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Construction of Recombinant E.coli Expressing Fusion Protein to Produce 1,3-Propanediol
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.
Digital Object Identifier (DOI): doi.org/10.5281/zenodo.1333050Procedia APA BibTeX Chicago EndNote Harvard JSON MLA RIS XML ISO 690 PDF Downloads 1654
 H. Miller, "Major fiber producers hop on corterra bandwagon," Int Fiber J, vol. 15, pp. 14-16, 2000.
 R. Rudie, "Cargill dow sows seeds of future fibers; will build $ 300 million PLA polymer plant," Int Fiber J, vol. 15, pp. 8-12, 2000.
 A.Nemeth, B. Kupcsulik, and B. Sevella, "1,3-Propanediol oxidoreductase production with Klebsiella pneumoniae DSM 2026," World J Microbiol Biotechnol, vol. 19, pp. 659-663, 2003.
 H. Biebl, K. Menzel, A.-P. Zeng, and W.-D. Deckwer, "Microbial production of 1,3-propanediol," Appl Microbiol Biotechnol, vol. 52, pp. 289-297, 1999.
 A.-P. Zeng, H. Biebl,, H. Schlieker, and W.-D. Deckwer, "Pathway analysis of glycerol fermentation by Klebsiella pneumoniae: regulation of reducing equivalent balance and product formation," Enz Microb Technol, vol. 15, pp. 770-779, 1993.
 S. Sattayasamitsathit, P. Prasertsan, and P. Methacanon, "Statistical optimization for simultaneous production of 1,3-propanediol and 2,3- butanediol using crude glycerol by newly bacterial isolate," Process Biochem vol. 46, pp. 608-614, 2011.
 M. Seyfried, R. Daniel, and G. Gottschalk, "Cloning, sequencing, and overexpression of the genes encoding coenzyme B12-dependent glycerol dehydratase of Citrobacter freundii," J Bacteriol, vol. 178, pp. 5793- 5796, 1996.
 B. Guenzel, S. Yonsel, and W.-D. Deckwer, "Fermentative production of 1,3-propanediol from glycerol by Clostridium butyricum up to a scale of 2 m3," Appl Microbiol Biot, vol. 36, pp. 288-294, 1991.
 I.T. Tong, H.H. Liao, and D.C. Cameron, "1,3-Propanediol Production by Escherichia coli Expressing Genes from the Klebsiella pneumoniae dha Regulon," Appl Environ Microbiol, vol. 57, pp. 3541-3546, 1991.
 T. Tobimatsu, M. Azuma, H. Matsubara, H. Takatori, T. Niida, K. Nishimoto, H. Satoh, R. Hayashi, and T. Toraya, "Cloning, sequencing, and high level expression of the genes encoding adenosylcobalamindependent glycerol dehydrase of Klebsiella pneumoniae," J Biol Chem, vol. 27, pp. 22352-22357, 1996.
 J. Sun, J. Heuvel, P. Soucaille, Y. Qu, and A.-P. Zeng, "Comparative genomic analysis of dha regulon and related genes for anaerobic glycerol metabolism in bacteria," Biotechnol Prog, vol. 19, pp. 263-272, 2003.
 W. Fenghuan, Q. Huijin, H. He, T. Tan, "High-level expression of the 1,3-propanediol oxidoreductase from Klebsiella pneumoniae in Escherichia coli," Mol Biotechnol, vol. 31, pp. 211-219, 2005
 L. A. Laffend and V. Nagarajan, "Bioconversion of a fermentable carbon source to 1,3-propanediol by a single microorganism," U.S. patent 6,025,184, 2000.
 M. Rasch, "The influence of temperature, salt and pH on the inhibitory effect of reuterin on Escherichia coli," Int J Food Microbiol, vol. 72, pp. 225-231, 2002.
 F. Barbirato, P. Soucaille, and A. Bories, "Physiologic Mechanisms involved in accumulation of 3-hydroxypropionaldehyde during fermentation of glycerol by Enterobacter agglomerans," Appl Environ Microbiol, vol. 62, pp. 4405-4409, 1996.
 B. Zhuge, C. Zhang, H. Fang, J. Zhuge, and K. Permaul, "Expression of 1,3-propanediol oxidoreductase and its isoenzyme in Klebsiella pneumoniae for bioconversion of glycerol into 1,3-propanediol," Appl Microbiol Biotechnol, vol. 87, pp. 2177-2184, 2010.
 P. Zheng, K. Wereath, J. Sun, J.vd. Heuvel, and A.-P. Zeng, "Overexpression of genes of the dha regulon and its effects on cell growth, glycerol fermentation to 1,3-propanediol and plasmid stability in Klebsiella pneumoniae," Process Biochem, vol. 41, pp. 2160-2169, 2006.
 C.E. Nakamura and G.M. Whited, "Metabolic engineering for the microbial production of 1,3-propanediol," Curr Opin Biotechnol, vol. 14, pp. 454-459, 2003.
 M. Emptage, S.L. Haynie, L.A. Laffend, J.P. Pucci, and G. Whited, "Process for biological of 1, 3-propanediol with high titer," U.S. Pat. 6514733, 2003.
 K. Arnold and L. Bordoli, "The SWISS-MODEL workspace: a web based environment for protein structure homology modeling," Bioinformatics, vol. 22(2), 195-201, 2006.
 R. A. Laskowski, "PDBsum: summaries and analyses of PDB structures," Nucleic Acids Res, vol. 29(1), pp. 221-222, 2001.
 R. A. Laskowski and J.D. Watson, "ProFunc: a server for predicting protein function from 3D structure," Nucleic Acids Res, 33(Web Server issue): W89-93, 2005
 J. Sambrook and D.W. Russel, "Molecular cloning: A laboratory manual, 3rd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 2001.
 D. Riesenberg, V. Schulz, W.A. Knorre, H.D. Pohl, D. Korz, E.A. Sanders, A. RoR, and W.D. Deckwer, "High cell density cultivation of Escherichia coli at controlled specific growth rate," J Biotechnol, vol. 20, pp. 17-28, 1999.
 U. K. Laemmli, "Cleavage of structural proteins during the assembly of the head of bacteriophage T4," Nature, vol. 227, pp. 680-685, 1970.
 R. Daniel, K. Stuertz, G. Gottschalk, "Biochemical and molecular characterization of the oxidative branch of glycerol utilization by Citrobacter freundii," J Bacteriol, vol. 177, pp. 4392-401, 1995.
 J. Hao, R. Lin, Z. Zheng, H. Liu, and D. Liu, "Isolation and characterization of microorganisms able to produce 1,3-propanediol under aerobic conditios," World J Microbiol Biotechnol, vol. 24, pp.1731-40, 2008a.