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2020 Fiscal Year Final Research Report

High throughput technique for measuring specific enzyme activities of a metabolic pathway

Research Project

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Project/Area Number 18K04850
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeMulti-year Fund
Section一般
Review Section Basic Section 27040:Biofunction and bioprocess engineering-related
Research InstitutionOsaka University

Principal Investigator

Toya Yoshihiro  大阪大学, 情報科学研究科, 准教授 (70582162)

Project Period (FY) 2018-04-01 – 2021-03-31
Keywords代謝経路 / 律速反応 / 酵素活性 / 反応速度論モデル / 代謝制御解析 / 代謝工学 / 解糖系
Outline of Final Research Achievements

In the field of bio-productions, identification of a rate-limiting step in the metabolic pathways is one of the important issues for enhancing the productivity. Although the maximum enzyme activity (Vmax) provides a clue for the identification, measuring the Vmax values of many reactions at once is a time-consuming task. Here, I developed a high-throughput method for simultaneously estimating the Vmax of the enzymes in a pathway using a time-course data of intermediate concentrations obtained from an in vitro experiment. The Vmax values of reactions in glycolysis of Escherichia coli were estimated with this protocol. Metabolic control analysis using the estimated Vmax values revealed a rate-limiting step. The validity was confirmed by additional in vitro and in vivo experiments.

Free Research Field

代謝工学

Academic Significance and Societal Importance of the Research Achievements

細胞内における代謝の仕組みを理解するために,これまで様々な方法が開発されてきた.なかでも代謝フラックス解析は,代謝経路の流れ (フラックス) を明らかにすることで,代謝の実態を評価するための方法として広く用いられてきた.一方で,なぜその代謝状態だったのかは,フラックスだけを見ているだけではわからなかった.本研究で推定が可能になった各酵素反応の最大反応速度 (Vmax) と実際のフラックスを比較することで,どの反応が酵素量レベルでの制限を受けているのか明らかにできる.また,代謝経路中でどの反応が経路全体のフラックスに強く影響するか詳細な仕組みを解明できるようになる.

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Published: 2022-01-27  

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