An ATP regeneration system is advantageous for industrial processes that are coupled with ATP-dependent enzymes. For ATP regeneration from AMP, a few methods have been reported; however, these methods employ multiple enzymes. To establish an ATP regeneration system using a single enzyme, we focused on class Ill polyphosphate kinase 2 (class III PPK2) that can synthesize ATP from AMP and polyphosphate. We constructed an ATP regeneration system from AMP using Deipr_1912, a class III PPK2 from Deinococcus proteolyticus NBRC 101906(T), coupled with aminoacyl proline (XaaPro) synthesis catalyzed by the adenylation domain of tyrocidine synthetase A (TycA-A). Using this system, 0.87 mM of (L)-Trp-(L)-Pro was successfully synthesized from AMP after 72 h. Farther, addition of inorganic pyrophosphatase from Escherichia coil to the coupling reaction increased the reaction rate by 14-fold to yield 6.2 mM (L)-Trp-(L)-Pro. When the coupling reaction was applied to whole-cell reactions in E. coli BL21(DE3)pepQ(-) putA(-), ATP was successfully regenerated from AMP by Deipr_1912, and 6.7 mM of (L)-Trp-(L)-Pro was produced after 24 h with the supplementation of 10 mM AMP. In addition, by altering the substrate amino acid of TycA-A, not only (L)-Trp-(L)-Pro, but also various other (L)-Xaa-(L)-Pro (Xaa = Val, Leu, Met, or Tyr) were produced using the whole-cell reaction incorporating ATP regeneration. Therefore, a production method for Xaa-Pro employing the adenylation domain of a nonribosomal peptide synthetase was established by introducing an ATP regeneration system that utilizes class III PPK2 with pyrophosphatase. (C) 2017, The Society for Biotechnology, Japan. All rights reserved.