Facile production of d-arabinitol from waste cells via artificial in vitro enzymatic cascade

d-Arabinitol is a high-value platform chemical widely applied in food, cosmetic, and pharmaceutical industries, yet conventional production suffers from low conversion efficiency, prolonged cycles, and considerable environmental hazards. Herein, an artificial in vitro enzymatic cascade was developed consisting of Thermotoga maritima ribose-5-phosphate isomerase (RPI) and d-arabinitol 2-dehydrogenase (Ar2DH), plus Thiobacillus sp. formate dehydrogenase (FDH), for efficient d-arabinitol production from d-ribose. Systematic optimization of RPI, Ar2DH, formate, and NAD⁺ loadings yielded 9.9 g/L d-arabinitol from 10.4 g/L d-ribose within 4 h, corresponding to 93.9% molar conversion. Scale-up with 102.1 g/L d-ribose as substrate, 71.8 g/L of d-arabinitol was obtained with a molar conversion rate of 69.4%. Moreover, 3.0 g/L d-arabinitol was synthesized using 70 g/L Escherichia coli waste cells as feedstock. In conclusion, this enzymatic cascade demonstrates a rapid, sustainable, and eco-friendly approach to produce d-arabinitol, providing a promising strategy for upgrading industrial waste biomass in the biomanufacturing of bio-based products.