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.

Automated summary

An in vitro synthetic enzymatic biosystem was designed and constructed for the thermodynamics-driven production of D-allulose from low-cost starch, achieving high product yields. This strategy could significantly reduce the production cost of D-allulose and provide a promising alternative for the cost-efficient production of other rare sugars on an industrial scale.