Gene Cloning Of Glycerol Dehydratase And 1,3-Propanediol Oxidoreductase From Klebsiella Pneumoniae And Expression In Escherichia Coli

1,3-propanediol is an important chemical industry material. It can be used as solving solution, resisting frost solution, protecting solution, delicacy chemical industry material and a monomer of new polyester: polytrimethylene terephthalate (PTT) and polyethylene terephthalate. As an engineering thermoplastic, polytrimethylene terephthalate (PTT) combines the rigidity, in addition to benefits of softness, stretch recovery, vibrant color, with stain and heat resistances. Currently, nylon carpet fiber, membrane, engineering plastics and costume field are known for its resiliency. It was regarded as being widely used chemical indudstry material in 21 century and more and more researchers pay attention to that.At present, the primary method to produce 1,3-propanediol is chemosynthesis method. Due to it's high cost and severe pollution to environment, microbiological approach has become the popular method to produce 1,3-propanediol.The genes encoding glycerol dehydratase (DHAB) and 1,3-propanediol oxidoreductase (DHAT) were amplified from total DNA of Klebsiella pneumoniae by PCR. The genes of dhaB and dhaT were inserted into expression vector pSE380 and then transformed into E.coli JM109. The recombinant strains were induced to express DHAB and DHAT with IPTG SDS-PAGE showed that the relative molecular weights of the dhaB expression product were about 63KD, 61KD, 22KD, 16KD and 43KD for dhaT.The recombinant plasmid for the production of 1,3-propanediol was constructed by combining dhaB and dhaT with ribosomal binding sites and was placed under the control of trc promoter forming a polycistron expression vector.This plasmid was transformed into E.coli JM109. Recombinant strains produced 0.27% 1,3-propanediol (g/L) from 0.8% glycerol under the condition of 200rpm, 37癈 and 44 hours. However, the wild-type E.coli JM109 didn't produce 1,3-propanediol. This research successfully introduce the novel pathway for fermenting glycerol to 1,3-propanediol in E.coli by expressing Klebsiella pneumoniae genes encoding DHAB and DHAT, which was reported for the first time in this country.