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Scalable continuous evolution for the generation of diverse enzyme variants encompassing promiscuous activities

Author

Listed:
  • Gordon Rix

    (University of California)

  • Ella J. Watkins-Dulaney

    (California Institute of Technology)

  • Patrick J. Almhjell

    (California Institute of Technology)

  • Christina E. Boville

    (California Institute of Technology
    Aralez Bio)

  • Frances H. Arnold

    (California Institute of Technology
    California Institute of Technology)

  • Chang C. Liu

    (University of California
    University of California
    University of California)

Abstract

Enzyme orthologs sharing identical primary functions can have different promiscuous activities. While it is possible to mine this natural diversity to obtain useful biocatalysts, generating comparably rich ortholog diversity is difficult, as it is the product of deep evolutionary processes occurring in a multitude of separate species and populations. Here, we take a first step in recapitulating the depth and scale of natural ortholog evolution on laboratory timescales. Using a continuous directed evolution platform called OrthoRep, we rapidly evolve the Thermotoga maritima tryptophan synthase β-subunit (TmTrpB) through multi-mutation pathways in many independent replicates, selecting only on TmTrpB’s primary activity of synthesizing l-tryptophan from indole and l-serine. We find that the resulting sequence-diverse TmTrpB variants span a range of substrate profiles useful in industrial biocatalysis and suggest that the depth and scale of evolution that OrthoRep affords will be generally valuable in enzyme engineering and the evolution of biomolecular functions.

Suggested Citation

  • Gordon Rix & Ella J. Watkins-Dulaney & Patrick J. Almhjell & Christina E. Boville & Frances H. Arnold & Chang C. Liu, 2020. "Scalable continuous evolution for the generation of diverse enzyme variants encompassing promiscuous activities," Nature Communications, Nature, vol. 11(1), pages 1-11, December.
    • Handle: RePEc:nat:natcom:v:11:y:2020:i:1:d:10.1038_s41467-020-19539-6
    DOI: 10.1038/s41467-020-19539-6
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    Cited by:

    1. Dae-yeol Ye & Myung Hyun Noh & Jo Hyun Moon & Alfonsina Milito & Minsun Kim & Jeong Wook Lee & Jae-Seong Yang & Gyoo Yeol Jung, 2022. "Kinetic compartmentalization by unnatural reaction for itaconate production," Nature Communications, Nature, vol. 13(1), pages 1-10, December.
    2. Rosario Vanella & Christoph Küng & Alexandre A. Schoepfer & Vanni Doffini & Jin Ren & Michael A. Nash, 2024. "Understanding activity-stability tradeoffs in biocatalysts by enzyme proximity sequencing," Nature Communications, Nature, vol. 15(1), pages 1-14, December.
    3. 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.
    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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