Fig. 3: Biosynthesis of QA.
a, Four S. officinalis enzymes enable biosynthesis of QA (4) in N. benthamiana. b, Products generated by transient expression of CYP716A378 (C-28 oxidase) and CYP716A379 (C-28,16α oxidase) in N. benthamiana. GC–MS TICs of leaf extracts coexpressing SobAS1 with either CYP716A378 or CYP716A379 are shown, along with a control (leaf expressing only AstHMGR) and the following commercial standards: bA (1, β-amyrin), OA (2, oleanolic acid) and EA (3, echinocystic acid). Mass spectra of bA (1), OA (2) and EA (3) for leaf extracts expressing SobAS1 with either CYP716A378 or CYP716A379 and for relevant commercial standards are also shown. c, Transient expression of CYP72A984 (C-23 oxidase) in N. benthamiana. LC–MS extracted ion chromatograms (EICs) of leaf extracts coexpressing CYP72A984 with the minimal gene set for 3 (SobAS1 and CYP716A379), along with a control (leaf expressing only AstHMGR) and a QA (4) commercial standard. EICs displayed are at m/z 485.3267 (calculated [M − H]− of QA (4)). MS and MS/MS spectra of QA (4) from the commercial standard and leaf extracts coexpressing SobAS1, CYP716A379 and CYP72A984 are also shown. Formation of another peak (4′) putatively identified as gypsogenic acid is also observed when CYP72A984 is coexpressed with SobAS1 and CYP716A379 (MS/MS shown in Supplementary Fig. 25).
