Continuous Evolution of Protein through T7 RNA Polymerase-Guided Base Editing in <i>Corynebacterium glutamicum</i>

<i>In vivo</i> 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 <i>Corynebacterium glutamicum</i> 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.