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

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 d-allulose from 30 g L 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 (0.53 g g ). Fine-tuning of carbon flux caused a 48% reduction in d-fructose accumulation to 1.47 ± 0.15 g L . After implementing fed-batch co-substrate strategy, the d-allulose titer reached 15.80 ± 0.31 g L (0.62 g g ) 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.