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

• Published in: Journal of agricultural and food chemistry 2024-06, Vol.72 (25), p.14216-14228
• Main Authors: Zhao, Jingyu, Sun, Huanhuan, Wang, Gege, Wang, Qi, Wang, Yipeng, Li, Qingbin, Bi, Shuangyu, Qi, Qingsheng, Wang, Qian
• Format: Article
• Language: English
• Subjects: biosensors; Biotechnology and Biological Transformations; chemoreceptors; Corynebacterium glutamicum; DNA; Escherichia coli; evolution; food chemistry; gene expression; hypersensitivity; protein degradation; synthetic biology; Tobacco etch virus
• ISSN: 0021-8561; 1520-5118; 1520-5118
• DOI: 10.1021/acs.jafc.4c00041

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.