Abstract
The occurrence of random mutations can increase the diversity of the
genome and promote the evolutionary process of organisms. High
efficiency mutagenesis techniques significantly accelerate the
evolutionary process. In this work, we describe a targeted in vivo
mutagenesis system to significantly increase mutation frequency and
generate mutations across all four nucleotides. We constructed different
DNA-modifying enzyme-PmCDA1-T7 RNA polymerase fusion proteins, achieved
targeted mutagenesis by flanking the target gene with T7 promoters, and
tuned the mutation spectra by introducing different DNA-modifying
enzymes. With the mutagenesis fusion proteins, the mutation frequency of
the target gene could reach 5.13x10-3, and the proportion of non-C→T
mutations is 10~11-fold higher than the cytidine-based
evolutionary tools. We also demonstrated that our mutagenesis tools
could be used to evolve the essential enzyme in the β-carotene
biosynthesis process and generate mutations with different types.