产甘油假丝酵母与酿酒酵母胞浆3-磷酸甘油脱氢酶基因的功能比较

Comparative Characterization of Genes Encoding Glycerol 3-phosphate Dehydrogenase From Candida glycerinogenes and Saccharomyces cerevisiae

胞浆3-磷酸甘油脱氢酶(GPD)是酿酒酵母细胞甘油合成过程中的关键限速酶.尽管高产甘油菌株产甘油假丝酵母基因组中编码该酶的基因CgGPD已经被克隆出来,但是具体的功能,特别是与酿酒酵母GPD1和GPD2基因的功能比较值得进一步研究.以酿酒酵母渗透压敏感型的gpd1/gpd2和gpd1突变株为宿主,分别导入CgGPD、GPD1和GPD2基因,比较分析了CgGPD、GPD1和GPD2基因在高渗透压胁迫条件下和厌氧环境中的表达调控,及其对细胞甘油合成能力的影响.研究发现,GPD1基因受到渗透压诱导表达,GPD2基因在细胞厌氧条件下起着氧化还原平衡调节作用,而CgGPD基因不仅能够在渗透压胁迫条件下通过过量快速合成甘油调节渗透压平衡,而且能够在厌氧培养环境中互补GPD2基因的缺失,使gpd1/gpd2缺失突变株能够正常生长,同时提高了突变株的甘油合成能力.结果表明,CgGPD基因在gpd1/gpd2缺失突变株中既具有GPD1基因的功能,又能发挥GPD2基因的功能.

NAD^＋-dependent glycerol 3-phosphate dehydrogenase （GPD） are rate limiting for glycerol production in Saccharomyces cerevisiae. Recently, the gene CgGPD encoding glycerol 3-phosphate dehydrogenase homologous to GPO genes in other yeasts was cloned from Candida glycerinogenes WL2002-5, an excellent industrial glycerol producer. However, the knowledge about CgGPD expression regulation, especially difference with GPD1 and GPD2 from S. cerevisiae is so less. A functional comparison of CgGPD from C. glycerinogenes with GPD1 and GPD2 from S. cerevisiae was undertaken, using S. cerevisiae gpd1/gpd2 and gpdl osmosensitive mutants as expression systems. The functions of three indicated genes in S. cerevisiae was characterized for osmoregulation under high osmotic stress and redox regulation under anaerobic condition with various transformants. The results showed that gpd1/gpd2 mutants harbouring CgGPD and GPD1 can restore osmotolerance and increase glycerol production ability under hyperosmotic stress but mutant expressed GPD2 can not. When cells were cultured under the anaerobic condition, the growth pattern of mutants harbouring CgGPD and GPD2 are smilar, however the mutant harbouring GPD1 grows slowly and the growth of the controll supresses thoroughly. Furthermore, gpd1/gpd2 mutant employing either CgGPD or GPD2 can increase glycerol production ability and improve GPD specific enzyme activity in anaerobic incubation, but gpd1/gpd2 mutant expressed GPD1 has no the similar results under the same condition. This indicated that CgC, PD may involve in both osmoregulation and redox balance and can complement GPD1 and GPD2 in gpd1/gpd2 mutant.