Enhanced assessment of water quality for both nitrate and nitrite using engineered E. coli with para-aminobenzoic acid biosynthesis

Background: Monitoring nitrate and nitrite levels in water is vital for protecting human health, aquatic ecosystems, and regulatory compliance. However, traditional detection methods often involve environmentally harmful chemicals. This study introduces a sustainable alternative by leveraging metabolically engineered E. coli to biosynthesize para-aminobenzoic acid (PABA) via the shikimate pathway, replacing conventional sulfonamides in the Griess reaction. This approach significantly reduces environmental impact while maintaining high analytical performance. Results: This study introduces a sustainable approach for simultaneously detecting nitrate and nitrite in water using a combination of E. coli strains DH5α and BL21. Metabolically engineered E. coli BL21 produces PABA via the shikimate pathway, replacing synthetic chemicals in the modified Griess reaction. The modified Griess reaction, utilizing PABA, achieved a high sensitivity detection limit of 0.57 μM with excellent selectivity for nitrite over other ions. Recognizing the importance of portability for on-site, real-time water quality assessment, we developed a paper-based detection system utilizing lyophilized cell supernatant. To enhance portability, we developed a paper-based method for detecting nitrite using lyophilized cell supernatant. This approach confirmed successful nitrite detection through a clear colorimetric response, enabling immediate and quantitative analysis of nitrate and nitrite. Validation with real-world water samples yielded a recovery rate of 90–100 %, comparable to the Griess Reagent, confirming the effectiveness and reliability of the proposed sensors for environmental monitoring. By integrating the capabilities of two E. coli strains, this dual-detection system uniquely allows simultaneous quantification of nitrate and nitrite in a single sample, significantly advancing the field of water quality monitoring. Significance and novelty: This study demonstrates a sustainable, high-sensitivity solution for water quality monitoring by combining microbial metabolic engineering with a portable, paper-based detection platform. The approach meets EPA standards, minimizes environmental impact, and provides a practical tool for field-testing, underscoring the potential of engineered microbes for eco-friendly and effective environmental monitoring.