Structure-Guided Redesign of Terminal Deoxynucleotidyl Transferase Enables Scalable Enzymatic DNA Synthesis for Data...

Area of Science:

• Biotechnology
• Molecular Biology
• Data Storage

Background:

• DNA offers high information capacity and stability, making it ideal for next-generation data storage.
• Enzymatic DNA synthesis is a sustainable production method, but limited by enzyme efficiency and stability.
• Terminal deoxynucleotidyl transferase (TdT) enzyme's low catalytic efficiency and aggregation-induced inactivation hinder practical DNA synthesis scalability.

Purpose of the Study:

• To overcome limitations of TdT for scalable and cost-effective DNA data storage.
• To develop a structure-guided enzyme design framework for improved polymerase performance.
• To enhance both solubility and catalytic efficiency of TdT for practical molecular manufacturing.

Main Methods:

• Computational redesign of aggregation-prone regions to enhance enzyme solubility.
• Targeted active-site engineering to improve catalytic efficiency with specific nucleotide analogs (3'-ONH2-dNTPs).
• Structure-guided enzyme design framework applied to terminal deoxynucleotidyl transferase (TdT).

Main Results:

• Engineered TdT variant (HL2-LKI) achieved 3.4 g L-1 soluble expression without fusion tags.
• High polymerization efficiency (99.9%) and DNA writing fidelity (98.9%) were demonstrated.
• Enzyme production cost reduced to approximately $0.7 g-1, a seven-order-of-magnitude decrease.

Conclusions:

• A generalizable strategy was established for transforming aggregation-limited enzymatic polymerization into scalable processes.
• The engineered TdT variant significantly advances the practical implementation of DNA as an information material.
• This work paves the way for low-cost molecular manufacturing using enzymatic polymerization.