Rapid and Continuous Directed Evolution in Vibrio natriegens Utilizing an In Vivo Hypermutation System

Genetic diversification serves as the foundation for in vivo molecular evolution. The fusion of a cytidine deaminase with T7 RNA polymerase (MutaT7) enables efficient diversification of DNA sequences downstream of the T7 promoter in model microbial hosts E. coli and S. cerevisiae. Vibrio natriegens, currently the fastest-growing nonpathogenic bacterium with a doubling time half that of E. coli, emerged as a promising alternative chassis for synthetic biology. Here, we report the establishment of MutaT7 in V. natriegens (dubbed VnMutaT7), achieving rapid evolution of the model protein TEM-1 to confer high-level ceftazidime resistance in just 36 h. This work establishes the potential of V. natriegens as an accelerated platform for protein engineering.