DNA合成技术是支撑合成生物学发展的关键，目前长链DNA（>200bp）合成几乎完全依托固相亚磷酰胺化学法合成寡核苷酸的拼接而成，而酶法合成DNA有望解决长链DNA从头合成这一难题。末端脱氧核苷酸转移酶（TdT）是酶促合成DNA的天然候选酶，但因其合成效率低还无法满足商业化应用。我们前期工作发现不同物种来源TdT酶对修饰碱基有着不同的催化活性，并已建立了新型酶活性高通量检测系统。本项目拟从TdT蛋白出发，采用理性设计和定向进化结合的方法对TdT进行重新设计改造，通过酵母表面展示技术和cell-free表达系统快速获得TdT蛋白，利用高通量系统筛选出利用3’-O修饰可逆终止碱基高效合成DNA的TdT酶，旨在回答TdT酶对脱氧核苷酸亲核攻击与聚合作用的调控机制是什么，最终实现酶法从头合成长链DNA的目标。本项目的完成将大幅降低DNA合成成本，成为推动合成生物学飞速发展的核心驱动力。

DNA synthesis is the key to support the development of synthetic biology. Long-chain DNA (>200bp) synthesis almost completely relies on the assembly of oligonucleotides synthesized by solid phase phosphoramidite chemistry. Enzymatic DNA synthesis is expected to solve the bottleneck of de novo synthesis of long-chain DNA. Terminal deoxynucleotidyl transferase (TdT) is a natural candidate for enzymatic synthesis of DNA, but it is not yet commercially viable due to its low synthesis efficiency. Our previous work found that TdT enzymes from the different species have different catalytic activities for modified bases, and a high-throughput systems for enzyme activity has been established. This project intends to redesign TdT by rational design and directed evolution. TdT protein is rapidly obtained by yeast surface display technology and cell-free expression system, then the high-throughput system is used to screen the ideal TdT of utilizing 3'-O-modified base. We will answer the mechanism of TdT enzyme on nucleophilic attack and polymerization, and achieve enzymatic de novo synthesis of long-chain DNA. The completion of this project will greatly reduce the cost of DNA synthesis and become the core driving force for the rapid development of synthetic biology.