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

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.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%.The present study reports a novel strategy for high-yield d-allulose production from low-cost substrate. © 2023 Society of Chemical Industry.