Development of an efficient pathway construction strategy for rapid evolution of the biodegradation capacity of Pseudomonas putida KT2440 and its application in bioremediation

In this work, we developed an efficient pathway construction strategy, consisting of DNA assembler-assisted pathway assembly and counterselection system-based chromosomal integration, for the rapid and efficient integration of synthetic biodegradation pathways into the chromosome of Pseudomonas putida KT2440. Using this strategy, we created a novel degrader capable of complete mineralization of γ-hexachlorocyclohexane (γ-HCH) and 1,2,3-trichloropropane (TCP) by integrating γ-HCH and TCP biodegradation pathways into the chromosome of P. putida KT2440. Furthermore, the chromosomal integration efficiencies of γ-HCH and TCP biodegradation pathways were improved to 50% and 41.6% in P. putida KT2440, respectively, by the inactivation of a type I DNA restriction-modification system. The currently developed pathway construction strategy coupled with the mutant KTUΔhsdRMS will facilitate implantation of heterologous catabolic pathways into the chromosome for rapid evolution of the biodegradation capacity of P. putida. More importantly, the successful removal of γ-HCH (10 mg/kg soil) and TCP (0.2 mM) from soil and wastewater within 14 days, respectively, highlighted the potential of the novel degrader for in situ bioremediation of γ-HCH- and TCP-contaminated sites. Moreover, chromosomal integration of gfp made the degrader to be monitored easily during bioremediation. In the future, this strategy can be expanded to a broad range of bacterial species for widespread applications in bioremediation.