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 CrtE , CrtI , and CrtYB genes in yeast to
increase β-carotene yields.