Production Of1,3-Propanediol By Recombinant Bacteria

1,3-propanediol (1,3-PD) is an important chemical material and can be used as monomer to produce polyesters, polyethers and polyurethanes. Its biosynthetic method arouses our more and more interests. As one of the main producers of1,3-PD, Klebsiella pneumoniae can convert glycerol to1,3-PD by two enzymes, glycerol dehydratase (GDHt) and1,3-propanediol oxidoreductase (PDOR).The gene (dhaT) enconding1,3-propanediol oxidoreductase of Klebsiella pneumoniae was cloned and expressed in E.coli. When the1,3-propanediol oxidoreductase was expressed directly in cytoplasm, the recombinant enzyme was mostly in soluble form, and exhibited high activity (96.8U/ml broth). After purification by affinity chromatography, the recombinant enzyme showed maximum activity at pH9.5and30℃.The dhaB gene which encodes glycerol dehydratase was cloned and expressed in E. coli. Three recombinant expression plasmids were constructed. After induced by IPTG, SDS-PAGE analysis revealed that both co-expression and fusion expression systems expressed the recombinant glycerol dehydratase in different forms with predict molecular weights of64kDa (α),22kDa (β),16kDa (γ) and99kDa (fusion protein), only a subunit was expressed in direct expression system. Enzyme assay showed that the activities of three heterologous expression products were27.4,2.3, and0.2U/mg respectively. The enzymatic specific activity of glycerol dehydratase could reach90U/mg after purification. The gene (gdrAB) encoding reactiviting facator for glycerol dehydratase was also cloned and expressed in E. coli. As the molecular chaperon of glycerol dehydratase, the reactiviting factor could reactivate the inactivated holoenzyme in the presence of ATP and Mg2+(or Mn2+) and make the glycerol dehydratase convert glycerol to3-hydroxypropionaldehyde effectively.Two incompatible plasmids were used to coexpress the dha regulon (dhaB and dhaT) and glycerol dehydratase reactivating factor (gdrAB) in E. coli. After cloning dhaB, dhaT and gdrAB into two incompatible plasmids carrying different antibiotic resistance genes, the recombinant plasmids could coexist stably in E. coli in the presence of two selective pressures. The glycerol dehydratase,1,3-propanediol oxidoreductase and glycerol dehydratase reactivating factor were coexpressed in E. coli in high levels. In a fed-batch fermentation with glycerol and glucose, the recombinant E. coli containing the two incompatible plasmids consumed14.3g/1of glycerol and produced8.6g/11,3-propanediol. In the substitution case of dhaT by yqhD, which encoding alcohol dehydrogenase from E. coli (the isoenzyme of PDOR) for, the final1,3-propanediol concentration of the fermentation broth from recombinant E. coli could reach13.2g/1.In order to improve the productivity and efficiency of K pneumoniae, recombinant expression plasmid pKP-28a-d/daBY was constructed and expressed in K. pneumoniae. Activities of key enzymes of the recombinant K. pneumoniae increased significantly in the1,3-PD fermentation under both anaerobic and microaerobic conditions.The batch culture of recombinant K. pneumoniae under microaerobic condition was studied. The conversion rate of glycerol was a slight lower than that under anaerobic condition, but the concentration and productivity of1,3-PD enhanced. Glucose (8g/1) added at the beginning of microaerobic fermentation, increased the productivity more than30%while glucose fed with the ratio of1:8to glycerol improved the conversion maximum to16%. And when the two steps were combined in fed-batch fermentation of recombinant K. pneumoniae under microaerobic condition, the indexes of1,3-PD concentration, conversion rate and productivity were19.2%,8.3%and19.2%respectively higher than those of K. pneumoniae in the same condition, and were25%,20.4%and85.6%respectively higher than those of K. pneumoniae under anerobic condition using glycerol as the only substrate. Moreover, the concentration of lactic acid was very low.In order to improve the tolerance to product inhibition of recombinant K. pneumoniae so as to increase the yield of1,3-PD, UV/LiCl mutaiton associating with domestication was performed. The excellent mutant M-5, which could tolerate higher1,3-PD concentration than parent strain was obtained. The fed-batch fermentation fo1,3-PD by mutant M-5was studied. The1,3-PD concentration in fermentation broth from mutant M-5was24%higher than that of parent strain’s, and the acetic acid concentration increased while the ethanol concentration decreased. Compared with the fermentation by wild-type K. pneumoniae under anaerobic conditon, the indexes of1,3-PD concentration and productivity of mutant M-5increased56.1%and131.5%, respectively. The final1,3-PD concentration of fermentation broth with fed-batch fermentation in30L fermentor could maintain87g/1stably.Three ethanol deficient mutants were obtained by knocking out the aldH gene of aldehyde dehydrogenase of K. pneumoniae. In the fed-batch fermentation of1,3-PD by mutant A-6, whose ethanol concentration was lowest in flask culture selection, cell growth and metabolism ceased during12-16h, no more1,3-PD produced. Compared with parent strain in the corresponding period, ethanol concentration decreased37%while acetic acid concentration doubled and the cell growth was inhibited obviously. The situation may be improved by domestication of tolerance to acetic acid.