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.