Enhanced Synthesis of Rare <sc>d</sc>-Allose from <sc>d</sc>-Glucose by Positively Pulling and Forcing Reversible Epimerization in Engineered Escherichia coli

d-Allose has great potential for application in the food and pharmaceutical industries owing to its remarkable physiological properties. Most studies on d-allose production have primarily focused on enzyme catalysis using the Izumoring strategy, which typically requires the use of expensive d-allulose as a substrate. Herein, a metabolically engineered strain of Escherichia coli was developed to synthesize d-allose directly from inexpensive d-glucose. The synthesis pathway was systematically optimized through a modular metabolic engineering. The functionality of the isomerases involved in the conversion of d-allulose to d-allose was confirmed in vivo, while the byproduct and transporter pathways were blocked to positively pull the reversible epimerization. Gene knockouts were employed to weaken glycolytic pathways, redirecting the carbon flux toward product synthesis. Additionally, the nonphosphorylated transport of d-glucose was introduced to enhance substrate utilization. In fed-batch fermentation, the engineered strain achieved a d-allose titer of 4.17 g/L, with a yield of 0.103 g/g from d-glucose. Our achievements are expected to advance the industrial production of d-allose, and this strategy is also applicable for producing other rare sugars.