Evolving a terminal deoxynucleotidyl transferase for commercial enzymatic DNA synthesis
Stephanie M. Forget, M. Krawczyk, Anders M. Knight, Charlene Ching, Rachelle A. Copeland, Niusha Mahmoodi, Melissa Mayo, James Nguyen, Amanda Tan, Mathew Miller, Jonathan Vroom, Stefan Lutz
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.