安全酵母Candida glycerinogenesis食品级甘油合成关键基因的研究

Study on key glycerol synthesis genes from Candida glycerinogenesis to production food grade glycerol

为了研究酵母甘油合成关键酶基因在食品级甘油合成中的重要作用,以产甘油假丝酵母基因组为模板,设计引物,克隆获得甘油合成关键酶基因3-磷酸-甘油脱氢酶基因(CgGPD),并在大肠杆菌中通过tac启动子串联表达来自酿酒酵母的3-磷酸甘油酯酶基因(ScGPP2)。经IPTG诱导培养后,3-磷酸-甘油脱氢酶(CgGPD)的比酶活达到了60mU/mg。在30%的葡萄糖浓度下,经过48h的摇瓶发酵,可合成甘油2.52g/L。表明3-磷酸甘油酯酶在甘油合成过程中起了关键作用,过表达CgGPD基因有助于食品级甘油大量合成,从而降低下游分离纯化的难度,可从分子水平进一步提高甘油的产量。

To research the key genes in glycerol synthesis mechanism, Candida glycerinogenesis, a safety industrial yeast strains, harbo-ring the perfect ability to yield high production of food-grade glycerol, was used to obtain the key gene. In order to further increasing production and reducing by-products, the research of the key genes in glycerol synthesis mechanism is the necessary. In this study, CgGPD, encoding the key enzyme glycerol 3 phosphate dehydrogenase, was cloned, using the genome of C. glycerinogenesis template. The serial analysis of gene expression with ScGPP2 from Saccharomyces cerevisiae by tac promoter in Escherichia coll. After 48h fermentation, the specific activity of CgGPD reached 60 mU/mg and the production of glycerol reached 2. 52 g/L under 30 g/L glucose. The study showed that CgGPD plays a key role in the synthesis of glycerol, and over expression of CgGPD is beneficial to glycerol synthesis. The results provide a theoretical basis for metabolic regu-lation by over-expression of CgGPD to improved production of process food-grade glycerol and reduce the difficulty of downstream separation and purification, to further increase the molecular level of food-grade glycerol production.