Efflux pump knockout minimizes crosstalk and boosts response in transcription factor-based biosensors

Cellular transport systems are key determinants of intracellular molecule concentrations and can be utilized to modulate the responsiveness of transcription factor (TF)-based biosensors. Efflux pumps facilitate the export of small molecules; therefore, their activity can directly compromise the accuracy of biosensor-based assays. We engineered a cellular export machinery to enhance the responsiveness and reduce crosstalk of a DmpR-based biosensor, which employs the phenol-responsive TF DmpR to detect intracellular phenolic ligands but is affected by ligand diffusion between cells. To overcome this limitation, efflux pump genes were knocked out to minimize ligand diffusion and improve signal fidelity.

Among the efflux pumps tested, deletion of mdtA was found to promote effective intracellular accumulation of phenolic compounds. This strategy not only increased biosensor sensitivity by up to 19-fold but also reduced false positives during enzyme screening by suppressing intercellular diffusion of enzymatic products. In the mock library experiment, the proportion of false positives relative to the total positive cells was 74% in the wild-type strain, whereas it was only 5% in the ΔmdtA strain. To demonstrate the applicability of this approach to an enzyme screening platform, we targeted penicillin G acylase (PGA), an enzyme useful for producing semi-synthetic antibiotics. Knockout of mdtA effectively reduced false positives during flow cytometry-based high-throughput screening. Using this biosensor platform, several PGA variants with improved catalytic activity were successfully identified from a random mutagenesis library.

Overall, host engineering to adjust cellular conditions and ligand concentrations provides a versatile approach to enhance the sensitivity, precision, and efficiency of single-cell-based enzyme screening platforms.