Construction of Recombinant E.coli Expressing Fusion Protein to Produce 1,3-Propanediol

In this study, a synthetic pathway was created by assembling genes from Clostridium butyricum and Escherichia coli in different combinations. Among the genes were dhaB1 and dhaB2 from C. butyricum VPI1718 coding for glycerol dehydratase (GDHt) and its activator (GDHtAc), respectively, involved in the conversion of glycerol to 3-hydroxypropionaldehyde (3-HPA). The yqhD gene from E.coli BL21 was also included which codes for an NADPHdependent 1,3-propanediol oxidoreductase isoenzyme (PDORI) reducing 3-HPA to 1,3-propanediol (1,3-PD). Molecular modeling analysis indicated that the conformation of fusion protein of YQHD and DHAB1 was favorable for direct molecular channeling of the intermediate 3-HPA. According to the simulation results, the yqhD and dhaB1 gene were assembled in the upstream of dhaB2 to express a fusion protein, yielding the recombinant strain E. coliBL21 (DE3)//pET22b+::yqhD-dhaB1_dhaB2 (strain BP41Y3). Strain BP41Y3 gave 10-fold higher 1,3-PD concentration than E. coliBL21 (DE3)//pET22b+::yqhD-dhaB1_dhaB2 (strain BP31Y2) expressing the recombinant enzymes simultaneously but in a non-fusion mode. This is the first report using a gene fusion approach to enhance the biological conversion of glycerol to the value added compound 1,3- PD.

Bioconversion of Biodiesel Derived Crude Glycerol by Immobilized Clostridium pasteurianum: Effect of Temperature

Batch fermentation of 5, 10 and 25 g/L biodiesel derived crude glycerol was carried out at 30, 37 and 450C by Clostridium pasteurianum cells immobilized on silica. Maximum yield of 1,3-propanediol (PDO) (0.60 mol/mol), and ethanol (0.26 mol/mol) were obtained from 10 g/L crude glycerol at 30 and 370C respectively. Maximum yield of butanol (0.28 mol/mol substrate added) was obtained at 370C with 25 g/L substrate. None of the three products were detected at 45oC even after 10 days of fermentation. Only traces of ethanol (0.01 mol/mol) were detected at 450C with 5 g/L substrate. The results obtained for 25 g/L substrate utilization were fitted in first order rate equation to obtain the values of rate constant at three different temperatures for bioconversion of glycerol. First order rate constants for bioconversion of glycerol at 30, 37 and 45oC were found to be 0.198, 0.294 and 0.029/day respectively. Activation energy (Ea) for crude glycerol bioconversion was calculated to be 57.62 kcal/mol.