The 21st century has witnessed rapid advancements in synthetic biology, with DNA synthesis emerging as a foundational technology. Conventional phosphoramidite-based methods face significant limitations, including short DNA elongation lengths (<300 nt), hazardous chemical waste, and low stepwise incorporation efficiency. Enzymatic DNA synthesis using terminal deoxynucleotidyl transferase (TdT) offers a promising alternative, enabling kilobase-scale assembly with greater efficiency and minimal environmental impact. Here, we identified Bos taurus TdT (BtTdT) through UniProt database mining as a catalytically active scaffold for natural and 3′-modified dNTPs. Comprehensive characterization of BtTdT's enzymatic properties—including pH, temperature, metal ion dependence, and substrate specificity—revealed its optimal conditions. Truncation of the BRCT domain generated variants with enhanced activity compared to wild-type BtTdT. Guided by AlphaFold3-predicted structural models, we engineered a quintuple mutant (M5: Bt15AA R336L/K338G/L397M/E456S/D395G ) optimized for 3′–ONH 2 –dNTP incorporation. M5 exhibited 30-fold activity enhancement relative to the triple mutant M3 (Bt15AA R336L/K338G/L397M ) and achieved stepwise incorporation efficiency exceeding 98% in de novo synthesis of 10-nt ssDNA, demonstrating its potential for scalable enzymatic DNA synthesis. This work establishes a rational framework for TdT engineering through rational domain truncation and computational design, showing potential toward industrial-scale enzymatic DNA manufacturing.
Zhang et al. (2026) studied this question.