Engineered TtgR-Based Whole-Cell Biosensors for Quantitative and Selective Monitoring of Bioactive Compounds.

Area of Science:

• Microbiology
• Molecular Biology
• Biotechnology

Background:

• TtgR, a transcriptional repressor from Pseudomonas putida, regulates multidrug resistance gene expression.
• TtgR's broad ligand specificity makes it a candidate for developing transcription factor (TF)-based biosensors.

Purpose of the Study:

• To construct and characterize TF-based biosensors in Escherichia coli using TtgR and its native promoter (PttgABC).
• To engineer TtgR variants with enhanced selectivity and specificity for flavonoid detection.
• To investigate TtgR-flavonoid interactions using computational methods.

Main Methods:

• Construction of biosensors in E. coli by coupling TtgR and PttgABC with egfp reporter gene.
• Genetic engineering of the TtgR-binding pocket to create variants with altered ligand specificities.
• Computational structural analysis and ligand docking to elucidate interaction mechanisms.

Main Results:

• Developed a biosensor responsive to diverse flavonoids by combining TtgR, PttgABC, and egfp.
• Engineered TtgR variants displayed modified sensing profiles, allowing for tailored biosensor responses.
• Biosensors utilizing wild-type TtgR and the N110F mutant accurately quantified resveratrol and quercetin at 0.01 mM.

Conclusions:

• The study presents novel TtgR-based biosensors for flavonoid detection with potential for broad applications.
• Engineered TtgR variants offer a platform for developing biosensors with specific ligand responses.
• This work provides a foundation for future applications in synthetic biology and metabolic engineering.