1,3-丙二醇氧化还原酶同功酶基因yqhD在肺炎克雷伯氏菌中的表达研究

Expression of Gene yqhD Encoding 1,3-peopanediol Oxidoreductase Isoenzyme in Klebsiella pnemoniae

以大肠杆菌(Escherichia coli)基因组为模板,PCR扩增出1,3-丙二醇氧化还原酶同功酶基因yqhD,经测序确认,将yqhD基因与四环素抗性基因TetR同时插入表达载体pUC18构建重组质粒pUC18-yqhD-TetR,重组质粒转化Klebsiella pneumoniae ME308;37℃,经1.0mmol/LIPTG诱导8h,重组肺炎克雷伯氏菌中1,3-丙二醇氧化还原酶同功酶的酶活力达到4.16U/mg,而对照菌株的酶活仅为0.62U/mg;将重组菌在摇瓶中进行微氧发酵培养,经IPTG诱导,可将60g/L甘油转化为33.8g/L1,3-丙二醇。

The gene yqhD from Escherichia coli encoding 1,3-propanediol oxidoreductase isoenzyme was amplified by PCR. After combining yqhD and the tetracycline resistant gene Tet^R, they were connected to plasmid pUC18 to yield the recombinant plasimid pUC18-yqhD-Tet^R. The recombinant plasmid was transformed into E. coli DH5a and Klebsiella pneumoniae ME308 separately and the 1,3-propanediol oxidoreduetase isoenzyme was successfully expressed. The 1,3-propanediol oxidoreductase isoenzyme of recombinant Klebsiella pneumoniae reached 4.16 U/mg under the induction of IPTG at 37 ℃ for 8 h, while at the same condition the enzyme activity of the original strain was only 0. 62 U/mg. In the fermentation process of recombinant Klebsiella pneumoniae, 60 g/L glycerol was transformed into 33.8 g/L 1,3-propanediol.