Construction of a hybrid strain which is capable of mineralizing each components of a benzene, toluene, and p-xylene mixture simultaneously was attempted by redesigning the metabolic pathway of Pseudomonas putida. Genetic and biochemical analyses of the tod and the tol pathways revealed that dihydrodiols formed from benzene, toluene, and p-xylene by toluene dioxygenase in the tod pathway could be channeled into the tol pathway by the action of cis-p-toluate dihydrodiol dehydrogenase, consequently leading to complete mineralization of a benzene, toluene, and p-xylene mixture. cis-p-Toluate dihydrodiol dehydrogenase encoded in the TOL plasmid pWW0 recognized cis-benzene dihydrodiol, cis-toluene dihydrodiol, and cis-p-xylene dihydrodiol as substrates, forming catechol as an in vivo reaction product. Consequently, a hybrid strain of P. putida TB101 was constructed by introduction of the TOL plasmid into P. putida F39/D, a blocked mutant of P. putida F1, which is unable to transform dihydrodiols to corresponding catechols. The metablic fluxes of benzene, toluene, and p-xylene were redirected from the tod to the tol pathway at the level of dihydrodiol, resulting in the mineralization of the three components of benzene, toluene, and p-xylene mixture. P. putida TB101, however, showed such problems in the degradation of a benzene, toluene, and p-xylene mixture as low degradation rate of benzene and delayed initiation of benzene degradation. These problems were solved by constructing the second hybrid strain of P. putida TB103. P. putida TB103 was constructed by cloning the todC1C2BA genes encoding toluene dioxygenase in the broad-host-range vector RSF1010 and by introducing the resulting plasmid pTOD037 into P. putida mt-2 which harbors the TOL plasmid pWW0. The degradation rates of benzene, toluene, and p-xylene by P. putida TB103 were increased by about 9.3, 3.7, and 1.4-fold, respectively, compared with those by P. putida TB101. Apparently, this improved capability of P. putida TB103 for the degradation of a benzene, toluene, and p-xylene mixture resulted from the amplification of the todC1C2BA genes. Furthermore, a relatively long lag period for benzene degradatoin observed when P. putida TB101 was used for the degradation at low dissolved oxygen tension disappeared when P. putida TB103 was employed. The absence of the lag period was mainly due to the fact that the expression of toluene dioxygenase genes in P. putida TB103 is controlled by the Su promoter of RSF1010 which is insensitive to the dissolved oxygen tension. The mineralization of nitrobenzene was also attempted by Pseudomonas putida TB 103 based on the hypothesis that cis-nitrobenzene dihydrodiol formed from nitrobenzene by the action of toluene dioxygenase may be channeled into the tol pathway. cis-p-Toluate dihydrodiol dehydrogenase again was observed to act a critical role in the mineralization of nitrobenzene through the hybrid pathway by catalyzing conversion of cis-nitrobenzene dihydrodiol produced during the initial catabolism of nitrobezene via the tod pathway to catechol, and by redirecting the metabolic route of cis-nitrobenzene dihydrodiol from the tod to the tol pathway.
