Metabolic Engineering of <i>Pseudomonas putida</i> for Simultaneous Degradation of Chlorpyrifos and 2,4-Dichlorophenoxyacetic Acid and Its Application in Wastewater Treatment

With the widespread use of synthetic pesticides and herbicides in agriculture, combined pollution caused by toxic and recalcitrant compounds has become an increasingly significant environmental concern. An organophosphorus pesticide chlorpyrifos (CP) and a prevalent organochlorine herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) are often codetected in agricultural soil and groundwater. This study utilized a scarless genome editing approach to insert the mpd, tfdABCDEF, gfp, and vgb genes into the genome of Pseudomonas putida KT2440, to generate strain KTU-G7 V. Unlike engineered strains for bioremediation of individual pollutants, KTU-G7 V can efficiently degrade 25 mg/L 2,4-D and 100 mg/L CP within 9 and 32 h, respectively. Stable isotope analysis showed that KTU-G7 V could fully convert 13C6-labeled 2,4-D into 13CO2 within 4 days. Under low-oxygen conditions, KTU-G7 V retained the ability to fully codegrade 2,4-D and CP within 24 h, suggesting the potential of KTU-G7 V for groundwater bioremediation. Also, KTU-G7 V emitted strong green fluorescence in sunlight, enabling real-time monitoring of in situ bioremediation process. The exogenous genes were also not lost in the KTU-G7 V genome after successive passage cultures, which outcompeted the plasmid-borne engineered strains. In real wastewater with toxic substances, KTU-G7 V achieved an 80% removal rate for 10 mg/L CP and 2,4-D mixtures within 12 h, demonstrating its applicability in real-world scenarios. These merits of KTU-G7 V make it an ideal candidate for in situ bioremediation of environments cocontaminated with CP and 2,4-D. More importantly, this strategy of combining a superior chassis with multipathways assembly may be utilized for the remediation of combined pollution.