Detection of\nShort-Chain Chlorinated Aliphatic Hydrocarbons\nthrough an Engineered Biosensor with Tailored Ligand Specificity

Short-chain chlorinated aliphatic\nhydrocarbons (SCAHs),\ncommonly\nused as industrial reagents and solvents, pose a significant threat\nto ecosystems and human health as they infiltrate aquatic environments\ndue to extensive usage and accidental spills. Whole-cell biosensors\nhave emerged as cost-effective, rapid, and real-time analytical tools\nfor environmental monitoring and remediation. While the broad ligand\nspecificity of transcriptional factors (TFs) often prohibits the application\nof such biosensors. Herein, we exploited a semirational transition\nligand approach in conjunction with a positive/negative fluorescence-activated\ncell sorting (FACS) strategy to develop a biosensor based on the TF\nAlkS, which is highly specific for SCAHs. Furthermore, through promoter-directed\nevolution, the performance of the biosensor was further enhanced.\nMutation in the −10 region of constitutive promoter P<i>alkS</i> resulted in reduced AlkS leakage expression, while\nmutation in the −10 region of inducible promoter P<i>alkB</i> increased its accessibility to the AlkS-SCAHs complex. This led\nto an 89% reduction in background fluorescence leakage of the optimized\nbiosensor, M2-463, further enhancing its response to SCAHs. The optimized\nbiosensor was highly sensitive and exhibited a broader dynamic response\nrange with a 150-fold increase in fluorescence output after 1 h of\ninduction. The detection limit (LOD) reached 0.03 ppm, and the average\nrecovery rate of SCAHs in actual water samples ranged from 95.87 to\n101.20%. The accuracy and precision of the proposed biosensor were\nvalidated using gas chromatography–mass spectrometry (GC–MS),\ndemonstrating the promising application for SCAH detection in an actual\nenvironment sample.