A microbial sensor for organophosphate hydrolysis exploiting an engineered specificity switch in a transcription...

Area of Science:

• Synthetic Biology
• Biotechnology
• Molecular Engineering

Background:

• Whole-cell biosensors require diverse transcription factors (TFs) for detecting various molecules.
• Existing TFs often lack specificity for target anthropogenic compounds.

Purpose of the Study:

• To engineer a novel specificity into an Acinetobacter TF, PobR, for sensing p-nitrophenol (pNP).
• To develop a smart microbial cell-based biosensor for detecting pNP and its derivatives.

Main Methods:

• Utilized homology modeling and ligand docking to identify TF binding pockets.
• Employed conservative mutations to engineer TF specificity.
• Constructed a whole-cell biosensor with a fluorescent reporter system.
• Developed a coupled assay involving phosphotriesterase (PTE) for insecticide hydrolysis detection.

Main Results:

• Engineered PobR variants with novel specificity for pNP, with two mutants switching specificity from 4-hydroxybenzoate (4HB).
• Developed a smart microbial cell (pNPmut1) responsive to pNP produced from paraoxon hydrolysis.
• Demonstrated fluorescence correlation with PTE catalytic efficiency.
• Achieved high selectivity (4HB vs. pNP) and sensitivity (∼2 μM pNP detection).

Conclusions:

• Successfully engineered a transcription factor for specific pNP detection.
• Created a functional whole-cell biosensor with potential applications in chemical sensing and environmental monitoring.
• Validated computational modeling approaches for TF engineering.