Construction Of 1, 3-Propanediol Recombinants

The purpose of the research is to screen out the enteric bacterial isolates for producing 1,3-propanediol based on efficient utilization of glycerol. After identification and chemical mutation, the strains with higher 1,3-propanediol yield was selected. Genes dhaB and dhaT encoding essential enzymes of 1,3-propanediol pathway were amplified by PCR technique using this chromosomal DNA as a template. Those two genes would be inserted into wild host cells yielding 1,3-propanediol recombinants in order to make them convert various kinds of substrates to 1,3-propanediol.Nine strains of enteric bacteria, capable of converting glycerol to 1.3-PD were screened. All of the strains were identified as Klebsiella pneumoniae. After chemical mutation, the production of 1,3-PD was improved. After beering optimized the fermentation the yield of 1,3-PD is up to 20 g/L.Structure Genes dhaB (4.6kb) and dhaT (1.16kb) encoding essential enzymes of 1,3-propanediol pathway were amplified by PCR technique using Klebsiella pneumoniae 1192-121# chromosomal DNA as a template. And the primers design refered to the sequence of Klebsiella pneumoniae ATCC 25955 publicated by NCBI. dhaB and dhaT were inserted into the downstream of tac promoter. Finally, recombinant plasmids pEtac-dhaT, pUCtac-dhaB and pUCtac-dhaB-dhaT were got. After transforming these recombinant plasmids into E. coli JM109, recombinants E. coli JM109 (pUCtac-dhaB), E. coli JM109 (pEtac-dhaT), E. coli JM109 (pUCtac-dhaB-dhaT) and E. coli JM109 (pUCtac-dhaB, pEtac-dhaT) were constructed.Specific enzymatic Activity of glycerol dehydratase in E. coli JM109 (pUCtac-dhaB) is 104 U·mg-1 protein. Specific enzymatic Activity of 1,3-propanediol oxidoreductase in E. coli JM109 (pEtac-dhaT) is 0.2 U·mg-1 protein. Specific enzymatic Activity of glycerol dehydratase and 1,3-propanediol oxidoreductase in E. coli JM109 (pUCtac-dhaB-dhaT) are 153 U·mg-1 protein and 0.17 U·mg-1 protein. Specific enzymatic Activity of glycerol dehydratase and 1,3-propanediol oxidoreductase in E. coli JM109 (pUCtac-dhaB, pEtac-dhaT) are 132 U·mg-1 protein and 0.08 U·mg-1 protein. SDS-PAGE electrophoresis profile of whole cell proteins indicated that glycerol dehydratase big subunit and 1,3-propanediol oxidoreductase were overexpressed and their molecular weight were of 64 kD and 40 kD. 1,3-propanediol fermentation test by E. coli JM109 (pEtac-dhaT), E. coli JM109 (pUCtac-dhaB), E. coli JM109 (pUCtac-dhaB-dhaT) and E. coli JM109 (pUCtac-dhaB, pEtac-dhaT) in 250 mL flask using 50 mL working volume and CaCO3 for pH buffering. Induced by IPTG, fermented 50 g/L glycerol, E. coli JM109 (pEtac-dhaT) yielded no 1,3-PD. E. coli JM109 (pUCtac-dhaB) yielded 1.1 g/L 1,3-PD. E. coli JM109 (pUCtac-dhaB-dhaT) yielded 18.2 g/L 1,3-PD. E. coli JM109 (pUCtac-dhaB, pEtac-dhaT) yielded 14.6 g/L 1,3-PD.Induced by IPTG, fermented 40 g/L glucose, E. coli JM109 (pEtac-dhaT) yielded no 1,3-PD. E. coli JM109 (pUCtac-dhaB) yielded 0.8 g/L 1,3-PD. E. coli JM109 (pUCtac-dhaB-dhaT) yielded 9.1 g/L 1,3-PD. E. coli JM109 (pUCtac-dhaB, pEtac-dhaT) yielded 8.2 g/L 1,3-PD.