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

DNA, with its exceptional information capacity and chemical stability, represents a promising material for next-generation data storage to meet the exponential growth of global digital information demands. Enzymatic DNA synthesis provides a sustainable route to DNA production. However, the practical scalability of the underlying polymerization chemistry has been fundamentally constrained by the low catalytic efficiency and aggregation-induced inactivation of terminal deoxynucleotidyl transferase (TdT) that is responsible for nucleotide polymerization. Here, we report a structure-guided enzyme design framework that overcomes these intrinsic limitations by decoupling solubility and catalytic performance in a processive polymerase. Computational redesign of aggregation-prone regions markedly enhances soluble expression, while targeted active-site engineering improves catalytic efficiency toward 3'-ONH₂-dNTPs used in enzymatic DNA synthesis. The resulting TdT variant HL2-LKI achieves 3.4 g L^-1 soluble expression in a 5 L fermenter without fusion tags and exhibits high polymerization efficiency (99.9%) and DNA writing fidelity (98.9%). This redesign reduces enzyme production costs to approximately $0.7 g^-1, nearly seven orders of magnitude lower than the catalog price of commercially available TdT. This work establishes a generalizable strategy for transforming aggregation-limited enzymatic polymerization reactions into scalable and low-cost molecular manufacturing processes, thereby advancing the practical implementation of DNA as an information material.