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Accessing unexplored regions of sequence space in directed enzyme evolution via insertion/deletion mutagenesis

Author

Listed:
  • Stephane Emond

    (University of Cambridge
    Evonetix Ltd, Coldhams Business Park)

  • Maya Petek

    (University of Cambridge)

  • Emily J. Kay

    (University of Cambridge
    Cancer Research UK Beatson Institute)

  • Brennen Heames

    (University of Cambridge
    Westfälische Wilhelms-Universität)

  • Sean R. A. Devenish

    (University of Cambridge
    Fluidic Analytics, The Paddocks Business Centre)

  • Nobuhiko Tokuriki

    (University of British Columbia)

  • Florian Hollfelder

    (University of Cambridge)

Abstract

Insertions and deletions (InDels) are frequently observed in natural protein evolution, yet their potential remains untapped in laboratory evolution. Here we introduce a transposon-based mutagenesis approach (TRIAD) to generate libraries of random variants with short in-frame InDels, and screen TRIAD libraries to evolve a promiscuous arylesterase activity in a phosphotriesterase. The evolution exhibits features that differ from previous point mutagenesis campaigns: while the average activity of TRIAD variants is more compromised, a larger proportion has successfully adapted for the activity. Different functional profiles emerge: (i) both strong and weak trade-off between activities are observed; (ii) trade-off is more severe (20- to 35-fold increased kcat/KM in arylesterase with 60-400-fold decreases in phosphotriesterase activity) and (iii) improvements are present in kcat rather than just in KM, suggesting adaptive solutions. These distinct features make TRIAD an alternative to widely used point mutagenesis, accessing functional innovations and traversing unexplored fitness landscape regions.

Suggested Citation

  • Stephane Emond & Maya Petek & Emily J. Kay & Brennen Heames & Sean R. A. Devenish & Nobuhiko Tokuriki & Florian Hollfelder, 2020. "Accessing unexplored regions of sequence space in directed enzyme evolution via insertion/deletion mutagenesis," Nature Communications, Nature, vol. 11(1), pages 1-14, December.
    • Handle: RePEc:nat:natcom:v:11:y:2020:i:1:d:10.1038_s41467-020-17061-3
    DOI: 10.1038/s41467-020-17061-3
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    Cited by:

    1. Remkes A. Scheele & Laurens H. Lindenburg & Maya Petek & Markus Schober & Kevin N. Dalby & Florian Hollfelder, 2022. "Droplet-based screening of phosphate transfer catalysis reveals how epistasis shapes MAP kinase interactions with substrates," Nature Communications, Nature, vol. 13(1), pages 1-14, December.
    2. Mireia Seuma & Ben Lehner & Benedetta Bolognesi, 2022. "An atlas of amyloid aggregation: the impact of substitutions, insertions, deletions and truncations on amyloid beta fibril nucleation," Nature Communications, Nature, vol. 13(1), pages 1-13, December.
    3. Dan Kozome & Adnan Sljoka & Paola Laurino, 2024. "Remote loop evolution reveals a complex biological function for chitinase enzymes beyond the active site," Nature Communications, Nature, vol. 15(1), pages 1-11, December.

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