Characterizing and Engineering a Succinate-Responsive Biosensor System in Escherichia coli

Area of Science:

• Metabolic Engineering
• Synthetic Biology
• Microbial Biotechnology

Background:

• Metabolic engineering aims for sustainable compound production but faces challenges like metabolic imbalance and limited regulatory tools.
• Transcription factor (TF)-based biosensors offer dynamic control of intracellular metabolites, but their limited diversity restricts applications.
• Developing biosensors responsive to central metabolic intermediates is crucial for versatile pathway control in metabolic engineering.

Purpose of the Study:

• To characterize a succinate-responsive biosensor system regulated by the IclR family TF, PcaR.
• To elucidate the dual-function mechanism of the PcaR biosensor system.
• To engineer a versatile biosensor for improved metabolic regulation and microbial production.

Main Methods:

• Characterization of the PcaR TF and its regulatory promoter.
• Site-directed mutagenesis and promoter engineering to elucidate PcaR's dual-function mechanism.
• Construction and screening of a succinate-responsive biosensor library using PcaR-succinate complex analysis.

Main Results:

• Fine-tuning PcaR expression restored promoter strength.
• A dual-function mechanism of PcaR was discovered and elucidated.
• A succinate-responsive biosensor library was established, identifying the P1-AII variant with a 33-fold improved dynamic range.
• A bifunctional regulatory circuit controlled by succinate was constructed.

Conclusions:

• The engineered PcaR biosensor system provides a robust tool for real-time metabolic monitoring.
• This system enables dynamic metabolic regulation and optimization of microbial production.
• The PcaR-based biosensor expands the toolkit for advanced metabolic engineering applications.