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In vivo continuous evolution of genes and pathways in yeast

Author

Listed:
  • Nathan Crook

    (The University of Texas at Austin)

  • Joseph Abatemarco

    (The University of Texas at Austin)

  • Jie Sun

    (The University of Texas at Austin)

  • James M. Wagner

    (The University of Texas at Austin)

  • Alexander Schmitz

    (The University of Texas at Austin)

  • Hal S. Alper

    (The University of Texas at Austin
    Institute for Cellular and Molecular Biology, The University of Texas at Austin)

Abstract

Directed evolution remains a powerful, highly generalizable approach for improving the performance of biological systems. However, implementations in eukaryotes rely either on in vitro diversity generation or limited mutational capacities. Here we synthetically optimize the retrotransposon Ty1 to enable in vivo generation of mutant libraries up to 1.6 × 107 l−1 per round, which is the highest of any in vivo mutational generation approach in yeast. We demonstrate this approach by using in vivo-generated libraries to evolve single enzymes, global transcriptional regulators and multi-gene pathways. When coupled to growth selection, this approach enables in vivo continuous evolution (ICE) of genes and pathways. Through a head-to-head comparison, we find that ICE libraries yield higher-performing variants faster than error-prone PCR-derived libraries. Finally, we demonstrate transferability of ICE to divergent yeasts, including Kluyveromyces lactis and alternative S. cerevisiae strains. Collectively, this work establishes a generic platform for rapid eukaryotic-directed evolution across an array of target cargo.

Suggested Citation

  • Nathan Crook & Joseph Abatemarco & Jie Sun & James M. Wagner & Alexander Schmitz & Hal S. Alper, 2016. "In vivo continuous evolution of genes and pathways in yeast," Nature Communications, Nature, vol. 7(1), pages 1-14, December.
    • Handle: RePEc:nat:natcom:v:7:y:2016:i:1:d:10.1038_ncomms13051
    DOI: 10.1038/ncomms13051
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    Cited by:

    1. Anna Zimmermann & Julian E. Prieto-Vivas & Charlotte Cautereels & Anton Gorkovskiy & Jan Steensels & Yves Peer & Kevin J. Verstrepen, 2023. "A Cas3-base editing tool for targetable in vivo mutagenesis," Nature Communications, Nature, vol. 14(1), pages 1-16, December.
    2. 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.
    3. Mary S. Morrison & Tina Wang & Aditya Raguram & Colin Hemez & David R. Liu, 2021. "Disulfide-compatible phage-assisted continuous evolution in the periplasmic space," Nature Communications, Nature, vol. 12(1), pages 1-14, December.

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