Continuous Evolution of Protein through T7 RNA Polymerase-Guided Base Editing in Corynebacterium glutamicum

In vivo targeted mutagenesis technologies are the basis for the continuous directed evolution of specific proteins. Here, an efficient mutagenesis system (CgMutaT7) for continuous evolution of the targeted gene in Corynebacterium glutamicum was developed. First, cytosine deaminase and uracil-DNA glycosylase inhibitor were sequentially fused to T7 RNA polymerase using flexible linkers to build the CgMutaT7 system, which introduces mutations in targeted regions controlled by the T7 promoter. After a series of optimizations, the resulting targeted mutagenesis system (CgMutaT7⁴) can increase the mutant frequency of the target gene by 1.12 × 10⁴-fold, with low off-target mutant frequency. Subsequently, high-throughput sequencing further revealed that the CgMutaT7⁴ system performs efficient and uniform C → T transitions in at least a 1.8 kb DNA region. Finally, the xylose isomerase was successfully continuously evolved by CgMutaT7⁴ to improve the xylose utilization, indicating that the CgMutaT7 system has great potential for applications in the continuous evolution of protein function and expression components.