Genetic Engineering Reconstruction Of Klebsiella Pneumoniae Of Producing 1, 3-propanediol

1,3-propanediol is acknowledged as one of six new petrochemical products currently in the world. Its main function is as an important monomer to synthesize a new type of polyester,polyether,polyurethane. 1,3-Propanediol is produced by two ways:chemical synthesis and microbial conversion. Producing 1,3-propanediol by microbial fermentation have many obvious advantages and become the focuses of research.For research convenience,the analytical method of key enzyme in metabolic pathway producing 1,3-PD in Klebsiella pneumoniae,Escherichia.coli(pHsh-dhaB-yqhD) was improved firstly.The relatively appropriate analytical condition was as follows:with the working power of 300 w, the crushing processes of Klebsiella pneumoniae and Escherichia.coli(pHsh-dhaB-yqhD) both took place for 1 sec after each 4 sec and the total crushing times were both 5 min.The most appropriate concentration of buffer phosphate employed in glycerol dehydratasele(GDHt) and buffer bicarbonate employed in 1,3-propanediol oxidoreductase(PDOR) and 1,3-propanediol oxidoreductase isoenzyme(YQHD) analysis were 0.045 mol/L and 0.1 mol/L. The most appropriate pH of buffer phosphate employed in GDHt analysis from Klebsiella pneumoniae and Escherichia.coli(pHsh-dhaB-yqhD) were 7.2 and 7 and the most appropriate pH of buffer bicarbonate employed in PDOR and YQHD analysis from Klebsiella pneumoniae and Escherichia.coli(pHsh-dhaB-yqhD) were 9.5 and 9. The most appropriate analytical temperature of GDHt and PDOR (YQHD) were 37℃and 45℃respectively.In the reductive branch, glycerol was firstly dehydrated to 3-hydroxypropionaldehyde and then reduced to 1,3-PD with the consumption of reducing power (NADH). If the over-expression of gene dhaB encoding glycerol dehydrase was achieved, the reducing power would be short and 3-hydroxypropionaldehyde would be accumulated, which was disadvantage to produce 1,3-propanediol. The structure gene yqhD from E.coli encoding 1,3-propanediol oxidoreductase isoenzyme(with the consumption of reducing power (NADPH))and the gene dhaB encoding glycerol dehydrase from Klebsiella pneumoniae was amplified using PCR method.The two genes were transferred into expression vector pEtac to construct a novel recombinant Klebsiella pneumoniae(pEtac-dhaB-tac-yqhD). Over -expression of yqhD and dhaB in Klebsiella pneumoniae was achieved with pEtac-dhaB-tac-yqhD.Through the antibiotic concentration gradient experiment, Klebsiella pneumoniae including pEtac on seed LB seed medium and fermentation medium when the kanamycin concentration was 30 mg/L at least. The growth curve of Klebsiella pneumoniae and Klebsiella pneumoniae (pEtac-dhaB-tac-yqhD) in the seed culture medium and the fermentation medium were drawn up respectivly. The Klebsiella pneumoniae and Klebsiella pneumoniae (pEtac-dhaB-tac-yqhD) entered the logarithmic growth midanaphase at about 7th hour. The fermentation result on the shake flask showed the increase of 1,3-propanediol yield by Klebsiella pneumoniae(pEtac-dhaB-tac-yqhD) by 25% compared with Klebsiella pneumoniae. The main by-products,acetic acid and butanediol decreased by 26.5% and 34% obviously.Acetic acid, alcohol and 1,3-propanedio formed from about the 7th hour while butanediol formed from the 20th hour. The optimal agitation speed of fermentor was 200 r/min and then 100 r/min from the 7th hour.The optimal ventilation of fermentor was 2 vvm and then 1vvm from the 7th hour. It was advantageous to the fermentation of Klebsiella pneumoniae(pEtac-dhaB-tac-yqhD) for keeping pH 7.0.