Skip to main content
SpringerLink
Log in

Cloning of Catechol 2,3-Dioxygenase Gene and Construction of a Stable Genetically Engineered Strain for Degrading Crude Oil

  • Original Article
  • Published:
Indian Journal of Microbiology Aims and scope Submit manuscript

Abstract

Pseudomonas putida strain BNF1 was isolated to degrade aromatic hydrocarbons efficiently and use phenol as a main carbon and energy source to support its growth. Catechol 2,3-dioxygenase was found to be the responsible key enzyme for the biodegradation of aromatic hydrocarbons. Catechol 2,3-dioxygenase gene was cloned from plasmid DNA of P. putida strain BNF1. The nucleotide base sequence of a 924 bp segment encoding the catechol 2,3-dioxygenase (C23O) was determined. This segment showed an open reading frame, which encoded a polypeptide of 307 amino acids. C23O gene was inserted into NotI-cut transposon vector pUT/mini-Tn5 (Kmr) to get a novel transposon vector pUT/mini-Tn5-C23O. With the helper plasmid PRK2013, the transposon vector pUT/mini-Tn5-C23O was introduced into one alkanes degrading strain Acinetobacter sp. BS3 by triparental conjugation, and then the C23O gene was integrated into the chromosome of Acinetobacter sp. BS3. And the recombinant BS3-C23O, which could express catechol 2,3-dioxygenase protein, was obtained. The recombinant BS3-C23O was able to degrade various aromatic hydrocarbons and n-alkanes. Broad substrate specificity, high enzyme activity, and the favorable stability suggest that the BS3-C23O was a potential candidate used for the biodegradation of crude oil.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Di Martino C, Lópezand NIR, Iustman LJ (2002) Isolation and characterization of benzene, toluene and xylene degrading Pseudomonas sp. selected as candidates for bioremediation. Int Biodeter Biodegrad 67:15–20

    Article  Google Scholar 

  2. Lindstrom JE, Braddock JF (2002) Biodegradation of petroleum hydrocarbons at low temperature in the presence of dispersant Corexit 9500. Mar Pollut Bul 44:739–747

    Article  CAS  Google Scholar 

  3. Pepi M, Cesaro A, Liut G, Baldi F (2005) An antarctic psychrotrophic bacterium Halomonas sp. ANT-3b, growing on n-hexadecane, produces a new emulsifying glycolipid. FEMS Microbiol Ecol 53:157–166

    Article  CAS  PubMed  Google Scholar 

  4. Aldrett S, Bonner JS, Mills MA, Autenrieth RL, Stephens FL (1997) Microbial degradation of crude oil in marine environments tested in a flask experiment. Water Res 31:2840–2848

    Google Scholar 

  5. Zídková L, Szőköl J, Rucká L, Pátek M, Nešvera J (2012) Biodegradation of phenol using recombinant plasmid-carrying ropolis strains Rhodococcus eryth. Int Biodeter Biodegrad 1–6. doi:10.1016/j.ibiod.2012.05.017

  6. Ibrahim MK (2003) Cloning and nucleotide sequence of catechol 2,3-dioxygenase gene from the naphthalene-degrading pseudomonas putida NA3. Int J Agric Biol 5:423–427

    CAS  Google Scholar 

  7. Han EM, Jung YH, On HY et al (1997) Tn5 tagging of the phenol-degrading gene on the chromosome of Pseudomonas putida. Mol Cells 7:40–44

    CAS  PubMed  Google Scholar 

  8. Harayama S, Rekik M (1989) Bacterial aromatic ring-cleavage enzyme are classified into two different gene families. J Biol Chem 263:15328–15333

    Google Scholar 

  9. Diaz E (2004) Bacterial degradation of aromatic pollutants: a paradigm of metabolic versatility. Int Microbiol 7:173–180

    CAS  PubMed  Google Scholar 

  10. Zahir ZA, Akhtar SS, Ahmad M, Nadeem SM, Saifullah (2012) Comparative effectiveness of Enterobacter aerogenes and Pseudomonas fluorescens for mitigating the depressing effect of brackish water on maize. Int J Agric Biol 14:337–344

    CAS  Google Scholar 

  11. Sambrook J, Fritisch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor, New York

    Google Scholar 

  12. Meyer S, Moser R, Neef A, Stahl U, Kämpfer P (1999) Differential detection of key enzymes of polyaromatic-hydrocarbon-degrading bacteria using PCR and gene probes. Microbiol 145:1731–1741

    Article  CAS  Google Scholar 

  13. Mezei LM, Storts DR (1994) Purification of PCR products. In: Griffin HG, Griffin AM (eds) PCR technology: current innovations. CRC, Boca Raton, p 21

    Google Scholar 

  14. Heinaru E, Truu J, Stottmeister U, Heinaru A (2000) Three types of phenol and p-cresol catabolism in phenol- and p-cresol-degrading bacteria isolated from river water continuously polluted with phenolic compounds. FEMS Microbiol Ecol 31:195–205

    Article  CAS  PubMed  Google Scholar 

  15. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  16. Nakai C, Kagamiyama H, Nozaki M et al (1983) Complete nucleotide sequence of the metapyrocatechase gene on the TOI plasmid of Pseudomonas putida mt-2. J Biol Chem 258:2923–2928

    CAS  PubMed  Google Scholar 

  17. Noh SJ, Kim Y, Min KH, Karegoudar TB, Kim CK (2000) Cloning and nucleotide sequence analysis of xylE gene responsible for meta-cleavage of 4-chlorocatechol from Pseudomonas sp. S-47. Mol Cells 10:475–479

    CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was financially supported by the Microbial Engineering Laboratory of Northwestern University and Technical Innovation Program of Xi’an of China (CX09017).

Conflict of interest

The authors declare that there are no conflicts of interest.

Author information

Authors and Affiliations

  1. Biology Department, Northwestern University, Xi’an, China

    Yun Xie, Feng Yu, Qi Wang & Wuling Chen

  2. Sinochem Chemical Industry Park, Taicang, China

    Xin Gu

Authors
  1. Yun Xie
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Feng Yu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Qi Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Xin Gu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Wuling Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Wuling Chen.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Xie, Y., Yu, F., Wang, Q. et al. Cloning of Catechol 2,3-Dioxygenase Gene and Construction of a Stable Genetically Engineered Strain for Degrading Crude Oil. Indian J Microbiol 54, 59–64 (2014). https://doi.org/10.1007/s12088-013-0411-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12088-013-0411-2

Keywords

Search

Navigation