Fig. 3

Engineering of the dual P450 module of CYP79A2 with CYP83B1 or CYP83A1. A The design of the expression constructs to examine the combinatorial effect of engineering the two P450s together. We paired the native and [ΔTM]-CYP79A2 with variants of the two CYP83s and co-expressed the P450 oxidoreductase ATR1 and glutathione transferase F11. B, D Production of phenylacetaldoxime (Phe-Ox, light colours) and S-phenylacetohydroxymoyl-L-glutathione (Phe-GSH, dark colours) by native and [ΔTM]CYP79A2 with native CYP83B1 and CYP83A1, respectively. C, E Production of Phe-Ox and Phe-GSH by [ΔTM]CYP79A2 with native and modified variants of CYP83B1 and CYP83A1, respectively. Bars are colour coded by the type of modification applied to CYP83 enzymes. All strains were grown in 12 biological replicates across two independent experiments. Error bars represent standard deviation from the mean and outliers were removed from the data. For C and E, Student’s upper-tailed t test denotes a significant increase in Phe-GSH titre compared to [ΔTM]CYP79A2 and native CYP83s with p value (with Holm adjustment), *** p < 0.001. Exact levels of Phe-Ox, Phe-GSH, Phe and OD600 are listed in Table S3