Engineering chimeric signaling proteins for microbial whole-cell biosensors: from design to deployment.

Microbial whole-cell biosensors (MWCBs) harness living cells to detect analytes and produce measurable outputs, enabling continuous, low-cost, and in situ sensing. Central to MWCB function are modular sensing architectures, which can be reprogrammed to respond to diverse signals. Particularly, two-component systems (TCSs) and allosteric transcription factors (aTFs) offer modular, engineerable frameworks for building chimeric proteins. Recent advances in domain swapping, fusion point selection, and protein engineering are expanding the versatility and specificity of these systems, presenting new opportunities for tailored and multiplexed detection. However, translating chimeric MWCBs into real-world applications still faces multiple hurdles. This review examines current strategies for engineering TCS- and aTF-based biosensors and outlines key opportunities and challenges for their deployment in applications such as diagnostics, environmental monitoring, and biomanufacturing.