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Real-time Monitoring of Reactions Performed Using Continuous-flow Processing: The Preparation of 3-Acetylcoumarin as an Example

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Optimization of a p-Coumaric Acid Biosensor System for Versatile Dynamic Performance.

Tian Jiang¹, Chenyi Li¹, Yajun Yan¹

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

ACS Synthetic Biology

|December 30, 2020

Summary

This study engineered a biosensor for metabolic engineering, enhancing its dynamic range and sensitivity. New components and hybrid promoters improve control over valuable compound synthesis.

Keywords:

PadR biosensor hybrid promoter p-coumaric acid

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Area of Science:

Metabolic Engineering
Synthetic Biology
Molecular Biology

Background:

Metabolic engineering aims to synthesize valuable compounds.
Transcriptional factor-based biosensors dynamically regulate metabolic pathways.
PadR regulator from Bacillus subtilis responds to p-coumaric acid.

Purpose of the Study:

Engineer the PadR regulator for improved biosensor performance.
Investigate novel components (yveF, yveG) for enhanced regulation.
Develop hybrid promoters for increased strength and "plug-and-play" functionality.

Main Methods:

Site-directed mutagenesis of PadR.
Construction and characterization of hybrid promoters.
Analysis of biosensor regulation by p-coumaric acid.

Main Results:

Identified yveF and yveG as positive regulators of the PadR-PpadC system.
Generated PadR mutants (K64A, H38A) with increased dynamic range and sensitivity.
Developed four hybrid promoters (P1, P2, P7, P9) with enhanced strength regulated by PadR.

Conclusions:

Engineered PadR variants and hybrid promoters expand biosensor applicability.
This work provides tools for advanced metabolic engineering.
The study demonstrates effective dynamic regulation of metabolic pathways.