3.4 Evolution of key enzymes in the β-carotene metabolic pathway using mutagenesis tools
To explore the potential of our mutagenesis tools in different evolutionary scenarios, such as enhancing the production of valuable compounds, we utilized them in the evolution of the CrtE ,CrtI and CrtYB genes in the β-carotene biosynthetic pathway. We selected the β-carotene-producing yeast strain as the chassis strain and added reverse T7 promoter sequences to CrtE ,CrtI , and CrtYB gene expression cassettes (Fig. 5). After the introduction of mutagenesis plasmids into the chassis strain and induction, we found that there were a few colonies of the strains exhibited different colors. We selected four colonies with distinct color changes and sequenced the target loci. The data suggested that diverse mutations occurred in the CrtE , CrtI , andCrtYB expression cassettes, including transition and transversion mutations. We analyzed these base conversions and found that most of them were C and G mutations, which was also consistent with the mutation characteristics of our mutagenesis tools mentioned above.
We then respectively introduced these single point mutations into the original β-carotene-producing strain and verified whether these strains could still show different colors. We observed that even without the mutagenic fusion proteins, the strains with point mutations still exhibited obvious color changes, indicating that these mutations can effectively alter colony colors and β-carotene yields.
We proved that our mutagenesis tools can generate random mutations in the key enzymes in the β-carotene biosynthetic pathway, resulting in increased β-carotene yields. These results demonstrated that our mutagenesis tools can be applied to the evolution of non-growth-limiting genes. Even in the absence of the growth pressure or selection, the mutagenesis fusion proteins were able to function robustly.