Abstract: Terminal deoxynucleotidyl transferase (TdT) is a unique DNA polymerase that catalyzes template-independent DNA synthesis. This distinctive enzymatic property makes TdT highly valuable for various biotechnological applications, particularly in de novo gene synthesis and data storage. To enhance the soluble expression of recombinant TdT in Escherichia coli, a rational design approach was employed. Hydrophobic residues in the Zonotrichia albicollis TdT N-terminal region (positions 131-149) were systematically replaced with four categories of hydrophilic amino acids (acidic, basic, neutral, and small side chains) using site-directed mutagenesis. Comparative analysis revealed that all eight engineered variants exhibited significantly higher soluble expression levels than the wild-type enzyme. Notably, the TdT-RK and TdT-KR mutants, in which hydrophobic residues were substituted with basic amino acids (arginine and lysine), demonstrated the most substantial improvements. These optimized variants enabled an increased induction temperature to 23°C while preserving full enzymatic activity. In shake-flask induction experiments, the TdT-KR variant achieved the highest soluble protein yield at 283.7 mg/L, a 1.6-fold increase compared to the wild-type. When scaled up to a 5 L bioreactor under optimized conditions derived from shake-flask experiments, the TdT-KR variant achieved a remarkable soluble protein yield of 2.7 g/L, representing a 2.3-fold enhancement over the wild-type protein. Additionally, the fermentation process was more efficient, reducing the production cycle by 4 hours. These significant improvements provide a strong foundation for the large-scale industrial production of TdT.