Shao Mingwei, Zhang Zhongnan, Jin Xiaofan, Ding Jun, Chen Guo-Qiang
School of Life Sciences, Tsinghua University, Beijing, China.
Department of Chemical Engineering, Tsinghua University, Beijing, China.
Nat Commun. 2025 Jul 1;16(1):6041. doi: 10.1038/s41467-025-61354-4.
Targeted mutagenesis systems are critical for protein evolution. Current deaminase-T7 RNA polymerase fusion systems enable gene-specific mutagenesis but remain limited to certain model organisms. Here, we develop an orthogonal transcription mutation system for in vivo hypermutation in both non-model organism Halomonas bluephagenesis and E. coli, achieving >1,500,000-fold increased mutation rates. By fusing deaminases with three phage RNA polymerases, this system uniformly introduces C:G to T:A and A:T to G:C mutations across target genes. The system demonstrates high specificity, minimal off-target effects, and high orthogonality between phage polymerases. We apply this system to rapidly evolve fluorescent proteins, chromoproteins, cytoskeletal proteins, cell division-related proteins, global sigma factor, and the LysE exporter within a single day of the mutagenesis process. Overall, the orthogonal transcription mutation system is a modular and versatile platform that accelerates protein evolution in the shortest period reported so far.
靶向诱变系统对蛋白质进化至关重要。目前的脱氨酶 - T7 RNA聚合酶融合系统能够实现基因特异性诱变，但仍局限于某些模式生物。在此，我们开发了一种正交转录突变系统，用于在非模式生物蓝色嗜盐菌和大肠杆菌中进行体内超突变，实现了超过1500000倍的突变率增加。通过将脱氨酶与三种噬菌体RNA聚合酶融合，该系统在整个靶基因上均匀地引入C:G到T:A以及A:T到G:C的突变。该系统具有高特异性、最小的脱靶效应以及噬菌体聚合酶之间的高正交性。我们应用该系统在诱变过程的一天内快速进化荧光蛋白、色素蛋白、细胞骨架蛋白、细胞分裂相关蛋白、全局σ因子和LysE输出蛋白。总体而言，正交转录突变系统是一个模块化且通用的平台，能在迄今为止报道的最短时间内加速蛋白质进化。
