Extended Data Fig. 2: Evolutionary strategy used to generate OE1.3 and OE1.4.

Structures showing the amino acid positions randomized during each round of evolution. The Me-His residue is shown in ball-and-stick representation and targeted residues are represented as spheres. a, The round 1 library was prepared by randomizing active-site residues in pairs Ala19/Ser22 (blue) Tyr45/Glu46 (orange) Tyr87/Trp88 (red) Met94/Ser95 (green) and Asp125/Gln128 (purple). b, The round 2 library was prepared by random mutagenesis of the entire gene. Variants with improved activity enabled the identification of ‘hot spots’, which were further interrogated using saturation mutagenesis. Residues Leu42/Glu46 (red) and Phe132/Leu133 (blue) were randomized simultaneously; all other positions were randomized individually (green). c, The round 3 library was prepared by saturation mutagenesis, using NNK degenerate codons to individually randomize 21 positions (see table). d, The round 4 library was prepared by saturation mutagenesis, using NNK degenerate codons to individually randomize 20 positions (see table). Libraries generated during rounds 1–3 were screened for activity towards fluorescein 2-phenylacetate. The round 4 library was screened for activity towards fluorescein (R)-2-phenylpropanoate.