Applied Microbiology and Biotechnology

, Volume 75, Issue 5, pp 1095–1101 | Cite as

Design of a secondary alcohol degradation pathway from Pseudomonas fluorescens DSM 50106 in an engineered Escherichia coli

  • Anett Kirschner
  • Josef Altenbuchner
  • Uwe T. Bornscheuer
Applied Genetics and Molecular Biotechnology
First Online:
Received:
Revised:
Accepted:

Abstract

The genes encoding an alcohol dehydrogenase, Baeyer–Villiger monooxygenase and an esterase from P. fluorescens DSM 50106, which seemed to be metabolically connected based on the sequence of the corresponding open reading frames, were cloned into one vector (pABE) and functionally expressed in Escherichia coli. Overall expression levels were quite low, however, using whole cells of E. coli JM109 pABE expressing the three recombinant enzymes, conversion of secondary alcohols (Cn) to the corresponding primary alcohols (Cn−2) and acetic acid via ketone and ester was possible. In this way, 2-decanol was almost completely converted within 20 h at 30°C. Thus, it could be shown that the three enzymes are metabolically connected and that they are most probably involved in alkane degradation via sub-terminal oxidation of the acyclic aliphatic hydrocarbons.

Keywords

Baeyer–Villiger monooxygenase Esterase Alcohol dehydrogenase Aliphatic alcohols Pseudomonas fluorescens Degradation pathway 
This is a preview of subscription content, log in to check access.

Notes

Acknowledgment

We thank the Fonds der Chemischen Industrie (Frankfurt, Germany) and the Studienstiftung des deutschen Volkes (Bonn, Germany) for stipends to Anett Kirschner.

References

  1. Abdel-Megeed A (2004) Psychrophilic degradation of long chain alkanes. Dissertation, Technical University Hamburg-Harburg, GermanyGoogle Scholar
  2. Ashraf W, Mihdhir A, Murell JC (1994) Bacterial oxidation of propane. FEMS Microbiol Lett 122:1–6PubMedCrossRefGoogle Scholar
  3. Atlas RM (1981) Microbial degradation of petroleum hydrocarbons: an environmental perspective. Microbiol Rev 45:180–209PubMedGoogle Scholar
  4. Britton LN (1984) Microbial degradation of aliphatic hydrocarbons. In: Gibson DT (ed) Microbial degradation of organic compounds, vol. 13. Marcel Dekker, New York, pp 89–129Google Scholar
  5. Forney FW, Markovetz AJ (1970) Subterminal oxidation of aliphatic hydrocarbons. J Bacteriol 102:281–282PubMedGoogle Scholar
  6. Hildebrandt P, Musidlowska A, Bornscheuer UT (2002) Cloning, functional expression and biochemical characterization of a stereoselective alcohol dehydrogenase from Pseudomonas fluorescens DSM50106. Appl Microbiol Biotechnol 59:483–487PubMedCrossRefGoogle Scholar
  7. Hundt K, Wagner M, Becher D, Hammer E, Schauer F (1998) Effect of selected environmental factors on degradation and mineralization of biaryl compounds by the bacterium Ralstonia pickettii in soil and compost. Chemosphere 36:2321–2335PubMedCrossRefGoogle Scholar
  8. Khalameyzer V, Fischer I, Bornscheuer UT, Altenbuchner J (1999) Screening, nucleotide sequence and biochemical characterization of an esterase from Pseudomonas fluorescens with high activity toward lactones. Appl Environ Microbiol 65:477–482PubMedGoogle Scholar
  9. Kirschner A, Altenbuchner J, Bornscheuer UT (2007) Cloning, expression and characterization of a Baeyer–Villiger monooxygenase from Pseudomonas fluorescens DSM 50106 in E. coli. Appl Microbiol Biotechnol 73:1065–1072PubMedCrossRefGoogle Scholar
  10. Laemmli UK (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 27:680–685CrossRefADSGoogle Scholar
  11. Leahy JG, Colwell RR (1990) Microbial degradation of hydrocarbons in the environment. Microbiol Rev 54:305–315PubMedGoogle Scholar
  12. Margesin R, Schinner F (1997) Bioremediation of diesel-oil-contaminated alpine soils at low temperatures. Appl Microbiol Biotechnol 47:462–468CrossRefGoogle Scholar
  13. Margesin R, Schninner T (2001) Biodegradation and bioremediation of hydrocarbons in extreme environments. Appl Microbiol Biotechnol 56:650–663PubMedCrossRefGoogle Scholar
  14. Stumpp T, Wilms B, Altenbucher J (2000) Ein neues, l-Rhamnose-induzierbares Expressionssystem für Escherichia coli. Biospektrum 1.00:33–36Google Scholar
  15. Urlacher V, Schmid RD (2006) Recent advances in oxygenase-catalyzed biotransformations. Curr Opin Chem Biol 10:156–161PubMedCrossRefGoogle Scholar
  16. van Beilen JB, Li Z, Duetz WA, Smits THM, Witholt B (2003) Diversity of alkane hydroxylase systems in the environment. Oil Gas Sci Technol Rev IFP 58:427–440CrossRefGoogle Scholar
  17. Whyte LG, Hawari J, Zhou E, Bourbonnière L, Inniss WE, Greer CW (1998) Biodegradation of variable-chain-length alkanes at low temperatures by a psychrophilic Rhodococcus sp. Appl Environ Microbiol 64:2578–2584PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Anett Kirschner
    • 1
  • Josef Altenbuchner
    • 2
  • Uwe T. Bornscheuer
    • 1
  1. 1.Department of Biotechnology and Enzyme Catalysis, Institute of BiochemistryGreifswald UniversityGreifswaldGermany
  2. 2.Institute of Industrial GeneticsStuttgart UniversityStuttgartGermany

Personalised recommendations