Enzymatic Synthesis of L-threo-β-Hydroxy-α-Amino Acids via Asymmetric Hydroxylation Using 2-Oxoglutarate-Dependent Hydroxylase from Sulfobacillus thermotolerans Strain Y0017

Ryotaro Hara, Yuta Nakajima, Hiroaki Yanagawa, Ryo Gawasawa, Izumi Hirasawa, Kuniki Kino*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

β-Hydroxy-α-amino acids are useful compounds for pharmaceutical development. Enzymatic synthesis of β-hydroxy-α-amino acids has attracted considerable interest as a selective, sustainable, and environmentally benign process. In this study, we identified a novel amino acid hydroxylase, AEP14369, from Sulfobacillus thermotolerans Y0017, which is included in a previously constructed CAS-like superfamily protein library, to widen the variety of amino acid hydroxylases. The detailed structures determined by nuclear magnetic resonance and X-ray crystallography analysis of the enzymatically produced compounds revealed that AEP14369 catalyzed threo- β-selective hydroxylation of L-His and L-Gln in a 2-oxoglutarate-depend-ent manner. Furthermore, the production of L-threo-β-hydroxy-His and L-threo-β-hydroxy-Gln was achieved using Escherichia coli expressing the gene encoding AEP14369 as a whole-cell biocatalyst. Under optimized reaction conditions, 137 mM (23.4 g liter21) L-threo- β-hydroxy-His and 150 mM L-threo- β-hydroxy-Gln (24.3 g lit-er21) were obtained, indicating that the enzyme is applicable for preparative-scale production. AEP14369, an L-His/L-Gln threo- β-hydroxylase, increases the availability of 2-oxoglutarate-dependent hydroxylase and opens the way for the practical production of β-hydroxy-α-amino acids in the future. The amino acids produced in this study would also contribute to the structural diversification of pharmaceuticals that affect important bioactivities. IMPORTANCE Owing to an increasing concern for sustainability, enzymatic approaches for producing industrially useful compounds have attracted considerable attention as a powerful complement to chemical synthesis for environment-friendly synthesis. In this study, we developed a bioproduction method for β-hydroxy-α-amino acid synthesis using a newly discovered enzyme. AEP14369 from the moderate thermophilic bacterium Sulfobacillus thermotolerans Y0017 catalyzed the hydroxylation of L-His and L-Gln in a regioselective and stereoselective fashion. Furthermore, we biotechnologically synthesized both L-threo-β-hydroxy-His and L-threo-β-hydroxy-Gln with a titer of over 20 g liter21 through whole-cell bioconversion using recombinant Escherichia coli cells. As β-hydroxy-α-amino acids are important compounds for pharmaceutical development, this achievement would facilitate future sustainable and economical industrial applications.

Original languageEnglish
Pages (from-to)1-10
Number of pages10
JournalApplied and Environmental Microbiology
Volume87
Issue number20
DOIs
Publication statusPublished - 2021 Sep
Externally publishedYes

Keywords

  • 2-oxoglutarate-dependent hydroxylase
  • CAS-like superfamily
  • L-threo-β-hydroxy-Gln
  • L-threo-β-hydroxy-His
  • asymmetric hydroxylation
  • dioxygenases
  • β-hydroxy-α-amino acid

ASJC Scopus subject areas

  • Biotechnology
  • Food Science
  • Ecology
  • Applied Microbiology and Biotechnology

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