Integrated Protein Engineering and Expression Balancing Enable Efficient <scp>d</scp>-Allulose Production

Allulose, a rare sugar with promising applications in food and health industries, can be efficiently synthesized via biocatalysis using d-allulose 3-epimerase (DAE). However, challenges in enzyme thermostability and cascade efficiency limit industrial scalability with low-cost substrates. Here, we engineered a thermostable DAE from <i>Clostridium cellulolyticum</i> through semirational design, yielding a triple mutant (F155Y/D281G/C289R) with a 12.0 °C increase in melting temperature (Tm to 73.2 °C), optimal temperature shifted to 60 °C, and a conversion rate improved from 27.5 to 33.3%─surpassing reported rates. Integrated with a modular coexpression system for expression-level balancing between DAE and thermophilic glucose isomerase, our one-pot cascade achieved high d-allulose titers from glucose (18.5%), F42 fructose syrup (17.4%), and corncob hydrolysate (7.5%)─outperforming literature benchmarks in yield and sustainability. This work advances green biomanufacturing by converting agricultural residues into high-value sugars, offering a scalable alternative to traditional methods with reduced costs and enhanced process continuity.