An orthogonal transcription mutation system generating all transition mutations for accelerated protein evolution in vivo

Abstract 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.