Figure legends
Fig. 1 PmCDA1-T7 RNAP fusion increased the mutation frequency of the target gene. (A) Schematic view of the design of the yeast mutagenesis tool. PmCDA1-T7 RNAP could be recruited to the target gene by the T7 promoter. T7 RNAP would move along the target gene and PmCDA1 could generate random mutations on the target gene. The target gene in this work is the CAN1 gene, and the T7 promoter is upstream of CAN1 . (B) Construction of PmCDA1-T7 RNAP with different linker lengths and the mutation effects (C ) after 24 h of induction. (D) The mutation rates at CAN1in strains expressing PmCDA1-T7 RNAP with different linkers and without PmCDA1-T7 RNAP (null). (E) The proportions of different types of mutations at CAN1 . (F) Distribution of mutations atCAN1 generated by PmCDA1-T7 RNAP fusions with different linker lengths. Values represent the mean and standard deviation of three biologically independent replicates.
Fig. 2 The introduction of DNA-modifying enzymes improved the mutation effect. (A) Fusion proteins were designed in five constructions, where Cons. represents the construction. (B) The mutation rates at CAN1 in yeasts with different mutagenesis fusions. Null denotes strains without mutagenesis fusions. (C)Fraction of different base substitutions occurring in strains with different mutagenesis fusions. Values represent the mean and standard deviation of three biologically independent replicates.
Fig. 3 Screening of DNA-modifying enzymes to tune the mutation spectra. (A) Six other candidates were selected for their involvement in the DNA repair process. DNA-modifying enzymes could significantly alter the mutation rate (B ) and base conversion types (C ).
Fig. 4 Dual T7 promoters increased the mutation frequencies.(A) The structure of the dual T7 promoter system. The two T7 promoters flanking the CAN1 gene were in the reverse direction.(B) The mutation rates in the dual T7 promoter system were obviously higher than in the single promoter system after 24 h of induction. (C) The addition of the second T7 promoter barely changed the proportions of different mutation types. MAG1, EXO1 and REV3 represent strains expressing these fusions with single T7 promoter, while MAG1*, EXO1* and REV3* represent strains with dual T7 promoters. Values represent the mean and standard deviation of three biologically independent replicates. (Student’ s t-test, **P<0.01).
Fig. 5 Application of mutagenesis tools to the evolution of key enzymes in the β-carotene biosynthetic pathway. The transcription units of the essential genes in the β-carotene biosynthetic process were flanked by dual T7 promoters. After introducing mutagenesis plasmids and induction, some of the colonies showed apparent color changes. TU denotes transcription unit, including promoter, coding region and terminator. Original strain denotes the β-carotene-producing strain containing T7 promoters and mutagenesis proteins, but without induction.