甲酸脱氢酶在Klebsiella pneumoniae中的表达和功能分析

Expression and characterization of formate dehydrogenase gene in Klebisella pneumoniae

摘要:

在甘油厌氧发酵生产1,3-丙二醇的过程中，需要消耗还原当量NADH，NADH的有效供给决定了1,3-丙二醇的产量和得率。采用PCR方法从Candida boidinii基因组中克隆编码甲酸脱氢酶基因fdh，将fdh基因片段插入载体pMAL^TM-p2X中，构建表达载体pMALsup>TM-p2X-fdh，并转入1,3-丙二醇生产菌Klebsiella pneumoniae YMU2，获得重组菌Klebsiella pneumoniae F-1。研究了重组质粒的稳定性和IPTG诱导fdh基因过量表达的条件。结果表明，重组质粒具有良好的稳定性；fdh基因表达的蛋白分子量为40.2kDa；IPTG诱导表达研究表明，在IPTG浓度为0.5mmol/L时，诱导4h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5.47U/mg；与出发菌株K. pneumoniae YMU2相比，重组菌F-1合成1,3-丙二醇的浓度提高了12.5％。

Abstract:

Glycerol can be converted to 1,3-propanediol by the anaerobic fermentation of Klebsiella pneumoniae, during which reducing equivalent NADH was consumed. Therefore, the availability of NADH would be critical for the yield of 1,3-propanediol. In this paper, formate/formate dehydrogenase system was used for the regeneration of in vivo 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. From the transformant the fdh gene was separated and inserted into pMAL^TM-p2X to construct expression vector pMAL^TM-p2X-fdh, which was transformed into Klebsiella pneumoniae YMU2 and a recombinant strain Klebsiella pneumoniae F-1 was obtained. The plasmid stability of strain F-1 and the conditions of fdh expression induced by IPTG were studied. It was demonstrated that the plasmid had good stability, and 0.5mmol/L IPTG would induce the expression of protein encoded by fdh gene with the molecular weight of 40.2kDa. The enzyme activity reached 5.47U/mg crude protein when K. pneumoniae F-1 was induced for 4h by 0.5mmol/L IPTG. Compared with that of the parent strain K. pneumoniae YMU2, the yield of 1,3-propanediol of recombinant strain F-1 increased by 12.5% in the anaeribic bioreactor.