研究组在大肠杆菌中描述了一个基于噬菌体T7 （T7- oracle）复制体控制表达的正交DNA复制系统。该系统复制环状质粒，实现高转化效率并无缝集成到标准分子生物学工作流程中。T7 DNA聚合酶的工程产生了变异蛋白，在体内每个碱基的突变率为1.7×10−5个替换，比基因组突变率高10万倍。课题组人员通过扩大TEM-1 β-内酰胺酶的底物范围，并在不到一周的时间内将抗临床相关的单巴坦和头孢菌素抗生素的活性提高5000倍，证明了T7复制体的持续进化。
研究人员表示，在不损害宿主基因组完整性的情况下对指定基因进行连续超突变的系统可以大大加速新的或增强的蛋白质功能的进化。
附：英文原文
Title: An orthogonal T7 replisome for continuous hypermutation and accelerated evolution in E. coli
Author: Christian S. Diercks, Philipp Sondermann, Cynthia Rong, Thomas G. Gillis, Yahui Ban, Celine Wang, David A. Dik, Peter G. Schultz
Issue&Volume: 2025-08-07
Abstract: Systems that perform continuous hypermutation of designated genes without compromising the integrity of the host genome can substantially accelerate the evolution of new or enhanced protein functions. We describe an orthogonal DNA replication system in Escherichia coli based on the controlled expression of the replisome of bacteriophage T7 (T7-ORACLE). The system replicates circular plasmids that enable high transformation efficiencies and seamless integration into standard molecular biology workflows. Engineering of T7 DNA polymerase yielded variant proteins with mutation rates of 1.7 × 105 substitutions per base in vivo—100,000-fold above the genomic mutation rate. We demonstrated continuous evolution using the T7 replisome by expanding the substrate scope of TEM-1 β-lactamase and increasing activity 5000-fold against clinically relevant monobactam and cephalosporin antibiotics in less than 1 week.
DOI: adp9583
Source: https://www.science.org/doi/10.1126/science.adp9583