共表达1,3-丙二醇氧化还原酶与甲酸脱氢酶对Klebsiella pneumoniae生理代谢的影响

针对1,3-丙二醇发酵过程中中间代谢产物3-羟基丙醛致死性的现象,在野生型的克雷伯氏肺炎杆菌中同时强化表达1,3-丙二醇氧化还原酶和甲酸脱氢酶,在增强1,3-丙二醇氧化还原酶酶活的同时,促进NADH的合成,最终降低3-羟基丙醛积累.利用PCR技术分别从克雷伯氏肺炎杆菌和甲醇酵母Candida boidinii基因组中扩增出基因dhaT和fdh,通过表达载体pDK获得1,3-丙二醇氧化还原酶和甲酸脱氢酶共表达的菌株K.p/pTF在IPTG的诱导下,基因工程菌K.p/pTF1,3-丙二醇氧化还原酶和甲酸脱氢酶的酶活分别提高了2.8倍和3.5倍,而NADH含量提高了1.3倍.在有氧条件下批式发酵培养,基因工程菌K.p/pTF可以利用以50g/L的初始甘油为底物正常进行发酵,3-羟基丙醛的最高积累浓度较野生菌降低了50.1%,有效避免了发酵的异常中止.

The effect of co-overexpression of fdh and dhaT on physiological metabolism in Klebsiella pneumoniae

1,3-propanediol oxidoreductase (PDOR) and formate dehydrogenase (fdh) were co-overexpressed in the wild type Klebsiella pneumoniae to enhance the NADH production and decrease the 3-HPA accumulation which may cause an irreversible cessation of the 1,3-propanediol fermentation process. dhaT gene encoded PDOR from K. pneumoniae and fdh gene encoded fdh from Candida boidinii were both amplified and then overexpressed in K. pneumoniae. For the constructed strain, the activity of PDOR, fdh, and NADH were 2.8, 3.5, and 1.3 fold higher than the wild type strain, respectively. In the batch fermentation with initial 50 g/L glycerol, 3-HPA concentration decreased by 50.1%, which effectively avoided the irreversible cessation of the fermentation process with strain K.p/pTF.