Highly efficiency production of D-allulose from inulin using curli fiber multi-enzyme cascade catalysis

D-Allulose is a rare sugar with numerous physiological benefits and low caloric content, which can be obtained from inulin through enzymatic catalysis. In this study, we combined D-allulose 3-epimerase, exo- and endo-inulinases (EXINU and ENINU) with the NGTag/NGCatcher/CsgA system to accelerate D-allulose accumulation from inulin. Molecular dynamics simulations were used to screen linkers of appropriate length for ENINU. In vitro, we successfully observed the assembled NGCatcher_ENINU_CsgA, NGTag_EXINU, and DAERK fibers using fluorescent labelling with GFP, YFP, and mCherry. The optimal pH and temperature of the tagged variants were comparable to those of the wild-type, and the MD simulations showed that NGCatcher_ENINU_CsgA had improved stability in the working environment of EXINU. D-Allulose accumulation rate of the assembled enzymes cascade (NGCatcher_ENINU_CsgA/NGTag_EXINU_CsgA/DAERK) reached 0.25 g/L min⁻¹ (1.25 mg_D-allulose mg_DAERK⁻¹ min⁻¹) at an inulin concentration of 100 g/L. The assembled system greatly improves the high-valued productions of rare sugars from cheap biomass.