Figure 3.

Screening of T. elegans P450s for oxygenation of tabersonine.

TEX‐like (a) and T3O‐like (b) gene candidates and GFP were transiently overexpressed in tobacco leaves in the presence of tabersonine and reaction products were analyzed by LC–MS. Positive differential mass chromatograms of oxygenated tabersonine products at m/z 353 of P450 candidates are compared with GFP (negative control) and standards: 16‐hydroxytabersonine (green line), pachysiphine (blue line), lochnericine (red line), and 2,3‐epoxytabersonine (orange line). Metadata associated with bootstrap nodes are represented by a circle whose fill color scales proportionally with the bootstrap value (lower value, purple; higher value yellow), while branch lengths scale proportionally with substitutions per site value. Tel01G.1927 is the CYP71 P450 used to root the phylogenetic trees.

Expanded HSQC spectrum (¹H–¹³C correlation, focused on ¹H 2.7–3.8 ppm and ¹³C 44–58 ppm) of the purified reaction product of Tel01G.15088 (c) characterized as (−)‐pachysiphine (d). Full NMR data are available in Figures [Link], [Link]. GFP, green fluorescent protein; T3O, tabersonine 3‐oxygenase; TEX, tabersonine‐6,7‐epoxydase.