Stoichiometric Regeneration of ATP by A NAD(P)/CoA‐free and Phosphate‐balanced In Vitro Synthetic Enzymatic Biosystem

Abstract The regeneration of adenosine triphosphate is crucial for establishing a cost‐effective biosystem in which energy‐dependent processes are involved. Here, an in vitro synthetic enzymatic biosystem was designed to utilize low‐cost starch‐based material as energy source for the stoichiometric regeneration of ATP. This system required neither NAD(P) nor coenzyme A, and theoretically produced 3 ATPs per glucose equivalent of starch. When integrated with the L‐theanine‐producing enzymatic reaction which consumes 1 ATP to generate 1 L‐theanine molecule, our system resulted in a near‐theoretical yield of 2.94 L‐theanine per glucose equivalent. Furthermore, inorganic phosphate concentration of the system was nearly constant throughout the reaction period, suggesting the homeostatic reaction environment for enzymatic processes. This work provides a useful strategy for cost‐effective ATP regeneration which facilitates the production of ATP‐dependent value‐added chemicals.