Production of aminoacyl prolines using the adenylation domain of nonribosomal peptide synthetase with class III polyphosphate kinase 2-mediated ATP regeneration

Abstract

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 III 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 101906T, coupled with aminoacyl proline (Xaa-Pro) synthesis catalyzed by the adenylation domain of tyrocidine synthetase A (TycA-A). Using this system, 0.87mM of l-Trp-l-Pro was successfully synthesized from AMP after 72h. Farther, addition of inorganic pyrophosphatase from Escherichia coli to the coupling reaction increased the reaction rate by 14-fold to yield 6.2mM 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 6.7mM of l-Trp-l-Pro was produced after 24h with the supplementation of 10mM 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.