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bioRxiv · 10.64898/2025.12.29.696827

Semi-rational Engineering of Terminal Deoxynucleotidyl Transferase for High-efficiency Enzymatic de Novo DNA Synthesis

Zhang, C.·He, Z.·Gao, Z.·Meng, C.·Peng, W.·Du, L.·Li, S.
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Abstract

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 BtTdTs 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: Bt15AAR336L/K338G/L397M/E456S/D395G) optimized for 3'-ONH2-dNTP incorporation. M5 exhibited 30-fold activity enhancement relative to the triple mutant M3 (Bt15AAR336L/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.

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Zhang, C., He, Z., Gao, Z., Meng, C., Peng, W., Du, L., Li, S.. 2025-12-29. Semi-rational Engineering of Terminal Deoxynucleotidyl Transferase for High-efficiency Enzymatic de Novo DNA Synthesis. https://doi.org/10.64898/2025.12.29.696827

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