Creating a novel halotolerant whole-cell biosensor for efficient degradation and precise quantification of p-nitrophenol-substituted organophosphate pesticides in hypersaline ecosystems

The previously constructed biosensors for organophosphate pesticides (OPs) detection cannot be used to measure OPs concentrations under high salt conditions. In this study, a novel halotolerant whole-cell biosensor J9U-pBBR- pobRA - gfp , which consisted of a p -nitrophenol (pNP)-responsive transcription regulator PobR and its cognate promoter fused to a reporter GFP, was constructed from a salt-tolerant chassis Halomonas cupida J9 for pNP-substituted OPs detection in high-salinity environments. Through dynamic regulation of PobR expression levels, the leaky fluorescence was significantly reduced. Furthermore, the optimized biosensing circuits were introduced into an OP-degrading chassis J9U- mpd , to generate dual-functional biosensors that could degrade 40 μM methyl parathion (MP) within 80 min in high-salinity media. The biosensors exhibited broad specificity towards pNP-substituted OPs (e.g., MP and fenitrothion), while showing no response to non-pNP-substituted OPs like chlorpyrifos. Dose-response curves indicated that the three biosensors J9U- mpd -pBBR-P3 (P15 or P17) pobRA - gfp maintained a linear detection range of 0.1–60 μM for pNP and 0.1–20 μM for MP in high-saline media. Biosensors J9U- mpd -pBBR-P3 (or P17) pobRA - gfp achieved a low detection limit (LOD) of 0.1 μM for both pNP and MP in seawater and high-salinity river water, and showed a LOD of 0.019 mg/kg for pNP and 0.026 mg/kg for MP in saline-alkali soil. pNP and MP concentrations estimated by the biosensors J9U- mpd -pBBR-P3 (or P17) pobRA - gfp matched well with pollutant concentrations measured by HPLC analysis, demonstrating the reliability of the biosensor detection results. The halotolerant biosensor can be applied to efficient degradation and precise quantification of OPs in hypersaline aquatic and terrestrial ecosystems. Our strategy of combining synthetic biology with extremophile chassis may be utilized to create novel biosensors for pollutant detection in extreme environments.