Figure 9. LC-qTOF-MS analysis of 13R-manoyl oxide-derived diterpenoids obtained by transient expression of combinations of C.forskohlii CYP and ACT encoding genes in N. benthamiana leaves.
Total ion chromatograms (TIC) from extracts expressing the 13R-manoyl oxide biosynthesis genes (CfCXS, CfGGPPS, CfTPS2, CfTPS3) in combination with (from the top) water (-), CfCYP76AH15 + CfCYP76AH11+ CfCYP76AH16 + CfACT1-6, and CfCYP76AH15 + CfCYP76AH11 + CfCYP76AH16 + CfACT1-8 are shown. Hydroxylated and acetylated 13R-manoyl oxide-derived diterpenoids (marked with grey bars) and their identity, including their molecular formulas, was confirmed by their accurate mass (5 ppm tolerance, Supplementary file 1). Compounds present in trace amounts are not marked. The identity of deacetylforskolin (13b) and forskolin (16c) was confirmed by comparison to authentic standards. No 13R-manoyl oxide-derived diterpenoids were detected in the water control (−). For all combinations, extracts from leaves of three different N. benthamiana plants have been analyzed and representative chromatograms are shown.