| Summary: | Adenosine triphosphate (ATP) acts as a crucial energy currency in vivo, and it is a widely used energy and/or phosphate donor for enzyme-catalyzed reactions in vitro. In this study, we established an in vitro multi-enzyme cascade system for ATP production. Using adenosine and inorganic polyphosphate (polyP) as key substrates, we combined adenosine kinase and two functionally distinct polyphosphate kinases (PPKs) in a one-pot reaction to achieve chain-like ATP regeneration and production. Several sources of PPK were screened and characterized, and two suitable PPKs were selected to achieve high rates of ATP production. Among these, Sulfurovum lithotrophicum PPK (SlPPK) exhibited excellent activity over a wide pH range (pH 4.0–9.0) and synthesized ATP from ADP using short-chain polyP. Furthermore, it had a half-life > 155.6 h at 45 °C. After optimizing the reaction conditions, we finally carried out the coupling-catalyzed reaction with different initial adenosine concentrations of 10, 20, and 30 mM. The highest yields of ATP were 76.0, 70.5, and 61.3%, respectively. Graphical Abstract: [Figure not available: see fulltext.] © 2021, The Author(s).
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