Optimization of Expression and Thermostability of Terminal Deoxynucleotidyl Transferase through Iterative Mutagenesis and Computational Design

Abstract Terminal deoxynucleotidyl transferase (TdT) is a template-independent polymerase that catalyzes the addition of deoxynucleoside triphosphates to the 3'-terminus of a DNA strand. While TdT is a key enzyme for developing enzymatic DNA synthesis technologies, its inherent low thermal stability presents a significant limitation. This study aims to improve the thermostability of TdT from Mus musculus through a combination of site-saturation mutagenesis and rational design. Residues for saturation mutagenesis were identified using B-factor analysis and the B-FITTER program, while promising substitutions for rational design were selected using Foldit, ProteinMPNN and CARBonAra. Through several iterations of mutagenesis, we obtained two highly promising variants. The first one, dubbed A4, obtained solely through saturation mutagenesis, showed a 26-fold higher expression level than the WT protein. The second one, dubbed mutant 275 , demonstrated exceptional stability, showing no significant loss of activity after 180 minutes of incubation at 45°C — conditions under which the wild-type enzyme's half-life was less than 2 minutes. This corresponds to a >120-fold increase in stability. Additionally, its melting temperature (Tm) was increased by 6.5°C. Moreover, mutant 275 demonstrated a 4- to 6-fold increase in catalytic activity at 37 °C. This significant enhancement in thermostability was achieved after four rounds of iterative mutagenesis and is attributed to the formation of a stabilizing salt bridge network on the protein surface, distant from the active site. The obtained thermostable TdT variants serve as robust scaffolds for further engineering to improve activity towards the 3'-reversibly blocked nucleotides required for next-generation enzymatic DNA synthesis.