Fig. 2: Directed enzyme evolution of a ketone synthase.
a, Activity and selectivity as a function of evolution starting with wild-type P450LA1 (Wt). Biotransformations were performed with 0.625 µM P450 enzyme variant, 5 mM trans-β-methylstyrene (1), 5 mM NADH cofactor and 1 vol% isopropanol in reaction buffer. The reactions were shaken at room temperature for 2 h before UHPLC analysis. The TTN was calculated by dividing the concentration of the ketone produced by the enzyme concentration used. TTN data are shown as a bar graph as a mean of six experiments (experimental triplicates of biological duplicates, n = 6). The individual data points are shown as white circles. The ketone selectivity was determined as the proportion of ketone in relation to all oxidation products formed (including epoxides and allylic oxidation products) and the data points are shown as black dots. b, Trajectory of the directed evolution experiment, showing the amino acid substitutions in each round. Inset: key data regarding the entire directed evolution experiment starting from Wt. c, Epoxides are not intermediates in the reaction as they are not converted by the ketone synthase via isomerization into the corresponding ketones. d, Homology model of P7E with the mutations introduced to generate the ketone synthase shown as purple spheres. The haem cofactor is shown as black sticks.
