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

Whole-cell biosensors (WCBs) offer a cost-effective and user-friendly platform for detecting toxic mercury ions (Hg). The Tn501 MerR protein has been widely employed in WCBs for Hg 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 Hg). 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 Hg). 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 Hg sensing in MerR. The resulting mutant MerR E (V124E) showed a significantly enhanced Hg response and gained the ability to respond to methylmercury chloride (MeHgCl). The MerR E-based WCB enabled naked-eye detection of Hg 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.