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. 2017 Mar 14;6:e23001. doi: 10.7554/eLife.23001

Figure 3. GC-MS analysis of 13R-manoyl oxide (1) derived diterpenoids obtained by transient expression of CYP76AHs from C.

forskohlii in N. benthamiana leaves. (A) GC-MS total ion chromatograms (TIC) of extracts from N. benthamiana transiently expressing CfCXS, CfGGPPS, CfTPS2 and CfTPS3 (13R-manoyl oxide biosynthesis) genes in combination with water (-), CfCYP76AH15, CfCYP76AH17, CfCYP76AH8, CfCYP76AH11 or CfCYP76AH16. 1-eicosene was used as internal standard (IS). 13R-manoyl oxide (1) was identified only in (-), indicating that it is further metabolized by the CfCYP76AH15, CfCYP76AH17, CfCYP76AH8, CfCYP76AH11 and CfCYP76AH16 enzymes. (B) m/z spectrum of 11-oxo-13R-manoyl oxide (2). (C) m/z spectrum of 9-hydroxy-13R-manoyl oxide (3a). The structure of both compounds was verified by NMR analysis (Tables 1 and 2). The compounds have been identified previously in C. forskohlii as putative intermediates in the in planta biosynthesis of forskolin (Asada et al., 2012). For each combination, extracts from leaves of three different N. benthamiana plants have been analyzed and representative chromatograms are shown.

DOI: http://dx.doi.org/10.7554/eLife.23001.008

Figure 3.

Figure 3—figure supplement 1. LC-qTOF-MS analysis of 13R-manoyl oxide-derived diterpenoids obtained by transient expression of C. forskohlii CYP76AH encoding genes in N. benthamiana leaves.

Figure 3—figure supplement 1.

Total ion chromatograms (TIC) of extracts expressing the 13R-manoyl oxide biosynthesis genes (CfCXS, CfGGPPS, CfTPS2, CfTPS3) in combination with water (-), CfCYP76AH8, CfCYP76AH17, CfCYP76AH15, CfCYP76AH11 or CfCYP76AH16 are shown. 13R-manoyl oxide-derived oxygenated compounds formed (marked with grey bars) and their identity including their molecular formulas was confirmed by their accurate mass (5 ppm tolerance, Supplementary file 1). The identity of 1,11-dihydroxy-13R-manoyl oxide (5d) and 9-deoxydeactylforskolin (10b) was confirmed by NMR analysis (Figure 4 and Tables 1 and 2). No 13R-manoyl oxide-derived diterpenoids were detected in the water control (-). For each combination, extracts from leaves of three different N. benthamiana plants have been analyzed and representative chromatograms are shown.
DOI: http://dx.doi.org/10.7554/eLife.23001.009
Figure 3—figure supplement 2. GC-MS analysis of 13R-manoyl oxide-derived diterpenoids following transient expression in N. benthamiana leaves of the C. forskohlii gene encoding CfCYP71D281 together with genes encoding the required enzymes for biosynthesis of 13R-manoyl oxide (CfCXS, CfGGPPS, CfTPS2, CfTPS3).

Figure 3—figure supplement 2.

(A) GC-MS total ion chromatograms (TIC) of extracts from N. benthamiana transiently expressing 13R-manoyl oxide biosynthesis genes in combination with water (-) or CfCYP71D381, respectively. 1-Eicosene was used as internal standard (IS) and 13R-manoyl oxide (1) was identified in both (-) and the CfCYP71D381 samples. Compounds 3b and 3c were identified in extracts from N. benthamiana leaves expressing CfCYP71D381 together with the genes in 13R-manoyl oxide biosynthesis. CfCYP71D381 efficiently converted compound 1 to a mixture of two mono-hydroxylated 13R-manoyl oxide derivatives (3b and 3c). Structural elucidation by NMR (Figure 4 and Tables 1 and 2) showed hydroxylation of 1 at positions C-2 (3b) and C-19 (3c). These hydroxylation positions do not coincide with those found in forskolin and to our knowledge have not been observed in other diterpenoids known from C. forskohlii. (B) m/z spectrum of 2-hydroxy-13R-manoyl oxide (3b). (C) m/z spectrum of 19-hydroxy-13R-manoyl oxide (3c). For each combination, extracts from leaves of three different N. benthamiana plants have been analyzed and representative chromatograms are shown.
DOI: http://dx.doi.org/10.7554/eLife.23001.010