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.