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

Benzene dioxygenase (BDO; EC 1.14.12.3) from Pseudomonas putida ML2 dihydro xylates benzene to produce cis-1,2-dihydroxy-cyclohexa-3,5-diene. As well a s oxidising benzene and toluene, cell-free extracts of Escherichia coli JM1 09 expressing recombinant BDO oxidised cyclohexene, 1-methylcyclohexene and 3-methylcyclohexene. In an attempt to construct a novel metabolic pathway for the degradation of cyclohexene (via an initial BDO-mediated dihydroxyla tion of cyclohexene), cis-1,2-cyclohexanediol-degrading bacteria were isola ted by enrichment culture. The bedC1C2BA genes encoding BDO (under the cont rol of the tac promoter) were sub-cloned into pLAFR5, successfully conjugat ed into seven of the Gram-negative cis-1,2-cyclohexanediol-degrading isolat es and stably maintained and expressed in three of them. However, despite t heir ability to grow on cis-1,2-cyclohexanediol as sole carbon source, expr ess an active BDO and oxidise cyclohexene, none of the three strains was ab le to grow on cyclohexene as sole carbon source. Analysis revealed that BDO oxidised cyclohexene to a mixture of two products, a monohydroxylated (2-c yclohexen-1-ol) product and a dihydroxylated (cis-1,2-cyclohexanediol) prod uct; and failure to grow on cyclohexene was attributed to the toxicity of m etabolic intermediates accumulating from the 2-cyclohexen-1-ol metabolism.