Creating a Halotolerant Degrader for Efficient Mineralization of p-Nitrophenol-Substituted Organophosphorus Pesticides in High-Saline Wastewater

作     者：Liu, Yujie Xiong, Weini Jiang, Yuting Meng, Yan Zhao, Wanwan Yang, Chao Liu, Ruihua 

作者机构：Nankai Univ Coll Life Sci Key Lab Mol Microbiol & Technol Minist Educ Tianjin Peoples R China Nankai Univ Coll Life Sci Dept Biochem & Mol Biol Tianjin Peoples R China 

出 版 物：《BIOTECHNOLOGY AND BIOENGINEERING》 (Biotechnol. Bioeng.)

年 卷 期：2025年第122卷第4期

页      面：936-947页

核心收录：

学科分类：0710[理学-生物学] 07[理学] 09[农学] 0836[工学-生物工程] 

基　　金：This work was supported by the National Key Research and Development Program of China (2023YFE0104900-4). [2023YFE0104900-4] National Key Research and Development Program of China 

主　　题：bioaugmentation complete mineralization Halomonas cupida high-saline wastewater organophosphorus pesticides 

摘      要：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 beta-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