Fig. 2: The outcome of two rounds of progressively stringent directed evolution and mutations analysis.

a The total inversion efficiency of the mutants generated in the two rounds of directed evolution. The orange and green represent the mutants produced in the first and second rounds of directed evolution, respectively. The error bars represent standard deviation of three biological replicates. b The co-inversion efficiency of three markers for the mutants generated during the two rounds of directed evolution. The efficiency is calculated following 12 h of induction. The orange and green represent the mutants produced in the first and second rounds of directed evolution, respectively. The error bars represent standard deviation of three biological replicates. c Yeast colonies containing the wild-type Rci, Rci51, or Rci51-5 were grown on various selective media after inducing 12 h, respectively. Compared to the Rci and Rci51, more colonies of Rci51-5 were grown on selective media as the number of selection markers increased. The pictures were taken after 3 days incubation at 30 °C. d Sequence alignment of Rci51-5 and the wild-type Rci. The mutant Rci51-5 identified five nonsynonymous mutations (red rectangle). e The relative multisite inversion efficiency of mutants containing single or combinations of mutations. ‘+’ indicates the introduction of specific mutations in Rci sequence; ‘-’ indicates their absence. The error bars represent standard deviation of three biological replicates. f Structural details of the inversion enzyme/DNA system by AlphaFold3. Two sfxa101 DNA(s) are shown in the protein-nucleic acid complex with four Rci51-5 monomers (the four are in blue, green, yellow and magenta cartoon, respectively). Mutation residues are depicted in spheres. The dashed box marks the region zoomed in and showed in detail (Rci51-5 and the wild-type Rci are in red and black, respectively). The hydrogen bonds and salt bridges are depicted with yellow and magenta dashed lines, respectively. Source data for this figure are provided as a Source Data file.