Expression of benzene dioxygenase from Pseudomonas putida ML2 in cis-1,2-cyclohexanediol-degrading pseudomonads

R. J. Swift, S. F. Carter, D. A. Widdowson, J. R. Mason, D. J. Leak

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

Benzene dioxygenase (BDO; EC 1.14.12.3) from Pseudomonas putida ML2 dihydroxylates benzene to produce cis-1,2-dihydroxy-cyclohexa-3,5-diene. As well as oxidising benzene and toluene, cell-free extracts of Escherichia coli JM109 expressing recombinant BDO oxidised cyclohexene, 1-methylcyclohexene and 3-methylcyclohexene. In an attempt to construct a novel metabolic pathway for the degradation of cyclohex-ene (via an initial BDO-mediated dihydroxylation of cyclohexene), cis-1,2-cyclohexanediol-degrading bacteria were isolated by enrichment culture. The bedC1C2BA genes encoding BDO (under the control of the tac promoter) were sub-cloned into pLAFR5, successfully conjugated into seven of the Gram-negative cis-1,2-cyclo-hexanediol-degrading isolates and stably maintained and expressed in three of them. However, despite their ability to grow on cis-1,2-cyclohexanediol as sole carbon source, express an active BDO and oxidise cyclohexene, none of the three strains was able to grow on cyclohexene as sole carbon source. Analysis revealed that BDO oxidised cyclohexene to a mixture of two products, a monohydroxylated (2-cyclohexen-1-ol) product and a dihydroxylated (cis-1,2-cyclohexanediol) product; and failure to grow on cyclohexene was attributed to the toxicity of metabolic intermediates accumulating from the 2-cyclohexen-1-ol metabolism.

Original languageEnglish
Pages (from-to)721-726
Number of pages6
JournalApplied Microbiology and Biotechnology
Volume55
Issue number6
DOIs
Publication statusPublished - 1 Jun 2001

Fingerprint

Dioxygenases
Pseudomonas putida
Benzene
benzene 1,2-dioxygenase
Gene encoding
Carbon
Metabolism
Escherichia coli
Toluene
Toxicity
Bacteria
Degradation
Metabolic Networks and Pathways
Cell Extracts
1,2-cyclohexanediol
cyclohexene
Genes

ASJC Scopus subject areas

  • Biotechnology
  • Microbiology
  • Bioengineering

Cite this

Expression of benzene dioxygenase from Pseudomonas putida ML2 in cis-1,2-cyclohexanediol-degrading pseudomonads. / Swift, R. J.; Carter, S. F.; Widdowson, D. A.; Mason, J. R.; Leak, D. J.

In: Applied Microbiology and Biotechnology, Vol. 55, No. 6, 01.06.2001, p. 721-726.

Research output: Contribution to journalArticle

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