非特异性二元醇氧化还原酶的克隆与表达

Cloning and expression of non-specific diol oxidoreductase

利用PCR技术分别从克雷伯氏产酸杆菌、克雷伯氏肺炎杆菌中扩增出基因yqhD,编码非特异性二元醇氧化还原酶(OX)。并利用强启动子使重组质粒OX1-pDK、OX2-pDK在生产菌株K.pneumoniaeAC01中得到高效表达。DNA测序结果表明,扩增的序列与已报道的yqhD阅读框同源性分别为80.84%和80.67%。在有氧条件下摇瓶发酵培养,含OX1-pDK的基因工程菌1,3-PDO产量比对照菌株增长了8.18%,含OX2-pDK的基因工程菌1,3-PDO产量比对照菌株增长了14.33%。

The non-specific diol oxidoreductase encoding genes(yqh D,1.16 kb) from Klebsiella oxytoca,Klebsiella pneumoniae were amplified by PCR technique.The genes of yqh D was inserted in pDK to yield the recombinant expression vector OX1-pDK,OX2-pDK.Over-expression of yqh D in Klebsiella pneumoniae AC01 was achieved by strong promoter.DNA sequence analysis showed that nucleotide sequence identity of yqh D gene from Klebsiella oxytoca with the nucleotide sequence being reported was 80.84%,yqh D gene from Klebsiella pneumoniae with the nucleotide sequence being reported was 80.67%,respectively.Fermentation process under aerobic condition showed that after induction with 1.0 mmol/L IPTG,1,3-PD yield of the recombinant strain with OX1-pDK was 8.18% higher than that of the wild strain,and carbon recovery increased 9.93%.1,3-PD yield of the recombinant strain with OX2-pDK was 14.33% higher than that of the wild strain,and carbon recovery increased by 5.10%.