Environmental bacteria such as Pseudomonas putidaoften receive new metabolic genes and pathways through various HGT mechanisms, thereby expanding their biochemical network towards new substrates. However, for achieving durable implantation, the new activities need to adjust various enzymatic, regulatory and spatial parameters to the existing molecular landscape of the recipient cells. The process through which the TOL plasmid pWW0 of P. putida mt-2 has ended up as a stable—yet autonomous—component of the metabolic complement of the strain, exposes a number of conflicts between the incoming biochemical module for degradation of toluene/m-xylene and the already existing route for benzoate catabolism. To solve these, in particular the critical routing of shared catechol intermediates through either an ortho or metacleaving pathway, diverse evolutionary patches seem to have emerged that alleviate the ensuing physiological stress. To gain further insights on how the new toluene/m-xylene-degrading routes can cohabit with the preceding network, the upper and the lowerpathways of the TOL plasmid were excised from their natural DNA context, reassembled in a conjugative vector and reintroduced in the plasmid-cured variant of P. putida mt-2 named KT2440. Adaptive laboratory evolution of the resulting strain for growth on m-xylene delivered variants that had acquired significant changes in the regulatory devices that rule transcription of the upper and the loweroperons. Taken together, the data exposed a complex series of tradeoffs between the new genes and the host's standing metabolism which could inform more effective approaches for designing heterologous gene expression in e.g. metabolic engineering.