Directed evolution of linker helix as an efficient strategy for engineering LysR-type transcriptional regulators as whole-cell biosensors

Whole-cell biosensors based on transcriptional regulators are powerful tools for rapid measurement, highthroughput 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 LysGE58V variant outperformed the wild-type LysG in enzyme high-throughput screening and dynamic regulation of L-lysine biosynthetic pathway. LH mutations are suggested to affect DNA binding and facilitate transcriptional activation upon effector binding. LH engineering was also successfully applied to optimize another LTTR BenM for biosensing. Since LTTRs represent the largest family of prokaryotic transcriptional regulators with highly conserved structures, LH engineering is an efficient and universal strategy for development and optimization of whole-cell biosensors.

Automated summary

Optimizing the effector-binding domain of transcriptional regulators can improve the performance of whole-cell biosensors, enabling high-throughput screening and dynamic regulation of metabolic pathways. Molecular dynamics simulation revealed the crucial role of a short linker helix in protein conformational change, and directed evolution successfully produced variants with extended operational range and unaltered effector specificity. This study highlights the potential of linker helix engineering as an efficient and universal strategy for developing and optimizing whole-cell biosensors.