Biosensor Guided Polyketide Synthases Engineering for Optimization of Domain Exchange Boundaries

Elias Englund, Matthias Schmidt, Alberto A. Nava, Sarah Klass, Leah Keiser, Qingyun Dan, Leonard Katz, Satoshi Yuzawa and Jay D. Keasling ()
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Elias Englund: Joint BioEnergy Institute
Matthias Schmidt: Joint BioEnergy Institute
Alberto A. Nava: Joint BioEnergy Institute
Sarah Klass: Joint BioEnergy Institute
Leah Keiser: Joint BioEnergy Institute
Qingyun Dan: Joint BioEnergy Institute
Leonard Katz: Joint BioEnergy Institute
Satoshi Yuzawa: Joint BioEnergy Institute
Jay D. Keasling: Joint BioEnergy Institute

Nature Communications, 2023, vol. 14, issue 1, 1-12

Abstract: 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.

Date: 2023
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DOI: 10.1038/s41467-023-40464-x

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