ATP Regeneration from Pyruvate in the PURE System

The "Protein synthesis Using Recombinant Elements" ("PURE") system is a minimal biochemical system capable of carrying out cell-free protein synthesis using defined enzymatic components. This study extends PURE by integrating an ATP regeneration system based on pyruvate oxidase, acetate kinase, and catalase. The new pathway generates acetyl phosphate from pyruvate, phosphate, and oxygen, which is used to rephosphorylate ATP in situ. Successful ATP regeneration requires a high initial concentration of ∼10 mM phosphate buffer, which surprisingly does not affect the protein synthesis activity of PURE. The pathway can function independently or in combination with the existing creatine-based system in PURE; the combined system produces up to 233 μg/mL of mCherry, an enhancement of 78% compared to using the creatine system alone. The results are reproducible across multiple batches of homemade PURE and importantly also generalize to commercial systems such as PURExpress from New England Biolabs. These results demonstrate a rational bottom-up approach to engineering PURE, paving the way for applications in cell-free synthetic biology and synthetic cell construction.