improved glycerol production signifcantly. In osmosensitive mutant, expresion of CgGPD including regulatory sequence increased cells osmotic tolernace and growth profile of transformants restored similar to wide type strain under the high osmotic stress condition. Furthermore, mutants harbouring CgGPD accumulated the intracellular glycerol content markedly and GPD specific enzyme activity increased abruptly when exposed to high osmolarity medium. [Conclusion] CgGPD from C. glycerinogenes compensate the GPD1 in S. cerevisiae functionally.