在Klebsiella pneumoniae醛脱氢酶失活菌中构建NADH再生系统

生物法生产1,3-丙二醇（1,3-Propanediol，1,3-PD）是当前工业生物技术研究的热点之一，生产过程中，需要消耗还原当量NADH，NADH的有效供给决定了1,3-PD的产量和得率。本文采用PCR的方法从Candida boidinii基因组中克隆编码fdh的基因，将该基因片段插入载体pMALTM-p2X，构建表达载体pMALTM -p2X-fdh，并转入醛脱氢酶失活菌Klebsiella pneumoniae DA-1HB，获得重组菌Klebsiella pneumoniae DAF-1。在IPTG浓度0.5 mmol/L时，诱导3 h后甲酸脱氢酶表达明显；发酵过程中甲酸脱氢酶比酶活达到4.82 U/mg；与出发菌株K. pneumoniae DA-1HB相比，重组菌DAF-1合成1,3-丙二醇的浓度提高了19.2%?。

Construction of NADH regeneration system in Klebisella pneumoniae with inactivation of aldehyde dehydrogenase

The microbial production of 1,3-Propanediol is of interest in the field of industry biotechnology, during which reducing equivalent NADH was consumed. Therefore, the available NADH would be critical for the yield of 1,3-Propanediol. In the present study, formate / formate dehydrogenase system was used for the generation in vivo of NADH and the improvement of 1,3-Propanediol.production. Formate Dehydrogenase gene (fdh) was amplified from Candida boidinii genome by PCR and the purified PCR product was inserted into the vector pMD18-T Simple to construct plasmid pMD18-T Simple-fdh, which was transformed into Escherichia coli DH5α and recombinants were selected by blue-white selection. After transformant the fdh gene was separated and inserted into pMALTM-p2X to construct expression vector pMALTM-p2X-fdh, which was transformed into DA-1HB, an engineered strain from Klebsiella pneumoniae by inactivating aldehyde dehydrogenase. Thus, a recombinant strain Klebsiella pneumoniae DAF-1 was obtained. The enzyme activity reached 4.82 U/mg crude protein after K. pneumoniae DAF-1 was induced for 3 h by 0.5mmol/L IPTG. Compared with that of the parent strain DA-1HB, the yield of 1,3-propanediol of recombinant strain DAF-1 was increased by 19.2? % in the anaeribic bioreactor.