Efficient and Low-Cost Error Removal in DNA Synthesis by a High-Durability MutS

Area of Science:

• Biotechnology
• Molecular Biology
• Synthetic Biology

Background:

• Enzyme-based error correction is crucial for de novo DNA synthesis.
• Instability of enzymes like MutS limits DNA synthesis throughput and efficiency.

Purpose of the Study:

• To introduce Improved MICC (iMICC) for efficient, low-cost error correction in gene synthesis.
• To enhance the durability of MutS protein for prolonged enzyme activity.

Main Methods:

• Engineered a MutS protein by establishing a disulfide bond (L157C-G233C) to increase durability.
• Stored the engineered E. coli MutS (eMutS) protein using cellulose-bound 4 °C storage.
• Applied iMICC in the synthesis of Cas9 and xylose reductase (XR) homologues.

Main Results:

• Prolonged shelf life of eMutS from 7 to 49 days, extended to 63 days with specialized storage.
• Reduced error frequency to 0.64/Kb (Cas9) and 0.41/Kb (XR).
• Achieved 72.1% (Cas9) and 86.4% (XR) error-free assembled fragments, with a 37.6-fold increase in base accuracy.

Conclusions:

• iMICC offers a more efficient, robust, and cost-effective alternative to wild-type eMutS and CorrectASE.
• The engineered MutS protein significantly improves accuracy and reduces costs in industrial DNA synthesis.
• iMICC demonstrates broad applicability for large-scale gene synthesis.