Design of a Survival-Fluorescence Cascade Screening Circuit (uFAST) for an Erythritol-Specific BmoR-Based Biosensor

Rapid, specific quantification of erythritol in complex matrices remains challenging due to its structural similarity to sugars like sucrose. To overcome this, we developed a survival-fluorescence cascade screening circuit (uFAST) to engineer a selective, σ 54 -dependent BmoR-based biosensor. This circuit integrated SacB-mediated negative and mCherry-mediated positive selection to rapidly isolate erythritol-specific mutants. Guided by structural analysis, we optimized BmoR, achieving a 6.03-fold increase in GFP output and a 2.09-fold improvement in apparent affinity ( K m ). Coupled with a VioABCE-based visual module, the biosensor enabled accurate erythritol quantification in complex matrices, matching HPLC results. Integrated with an erythritol production module and fluorescence-activated droplet sorting (FADS), this platform screened an ARTP-mutagenized library, identifying a strain with 2.88-fold higher titer, yielding 3.51 g/L erythritol in fermentation. The uFAST circuit provides a generalizable framework for developing highly specific biosensors, offering an efficient tool for erythritol detection and strain improvement.