利用PDOR同工酶基因yqhD对产1,3-丙二醇克雷伯氏杆菌进行基因工程改造

由于Klebsiella pneumoniae 1,3-丙二醇合成途径中，加强甘油脱水酶基因表达，导致因NADH供应不足使3-羟基丙醛累积，并对菌体生长及1,3-丙二醇合成造成负面影响。为改善Klebsiella pneumoniae 1,3-丙二醇合成途径，本文利用PCR技术从大肠杆菌(Escherichia coli)中扩增出以NADPH 为辅酶的1,3-丙二醇氧化还原酶同工酶编码基因yqhD，从克雷伯氏杆菌中扩增出2.66kb的甘油脱水酶基因（dhaB），构建了产1,3-丙二醇关键酶基因的串联载体pEtac-dhaB-tac-yqhD，并将其转入到野生克雷伯氏杆菌(Klebsiella pneumoniae)中，重组载体得到了表达。通过初步发酵，重组后的克雷伯氏杆菌产量比原始菌高20%左右，副产物中乙酸和丁二醇分别下降30%左右。

Genetic engineering reconstruction of Klebsiella pneumoniae producing 1,3-propanediol by the gene yqhD encoding 1,3-propanediol oxidoreductase isoenzyme

In the reductive branch, glycerol is first dehydrated to 3-hydroxypropionaldehyde that is then reduced to 1,3-PD under the consumption of reducing power (NADH). If over-expression of the gene dhaB encoding glycerol dehydrase is achieved,the reducing power will be scarce and 3-hydroxypropionaldehyde will be accumulated,which is disadvantage to produce 1,3-propanediol.The structure gene yqhD from E.coli encoding 1,3-propanediol oxidoreductase isoenzyme（under the consumption of reducing power (NADPH)）and the gene dhaB encoding glycerol dehydrase from Klebsiella pneumoniae was amplified using PCR method.The two gene were transferred into expression vector pEtac to construct a novel recombinant Klebsiella pneumoniae (pEtac-dhaB-tac-yqhD).Over-expression of yqhD and dhaB in Klebsiella pneumoniae was achieved with pEtac-dhaB-tac-yqhD.The fermentation result on aerobic conversion showed the increase of 20 % of 1,3-propanediol yield by Klebsiella pneumoniae(pEtac-dhaB-tac-yqhD) was obtained compared with Klebsiella pneumoniae.The main by-products,acetic acid and butanediol decreased estrogen receptors 30% obviously.