Engineering an allosteric transcription factor to respond to new ligands
Open accessAbstract
Genetic regulatory proteins inducible by small molecules are useful synthetic biology tools as sensors and switches. Bacterial allosteric transcription factors (aTFs) are a major class of regulatory proteins, but few aTFs have been redesigned to respond to new effectors beyond natural aTF-inducer pairs. Altering inducer specificity in these proteins is difficult because substitutions that affect inducer binding may also disrupt allostery. We engineered an aTF, the Escherichia coli lac repressor, LacI, to respond to one of four new inducer molecules: fucose, gentiobiose, lactitol and sucralose. Using computational protein design, single-residue saturation mutagenesis or random mutagenesis, along with multiplex assembly, we identified new variants comparable in specificity and induction to wild-type LacI with its inducer, isopropyl β-D-1-thiogalactopyranoside (IPTG). The ability to create designer aTFs will enable applications including dynamic control of cell metabolism, cell biology and synthetic gene circuits.
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Even in a highly structured protein, "communication" between sites is likely mediated by multiple paths.
“Since even in a highly structured protein “communication” between sites is likely mediated by multiple paths (Guo et al., 2015; Taylor et al., 2016), the questions of whether a given path can be rigorously validated against experimental data, or whether its specific role can be rationalized, need to be critically evaluated.”
From Allostery in Its Many Disguises: From Theory to Applications (2019) · cites this paper as Taylor et al., 2016
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- Authors
- Taylor, N. D., Garruss, A. S., Moretti, R., Chan, S., Arbing, M. A., Cascio, D., Rogers, J. K., Isaacs, F. J., Kosuri, S., Baker, D., Fields, S., Church, G. M., & Raman, S.
- Affiliations
- Harvard University (US)
- Howard Hughes Medical Institute (US)
- University of California, Los Angeles (US)
- Yale University (US)
- Corresponding author
- Raman, Srivatsan
- ORCID
- Venue
- Nature Methods
- Published
- 2015-12-21
- Volume
- 13
- Issue
- 2
- Pages
- 177-183
- ISSN
- 1548-7091 (ISSN-L)
- 1548-7105
- PMC
- PMC4907361
- OpenAlex
- W2260617737
- Cited by
- 328
- References
- 60
- Citations by year
- 2026: 24 · 2025: 26 · 2024: 30 · 2023: 27 · 2022: 39 · 2021: 42 · 2020: 26 · 2019: 35 · 2018: 32 · 2017: 24 · 2016: 23
- FWCI
- 11.44
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature Methods (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- OSTI OAI (U.S. Department of Energy Office of Scientific and Technical Information) (submittedVersion)
- eScholarship (California Digital Library) (submittedVersion)
- Funders
- Howard Hughes Medical Institute
- U.S. Department of Energy (DE‐ AC02‐06CH11357, DE‐FG02‐, DEFG0202ER63445, AC02‐06CH11357, P41 GM103403, FG02-02ER63445, P41 RR015301, 06CH11357, DEFC0202ER63421, FC02-02ER63421, DE-AC02-)
- National Institutes of Health (DE-FC02-02ER63421, DE-AC02–06CH11357, DE-FG02-02ER63445, GM103403, GM103533)
- National Human Genome Research Institute (T32HG002295)
- National Institute of General Medical Sciences (GM103533, P41GM103533, DE-AC02-06CH11357, GM-103403, P41GM103403)
- National Center for Research Resources (P41RR015301)
- Argonne National Laboratory (DE- AC02-06-CH11357, DE-AC02, AC02–06CH11357, P41 RR015301, P41 GM103403, 06CH11357)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gene Regulatory Network Analysis
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial Metabolic Engineering and Bioproduction
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
- Keywords
- allosteric transcription factors, lac repressor, LacI, inducer specificity, synthetic gene circuits, protein engineering, fucose, gentiobiose, lactitol, sucralose, IPTG, allosteric regulation, Escherichia coli
- MeSH terms
- Allosteric Regulation, Disaccharides, DNA, Bacterial, Escherichia coli, Fucose, Genetic Engineering, Models, Molecular, Mutation, Protein Binding, Protein Conformation, Sucrose, Sugar Alcohols, Gene Expression Regulation, Bacterial, Escherichia coli Proteins, Lac Repressors
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