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Orthogonal glycolytic pathway enables directed evolution of noncanonical cofactor oxidase

Author

Listed:
  • Edward King

    (University of California Irvine)

  • Sarah Maxel

    (Department Chemical and Biomolecular Engineering University of California Irvine)

  • Yulai Zhang

    (Department Chemical and Biomolecular Engineering University of California Irvine)

  • Karissa C. Kenney

    (University of California Irvine)

  • Youtian Cui

    (Genome Center, University of California Davis)

  • Emma Luu

    (Genome Center, University of California Davis)

  • Justin B. Siegel

    (Genome Center, University of California Davis
    Molecular Medicine University of California, Davis
    Department of Biochemistry and Molecular Medicine University of California, Davis)

  • Gregory A. Weiss

    (University of California Irvine
    University of California Irvine
    University of California Irvine)

  • Ray Luo

    (University of California Irvine
    Department Chemical and Biomolecular Engineering University of California Irvine
    University of California Irvine
    University of California Irvine)

  • Han Li

    (Department Chemical and Biomolecular Engineering University of California Irvine
    University of California Irvine)

Abstract

Noncanonical cofactor biomimetics (NCBs) such as nicotinamide mononucleotide (NMN+) provide enhanced scalability for biomanufacturing. However, engineering enzymes to accept NCBs is difficult. Here, we establish a growth selection platform to evolve enzymes to utilize NMN+-based reducing power. This is based on an orthogonal, NMN+-dependent glycolytic pathway in Escherichia coli which can be coupled to any reciprocal enzyme to recycle the ensuing reduced NMN+. With a throughput of >106 variants per iteration, the growth selection discovers a Lactobacillus pentosus NADH oxidase variant with ~10-fold increase in NMNH catalytic efficiency and enhanced activity for other NCBs. Molecular modeling and experimental validation suggest that instead of directly contacting NCBs, the mutations optimize the enzyme’s global conformational dynamics to resemble the WT with the native cofactor bound. Restoring the enzyme’s access to catalytically competent conformation states via deep navigation of protein sequence space with high-throughput evolution provides a universal route to engineer NCB-dependent enzymes.

Suggested Citation

  • Edward King & Sarah Maxel & Yulai Zhang & Karissa C. Kenney & Youtian Cui & Emma Luu & Justin B. Siegel & Gregory A. Weiss & Ray Luo & Han Li, 2022. "Orthogonal glycolytic pathway enables directed evolution of noncanonical cofactor oxidase," Nature Communications, Nature, vol. 13(1), pages 1-14, December.
    • Handle: RePEc:nat:natcom:v:13:y:2022:i:1:d:10.1038_s41467-022-35021-x
    DOI: 10.1038/s41467-022-35021-x
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    References listed on IDEAS

    as
    1. Lara Sellés Vidal & James W. Murray & John T. Heap, 2021. "Versatile selective evolutionary pressure using synthetic defect in universal metabolism," Nature Communications, Nature, vol. 12(1), pages 1-15, December.
    2. Xueying Wang & Yanbin Feng & Xiaojia Guo & Qian Wang & Siyang Ning & Qing Li & Junting Wang & Lei Wang & Zongbao K. Zhao, 2021. "Creating enzymes and self-sufficient cells for biosynthesis of the non-natural cofactor nicotinamide cytosine dinucleotide," Nature Communications, Nature, vol. 12(1), pages 1-9, December.
    3. Sarel J Fleishman & Andrew Leaver-Fay & Jacob E Corn & Eva-Maria Strauch & Sagar D Khare & Nobuyasu Koga & Justin Ashworth & Paul Murphy & Florian Richter & Gordon Lemmon & Jens Meiler & David Baker, 2011. "RosettaScripts: A Scripting Language Interface to the Rosetta Macromolecular Modeling Suite," PLOS ONE, Public Library of Science, vol. 6(6), pages 1-10, June.
    4. Linyue Zhang & Edward King & William B. Black & Christian M. Heckmann & Allison Wolder & Youtian Cui & Francis Nicklen & Justin B. Siegel & Ray Luo & Caroline E. Paul & Han Li, 2022. "Directed evolution of phosphite dehydrogenase to cycle noncanonical redox cofactors via universal growth selection platform," Nature Communications, Nature, vol. 13(1), pages 1-12, December.
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    Cited by:

    1. Enrico Orsi & Lennart Schada von Borzyskowski & Stephan Noack & Pablo I. Nikel & Steffen N. Lindner, 2024. "Automated in vivo enzyme engineering accelerates biocatalyst optimization," Nature Communications, Nature, vol. 15(1), pages 1-14, December.

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