Identification of a novel dioxygenase involved in metabolism of o-xylene, toluene, and ethylbenzene by Rhodococcus sp. strain DK17

Rhodococcus sp. strain DK17 is able to grow on o-xylene, benzene, toluene, and ethylbenzene. DK17 harbors at least two megaplasmids, and the genes encoding the initial steps in alkylbenzene metabolism are present on the 330-kb pDK2. The genes encoding alkylbenzene degradation were cloned in a cosmid clone and sequenced completely to reveal 35 open reading frames (ORFs). Among the OMFs, we identified two nearly exact copies (one base difference) of genes encoding large and small subunits of an iron sulfur protein terminal oxygenase that are 6 kb apart from each other. Immediateiy downstream of one copy of the dioxygenase genes (akbA1_a and akbA2_a) is a gene encoding a dioxygenase ferredoxin component (akbA3), and downstream of the other copy (akbA1_b and akbA2_b) are genes putatively encoding a meta-cleavage pathway. RT-PCR experiments show that the two copies of the dioxygenase genes are operonic with the downstream putative catabolic genes and that both operons are induced by o-xylene. When expressed in Escherichia coli, AkbA1_a-AkbA2_a-AkbA3 transformed o-xylene into 2,3- and 3,4-dimethylphenol. These were apparently derived from an unstable o-xylene cis-3,4-dihydrodiol, which readily dehydrates. This indicates a single point of attack of the dioxygenase on the aromatic ring. In contrast, attack of AkbA1_a-AkbA2_a-AkbA3 on ethylbenzene resulted in the formation of two different cis-dihydrodiols resulting from an oxidation at the 2,3 and the 3,4 positions on the aromatic ring, respectively.