Synthetically primed growth of Pseudomonas putida on 2,4-dinitrotoluene as sole carbon and nitrogen source

While current genetic tools easily enable transfer of metabolic genes among bacteria, their effective nesting in the recipients depends on biochemical and regulatory compatibilities of the introduced pathway with those of the host. This issue becomes evident in e.g. attempts to engineer bacteria to degrade 2,4-dinitrotoluene (DNT), a xenobiotic compound naturally broken down-if quite ineffectively-by Burkholderia sp. DNT through the so-called dnt route. That despite multiple efforts no strain engineered with a complete set of dnt genes has been able to grow on DNT as sole carbon and nitrogen source suggests that new hosts need to go through a mutual chassis-implant adaptation process for successful degradation of this xenobiotic. To explore a possible roadmap for this to happen we have applied Adaptive Laboratory Evolution (ALE) to a Pseudomonas putida strain designed to carry an active dnt pathway but initially unable to grow on DNT. Over 315 days of selective subculturing, evolved strains emerged that could metabolize DNT as the sole growth substrate. Genetic and phenotypic analyses of the best-performing isolate revealed a large number of adaptations that improved stress tolerance and fine-tuned to host's metabolic context to the newly introduced route. These results expose the occurrence of a sort of molecular negotiation between the incoming genes and the pre-existing molecular network of the host before cells entirely integrate the new pathway into their biochemical complement.