Skip to main content
SpringerLink
Log in

High-level expression of the 1,3-propanediol oxidoreductase from klebsiella pneumoniae in escherichia coli

  • Research
  • Published:
Molecular Biotechnology Aims and scope Submit manuscript

Abstract

As one of four key enzymes in glycerol dismutation process, 1,3-propanediol oxidoreductase (EC.1.1.1.202) is important in converting glycerol to 1,3-propanediol in Klebsiella pneumoniae. The dhaT gene encoding 1,3-propanediol oxidoreductase was amplified by polymerase chain reaction (PCR) using the genome DNA of K. pneumoniae as template, and then cloned into cloning vector pMD18-T. After DNA sequence was determined, the dhaT gene was subcloned into Escherichia coli expression vector pET-22b (+) and pET-28a (+). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that both the recombinant E. coli BL21 (DE3) (pET-22b (+)-dhaT) and E. coli BL21(DE3)(pET-28a (+)-dhaT) expressed predicted 42-kDa 1,3-propanediol oxidoreductase after induced by isopropyl-β-d-thiogalactopyranoside (IPTG), and the recombinant enzyme of E. coli BL21 (DE3) (pET-28a (+)-dhaT) was mostly in soluble form, and exhibited high activity (96.8 U/mL culture). The recombinant enzyme was purified and biochemically characterized. The apparent K m values of the enzyme for 1,3-propanediol and NAD+ were 8.5 and 0.21 mM, respectively. The enzyme had maximum activity at pH 9.5 and 30°C.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Witt, U., Müller, R. J., Widdecke, H., and Deckwer, W. D. (1994) Synthesis, properties and biodegradability of polyesters based on 1,3-propanediol. Makromol. Chem. Phys. 195, 793–802.

    Article  CAS  Google Scholar 

  2. Powell, J. B., Slaugh, L. H., Forschner, T. C., et al. (1995) Process for preparing 1,3-propanediol. Patent US 5463144.

  3. Powell, J. B., Slaugh, L. H., Forschner, T. C., Semple, T. C., and Weider, P. R. (1995) Process for preparing 1,3-propanediol. Patent US 5463145.

  4. Nakamura, C. E., Gatenby, et al. (2000) Method for the production of 1,3-propanediol by recombinant microorganisms. Patent US 6013494.

  5. Homann, T., Tag, C., Biebl, H., Deckwer, W.-D., and Schink, B. (1990) Fermentation of glycerol to 1,3-propanediol by Klebsiella and Citrobacter strains. Appl. Microbiol. Biotechnol. 33, 121–126.

    Article  CAS  Google Scholar 

  6. Dabrock, B., Bahl, H., and Gottschalk, G. (1992) Parameters effecting solvent production by Clostridium pasteurianum. Appl. Environ. Microbiol. 58, 1233–1239.

    PubMed  CAS  Google Scholar 

  7. Douglas, C. C. and James, A. K. (1994) Conversion of glycerol from soydiesel production to 1,3-propanediol. Final Report National Biodiesel Development Board. Madison, UW-Madison.

    Google Scholar 

  8. Zeng, A. P., Biebl, H., Schlieker, H., and Decker, W. D. (1993) Pathway analysis of glycerol fermentation by Klebsiella pnrumoniae: regulation of reducing equivalent balance and product formation. Enzymol. Microb. Technol. 15, 770–779.

    Article  CAS  Google Scholar 

  9. Johnson, E. A. and Lin, E. C. C. (1987) Klebsiella pneumoniae 1,3-propanediol NAD oxydoreductase. J. Bacteriol. 169, 2050–2054.

    PubMed  CAS  Google Scholar 

  10. Nemeth, A., Kupcsulik, B., and Sevella, B. (2003) 1,3-Propanediol oxidoreductase production with Klebsiella pneumoniae DSM2026. World J. Microbiol. Biotechnol. 19, 659–663.

    Article  CAS  Google Scholar 

  11. Veiga-Da-Cunha, M. and Foster, M. A. (1992) 1,3-propanediol: NAD oxydoreductase of Lactobacillus brevis and Lactobacillus buchneri. Appl. Environ. Microbiol. 58, 2005–2010.

    PubMed  CAS  Google Scholar 

  12. Malaoui, H. and Marczak, R. (2000) Purification and characterization of the 1,3-propanediol dehydrogenase of Clostridium butyricum E5. Enzymol. Microb. Technol. 27, 399–405.

    Article  CAS  Google Scholar 

  13. Daniel, R., Boenigk, R., and Gottschalk, G. (1995) Purification of 1,3-propanediol dehydrogenase from Citrobacter freundii and cloning, sequencing, and overexpression of the corresponding gene in E. coli. J. Bacteriol. 177, 2151–2156.

    PubMed  CAS  Google Scholar 

  14. Luers, F., Seyfried, M., Daniel, R., and Gottschalk, G. (1997) Glycerol conversion to 1,3-propanediol by Clostridium pasteurianum: cloning and expression of the gene encoding 1,3-propanediol dehydrogenase. FEMS Microbiol. Lett. 154, 337–345.

    Article  PubMed  CAS  Google Scholar 

  15. Yuanyuan, Z., Yang, C., and Baishan, F. (2004) Cloning and sequence analysis of the dhaT gene of the 1,3-propanediol regulon from Klebsiella pneumoniae. Biotechnol. Lett. 26, 251–255.

    Article  PubMed  Google Scholar 

  16. Sambrook, J. and Russel, D. W. (2001) Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

    Google Scholar 

  17. Sanger, F., Nicklen, S., and Coulson, A. R. (1997) DNA sequencing with chain termination inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463–5467.

    Article  Google Scholar 

  18. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.

    Article  PubMed  CAS  Google Scholar 

  19. Zhu, M. M., Lawman, P. D., and Cameron, D. C. (2002) Improving 1,3-propanediol production from glycerol in a metabolically engineered Escherichia coli by reducing accumulation of sn-glycerol-3-phosphate. Biotechnol. Prog. 18, 694–699.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. College of Life Science and Technology, Beijing University of Chemical Technology, 100029, Beijing, China

    Tianwei Tan

Authors
  1. Wang Fenghuan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Qu Huijin
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Huang He
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Tianwei Tan
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Tianwei Tan.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Fenghuan, W., Huijin, Q., He, H. et al. High-level expression of the 1,3-propanediol oxidoreductase from klebsiella pneumoniae in escherichia coli . Mol Biotechnol 31, 211–219 (2005). https://doi.org/10.1385/MB:31:3:211

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1385/MB:31:3:211

Index Entries

Search

Navigation