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Biosensor Guided Polyketide Synthase Engineering for Optimization of Domain Exchange Boundaries
Open accessPreprint — published as 10.1038/s41467-023-40464-x · Paper page
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, the resulting PKS hybrids typically have reduced catalytic activities and are often insoluble due to misfolding. Here, we have developed a fluorescence-based biosensor method for detecting engineered PKSs with high solubility. The biosensor has been used to sort through PKS hybrids that had acyltransferase (AT) domains from other PKSs exchanged for the native AT with randomly assigned linker junctions. Importantly, we observed a significant correlation between activity and solubility. Evaluation of highly soluble mutants in vitro revealed new boundaries for AT domain exchanges that give a wild-type level of catalytic activity. Together, we have successfully developed an experimentally validated high-throughput method to efficiently screen active engineered PKSs that produce target molecules.
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Highly accurate protein structure prediction with AlphaFold
Jumper, J., Evans, R., Pritzel, A., Green, T., Figurnov, M., Ronneberger, O., Tunyasuvunakool, K., Bates, R., Žídek, A., Potapenko, A., et al. (2021) · Nature · doi:10.1038/s41586-021-03819-2 · Cited by 43,764
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Metadata
- Authors
- Englund, E., Schmidt, M., Nava, A., Dan, Q., Katz, L., Yuzawa, S., & Keasling, J.
- Affiliations
- Science for Life Laboratory (SE)
- Lawrence Berkeley National Laboratory (US)
- Joint BioEnergy Institute (US)
- Corresponding author
- Keasling, Jay
- Venue
- Nature Communications
- Published
- 2022-04-19
- Volume
- 14
- Issue
- 1
- Pages
- 4871-4871
- ISSN
- 2041-1723 (ISSN-L)
- 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
- gold
- doi.org
- 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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