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December 23, 2020 preprint Open access Aachen paper

Tunnel Engineering for Modulating The Substrate Preference in Decarbonylase P450BsβHI

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DOI: 10.21203/rs.3.rs-132813/v1 Full text (OA) OpenAlex

Abstract

Abstract An active site normally locates inside of enzymes, substrates should go through the tunnel to access the active site. Tunnel engineering is a powerful strategy for refining the catalytic properties of enzymes. Here, P450BsβHI (Q85H/ V170I) derived from hydroxylase P450Bsβ from Bacillus subtilis was chosen as study model, which is reported as a potential decarbonylase. However, this enzyme showed low decarboxylase activity towards long-chain fatty acids. Here, a tunnel engineering campaign was performed for modulating the substrate preference and improving the decarbonylase activity of P450BsβHI. The finally obtained BsβHI-F79A variant had a 15.2-fold improved conversion for palmitic acid; BsβHI-F173V variant had a 3.9-fold improved conversion for pentadecanoic acid. The study demonstrates how the substrate preference can be modulated by tunnel engineering strategy.

Authors 10

  1. Shuaiqi Meng Aachen Beijing Bioprocess Key Laboratory Institute of Biotechnology Germany Ruipeng An Beijing Bioprocess Key Laboratory Ulrich Schwaneberg Institute of Biotechnology Yu Ji Institute of Biotechnology

    Beijing University of Chemical Technology · RWTH Aachen University

    Affiliation as printed

    Beijing Bioprocess Key Laboratory, Beijing University of Chemical Technology, Beijing, 100029, PR China. Institute of Biotechnology, RWTH Aachen University, Worringerweg 3, Aachen 52074, Germany

    Beijing Bioprocess Key Laboratory, Beijing University of Chemical Technology, Beijing, 100029, PR China

    Beijing University of Chemical Technology

    Beijing University of Chemical Technology, Beijing, 100029, PR China

    Beijing University of Chemical Technology, Beijing, 100029, PR China Zhongyu Li Beijing Bioprocess Key Laboratory,

    Institute of Biotechnology, RWTH Aachen University, Worringerweg 3, Aachen 52074, Germany

    RWTH Aachen University, Worringerweg 3, Aachen 52074, Germany Ruipeng An Beijing Bioprocess Key Laboratory,

    Ulrich Schwaneberg Institute of Biotechnology, RWTH Aachen University, Worringerweg 3, Aachen 52074, Germany

    Yu Ji Institute of Biotechnology, RWTH Aachen University, Worringerweg 3, Aachen 52074, Germany

  2. Zhongyu Li Aachen Institute of Biotechnology Germany Ruipeng An Beijing Bioprocess Key Laboratory Ulrich Schwaneberg Institute of Biotechnology Yu Ji Institute of Biotechnology

    Beijing University of Chemical Technology · RWTH Aachen University

    Affiliation as printed

    Beijing Bioprocess Key Laboratory, Beijing University of Chemical Technology, Beijing, 100029, PR China

    Beijing Bioprocess Key Laboratory, Beijing University of Chemical Technology, Beijing, 100029, PR China. Institute of Biotechnology,

    Beijing University of Chemical Technology

    Beijing University of Chemical Technology, Beijing, 100029, PR China

    Beijing University of Chemical Technology, Beijing, 100029, PR China Zhongyu Li Beijing Bioprocess Key Laboratory,

    Institute of Biotechnology, RWTH Aachen University, Worringerweg 3, Aachen 52074, Germany

    RWTH Aachen University, Worringerweg 3, Aachen 52074, Germany Ruipeng An Beijing Bioprocess Key Laboratory,

    Ulrich Schwaneberg Institute of Biotechnology, RWTH Aachen University, Worringerweg 3, Aachen 52074, Germany

    Yu Ji Institute of Biotechnology, RWTH Aachen University, Worringerweg 3, Aachen 52074, Germany

  3. Ulrich Schwaneberg Aachen Institute of Biotechnology

    DWI – Leibniz Institute for Interactive Materials · RWTH Aachen University

    Affiliation as printed

    Institute of Biotechnology, RWTH Aachen University, Worringerweg 3, Aachen 52074, Germany. DWI-Leibniz Institute for Interactive Materials, Forckenbeckstraße 50, Aachen 52074, Germany

  4. Yu Ji Aachen Institute of Biotechnology

    RWTH Aachen University

    Affiliation as printed

    Institute of Biotechnology, RWTH Aachen University, Worringerweg 3, Aachen 52074, Germany

  5. Mehdi D. Davari Aachen Institute of Biotechnology Germany Ruipeng An Beijing Bioprocess Key Laboratory Ulrich Schwaneberg Institute of Biotechnology Yu Ji Institute of Biotechnology

    Beijing University of Chemical Technology · RWTH Aachen University

    Affiliation as printed

    Institute of Biotechnology, RWTH Aachen University, Worringerweg 3, Aachen 52074, Germany

    Beijing Bioprocess Key Laboratory, Beijing University of Chemical Technology, Beijing, 100029, PR China

    Beijing Bioprocess Key Laboratory, Beijing University of Chemical Technology, Beijing, 100029, PR China. Institute of Biotechnology,

    Beijing University of Chemical Technology

    Beijing University of Chemical Technology, Beijing, 100029, PR China

    Beijing University of Chemical Technology, Beijing, 100029, PR China Zhongyu Li Beijing Bioprocess Key Laboratory,

    RWTH Aachen University, Worringerweg 3, Aachen 52074, Germany Ruipeng An Beijing Bioprocess Key Laboratory,

    Ulrich Schwaneberg Institute of Biotechnology, RWTH Aachen University, Worringerweg 3, Aachen 52074, Germany

    Yu Ji Institute of Biotechnology, RWTH Aachen University, Worringerweg 3, Aachen 52074, Germany

  6. Fang Wang Aachen Institute of Biotechnology Germany Ruipeng An Beijing Bioprocess Key Laboratory Ulrich Schwaneberg Institute of Biotechnology Yu Ji Institute of Biotechnology

    Beijing University of Chemical Technology · RWTH Aachen University

    Affiliation as printed

    Beijing Bioprocess Key Laboratory, Beijing University of Chemical Technology, Beijing, 100029, PR China

    Beijing Bioprocess Key Laboratory, Beijing University of Chemical Technology, Beijing, 100029, PR China. Institute of Biotechnology,

    Beijing University of Chemical Technology

    Beijing University of Chemical Technology, Beijing, 100029, PR China

    Beijing University of Chemical Technology, Beijing, 100029, PR China Zhongyu Li Beijing Bioprocess Key Laboratory,

    Institute of Biotechnology, RWTH Aachen University, Worringerweg 3, Aachen 52074, Germany

    RWTH Aachen University, Worringerweg 3, Aachen 52074, Germany Ruipeng An Beijing Bioprocess Key Laboratory,

    Ulrich Schwaneberg Institute of Biotechnology, RWTH Aachen University, Worringerweg 3, Aachen 52074, Germany

    Yu Ji Institute of Biotechnology, RWTH Aachen University, Worringerweg 3, Aachen 52074, Germany

  7. Meng Wang Aachen Institute of Biotechnology Germany Ruipeng An Beijing Bioprocess Key Laboratory Ulrich Schwaneberg Institute of Biotechnology Yu Ji Institute of Biotechnology

    Beijing University of Chemical Technology · RWTH Aachen University

    Affiliation as printed

    Beijing Bioprocess Key Laboratory, Beijing University of Chemical Technology, Beijing, 100029, PR China

    Beijing Bioprocess Key Laboratory, Beijing University of Chemical Technology, Beijing, 100029, PR China. Institute of Biotechnology,

    Beijing University of Chemical Technology

    Beijing University of Chemical Technology, Beijing, 100029, PR China

    Beijing University of Chemical Technology, Beijing, 100029, PR China Zhongyu Li Beijing Bioprocess Key Laboratory,

    Institute of Biotechnology, RWTH Aachen University, Worringerweg 3, Aachen 52074, Germany

    RWTH Aachen University, Worringerweg 3, Aachen 52074, Germany Ruipeng An Beijing Bioprocess Key Laboratory,

    Ulrich Schwaneberg Institute of Biotechnology, RWTH Aachen University, Worringerweg 3, Aachen 52074, Germany

    Yu Ji Institute of Biotechnology, RWTH Aachen University, Worringerweg 3, Aachen 52074, Germany

  8. Meng Hua Qin Aachen Institute of Biotechnology Germany Ruipeng An Beijing Bioprocess Key Laboratory Ulrich Schwaneberg Institute of Biotechnology Yu Ji Institute of Biotechnology

    Beijing University of Chemical Technology · RWTH Aachen University

    Affiliation as printed

    Beijing Bioprocess Key Laboratory, Beijing University of Chemical Technology, Beijing, 100029, PR China

    Beijing Bioprocess Key Laboratory, Beijing University of Chemical Technology, Beijing, 100029, PR China. Institute of Biotechnology,

    Beijing University of Chemical Technology

    Beijing University of Chemical Technology, Beijing, 100029, PR China

    Beijing University of Chemical Technology, Beijing, 100029, PR China Zhongyu Li Beijing Bioprocess Key Laboratory,

    Institute of Biotechnology, RWTH Aachen University, Worringerweg 3, Aachen 52074, Germany

    RWTH Aachen University, Worringerweg 3, Aachen 52074, Germany Ruipeng An Beijing Bioprocess Key Laboratory,

    Ulrich Schwaneberg Institute of Biotechnology, RWTH Aachen University, Worringerweg 3, Aachen 52074, Germany

    Yu Ji Institute of Biotechnology, RWTH Aachen University, Worringerweg 3, Aachen 52074, Germany

  9. Kaili Nie Aachen Institute of Biotechnology Germany Ruipeng An Beijing Bioprocess Key Laboratory Ulrich Schwaneberg Institute of Biotechnology Yu Ji Institute of Biotechnology

    Beijing University of Chemical Technology · RWTH Aachen University

    Affiliation as printed

    Beijing Bioprocess Key Laboratory, Beijing University of Chemical Technology, Beijing, 100029, PR China

    Beijing Bioprocess Key Laboratory, Beijing University of Chemical Technology, Beijing, 100029, PR China. Institute of Biotechnology,

    Beijing University of Chemical Technology

    Beijing University of Chemical Technology, Beijing, 100029, PR China

    Beijing University of Chemical Technology, Beijing, 100029, PR China Zhongyu Li Beijing Bioprocess Key Laboratory,

    Institute of Biotechnology, RWTH Aachen University, Worringerweg 3, Aachen 52074, Germany

    RWTH Aachen University, Worringerweg 3, Aachen 52074, Germany Ruipeng An Beijing Bioprocess Key Laboratory,

    Ulrich Schwaneberg Institute of Biotechnology, RWTH Aachen University, Worringerweg 3, Aachen 52074, Germany

    Yu Ji Institute of Biotechnology, RWTH Aachen University, Worringerweg 3, Aachen 52074, Germany

  10. Luo Liu

    Beijing University of Chemical Technology

    Affiliation as printed

    Beijing University of Chemical Technology

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References 44