ArticleNucleic acids research2025
Evolving a terminal deoxynucleotidyl transferase for commercial enzymatic DNA synthesis.
Article in Nucleic acids research, 2025. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 12 papers.
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Who cites it
12 citing papers in PubMed.
- Stimuli-responsive vesicles in water-resistant nanofibers for molecular random access in DNA data storage.Nanoscale advances · 2026Article
- Semi-rational engineering of terminal deoxynucleotidyl transferase for high-efficiency enzymaticSynthetic and systems biotechnology · 2026Article
- Enzymatic depolymerization of laboratory waste DNA to a dGMP-containing pool and analytical assessment of dGTP in a crude-lysate guanylate kinase system.Molecular biology reports · 2026Article
- Advances in large DNA fragment assembly for microbial cell factory engineering.Quantitative biology (Beijing, China) · 2026Review
- Engineering a Virus-Derived X Family DNA Polymerase FvPolX for de novo DNA Synthesis.Advanced science (Weinheim, Baden-Wurttemberg, Germany) · 2026Article
- Optimization of Expression and Thermostability of Terminal Deoxynucleotidyl Transferase Through Iterative Mutagenesis and Computational Design.Applied biochemistry and biotechnology · 2026Article
- Aspergillus fumigatus PolX1 is an early ancestor of vertebrate terminal deoxynucleotidyl transferases.Nucleic acids research · 2026Article
- Phosphorothioate DNA modification by BREX type 4 systems in the human gut microbiome.Nature communications · 2026Article
- Enzymatic synthesis of key RNA therapeutic building blocks using simple phosphate donors.Nature communications · 2025Article
- From liquid-phase synthesis to chemical ligation: preparation of oligonucleotides and their backbone analogs in solution.Nucleic acids research · 2025Review
- An Update: Enzymatic Synthesis for Industrial Applications.Angewandte Chemie (International ed. in English) · 2025Review
- Ultrahigh-Throughput Activity Engineering of Promiscuous Amidases through a Fluorescence-Activated Cell Sorting Assay.ACS catalysis · 2025Article
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Authors and funding
12 authors.
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Abstract
Enzymatic DNA synthesis, using stepwise nucleotide addition catalyzed by template-independent polymerases, promises higher efficiency, quality, and sustainability than today's industry-standard phosphoramidite-based processes. We report the directed evolution of a terminal deoxynucleotidyl transferase that uses 3'-phosphate-blocked 2'-deoxynucleoside triphosphates (dNTPs) to control the polymerization reaction. Over 32 iterative rounds of laboratory evolution, 80 amino acid substitutions-constituting ∼20% of the coding protein sequence-were introduced. The engineered polymerase exhibits uniformly high catalytic activity, raising incorporation efficiency by 200-fold to >99% for dNTPs with a 3'-reversible terminator while reducing extension times by >600-fold to 90 s. The same enzyme variant displays improved enzyme robustness, as reflected in the 20°C increase in thermostability. Based on these performance characteristics, the engineered polymerase represents an operational prototype for biocatalytic DNA synthesis at a commercial scale.
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