Creating a Halotolerant Degrader for Efficient Mineralization of <i>p</i> ‐Nitrophenol‐Substituted Organophosphorus Pesticides in High‐Saline Wastewater

The bioaugmentation performance is severely reduced in the treatment of high-saline pesticide wastewater because the growth and degradation activity of pesticide degraders are significantly inhibited by high salt concentrations. In this study, a heterologous biodegradation pathway comprising the seven genes mpd/pnpABCDEF responsible for the bioconversion of p-nitrophenol (PNP)-substituted organophosphorus pesticides (OPs) into β-oxoadipate and the genes encoding Vitreoscilla hemoglobin (VHb) and green fluorescent protein (GFP) were integrated into the genome of a salt-tolerant chassis Halomonas cupida J9, to generate a genetically engineered halotolerant degrader J9U-MP. RT-PCR assays demonstrated that the nine exogenous genes are successfully transcribed to mRNA in J9U-MP. Gas chromatography analysis of methyl parathion (MP) and its intermediates demonstrated that the expressed MP hydrolase and PNP-degrading enzymes PnpABCD show obvious degradation activity toward the specific substrates in J9U-MP. Stable isotope analysis showed that J9U-MP is able to efficiently convert ¹³C₆-PNP into ¹³CO₂, demonstrating the complete mineralization of MP in high-salt media. J9U-MP is genetically stable during passage culture, and genomic integration of exogenous genes does not negatively influence the growth of J9U-MP. Under oxygen-limited conditions, VHb-expressing J9U-MP does not show obvious growth inhibition and a significant reduction in the MP degradation rate. A real-time monitoring system with enhanced GFP is used to track the motion and activity of J9U-MP during bioremediation. Moreover, 50 mg/L MP and its intermediates (i.e., PNP and HQ) were completely degraded by J9U-MP within 12 h in wastewater supplemented with 60 g/L NaCl. After 3 days of incubation, 25 mg/L ¹³C₆-PNP was converted into ¹³CO₂ by J9U-MP in wastewater supplemented with 60 g/L NaCl. Our results highlight the power of synthetic biology for creating new halotolerant pollutant-mineralizing strains. The strong competitive advantages of J9U-MP in high-salinity and low-oxygen environments make this degrader suitable for in situ bioaugmentation of OP wastewater.