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.