Irreversible biosynthesis of D‐allulose from D‐glucose in Escherichia coli through fine‐tuning of carbon flux and cofactor regeneration engineering

Abstract BACKGROUND As a rare hexose with low calories and various physiological functions, d ‐allulose has drawn increasing attention. The current industrial production of d ‐allulose from d ‐fructose or d ‐glucose is achieved via epimerization based on the Izumoring strategy; however, the inherent reaction equilibrium during reversible reaction limits its high conversion yield. Although the conversion of d ‐fructose to d ‐allulose could be enhanced via phosphorylation‐dephosphorylation mediated by metabolic engineering, biomass reduction and byproduct accumulation remain a largely unresolved issue. RESULTS After modifying the glycolytic pathway of Escherichia coli and optimizing the whole‐cell reaction condition, the engineered strain produced 7.57 ± 0.61 g L −1 d ‐allulose from 30 g L −1 d ‐glucose after 24 h of catalysis. By developing an ATP regeneration system for enhanced substrate phosphorylation, the cell growth inhibition was alleviated and d ‐allulose production increased by 55.3% to 11.76 ± 0.58 g L −1 (0.53 g g −1 ). Fine‐tuning of carbon flux caused a 48% reduction in d ‐fructose accumulation to 1.47 ± 0.15 g L −1 . After implementing fed‐batch co‐substrate strategy, the d ‐allulose titer reached 15.80 ± 0.31 g L −1 (0.62 g g −1 ) with a d ‐glucose conversion rate of 84.8%. CONCLUSION The present study reports a novel strategy for high‐yield d ‐allulose production from low‐cost substrate. © 2023 Society of Chemical Industry.