Total biosynthesis of the cyclic AMP booster forskolin from Coleus forskohlii
- University of Copenhagen, Denmark
- Center for Synthetic Biology “bioSYNergy”, Denmark
- Evolva A/S, Denmark
- Faculty of Health and Medical Sciences, University of Copenhagen, Denmark
- Science for Life Laboratory, KTH - Royal Institute of Technology, Sweden
Figures
Figure 1
Biosynthesis of forskolin in the root cork cells of C. forskohlii.
(A) Scheme showing the structures of the diterpene precursor 13R-manoyl oxide, deacetylforskolin and forskolin on a background of root cork cells with forskolin containing oil bodies. (B) Transcript …
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Figure 1—source data 1
cDNAs identified in the C. forskohlii root cork transcriptome and cloned during this work, with the GeneBank accession numbers.
- https://doi.org/10.7554/eLife.23001.004
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Figure 1—source data 2
Table of FPKM (Fragments Per Kilobase of transcript per Million mapped reads) values of the first 20 most abundant cDNAs identified in the root cork transcriptome library.
cDNAs involved in terpenoid metabolism are marked in bold.
- https://doi.org/10.7554/eLife.23001.005
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Figure 1—source data 3
Table of primers used in this study.
Construction of plasmids for expression of CfTPS2, CfTPS3, CfTPS1 is described in Andersen-Ranberg et al. (2016). U (uracil, marked in bold), represents the cleavage site, used in the USER cloning (Nour-Eldin et al., 2006).
- https://doi.org/10.7554/eLife.23001.006
Figure 2
13R-manoyl oxide oxide-derived hydroxylated products formed following transient expression of CfCYP76AHs in N.
benthamiana leaves. (A) Molecular formulas of the hydroxylated products obtained using different CfCYP76AHs. The number of hydroxylations of each compound was deduced from its accurate molecular …
Figure 3 with 2 supplements
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) …
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.
Total ion chromatograms (TIC) of extracts expressing the 13R-manoyl oxide biosynthesis genes (CfCXS, CfGGPPS, CfTPS2, CfTPS3) in combination with water (-), CfCYP76AH8, CfCYP76AH17, CfCYP76AH15, …
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).
(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. …
Figure 4
Structures of key compounds presented in this work.
(A) Compounds confirmed using authentic standards. (B) Compounds which structure was confirmed/identified by comparison of 13C NMR data with existing literature. (C) Compounds which structure was …
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Figure 4—source data 1
NMR spectra’s of selected 13R-manoyl oxide derived molecules.
- https://doi.org/10.7554/eLife.23001.012
Figure 5 with 1 supplement
LC-qTOF-MS analysis of 13R-manoyl oxide-derived diterpenoids obtained by transient expression of combinations of C. forskohlii CYP encoding genes, together with genes encoding the required enzymes for biosynthesis of 13R-manoyl oxide in N. benthamiana leaves.
Total ion chromatograms (TIC) of extracts expressing the 13R-manoyl oxide biosynthesis genes (CfCXS, CfGGPPS, CfTPS2, CfTPS3), in combination with (from the top) water (-), CfCYP76AH8 + CfCYP76AH11 …
Figure 5—figure supplement 1
LC-qTOF-MS analysis of 13R-manoyl oxide-derived diterpenoids obtained by transient expression of combinations of C. forskohlii CYP76AH encoding genes in N. benthamiana leaves.
Total ion chromatograms (TIC) of extracts expressing the 13R-manoyl oxide biosynthesis genes (CfCXS, CfGGPPS, CfTPS2, CfTPS3) in combination with (from the top) water (-), CfCYP76AH15 + CfCYP76AH11, …
Figure 6
GC-MS analysis of miltiradiene-derived diterpenoids obtained by transient expression of CfCYP76AH15 in N. benthamiana leaves.
(A) Total ion chromatograms (TIC) of extracts transiently expressing CfCXS, CfGGPPS, CfTPS1 and CfTPS3 (miltiradiene biosynthesis genes) in combination with water (-) or CfCYP76AH15. …
Figure 7
GC-MS analysis of 13R-manoyl oxide-derived diterpenoids obtained by transient expression of CYP76AHs in N. benthamiana leaves.
(A) Total ion chromatograms (TIC) of extracts transiently expressing CfCXS, CfGGPPS, CfTPS2 and CfTPS3 (13R-manoyl oxide biosynthesis genes) in combination with water (-), CfCYP76AH15, RoCYP76AH4, …
Figure 8
De novo biosynthesis of forskolin by transient expression of C. forskohlii genes in N. benthamiana as monitored by LC-MS-based extracted ion chromatograms (EIC).
To monitor both deacetylforskolin (13b) and forskolin (16c), the EIC were selected as the sum of m/z 433.2 ± 0.1 and m/z 391.2 ± 0.1. Chromatograms represent LC-MS analysis of extracts from leaves …
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…
Figure 10 with 1 supplement
Forskolin production in S. cerevisiae following stable genomic integration of codon-optimized C.forskohlii genes.
(A) Forskolin (16c) accumulation in a fermenter batch using the EVST21543 strain (expressing CfCYP76AH15, CfCYP76HA11, CfCYP76AH16 and CfACT1-8 encoding genes in the EFSC4498 S. cerevisiae strain, …
Figure 10—figure supplement 1
Comparison of metabolite profiles between fermenter grown yeast culture of the EVST21543 strain and C.forskohlii root extract analyzed by LC-MS.
Forskolin (16c) and 13R-manoyl oxide (1) were identified based on co-elution with standards and 9-hydroxy-13R-manoyl oxide (3a) was identified based on the presence of the [M+Na]+ ion, 329.2457 (C20H…
Figure 11
Phylogeny of known full-length CYP76AHs.
The enzymes used are listed below with their accession numbers or source of publication: CfCYP76AH15, KT382358; CfCYP76AH17, KT382360; CfCYP76AH8, KT382348; CfCYP76AH11, KT382349; CfCYP76AH16, …
Figure 12
Phylogenetic tree of CfACT encoding candidate genes together with BAHD family acyltransferase representatives from all clades according to D'Auria (2006).
Accession numbers of the non-Coleus forskohlii selected protein sequences are shown next to the tree taxon names, while C. forskohlii peptide accession numbers are provided in Figure 1—source data 1.…
Tables
Table 1
1H-NMR and 13C-NMR chemical shifts (Figure 4—source data 1) of novel oxygenated 13R-(+)-manoyl oxide-derived diterpenoids formed following transient expression of CYP encoding genes from C. …
| 19-hydroxy- 13R-manoyl oxide (3c)* | 2-hydroxy- 13R-manoyl oxide (3b)* | 1,11-dihydroxy- 13R-manoyl oxide (5d)* | ||||
|---|---|---|---|---|---|---|
| Pos. | 1H (nH; m; J(Hz)) | 13C | 1H (nH; m; J(Hz)) | 13C | 1H (nH; m; J(Hz)) | 13C |
| 1 | 0.89 (1H;m) 1.63 (1H; m) | 39.1 | 1.10 (1H; t(br); 11.9, 11.9) 1.77 (1H; m) | 51.3 | 3.49 (1H; dd;11.1, 4.5) | 79.0 |
| 2 | 1.44 (1H; m) 1.56 (1H; m) | 18.1 | 3.92 (1H; m) | 65.3 | 1.75 (1H; td; 13.5, 11.1, 3.9) 1.60 (1H; m) | 29.0 |
| 3 | 0.95 (1H; m) 1.78 (1H; m) | 35.8 | 0.76 (1H; t(br); 11.9, 11.9) 1.99 (1H; d(br); 11.9) | 48.2 | 1.47 (1H; dd; 13.6, 3.9) 1.39 (1H; td; 13.5, 3.6) | 39.6 |
| 4 | 38.5 | 34.9 | 33.4 | |||
| 5 | 1.10 (1H; dd; 2.3, 12.6) | 56.9 | 0.95 (1H; dd; 2.2, 12.4) | 55.9 | 0.84 (1H; dd; 11.3, 2.0) | 55.6 |
| 6 | 1.36 (1H; dd; 3.6, 12.6) | 20.1 | 1.68 (1H; m) | 19.7 | 1.47 (1H; m) | 20.2 |
| 1.75 (1H; m) | 1.27 (1H; m) | 1.64 (1H; m) | ||||
| 7 | 1.42 (1H; m) 1.83 (1H; dt; 3.3, 12.2) | 43.6 | 1.45 (1H; dd(br); 3.6, 12.5) 1.85 (1H; dt(br); 2.9, 12.5) | 43.2 | 1.48 (1H; m) 1.85 (1H; m) | 44.0 |
| 8 | 75.1 | 75.1 | 75.3 | |||
| 9 | 1.35 (1H; dd; 4.3, 12.0) | 55.7 | 1.40 (1H; dd; 4.2, 11.9) | 55.4 | 1.54 (1H; d; 5.8) | 55.8 |
| 10 | 37.3 | 38.7 | 43.8 | |||
| 11 | 1.48 (1H; m) 1.58 (1H; m) | 15.4 | 1.53 (1H; m) 1.61 (1H; m) | 15.6 | 4.38 (1H; br q; ≈8.6) | 65.6 |
| 12 | 1.78 (1H; m) 1.64 (1H; m) | 35.7 | 1.78 (1H; m) 1.66 (1H; m) | 35.5 | 2.02 (1H; dd; 14.3, 8.7) 2.27 (1H; dd; 14.3, 8.7) | 35.8 |
| 13 | 73.4 | 73.4 | 72.8 | |||
| 14 | 5.87 (1H; dd; 10.8, 17.4) | 147.7 | 5.87 (1H; dd; 10.8, 17.4) | 147.7 | 5.90 (1H; dd; 17.4, 10.8) | 147.1 |
| 15 | 4.92 (1H; dd; 1.5, 10.8) 5.14 (1H; dd; 1.5, 17.4) | 110.2 | 4.92 (1H; d; 10.8) 5.14 (1H; d; 17.4) | 110.3 | 4.94 (1H; dd; 10.7, 1.5) 5.17 (1H; dd; 17.4, 1.5) | 111.2 |
| 16 | 1.27 (3H; s) | 28.5 | 1.27 (3H; s) | 28.7 | 1.27 (3H; s) | 32.1 |
| 17 | 1.28 (3H; s) | 25.3 | 1.29 (3H; s) | 25.7 | 1.49 (3H; s) | 27.8 |
| 18 | 0.97 (3H; s) | 26.8 | 0.93 (3H; s) | 33.5 | 0.78 (3H; s) | 13.5 |
| 19 | 3.70 (1H; d; 10.9) 3.46 (1H; d; 10.9) | 65.4 | 0.85 (3H; s) | 22.2 | 0.85 (3H; s) | 32.8 |
| 20 | 0.78 (3H; s) | 15.7 | 0.84 (3H; s) | 16.5 | 0.79 (3H; s) | 21.1 |
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* 1H and 13C NMR data acquired at 600 and 150 MHz, respectively, in methanol-d4, at 300 K. s = singlet, d = doublet, t = triplet, m = multiplet, br = broad
Table 2
Structural identification of four oxygenated 13R-manoyl oxide-derived diterpenoids formed following transient expression of CYP encoding genes from C. forskohlii based on comparison of their 1H-NMR …
| 9-Deoxydeacetylforskolin (10b)† | 1,9-Dideoxydeacetylforskolin (7h)† | 11-oxo-13R-manoyl oxide (2)† | Coleorol (3a)† | |||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Pos. | 1H (nH; m; J(Hz)) | 13C | (Gabetta et al., 1989) | 1H (nH; m; J(Hz)) | 13C | (Gabetta et al., 1989) | 13C | (Gabetta et al., 1989) | 13C | (Asada et al., 2012) |
| 1 | 4.38 (1H; t; 2.8) | 71.6 | 71.2 | 2.45 (1H, d(br); 13.1) 0.78 (H; m) | 41.5 | 43.1 | 42.1 | 41.9 | 31.7 | 31.6 |
| 2 | 1.47 (1H; m) 2.14 (1H; m) | 25.8 | 25.6 | 1.78 (H; m) 1.40 (H; m) | 18.7 | 18.4 | 18.5 | 18.4 | 18.6 | 18.4 |
| 3 | 1.12 (1H; dt; 3.4, 13.2) 1.62 (1H; dt; 3.5, 13.5) | 36.4 | 36.3 | 1.36 (H; m) 1.15 (H; m) | 43.8 | 43.7 | 43.4 | 43.3 | 41.9 | 41.8 |
| 4 | 34.2 | 34.1 | 34.4 | 34.1 | 33.4 | 33.2 | 33.3 | 33.2 | ||
| 5 | 1.34 (1H; d; 2.1) | 47.5 | 47.4 | n.d. | 55.7 | 55.2 | 56.0 | 55.8 | 45.7 | 45.5 |
| 6 | 4.44 (1H; t; 2.6) | 70.8 | 70.2 | 4.39 (1H; m) | 70.4 | 70.2 | 19.8 | 19.7 | 19.5 | 19.4 |
| 7 | 3.68 (1H; d; 3.6) | 80.7 | 81.1 | 3.71 (1H; d; 3.8) | 81.0 | 80.7 | 39.6 | 39.4 | 36.6 | 36.4 |
| 8 | 80.0 | 78.5 | 80.1 | 79.9 | 77.5 | 77.2 | 78.0 | 77.8 | ||
| 9 | 3.32 (1H; s) | 58.0 | 58.2 | 2.59 (1H; s) | 65.5 | 65.4 | 66.9 | 66.7 | 75.3 | 75.2 |
| 10 | 42.2 | 41.7 | 38.0 | 37.8 | 37.3 | 37.1 | 41.1 | 40.9 | ||
| 11 | 207.7 | 207.6 | 206.3 | 205.7 | 207.7 | 207.1 | 21.1 | 21.0 | ||
| 12 | 2.63 (1H; d; 18.0) 2.69 (1H; d; 18.0) | 49.8 | 49.9 | 2.60 (1H; d; 18.1) 2.66 (1H; d; 18.1) | 50.0 | 49.8 | 50.4 | 50.2 | 31.6 | 31.5 |
| 13 | 75.1 | 74.8 | 75.1 | 75.1 | 75.1 | 74.4 | 72.9 | 72.8 | ||
| 14 | 5.94 (1H; dd; 10.8, 17.4) | 146.2 | 145.8 | 5.95 (1H; dd; 10.7, 17.4) | 146.9 | 146.4 | 146.9 | 146.0 | 147.4 | 147.3 |
| 15 | 5.04 (1H; d; 10.8) 5.14 (1H; d; 17.4) | 112.4 | 112.7 | 5.04 (1H; d; 10.7) 5.17 (1H; d; 17.4) | 112.3 | 112.1 | 112.3 | 111.9 | 110.1 | 110.0 |
| 16 | 1.30 (3H; s) | 31.5 | 31.5* | 1.28 (3H; s) | 31.6 | 33.2* | 31.4 | 31.2* | 28.9 | 28.8 |
| 17 | 1.54 (3H; s) | 24.1 | 24.5* | 1.50 (3H; s) | 23.5 | 31.4* | 28.1 | 27.9* | 27.0 | 29.9 |
| 18 | 1.38 (3H; s) | 33.1 | 18.2* | 0.97 (3H; s) | 33.4 | 23.9* | 15.6 | 15.5* | 33.7 | 33.6 |
| 19 | 1.21 (3H; s) | 23.7 | 23.6* | 1.21 (3H; s) | 24.0 | 23.7* | 21.8 | 21.6* | 21.5 | 21.4 |
| 20 | 1.01 (3H; s) | 18.5 | 32.8* | 1.30 (3H; s) | 17.2 | 16.7* | 33.6 | 33.5* | 17.0 | 16.8 |
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†1H and 13C NMR data acquired at 600 and 150 MHz, respectively,in methanol-d4, at 300 K. s = singlet, d = doublet, t = triplet, m = multiplet, br = broad
Additional files
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Supplementary file 1
Overview of 13R-manoyl oxide-derived diterpenoids identified in N. benthamiana, expressing combinations of C. forskohlii genes encoding CYPs and acetyltransferases together with genes encoding the required enzymes for biosynthesis of 13R-manoyl oxide (CfDXS, CfGGPPs, CfTP2 and CfTPS3).
GC-MS and LC-qTOF-MS chromatograms of the identified diterpenoids are shown in previous figures.
- https://doi.org/10.7554/eLife.23001.025
