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Cited by 22Microbial Natural Products and BiosynthesisMicrobial Metabolic Engineering and BioproductionEnzyme Catalysis and Immobilization

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Biosensor Guided Polyketide Synthases Engineering for Optimization of Domain Exchange Boundaries

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Englund, Elias, Schmidt, Matthias, Nava, Alberto A., Klass, Sarah, Keiser, Leah, Dan, Qingyun, Katz, Leonard, Yuzawa, Satoshi, Keasling, Jay D.·2023·Nature Communications
DOI 10.1038/s41467-023-40464-xPMC PMC10423236

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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.

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Metadata

Authors
Englund, E., Schmidt, M., Nava, A. A., Klass, S., Keiser, L., Dan, Q., Katz, L., Yuzawa, S., & Keasling, J. D.
Affiliations
  • Science for Life Laboratory (SE)
  • Lawrence Berkeley National Laboratory (US)
  • Joint BioEnergy Institute (US)
Corresponding author
Keasling, Jay D.
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2023-08-12
Volume
14
Issue
1
Pages
4871-4871
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-023-40464-x
PMC
PMC10423236
OpenAlex
W4385780829
Cited by
22
References
1
Citations by year
2026: 8 · 2025: 9 · 2024: 4 · 2023: 1
FWCI
3.41
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • Research Square (acceptedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • RWTH Publications (RWTH Aachen) (publishedVersion, other-oa)
  • eScholarship (California Digital Library) (submittedVersion, cc-by)
  • OSTI OAI (U.S. Department of Energy Office of Scientific and Technical Information) (submittedVersion, cc-by)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Technical University of Denmark, DTU Orbit (Technical University of Denmark, DTU) (submittedVersion, cc-by)
  • RWTH Publications (RWTH Aachen)
Funders
  • National Science Foundation (DEAC02-05CH11231, 2018253421, 2036849)
  • U.S. Department of Energy (-AC02-05CH11231, Contract No. DE-AC02-05-CH11231, 107514, 05CH11231, No. DE-AC02-05-CH11231, DE AC02 05CH11231, 2018253421, DE-AC02-)
  • Svenska Forskningsrådet Formas (2017-00335)
  • Office of Science (No. DE-AC02- 05-CH11231, 107514, DEAC02–05CH11231, AC02-05CH11231, DE-AC02, Contract No. DE-AC02-05CH11231)
  • Basic Energy Sciences (DE-AC02, Contract No. DE-AC02-05-CH11231, 05CH11231, DE-AC02-05CH11231., 107514, No. DE-AC02-05-CH11231, AC02–05CH11231)
  • Advanced Scientific Computing Research (DE-AC02-05CH11231, AC02-05CH11231, Contract No. DE-AC02-05CH11231)
  • Biological and Environmental Research (05CH11231, No. DE-AC02-05CH11231, DE-AC02 − 05CH11231, Contract No. DE-AC02-05CH11231, AC02-05CH11231)
  • Lawrence Berkeley National Laboratory (AC02‐05CH11231, DE-AC02-05CH11231, No. DE-AC02-05CH11231, Contract No. DE-AC02-05CH11231, 05CH11231)
Topics
  • Life Sciences › Immunology and Microbiology › Applied Microbiology and Biotechnology › Microbial Natural Products and Biosynthesis
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial Metabolic Engineering and Bioproduction
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Enzyme Catalysis and Immobilization
Keywords
domain exchange, acyltransferase domain, protein stability, protein misfolding, high-throughput screening
MeSH terms
Amino Acid Sequence, Polyketide Synthases

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