| 摘要： |
Enzyme-based error correction is a key step in de novo DNA synthesis, yet the inherent instability of error-correction enzymes such as MutS has hindered the throughput and efficiency of DNA synthesis workflows. Here we introduce a process called Improved MICC (iMICC), in which all error-correction steps of oligos and fragments within a complete gene-synthesis cycle are completed in a simple, efficient, and low-cost manner via a MutS protein engineered for high durability. By establishing a disulfide bond of L157C-G233C, full-activity shelf life of E. coil MutS (eMutS) was prolonged from 7 to 49 days and was further extended to 63 days via cellulose-bound 4 degrees C storage. In synthesis of 10 Cas9 homologues in-solution and 10 xylose reductase (XR) homologues on-chip, iMICC reduced error frequency to 0.64/Kb and 0.41/Kb, respectively, with 72.1% and 86.4% of assembled fragments being error-free. By elevating base accuracy by 37.6-fold while avoiding repetitive preparation of fresh enzymes, iMICC is more efficient and robust than the wild-type eMutS, and it is 6.6-fold more accurate and 26.7-fold cheaper than CorrectASE. These advantages promise its broad applications in industrial DNA synthesis. |
