Structure‐Guided Redesign of Terminal Deoxynucleotidyl Transferase Enables Scalable Enzymatic DNA Synthesis for Data Storage

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′‐ONH 2 ‐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.