Detection of Short-Chain Chlorinated Aliphatic Hydrocarbons through an Engineered Biosensor with Tailored Ligand Specificity.

Short-chain chlorinated aliphatic hydrocarbons (SCAHs), commonly used as industrial reagents and solvents, pose a significant threat to ecosystems and human health as they infiltrate aquatic environments due to extensive usage and accidental spills. Whole-cell biosensors have emerged as cost-effective, rapid, and real-time analytical tools for environmental monitoring and remediation. While the broad ligand specificity of transcriptional factors (TFs) often prohibits the application of such biosensors. Herein, we exploited a semirational transition ligand approach in conjunction with a positive/negative fluorescence-activated cell sorting (FACS) strategy to develop a biosensor based on the TF AlkS, which is highly specific for SCAHs. Furthermore, through promoter-directed evolution, the performance of the biosensor was further enhanced. Mutation in the -10 region of constitutive promoter P resulted in reduced AlkS leakage expression, while mutation in the -10 region of inducible promoter P increased its accessibility to the AlkS-SCAHs complex. This led to an 89% reduction in background fluorescence leakage of the optimized biosensor, M2-463, further enhancing its response to SCAHs. The optimized biosensor was highly sensitive and exhibited a broader dynamic response range with a 150-fold increase in fluorescence output after 1 h of induction. The detection limit (LOD) reached 0.03 ppm, and the average recovery rate of SCAHs in actual water samples ranged from 95.87 to 101.20%. The accuracy and precision of the proposed biosensor were validated using gas chromatography-mass spectrometry (GC-MS), demonstrating the promising application for SCAH detection in an actual environment sample.