Combined Engineering of Promoter Architecture and MobR Enables Rational Optimization of Whole-Cell Biosensors for Hydroxybenzoic Acid Derivatives

MobR, a MarR family transcriptional repressor from Comamonas testosteroni, responds to hydroxybenzoic acid derivatives and represents a promising regulatory component for transcription factor (TF)-based whole-cell biosensors. In this study, we developed a MobR-based whole-cell biosensor in Escherichia coli by coupling the native mobA promoter with an enhanced green fluorescent protein (eGFP) reporter. Although the wild-type biosensor responded to 3-hydroxybenzoic acid (3-HBA), 2,3-dihydroxybenzoic acid (2,3-DHBA), and 3,4-dihydroxybenzoic acid (3,4-DHBA), its fluorescence output and dynamic range were limited. To improve biosensor performance, we independently engineered both the promoter architecture and the MobR transcription factor. Promoter variants were generated through modification of regulatory sequences to enhance transcriptional output, whereas structure-guided mutagenesis targeting predicted ligand-interacting residues was employed to modulate ligand recognition. Combinatorial integration of the optimized promoter and MobR variants produced biosensors exhibiting a 2–3-fold increase in fluorescence output while maintaining ligand-dependent regulation. Engineered biosensors enabled reliable quantitative detection of hydroxybenzoic acid derivatives, with promoter engineering primarily enhancing transcriptional output and structure-guided MobR engineering enabling modulation of ligand responsiveness. Collectively, this work demonstrates that promoter architecture and transcription factor function can be engineered as complementary design parameters and subsequently integrated to rationally optimize TF-based whole-cell biosensors. This modular strategy provides a practical framework for enhancing transcriptional output while tuning ligand response profiles.