乙醇脱氢酶基因和乙醛脱氢酶基因的克隆及表达

Cloning and expression of adh2 and aldh6

利用表达载体pYES2表达乙醇脱氢酶2(Alcohol dehydrogenase 2,ADH2)和乙醛脱氢酶6(Acetaldehyde dehydrogenase 6,ALDH6),从而实现将乙醇直接转化为乙酸。以啤酒酵母总DNA为模板,通过PCR获得adh2基因和aldh6基因。分别构建表达载体pYES2-adh2、pYES2-aldh6和pYES2-aldh6-adh2,并电击导入宿主菌INVScl中。PCR测序结果上传GenBank获得登录号为JX901290、JX901291,与已公布序列相似性分别为96%和99%。重组菌扩大培养后移入到诱导培养基中,进行诱导表达。在含2%半乳糖诱导培养基诱导下4 h酶活达到最高,ADH酶活为0.49U/mg,是原始菌株的2.7倍,ALDH酶活为0.11 U/mg,为原始菌株的2倍。

In order to realize ethanol directly transform to acetic acid, the alcohol dehydrogenase 2 （adh2） and acetalde-hyde dehydrogenase 6 （aldh6） genes were cloned and expressed. The genomic DNA of Saccharomyces cerevisiae as tem-plate was extracted and adh2 and aldh6 genes were obtained by PCR. Two genes were inserted into expression vector pY-ES2. Then the expression vectors pYES2-adh2, pYES2-aldh6 and pYES2-aldh6-adh2 were constructed. They were trans- formed to INVScl. Sequence tests showed that the acquired fragments exhibited 96% and 99% homology to adh2 and al-dh6 gene sequences from GenBank report. Then these recombinants were grown on glucose and shift to inductive medium. The activities reached to the highest when the recombinants were induced for 4 hours. The activities of adh2 and aldh6 were 2.7-fold and 2-fold higher than those of the wild types, respectively.