Thermodynamics-Driven Production of Value-Added d‑Allulose from Inexpensive Starch by an In Vitro Enzymatic Synthetic Biosystem

d-Allulose, which can be used as a food additive or functional food, is an important low-calorie functional rare sugar. The current commercial production of d-allulose is performed through the epimerization of fructose by d-allulose 3-epimerase. However, due to the inherent reaction equilibrium of this conversion, this method suffers from a low conversion yield (lower than 40%), leading to a high production cost. In this study, an in vitro synthetic enzymatic biosystem based on phosphorylation-dephosphorylation enzymatic cascade conversion routes for the thermodynamics-driven production of d-allulose from low-cost starch was designed and constructed. By optimizing the reaction conditions, the yield of d-allulose from 10 g/L starch reached 88.2%. To investigate the potential use of this in vitro synthetic enzymatic biosystem for the production of d-allulose on an industrial scale, d-allulose was synthesized from 50 g/L starch (275 mM glucose equivalent) with a product yield of 79.2%. These results indicated that the product cost of d-allulose could be decreased significantly through this strategy. In addition to d-allulose, this thermodynamics-driven strategy may also provide a promising alternative for the cost-efficient production of many other rare sugars (e.g., tagatose, mannitol, and sorbitol) on an industrial scale.