Studies On Recombinant1,3-Propanediol Oxidoreductase Of Klehsiella Pneumoniae

1,3-propanediol(1,3-PD) is an important chemical material and its biosynthetic method arouses broad interests. In one of the main production stains——Klebsiella pneumoniae,1,3-propanediol oxidoreductase(PDOR) and glycerol dehydratase(GDHt) are two key enzymes responsible for the conversion from glycerol to1,3-propanediol.In this study, the coding gene dhaT of PDOR was cloned from K. pneumoniae. Expression vectors pET-28a-dhaT and pET-22b-dhaT were constructed to achieve prokaryotic expression in E.coli BL21(DE3). The inducer, induction time, induction temperature, protein localization and enzyme activity were assayed. The results suggested that both lactose and IPTG can be used as inducer. Although PDOR expressed at30℃for5hours showed certain enzyme activity, it displayed3.7times activity when induced at20℃for14hours.In order to accomplish the conversion from glycerol to1,3-PD, PDOR and GDHt were coexpressed in E.coli BL21(DE3) by construction of polycistronic expression vector pET-28a-dhaB1B2B3-dhaT and coexistence of two incompatible plasmids pET-28a-dhaBlB2B3and pET-22b-dhaT. The two enzymes showed certain activities under the two methods, respectively. The fermentation of E.coli BL21(pET-28a-dhaB1B2B3-dhaT) in5liter reactor could convert glycerol to1,3-PD, and reach0.95g/L concentration of1,3-PD.Considering. the limited aspects of E.coli expression strain, two new expression vecters pPk-dhaT and pPk-dhaB1B2B3-dhaT were constructed which can copy in K. pneumoniae. They over-expressed PDOR alone and coexpressed PDOR and GDHt in K. pneumoniae, respectively. Enzyme activity assay showed that the activity of GDHt increased85times and the activity of PDOR increased1.2times. The fermentation of recombinant K. pneumoniae was carried out in a5liter reactor. The concentration, productivity and conversion rate of1,3-PD could reach76.3g/L,1.97g/L-h and46%.