Engineering the thermal stability of a polyphosphate kinase by ancestral sequence reconstruction to expand the temperature boundary for an industrially applicable ATP regeneration system.

was improved, guided by an ancestral sequence reconstruction strategy. The optimal variant has a 4.5-fold longer half-life at 60°C than the wild-type enzyme, thus enabling the extension of the temperature boundary for ATP regeneration. The ability of this variant to regenerate ATP was well demonstrated during high-temperature enzymatic production of nucleotides.