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

Shin Suzuki, Ryotaro Hara, Kuniki Kino

    Research output: Contribution to journalArticle

    4 Citations (Scopus)

    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, 0.87 mM of L-Trp-L-Pro was successfully synthesized from AMP after 72 h. Farther, addition of inorganic pyrophosphatase from Escherichia coli 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.

    Original languageEnglish
    Pages (from-to)644-648
    Number of pages5
    JournalJournal of Bioscience and Bioengineering
    Volume125
    Issue number6
    DOIs
    Publication statusPublished - 2018 Jun 1

    Keywords

    • Adenylation domain
    • Aminoacyl proline
    • ATP regeneration
    • Nonribosomal peptide synthetase
    • Polyphosphate kinase

    ASJC Scopus subject areas

    • Biotechnology
    • Bioengineering
    • Applied Microbiology and Biotechnology

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