Metabolic Engineering Strategies for Rare d -Allulose Production from d -Glucose– d -Xylose Mixtures Using Escherichia coli

Abstract d-Allulose is a functional sweetener with tremendous potential in food and health industries. Recent studies of its biosynthesis have mainly focused on enzyme catalysis or microbial fermentation using single-sugar substrates. Herein, we constructed a cell factory that coutilizes d-glucose and d-xylose for the efficient biosynthesis of d-allulose. Specifically, the expression of key enzymes in the synthesis pathway, along with the knockout of PtsG, ensured the simultaneous conversion of both sugars to d-allulose. By inactivating PfkA and PfkB and inhibiting Zwf expression, carbon flux from the Embden–Meyerhof–Parnas and pentose phosphate pathways was redirected toward the synthesis pathway. A transhydrogenase cycle was introduced to optimize the cofactor supply, while byproduct pathways were blocked to prevent product loss. Finally, the functionality of the engineered Escherichia coli was confirmed through shake-flask fermentation using wheat straw hydrolysate as the substrate, achieving a titer of 3.33 g/L and a yield of 0.226 g/g.