黄孢原毛平革菌乙醇脱氢酶基因的克隆和表达

Molecular cloning and expression of alcohol dehydrogenase gene of Phanerochaete chrysosporium

乙醇是黄孢原毛平革菌(Phanerochaete chrysosporium)在限氧培养条件下重要的代谢物之一,为了更好的理解P.chrysosporium在低氧条件下的代谢机制,文章从P.chrysosporium中克隆到一个长1071 bp的乙醇脱氢酶基因PCAdh1 cDNA,该基因编码一个由356个氨基酸组成的蛋白质,它与其他生物的乙醇脱氢酶的氨基酸序列的相似性很低,但酶催化活性位点序列却高度保守。将PCAdh1在大肠杆菌中表达,并获得有酶活性的重组蛋白。纯化的蛋白质用于制备抗体。半定量RT-PCR和Western blot分析结果显示,在限氧条件的培养过程中,PCAdh1基因在mRNA水平和蛋白水平上都保持相对稳定,表明该基因的表达是组成型的;但从菌丝体提取的粗蛋白中的乙醇脱氢酶活性却随着培养时间的增加及氧气含量的持续降低而逐渐升高,这暗示P.chrysospo-rium中存在其他低氧诱导型乙醇脱氢酶基因的表达。

When Phanerochaete chrysosporium is grown under oxygen-limited condition, ethanol is one of the important metabolites. In order to understand the metabolic mechanism of P chrysosporium grown under oxygen-limited condition, a cDNA sequence （1 071 bp） designated ＂PCAdhl＂ encoding an alcohol dehydrogenase （ADH） was cloned from the filamentous white-rot fungus P chrysosporium. PCAdhl gene encodes a protein of 356 amino acid residues. Although the catalytic domain and coenzyme-binding domain were highly conserved, the protein sequence of PCAdhl showed a low level of similarity to other known ADH. The recombinant PCAdhl protein was expressed in Escherichia coli and its enzyme activity was detected. The protein was purified and used to prepare antibody. Semi-quantitative RT-PCR and Western blot demonstrated that the expression level of PCAdhl in P. chrysosporium remained stable despite the lowered oxygen content, indicating that the gene expression is constitutive. But with the reduction of oxygen content, the overall activity of ADH from the crude mycelia proteins was increased during the growing periods, implying that the expression of other Adh genes in P chrysosporium is inductive.