Whole-cell biosensors based on transcriptional regulators are powerful tools for rapid measurement, high-throughput screening, dynamic metabolic regulation, etc. To optimize the biosensing performance of transcriptional regulator, its effector-binding domain is commonly engineered. However, this strategy is encumbered by the limitation of diversifying such a large domain and the risk of affecting effector specificity. Molecular dynamics simulation of effector binding of LysG (an LysR-type transcriptional regulator, LTTR) suggests the crucial role of the short linker helix (LH) connecting effector- and DNA-binding domains in protein conformational change. Directed evolution of LH efficiently produced LysG variants with extended operational range and unaltered effector specificity. The whole-cell biosensor based on the best LysG