产甘油假丝酵母甘油合成关键酶编码基因的克隆

Cloning of the gene encoding a key enzyme involved in production of glycerol in Candida glycerinogenes

产甘油假丝酵母(Candida glycerinogenes)作为优良的甘油生产菌株已经成功应用于工业化生产。但相对于酿酒酵母,该菌株的耐高渗机理和甘油代谢的分子机制还不甚清楚。本文根据已公布的3-磷酸甘油脱氢酶基因的序列信息,设计出一组寡核苷酸,再运用简并PCR结合反向PCR技术从C.glycerinogenes的基因组DNA中获得了4900 bp的核苷酸序列,递交GenBank(No.EU186536)。该序列包含完整的编码胞浆3-磷酸甘油脱氢酶编码基因(CgGPD)开放阅读框及其上、下游调控序列。1167bp的开放阅读框编码388个氨基酸残基的蛋白。所演绎出氨基酸序列分析比对结果表明该基因产物的序列具有典型的胞浆3-磷酸甘油脱氢酶结构特征,但与已鉴定的相关基因存在中等程度的同源性并在相应的辅酶催化位点和底物结合位点区域具有高度的保守性,在氨基酸水平上与安格斯毕赤酵母的相似性最高,达到70.9%。该基因在Saccharomy cescerevisiae W303A中异源表达能够显著提高细胞的甘油合成能力。

Candida glycerinogenes WL2002-5, an excellent glycerol producer, has been used for industrial scale fermentation of glycerol by an aerobic process. However, our knowledge about glycerol biosynthesis at the molecular level and genetic background of this yeast species lags far behind those of model yeasts such as Saccharomyces cerevisiae et al. In this report, inverse primers, in conjunction with degenerated primers, were used to amplify the NAD^＋-dependent glycerol 3-phosphate dehydrogenase （GPD） encoding gene from C. glycerinogenes. The completed nucleotide sequence of the coding, as well as flanking genomic regions was determined （GenBank accession No. EU186536）. DNA sequence analysis revealed the present of the open reading frame （ORF） of 1 167 bp, encoding a polypeptide with 388-amino-acid with a molecular mass of 42 695 Da. The CgGPD did not exhibit significant sequence similarity with others described in other eukaryotic systems by comparative analysis. However, it consisted of two typical functional domains which belong to almost all eukaryotic GPDs： a co-enzyme binding domain in the N-terminal, and a catalytic domain. Moreover, some relevant features involved in initiation, regulation and stress response element of gene transcription were observed in the nucleotide sequence of the 5′-non-coding regions. Heterologous expression of CgGPD gene in S. cerevisiae improved its glycerol production significantly. In conclusion, the functional CgGPD has been cloned and identified successfully from C. glycerinogenes genome.