A Toxin-Antitoxin-Driven Biosensor Platform for the Directed Evolution of d -Allulose 3-Epimerase

Biosensor-assisted growth-coupled screening facilitates the directed evolution of enzymes. However, antibiotic resistance-dependent screening tools generally suffer from declining selection pressure. To overcome this limitation, we developed a toxin-antitoxin-based high-throughput screening platform (TASP) for engineering d-allulose 3-epimerase (DAE), driven by a redesigned d-allulose-responsive biosensor (DB). Engineering the pPsiA promoter expanded its dynamic range to 150 mM d-allulose and enhanced signal-to-noise ratio by 14.2-fold. The TASP incorporates the toxin-antitoxin (AtaT-AtaR) as a sustainable and cell-autonomous selection pressure, where mCherry is used as a reporter gene. This design precisely links DAE catalytic activity to cell growth phenotypes, enabling both visual monitoring and quantitative analysis to rapidly identify variants. Using TASP, we identified the mutant M4-2 (W14C/G67A/L151Y/F245 K), which exhibits a 5.2-fold increase in catalytic activity and a 3.5-fold increase in half-life at 60 °C. This work establishes a reliable platform for high-throughput screening of DAE variants and advances the development of growth-coupled selection strategies.