Expression and characterization of formate dehydrogenase gene in Klebisella pneumoniae

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.