Engineering Chimeric Chemoreceptors and Two-Component Systems for Orthogonal and Leakless Biosensing of Extracellular γ-Aminobutyric Acid.

ABSTRACT

Two-component systems (TCSs) sensing and responding to various stimuli outside and inside cells are valuable resources for developing biosensors with synthetic biology applications. However, the use of TCS-based biosensors suffers from a limited effector spectrum, hypersensitivity, low dynamic range, and unwanted signal crosstalk. Here, we developed a tailor-made Escherichia coli whole-cell γ-aminobutyric acid (GABA) biosensor by engineering a chimeric GABA chemoreceptor PctC and TCS. By testing different TCSs, the chimeric PctC/PhoQ showed the response to GABA. Chimera-directed evolution and introduction of the insulated chimeric pair PctC/PhoQ*PhoP* produced biosensors with up to 3.50-fold dynamic range and good orthogonality. To further enhance the dynamic range and lower the basal leakage, three strategies, engineering of PhoP DNA binding sites, fine-tuning reporter expression by optimizing transcription/translation components, and a tobacco etch virus protease-controlled protein degradation, were integrated. This chimeric biosensor displayed a low basal leakage, a large dynamic range (15.8-fold), and a high threshold level (22.7 g L-1). Finally, the optimized biosensor was successfully applied in the high-throughput microdroplet screening of GABA-overproducing Corynebacterium glutamicum, demonstrating its desired properties for extracellular signal biosensing.