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Adenosine 5'-triphosphate (ATP) is a substrate for the biological synthesis of various valuable compounds, such as pharmaceuticals and agrochemicals. To make these biocatalytic processes more economically valuable, several ATP regeneration systems comprising enzymes and phosphate donors were developed. Due to their ability to synthesize ATP from both 5'-diphosphate and adenosine 5'-monophosphate, polyphosphate kinase 2 class III (PPK2-III) enzymes such as Cytophaga hutchinsonii PPK (ChPPK) have attracted considerable attention in the construction of ATP regeneration systems. Furthermore, PPK2-III enzymes employ inorganic polyphosphate, which is a low-cost and stable substrate, as the phosphate donor. To facilitate the application of the PPK2-III ATP regeneration system in biocatalytic processes depending on ATP, a semirational strategy coupled with a high-throughput screening assay based on a fluorescent sensor was performed to engineer ChPPK. Saturation mutagenesis was performed on 16 critical residues of ChPPK. After screening approximately 4,800 colonies, nine single amino acid variants exhibited more than threefold increased activities than wild-type ChPPK. Beneficial mutations were subsequently recombined, resulting in a quadruple variant (ChPPK/A79G/S106C/I108F/L285P), compared with wild-type ChPPK, displaying 18.8-fold enhanced activity and better stability at an elevated temperature and under acidic conditions. The introduction of this variant ChPPK/A79G/S106C/I108F/L285P in the biological synthesis of nicotinamide mononucleotide catalyzed by phosphoribosyl pyrophosphate synthetase and nicotinamide phosphoribosyltransferase increased the yield by approximately threefold compared with wild-type ChPPK. Therefore, the ATP regeneration system described may have implications for bioconversions requiring ATP. IMPORTANCE The adenosine 5'-triphosphate (ATP) regeneration system can significantly reduce the cost of many biocatalytic processes. Numerous studies have endeavored to utilize the ATP regeneration system based on Cytophaga hutchinsonii PPK (ChPPK). However, the wild-type ChPPK enzyme possesses limitations such as low enzymatic activity, poor stability, and limited substrate tolerance, impeding its application in catalytic reactions. To enhance the performance of ChPPK, we employed a semi-rational design approach to obtain the variant ChPPK/A79G/S106C/I108F/L285P. The enzymatic kinetic parameters and the catalytic performance in the synthesis of nicotinamide mononucleotide demonstrated that the variant ChPPK/A79G/S106C/I108F/ L285P exhibited superior enzymatic properties than the wild-type enzyme. All data indicated that our engineered ATP regeneration system holds inherent potential for implementation in biocatalytic processes. [ABSTRACT FROM AUTHOR]