Discussion
The occurrence of random mutations can improve genetic diversity and play an important role in many fields.[1-4, 6, 9]To date, researchers have developed a handful of mutagenesis techniques to increase the mutation rate and mutation diversity[6]. These tools have produced achievable effects in various chassis cells such as E. coli , S. cerevisiae , and mammalian cells. In this work, we developed a targetedin vivo mutagenesis tool that can significantly improve the mutation rate and broaden the mutation spectrum by fusing DNA-modifying enzymes, cytidine deaminase and T7 RNAP.
We constructed different mutagenesis proteins that could accelerate the evolution of the target gene. We first constructed the plasmid containing the cytidine deaminase PmCDA1-T7 RNAP expression cassette and inserted T7 promoter sequence upstream of the target gene. PmCDA1-T7 RNAP fusions could raise the mutation frequency to about 1.0~1.2x10-3, and exhibited a strong bias toward C→T mutations, which is consistent with the mutation characteristic of PmCDA1.[21, 22] We hypothesized that enhancing the removal of mismatched U as well as the subsequent DNA repair processes would lead to diverse mutation types.[34, 35, 46] Therefore, we fused different DNA-modifying enzymes with PmCDA1 to improve the mutation effect. The results indicated that the introduction of DNA-modifying enzymes could indeed improve the mutation effect. Among these candidates, MAG1 and REV3 could significantly increase the diversity of mutations. In the mutations generated by MAG1-PmCDA1-T7 RNAP, C→T mutations accounted for 64.5%, followed by G→A mutations (19.2%), C→G mutations (12.5%), and G→C mutations (3.8%). The ratio of non-C→T mutations was 36.5%, which was 11-fold higher than that of PmCDA1-T7 RNAP. REV3-PmCDA1-T7 RNAP could also generate more diverse mutations, of which C→T mutations comprised about 29.6%, nearly 10-fold higher than that of PmCDA1-T7 RNAP. The mutation diversity is crucial to the evolutionary process and the wider mutation spectrum would help us to obtain desired strains more efficiently. Smolke et al. developed TRIDENT system and increased the ratio of non-C→T mutations to about 20%,[23]while Shoulders et al. fused evolved adenosine deaminase to T7 RNAP and developed MutaT7A→G and eMuataT7A→G, which could generate all transition mutations when being employed with cytidine-bearing mutators.[25] As our mutagenesis fusions could generate higher proportion of C→G and G→A mutations, they could play a complementary role with the above-mentioned tools. Except for the change on the mutation spectra, the addition of DNA-modifying enzymes could also raise the mutation frequency. EXO1-PmCDA1-T7 RNAP could increase the mutation rate up to 2.2x10-3, which is twice as high as PmCDA1-T7 RNAP.
We then added a reverse T7 promoter at the end of the target gene and compared the mutation effects with that of the single T7 promoter system. The introduction of the second T7 promoter could significantly increase the mutation frequency of the target gene. The mutation frequencies of strains with dual T7 promoters were about 1.5~2.5-fold higher than that in the single T7 promoter system. We hypothesized that the dual T7 promoters might increase the possibility of T7 RNAP binding to the T7 promoter, thus increasing the mutation rate. The proportions of different mutations generated in the dual T7 promoter system were basically the same as the single promoter system. Both Kim et al. and Shoulders et al. have found that the introduction of the second T7 promoter increased the ratios of G→A mutations.[22, 24] However, we did not observe apparent change of the frequency of G→A mutations in the dual T7 promoter system. Instead, we found that the ratios of some transversion mutations (G→T, G→C etc.) increased than that in the single promoter system. We suspect that this difference might be due to the different DNA repair mechanisms in the hosts (S. cerevisiae vs. E. coli ). Our mutators still showed bias toward C→T mutations, and we hypothesized that the introduction of other DNA-modifying enzymes and their combinations might further broaden the mutation spectra.
Finally, we applied our mutagenesis tools to the evolution of the key enzymes in the β-carotene biosynthetic pathway. After induction, we observed apparent color changes compared with the original strain. Most of the mutations were C and G mutations, which was consistent with the mutation characteristic of our mutators. These results demonstrated that our mutagenesis tools could evolve the non-growth-limiting genes and generate diverse genotypes even without the growth pressure or selection.
Our mutagenesis tools are inducible, so the mutation rate could be flexibly tuned by changing the inducer concentration and induction time. The expression level of our mutagenesis fusion proteins may also influence the mutagenic activity and needs further investigation. In addition, using more DNA-modifying enzymes or their combinations may further alter the mutation effect of the mutagenesis proteins, thus developing evolutionary tools with diverse mutation characteristics to meet different needs of the evolutionary process. Our mutagenesis tools are compatible with continuous evolution, with the help of biosensors or other screening techniques, researchers can efficiently obtain desired mutants. In addition, our mutagenesis tools can also work complementary to the single deaminase-bearing mutators that are biased toward generating transition mutations, effectively creating both transition and transversion mutations, and can be applied to many aspects, such as industrial strain breeding, protein engineering, and so on.