Replacing manual operation with bio-automation: A high-throughput evolution strategy to construct an integrated whole-cell biosensor for the simultaneous detection of methylmercury and mercury ions without manual sample digestion.

Methylmercury is primarily responsible for most food mercury pollution cases. However, most biosensors developed for mercury pollution analysis can only detect mercury ions. Although oxidative strong-acid digestion or microwave-assisted digestion can convert methylmercury into mercury ions, it is unsuitable for on-site detection. This study designed a bio-digestion gene circuit and integrated it into a mercury ion whole-cell biosensor，creating a novel on-site methylmercury detection method. Five alkyl mercury lyases from different bacterial genomes were screened via bioinformatics analysis, of which goMerB from Gordonia otitis showed the highest catalytic biological digestion efficiency. The goMerB site-specific saturation and random mutation libraries were constructed. After two rounds of high-throughput visualization screening, the catalytic activity of the mutant increased 2.5-fold. The distance between the three crucial amino acid sites and methylmercury changed in the mutant, which likely contributed to the enhanced catalytic efficiency. The optimized whole-cell biosensor showed a linear dynamic concentration range of 100 nM to 100 μM (R =0.991), satisfactory specificity, and interference resistance. The detection limit of the goMerBt6-MerR-RFP biosensor was 0.015 μM, while the limit of quantitation was 0.049 μM. This study demonstrated the application of synthetic biology for food safety detection and highlighted the future potential of "Lab in a Cell" for hazard analysis.