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

Authors: Rosarin Rujananon, Poonsuk Prasertsan, Amornrat Phongdara, Tanate Panrat, Jibin Sun, Sugima Rappert, An-Ping Zeng

Abstract:

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.

Keywords: Recombinant E.coli, 1, 3-propanediol, glycerol, fusion protein.

Digital Object Identifier (DOI): doi.org/10.5281/zenodo.1333050

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