Development of screening tool for amino acid hydroxylase using insufficient TCA-cycle complementation test and its application

2020 Fiscal Year Final Research Report

• Project/Area Number: 18K05400
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Review Section: Basic Section 38020:Applied microbiology-related
• Research Institution: Kyoto University
• Principal Investigator: Hara Ryotaro 京都大学, 農学研究科, 特定准教授 (70553535)
• Project Period (FY): 2018-04-01 – 2021-03-31
• Keywords: Screening / Hydroxylase / Enzyme / Hydroxy amino acid / Dioxygenase / 3-Hydroxyhistidine / 3-Hydroxyglutamine / Insufficient TCA-cycle

Outline of Final Research Achievements

To satisfy a need for a method to produce hydroxy amino acids, which are promising materials for pharmaceutical development, bioprocesses with environmentally friendly and high efficiency are promising. In this study, we aimed to develop a useful hydroxy amino acid production process by effective tools for screening and modifying the enzymes that play a central role in the bioprocess. In this study, we obtained three findings. First, we developed the screening system to complement the growth of engineered E. coli by amino acid hydroxylase. Then, in the production of trans-3-hydroxy-L-proline using the E. coli, the excessive degradation of the cosubstrate was suppressed. Furthermore, we found a novel enzyme that hydroxylates L-histidine and L-glutamine in a threo-selevtive manner.

Free Research Field

応用微生物学

Academic Significance and Societal Importance of the Research Achievements

アミノ酸誘導体は、医薬品開発における重要な原料である。しかし、実用的に生産可能なものは限られている。アミノ酸誘導体の中でも、特にヒドロキシアミノ酸の合成は、従来法では多段階反応を要するため、シンプルな生産法が求められている。本研究で得た酵素は、化学合成では困難な位置選択的かつ立体選択的な水酸化により、ヒドロキシアミノ酸の生産を可能とする。成果物であるL-threo-3-ヒドロキシヒスチジンやL-threo-3-ヒドロキシグルタミンは合成例がなく、分子多様性が求められる医薬品開発に貢献できると考える。