Work

Evolving a terminal deoxynucleotidyl transferase for commercial enzymatic DNA synthesis

Year: 2025

Type: article

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.

Source: Nucleic Acids Research

DOI: 10.1093/nar/gkaf115