Xylene monooxygenase catalyzes the multistep oxygenation of toluene and pseudocumene to corresponding alcohols, aldehydes, and acids in Escherichia coli JM101

Xylene monooxygenase of Pseudomonas putida mt-2 catalyzes the methylgroup h ydroxylation of toluene and xylenes. To investigate the potential of xylene monooxygenase to catalyze multistep oxidations of one methyl group, we tes ted recombinant Escherichia coli expressing the monooxygenase genes xylM an d xylA under the control of the alh regulatory system of Pseudomonas oleovo rans Gpo1, Expression of xylene monooxygenase genes could efficiently be co ntrolled by n-octane and dicyclopropylketone. Xylene monooxygenase was foun d to catalyze the oxygenation of toluene, pseudocumene, the corresponding a lcohols, and the corresponding aldehydes, For all three transformations O-1 8 incorporation provided stong evidence for a monooxygenation type of react ion, with gem-diols as the most likely reaction intermediates during the ox ygenation of benzyl alcohols to benzaldehydes. To investigate the role of b enzyl alcohol dehydrogenase (XylB) in the formation of benzaldehydes,;xylB was cloned behind and expressed in concert with xylMA. In comparison to E. coli expressing only xylMA, the presence of xylB lowered product formation rates and resulted in back formation of benzyl alcohol from benzaldehyde. I n P. putida mt-2 XylB may prevent the formation of high concentrations of t he particularly reactive benzaldehydes. In the case of high fluxes through the degradation pathways and low aldehyde concentrations, XylB may contribu te to benzaldehyde formation via the energetically favorable dehydrogenatio n of benzyl alcohols. The results presented here characterize XylMA as an a ble to catalyze the multistep oxygenation of toluenes.