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, Jay D Keasling

Journal: Nature communications 2023;14(1):4871

PMID: 37573440

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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.

Address: Joint BioEnergy Institute, Emeryville, CA, USA.; School of Engineering Sciences in Chemistry, Biotechnology and Health, Science for Life Laboratory, KTH - Royal Institute of Technology, Stockholm, Sweden.; Institute of Applied Microbiology, Aachen Biology and Biotechnology (ABBt), RWTH Aachen University, Aachen, Germany.; Biological Systems and Engineering Division, Lawrence Berkeley National laboratory, Berkeley, CA, USA.; Department of Chemical & Biomolecular Engineering, University of California, Berkeley, Berkeley, CA, USA.; QB3, University of California, Berkeley, Berkeley, CA, USA.; Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata, Japan.; Graduate school of Media and Governance, Keio University, Fujisawa, Kanagawa, Japan.; Joint BioEnergy Institute, Emeryville, CA, USA. [email protected].; Biological Systems and Engineering Division, Lawrence Berkeley National laboratory, Berkeley, CA, USA. [email protected].; Department of Chemical & Biomolecular Engineering, University of California, Berkeley, Berkeley, CA, USA. [email protected].; QB3, University of California, Berkeley, Berkeley, CA, USA. [email protected].; Department of Bioengineering, University of California, Berkeley, Berkeley, CA, USA. [email protected].; Center for Biosustainability, Danish Technical University, Lyngby, Denmark. [email protected].; Center for Synthetic biochemistry, Institute for Synthetic biology, Shenzhen Institute of Advanced Technology, Shenzhen, China. [email protected].

DOI: 10.1038/s41467-023-40464-x

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MeSH Terms: Polyketide Synthases, Amino Acid Sequence