Construction Of Novel Recombinant Capable Of Producing 1,3-propanediol

1, 3-Propanediol has numerous applications in the manufacture of polymers, foods, lubricants, and medicines. Industrial 1, 3-propanediol production has attracted attention as an important monomer to synthesize a new type of polyester, polytrimethylene terephthalate. 1, 3-propanediol is produced by two methods: chemical synthesis and microbial conversion. However, traditional chemical conversion of 1, 3-propanediol is difficult, which has a low selectivity and consequently its high price hinders the utilization of 1, 3-propanediol in polymerindustries. Interest has now been focused on the production of 1, 3-propanediol by recombinant strain fermentation.The lack of NADH in the glycerol metabolism inhibit the activity of 1, 3-propanediol oxidoreductase, and induce the accumulation of 3-hydroxypropionaldehyde, which can inhibit the activity of glycerol dehydratase, and prevent the cell growth and the production of 1, 3- PD. To resolve the problem, this research was to construct a recombinant strain which could convert glycerol to 1, 3-propanediol. The structure gene yqhD from a wild-type Escherichia coli encoding 1, 3-propanediol oxidoreductase isoenzyme and the structure gene dhaB from K. pneumoniae encoding glycerol dehydratase were amplified by using PCR method. The expression vector pEtac harboring yqhD gene and dhaB gene was transformed into E. coli JM109 to yield the recombinant strain E. coli JM109 (pEtac-dhaB-tac-yqhD). Fermentation experiments were performed by E. coli JM109(pEtac), E. coli JM109(pEtac-dhaB-tac-yqhD) showed that no 1, 3-propanediol was produced in E. coli JM109(pEtac), only the strain harboring both dhaB gene and yqhD gene converted glycerol to 1, 3-propanediol after induction by IPTG. The recombinant enzyme activity analysis showed that the specific enzymatic activity of glycerol dehydratase and 1, 3-propanediol oxidoreductase isoenzyme in E. coli JM109 (pEtac-dhaB-tac-yqhD) were 15 U/mg protein and 10 U/mg protein, respectively. While, the specific enzymatic activity of 1, 3-propanediol oxidoreductase in E. coli JM109(pEtac) were only 4 U/mg protein.While, the recombinant plasmid pEtac-dhaB-tac-yqhD harboring the genes encoding DHAB and YQHD was transformed into E. coli JM109. A stable two plasmid system was obtained via cotransformation of pEtac-dhaB-tac-yqhD and pUC-tac-dhaT into E. coli JM109, which convert glycerol by using two coenzyme (NADH, NADPH), and decrease the accumulation of 3-hydroxypropionaldehyde. And the specific enzymatic activity of glycerol dehydratase and 1, 3-propanediol oxidoreductase isoenzyme in E. coli JM109(pEtac-dhaB-tac -yqhD, pUC-tac-dhaT) were 23 U/mg protein and 12 U/mg protein, respectively. 1, 3-propanediol oxidoreductase isoenzyme activity of E. coli JM109 (pEtac-dhaB-tac-yqhD, pUC-tac-dhaT) was higher to that of E. coli JM109(pEtac-dhaB-tac-yqhD). The production of 1, 3-propanediol on 50 g/L glycerol by E. coli JM109( pEtac-dhaB-tac -yqhD) was 1.52 g/L and the production of 1, 3-propanediol by E. coli JM109(pEtac-dhaB-tac -yqhD, pUC-tac-dhaT) was 1.86 g/L after induction. Meanwhile, the fermentation result showed the increase of 22.3% of 1, 3-propanediol yield by E. coli JM109(pEtac-dhaB-tac- yqhD, pUC-tac-dhaT) was obtained compared with E. coli JM109(pEtac-dhaB-tac-yqhD). The orthogonal design method was then applied to further optimize the fermentation condition of the recombinant strain. A mathematical model was then developed to show the effect of each medium compositions and their interactions on the production of 1, 3-propanediol by recombinant strain E. coli JM109. The model estimated that, a maximal yield of 1, 3-propanediol could be obtained when the concentrations of glycerol, Vitamin B12, KH2PO4, Mg2+ and yeast extract were set at 20 g/L, 0.01 g/L, 7.5 g/L, 0.67 g/L and 5 g/L, respectively. The yield under the optimal parameters and process can reach 2.25 g/L.