Highly efficient selection-based directed evolution of MerR for enhanced Hg²⁺ sensing in whole-cell biosensors

Whole-cell biosensors (WCBs) offer a cost-effective and user-friendly platform for detecting toxic mercury ions (Hg²⁺). The Tn501 MerR protein has been widely employed in WCBs for Hg²⁺ sensing and shows potential for further functional enhancement. Here, we developed a selection-based directed evolution system for MerR optimization, which proceeded through three sequential steps. First, the antibiotic-based survival selection was used to directly eliminate ∼98.0 % of variants by selecting for high On state expression (i.e., reporter activation with Hg²⁺). The subsequent visual identification step further refined the library to the top 0.2 % of mutants with higher On state expression while maintaining low Off state activity (i.e., no activation without Hg²⁺). The dual-reporter verification served as the final step to identify optimal variants. This benchtop-compatible system, primarily relying on LB agar plates, demonstrated performance comparable to fluorescence-activated cell sorting (FACS). Using this system, we identified Val124 as a critical residue for Hg²⁺ sensing in MerR. The resulting mutant MerR E (V124E) showed a significantly enhanced Hg²⁺ response and gained the ability to respond to methylmercury chloride (MeHgCl). The MerR E-based WCB enabled naked-eye detection of Hg²⁺ at 5 nmol/L (compared to 100 nmol/L for the wild-type) and achieved improved sensitivity in real-world sample analysis, thereby expanding the sensing elements for WCB-based mercury detection. Moreover, this selection-based strategy shows promise for broader application in evolving other regulatory proteins.