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

Yusong Zou¹, Yuanxin Qian¹, Connor Parish¹

¹School of Chemical, Materials and Biomedical Engineering, College of Engineering, The University of Georgia, Athens, Georgia 30602, United States.

ACS Synthetic Biology

|August 8, 2025

Summary

Researchers engineered a novel succinate-responsive biosensor using the PcaR transcription factor (TF). This tool enhances metabolic engineering by enabling real-time monitoring and optimization of microbial production systems.

Keywords:

PcaR dynamic behavior genetic biosensors site-directed mutagenesis succinate

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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.