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Biosensor Guided Polyketide Synthases Engineering for Optimization of Domain Exchange Boundaries

Author

Listed:
  • Elias Englund

    (Joint BioEnergy Institute
    KTH - Royal Institute of Technology)

  • Matthias Schmidt

    (Joint BioEnergy Institute
    RWTH Aachen University
    Lawrence Berkeley National laboratory)

  • Alberto A. Nava

    (Joint BioEnergy Institute
    University of California, Berkeley)

  • Sarah Klass

    (Joint BioEnergy Institute
    Lawrence Berkeley National laboratory
    University of California, Berkeley)

  • Leah Keiser

    (Joint BioEnergy Institute
    University of California, Berkeley)

  • Qingyun Dan

    (Joint BioEnergy Institute
    Lawrence Berkeley National laboratory)

  • Leonard Katz

    (Joint BioEnergy Institute
    QB3, University of California, Berkeley)

  • Satoshi Yuzawa

    (Joint BioEnergy Institute
    Lawrence Berkeley National laboratory
    Keio University
    Keio University)

  • Jay D. Keasling

    (Joint BioEnergy Institute
    Lawrence Berkeley National laboratory
    University of California, Berkeley
    QB3, University of California, Berkeley)

Abstract

Type I modular polyketide synthases (PKSs) are multi-domain enzymes functioning like assembly lines. Many engineering attempts have been made for the last three decades to replace, delete and insert new functional domains into PKSs to produce novel molecules. However, inserting heterologous domains often destabilize PKSs, causing loss of activity and protein misfolding. To address this challenge, here we develop a fluorescence-based solubility biosensor that can quickly identify engineered PKSs variants with minimal structural disruptions. Using this biosensor, we screen a library of acyltransferase (AT)-exchanged PKS hybrids with randomly assigned domain boundaries, and we identify variants that maintain wild type production levels. We then probe each position in the AT linker region to determine how domain boundaries influence structural integrity and identify a set of optimized domain boundaries. Overall, we have successfully developed an experimentally validated, high-throughput method for making hybrid PKSs that produce novel molecules.

Suggested Citation

  • Elias Englund & Matthias Schmidt & Alberto A. Nava & Sarah Klass & Leah Keiser & Qingyun Dan & Leonard Katz & Satoshi Yuzawa & Jay D. Keasling, 2023. "Biosensor Guided Polyketide Synthases Engineering for Optimization of Domain Exchange Boundaries," Nature Communications, Nature, vol. 14(1), pages 1-12, December.
    • Handle: RePEc:nat:natcom:v:14:y:2023:i:1:d:10.1038_s41467-023-40464-x
    DOI: 10.1038/s41467-023-40464-x
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    References listed on IDEAS

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