Semi-rational engineering of terminal deoxynucleotidyl transferase for high-efficiency enzymatic de novo DNA...

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'-ONH₂-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.