Whole-cell Biosensor Engineering Based on the Transcription Factor XylS/Pm for Sensitive Detection of PCB Intermediate Chlorobenzoic Acid

Chlorobenzoic acids (Cba) are toxic chloride pollutants commonly released by chemical industries or formed during the degradation pathway of organic chlorides, such as polychlorinated biphenyls (PCBs). Here, we successfully developed a highly sensitive Escherichia coli whole-cell Cba biosensor, named XyF10 (MG1655(DE3)-Ps-XylS (Step13)-Pm1-EGFP), by comprehensive engineering the elements of Cba sensing genetic circuit from Pseudomonas putida. Compared to the native sensing system, XyF10 exhibited a significantly reduced detection limit (<1 mu M vs. 50 mu M) and an increased signal-to-noise ratio (11.7-fold vs. 4.4-fold), making it a highly efficient whole-cell sensor with the lowest detection limit for Cba reported to date. Structural analysis and molecular dynamics simulations of XylS and its variants revealed that the increased flexibility of a ring structure in XylS (Step13) variant promoted the sensing efficiency of XyF10 system by fascinating the formation of active dimers and enabled more stable binding with inducer. Furthermore, the XyF10 system displayed an obvious preference for meta-substituted Cba (3-Cba) as substrates, which was attributed by the stronger interaction between 3-Cba and the active site of XylS. Overall, our research not only advanced the Cba detection techniques for large-scale detection of Cba in the environment, but also provided potential tools for high-throughput screening of PCB degradation pathways and enzymes.