Core Viewpoint - The article discusses the development of a novel orthogonal transcription mutation system (OTM) by a research team led by Professor Chen Guoqiang from Tsinghua University, which significantly accelerates in vivo protein evolution and addresses limitations of traditional directed evolution methods [1][2]. Group 1: Technology Overview - The OTM system integrates three broadly host-compatible bacteriophage RNA polymerases and two deaminases, creating an efficient and modular protein evolution platform [2]. - The core principle involves RNA polymerase unwinding DNA to form single-stranded regions, allowing deaminases to edit exposed single-stranded DNA, achieving base mutations with a mutation efficiency increased by 1.5 million times compared to spontaneous mutations [2][3]. Group 2: Experimental Results - The research team successfully inserted bacteriophage promoters upstream and downstream of target genes, ensuring uniform distribution of mutations across target genes, overcoming previous biases in mutation distribution [3]. - The orthogonality experiments demonstrated that the OTM components could specifically recognize their respective promoters, avoiding cross-interference and enabling modular design [3]. Group 3: Applications and Future Prospects - The OTM system has shown great potential in applications, such as generating engineered strains with various colors by mutating fluorescent and pigment proteins, and creating unique shapes of engineered strains by targeting cytoskeletal and division-related proteins [3]. - In industrial applications, the system successfully evolved the σ70 global transcription regulator RpoD and lysine efflux protein LysE, enhancing L-arginine tolerance and transport capabilities [3][5]. - The technology has broad expansion potential, with possibilities to integrate other types of deaminases to enrich mutation diversity and explore compatibility in other non-model organisms [5].

Core Viewpoint - The article discusses the development of an orthogonal transcription mutation system (OTM) that significantly enhances the efficiency and specificity of protein evolution, addressing limitations in traditional methods and expanding applications in both model and non-model organisms [2][6]. Group 1: System Development - The OTM system integrates three broadly compatible phage RNA polymerases with two deaminase variants, creating a modular platform for protein evolution [3]. - The core design allows RNA polymerase to unwind DNA, enabling deaminases to edit exposed single-stranded DNA, achieving base mutations of C:G to T:A and A:T to G:C types [3][4]. - The system can complete directed evolution of proteins in just one day, with mutation efficiency improved by 1.5 million times compared to spontaneous mutations [3][6]. Group 2: Functional Capabilities - The research team successfully constructed dual-function orthogonal transcription mutation elements that can introduce both C:G to T:A and A:T to G:C mutations simultaneously [4]. - The system demonstrates orthogonality, allowing different phage RNA polymerases to specifically recognize their respective promoters, minimizing cross-interference [5]. - The OTM system shows excellent performance in both non-model organisms (e.g., Halomonas bluephagenesis) and model organisms (e.g., E. coli), overcoming previous efficiency challenges in non-model industrial strains [5][6]. Group 3: Practical Applications - The system has been applied to mutate fluorescent proteins and pigment proteins, resulting in engineered strains with various colors [5]. - Targeted mutations in cytoskeletal and division-related proteins led to engineered strains with diverse morphologies, providing new insights for morphological engineering [5]. - In industrial applications, the system successfully evolved σ70 global transcription regulator RpoD and lysine export protein LysE, enhancing L-arginine tolerance and transport capabilities [5][6]. Group 4: Future Prospects - The OTM system has significant potential for further development, including integrating other types of deaminases to diversify mutation libraries [6]. - Future exploration may focus on the system's compatibility and applicability in other non-model organisms [6]. - The mutation system can accelerate the optimization of key enzymes in biomanufacturing, contributing to the advancement of a green bio-economy [6].