[Expression and characterization of formate dehydrogenase gene in Klebisella pneumoniae].
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 DH5alpha and recombinants were selected by blue-white selection. From the transformant the fdh gene was separated and inserted into pMALTM-p2X to construct expression vector pMAL-p2X-fdh, which was transformed into Klebsiella pneumoniae YMU2 and a recombinant strain Klebsiella pneumoniae F-l was obtained. The plasmid stability of strain F-l 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-I 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.
Huang
Zhi-hua
ZH
Department of Chemical Engineering, Tsinghua University, Beijing 100084, China. hzh917@163.com
Zhang
Yan-ping
YP
Liu
Ming
M
Wang
Bao-guang
BG
Cao
Zhu-an
ZA
D002175:Candida / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005560:Formate Dehydrogenases / Q000235:genetics* / Q000502:physiology; D007711:Klebsiella pneumoniae / Q000235:genetics*; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis
D011994:Recombinant Proteins; D005560:Formate Dehydrogenases