ArticleProceedings of the National Academy of Sciences of the United States of America2003
Targeted gene evolution in Escherichia coli using a highly error-prone DNA polymerase I.
Article in Proceedings of the National Academy of Sciences of the United States of America, 2003. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 82 papers.
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Who cites it
82 citing papers in PubMed, 168 citations in OpenAlex.
- Harnessing the central dogma: Enzyme-driven gene diversification strategies for in cellulo continuous directed evolution.Fundamental research · 2026Review
- Exploring the Evolutionary Landscape with Targeted In Vivo Hypermutations.Biomedicines · 2026Review
- AUXIN RESPONSE FACTOR thermostability.Nature communications · 2026Article
- Highly mutagenic continuous evolution in E. coli using a Φ29-based orthogonal replication system.Nature biotechnology · 2026Article
- Synthetic Biology of Plants and Microbes for Agriculture, Environment, and Future Applications.Chemical reviews · 2026Review
- Design and validation of an inducible and curable EvolvR system for directed evolution inFrontiers in bioengineering and biotechnology · 2026Article
- Recent advances in targeted mutagenesis to expedite the evolution of biological systems.Journal of microbiology (Seoul, Korea) · 2025Review
- Harnessing microbial heterogeneity for improved biosynthesis fueled by synthetic biology.Synthetic and systems biotechnology · 2025Review
- Development of a new affinity maturation protocol for the construction of an internalizing anti-nucleolin antibody library.Scientific reports · 2024Article
- Ultrahigh-throughput screening-assisted in vivo directed evolution for enzyme engineering.Biotechnology for biofuels and bioproducts · 2024Article
- Evolution and synthetic biology.Current opinion in microbiology · 2023Review
- Expanded MutaT7 toolkit efficiently and simultaneously accesses all possible transition mutations in bacteria.Nucleic acids research · 2023Article
- A primer to directed evolution: current methodologies and future directions.RSC chemical biology · 2023Review
- Article
- A CRISPR-guided mutagenic DNA polymerase strategy for the detection of antibiotic-resistant mutations inMolecular therapy. Nucleic acids · 2022Article
- Hypermutation of specific genomic loci of Pseudomonas putida for continuous evolution of target genes.Microbial biotechnology · 2022Article
- Directed evolution of phosphite dehydrogenase to cycle noncanonical redox cofactors via universal growth selection platform.Nature communications · 2022Article
- In vivo hypermutation and continuous evolution.Nature reviews. Methods primers · 2022Article
- Temperature effect on polymerase fidelity.The Journal of biological chemistry · 2021Article
- Systems for in vivo hypermutation: a quest for scale and depth in directed evolution.Current opinion in chemical biology · 2021Review
22 more citing papers are in PubMed but not listed here.
Corrections and comments
PubMed lists nothing against this paper. Absence here is not a guarantee, only a check that was made.
Authors and funding
4 authors at 1 institution in 1 country.
Funding
Abstract
We present a system for random mutagenesis in Escherichia coli for the evolution of targeted genes. To increase error rates of DNA polymerase I (Pol I) replication, we introduced point mutations in three structural domains that govern Pol I fidelity. Expression of error-prone Pol I in vivo results in strong mutagenesis of a target sequence encoded in a Pol I-dependent plasmid (8.1 x 10-4 mutations per bp, an 80,000-fold increase), with a preference for plasmid relative to chromosome sequence. Mutagenesis is maximal in cultures maintained at stationary phase. Mutations are evenly distributed and show a variety of base pair substitutions, predominantly transitions. Mutagenesis extends at least 3 kb beyond the 400-500 nt reportedly synthesized by Pol I. We demonstrate that our error-prone Pol I can be used to generate enzymes with distinct properties by generating TEM-1 beta-lactamase mutants able to hydrolyze a third-generation lactam antibiotic, aztreonam. Three different mutations contribute to aztreonam resistance. Two are found in the extended-spectrum beta-lactamases most frequently identified in clinical isolates, and the third (G276R) has not been previously described. Our system of targeted mutagenesis in E. coli should have an impact on enzyme-based applications in areas such as synthetic chemistry, gene therapy, and molecular biology. Given the structural conservation between polymerases, this work should also provide a reference for altering the fidelity of other polymerases.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.