Metabolic engineering of Pseudomonas putida for the simultaneous biodegradation of benzene, toluene, and p‐xylene mixture

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Abstract
For the complete biodegradation of a mixture of benzene, toluene, and p―xylene (BTX), a critical metabolic step that can connect two existing metabolic pathways of aromatic compounds (the tod and the tol pathways) was determined. Toluate―cis‐glycol dehydrogenase in the tol pathway was found to attack benzene―cis―glycol, toluene―cis―glycol, and p―xylene―cis―glycol, which are metabolic intermediates of the tod pathway. Based on this observation, a hybrid strain, Pseudomonase putida TB101, was constructed by introduction of the TOL plasmid pWW0 into P. putida F39/D, a derivative of P. putida F1, which is unable to transform cis―glycol compounds to corresponding catechols. The metabolic flux of BTX into the tod pathway was redirected to the tol pathway at the level of cis―glycol compounds by the action of toluate―cis―glycol dehydrogenase in P. putida TB101, resulting in the simultaneous mineralization of BTX mixture without accumulation of any metabolic intermediates. The profile of specific degradation rates showed a similar pattern as that of the specific growth rate of the microorganism, and the maximum specific degradation rates of benzene, toluene, and p‐xylene were determined to be about 0.27, 0.86, and 2.89 mg/mg biomass/h, respectively. P. putida TB101 is the first reported microorganism that mineralizes BTX mixture simultaneously. © 1994 John Wiley & Sons, Inc.