Efficient and Low-Cost Error Removal in DNA Synthesis by a High-Durability MutS.,ACS Synthetic Biology

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Efficient and Low-Cost Error Removal in DNA Synthesis by a High-Durability MutS.
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2020-03-13 , DOI: 10.1021/acssynbio.0c00079
Jia Zhang _{1,

2,

3} , Yefei Wang ₄ , Baihui Chai _{1,

2,

3} , Jichao Wang _{3,

5} , Lulu Li ₆ , Min Liu _{1,

2,

3} , Guang Zhao _{3,

5} , Lishan Yao _{3,

4} , Xiaolian Gao _{6,

7} , Yifeng Yin ₁ , Jian Xu _{1,

2,

3}

Affiliation

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. coli MutS (eMutS) was prolonged from 7 to 49 days and was further extended to 63 days via cellulose-bound 4 °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.

中文翻译：

高耐用性MutS在DNA合成中的高效且低成本的错误去除。

基于酶的错误校正是从头进行DNA合成的关键步骤，但是错误校正酶（例如MutS）固有的不稳定性阻碍了DNA合成工作流程的吞吐量和效率。在这里，我们介绍了一种称为改进的MICC（iMICC）的过程，其中通过完整的基因工程改造的MutS蛋白，以简单，高效且低成本的方式完成了完整基因合成周期中寡核苷酸和片段的所有错误校正步骤。耐用性。通过建立L157C-G233C的二硫键，大肠杆菌MutS（eMutS）的全活性货架期从7天延长至49天，并通过与纤维素结合的4°C储存进一步延长至63天。在芯片上合成10个Cas9同系物溶液和10个木糖还原酶（XR）同系物时，iMICC将错误频率分别降低至0.64 / Kb和0.41 / Kb，分别为72.1％和86。4％的组装碎片没有错误。通过将碱基准确度提高37.6倍，同时避免重复制备新鲜酶，iMICC比野生型eMutS更加有效和强大，它的准确性比CorrectASE高6.6倍，便宜26.7倍。这些优点保证了其在工业DNA合成中的广泛应用。

更新日期：2020-04-23

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