MODIFIED CELL

Abstract

The present disclosure relates to nucleic acids that encode enzyme activities involved in the synthesis of lathyranes, intermediates in the synthesis of lathyranes and also compounds derived from lathyranes such as tiglianes, daphnanes and ingenanes; cells transformed with the nucleic acid molecules and vectors comprising the nucleic acid molecules.

Claims

1. An isolated cell transformed or transfected with an expression vector adapted to express a nucleic acid molecule comprising (a) the nucleotide sequence of SEQ ID NO: 3, (b) a nucleotide sequence comprising at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 3 and encoding a polypeptide that has casbene-9-oxidase activity, (c) a nucleic acid sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, or (d) a nucleic acid sequence encoding a polypeptide that is greater than 96% identical to the amino acid sequence of SEQ ID NO: 2 and has casbene-9-oxidase activity.

2.-5. (canceled)

6. The isolated cell according to claim 1, wherein said isolated cell is transformed with at least one vector comprising a nucleotide molecule selected from the group consisting of: i) the nucleotide sequence of SEQ ID NO: 3, or a nucleotide sequence comprising at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 3 and encodes a polypeptide that has casbene-9-oxidase activity; and ii) the nucleotide sequence of SEQ ID NO: 6, or a nucleotide sequence comprising at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 6 and encodes a polypeptide that has casbene synthase activity; and iii) the nucleotide sequence of SEQ ID NO: 4, or a nucleotide sequence comprising at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 4 and encodes a polypeptide that has casbene 5,6-oxidase activity.

7. The isolated cell according to claim 1, wherein said isolated cell transformed with at least one vector comprising a nucleotide molecule selected from the group consisting of: i) the nucleotide sequence of SEQ ID NO: 3, or a nucleotide sequence comprising at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 3 and encodes a polypeptide that has casbene-9-oxidase activity; and ii) the nucleotide sequence of SEQ ID NO: 6, or a nucleotide sequence comprising at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 6 and encodes a polypeptide that has casbene synthase activity; and iii) the nucleotide sequence of SEQ ID NO: 5, or a nucleotide sequence comprising at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 5 and encodes a polypeptide that has casbene 5,6-oxidase activity.

8. The isolated cell according to claim 1, wherein said isolated cell is further transformed or transfected with an expression vector adapted to express a nucleic acid molecule encoding a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 1 or 2, or an amino acid sequence comprising at least 90% identity to the amino acid sequence of SEQ ID NO: 1 or 2 that has casbene-9-oxidase activity.

9.-10. (canceled)

11. The isolated cell according to claim 1, wherein said isolated cell is a microbial cell.

12. The isolated cell according to claim 11, wherein said microbial cell is a bacterial cell.

13. The isolated cell according to claim 11, wherein said microbial cell is a yeast cell.

14. (canceled)

15. A cell culture comprising the yeast cell according to claim 13.

16. A plant transformed with a nucleic acid transcription cassette comprising a nucleotide sequence selected from the group consisting of: i) the nucleotide sequence of SEQ ID NO: 3; or ii) a nucleotide sequence comprising at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 3 and encodes a polypeptide that has casbene-9-oxidase activity.

17. The plant according to claim 16, wherein said plant further comprises a transcription cassette comprising a nucleotide sequence selected from the group consisting of: i) the nucleotide sequence of SEQ ID NO: 6; or ii) a nucleotide sequence comprising at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 6 and encodes a polypeptide that has casbene synthase activity.

18. The plant according to claim 16, wherein said plant further comprises a transcription cassette comprising a nucleotide sequence selected from the group consisting of: i) the nucleotide sequence of SEQ ID NO: 4, or ii) a nucleotide sequence comprising at least 5090% sequence identity to the nucleotide sequence of SEQ ID NO: 4 and encodes a polypeptide that has casbene-5,6-oxidase activity.

19. The plant according to claim 16, wherein said plant further comprises a transcription cassette comprising a nucleotide sequence selected from the group consisting of: i) the nucleotide sequence of SEQ ID NO: 5; or ii) a nucleotide sequence comprising at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 5 and encodes a polypeptide that has casbene-5,6-oxidase activity.

20. The plant according to claim 16, wherein said plant is from the Solanaceae family.

21. (canceled)

22. A process for the manufacture of a lathyrane diterpene, or intermediates thereof, comprising: i) culturing the cell of claim 15 in a cell culture medium supplemented with a compound selected from the group consisting of casbene, 6-hydroxy-5-keto-casbene, 5-keto-casbene, 5-hydroxy-casbene, and 9-hydroxy-casbene, wherein the cell expresses casbene 9-oxidase; and optionally ii) isolating or purifying synthesized compounds from the cell and/or cell culture medium.

23. A process for the manufacture of 9-keto casbene, comprising: i) culturing the cell of claim 15 in cell culture medium, wherein the cells comprise an endogenous pool of geranylgeranyl disphosphate and express a casbene oxidase and a casbene synthase; and optionally ii) isolating or 9-keto-casbene from the cell or cell culture medium.

24. A process or the manufacture of a lathyrane diterpene, or intermediates thereof, comprising the steps: i) culturing the cell of claim 15 in cell culture medium, wherein the cells comprise an endogenous pool of geranylgeranyl disphosphate and express a casbene-9-oxidase, a casbene synthase, and a casbene-5,6-oxidase; and optionally ii) isolating or purifying synthesized compounds from the cell and/or the cell culture medium.

25. (canceled)

26. The process according to claim 22, wherein said compound is jolkinol C or epi-jolkinol.

27. An isolated polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence comprising greater than 96% amino acid sequence identity to SEQ ID NO: 2.

28. (canceled)

29. A nucleic acid molecule encoding the polypeptide according to claim 27.

30. The nucleic acid molecule according to claim 29, wherein said nucleic acid molecule is part of an expression vector adapted for expression of said nucleic acid molecule.

31.-34. (canceled)

Description

[0087] An embodiment of the invention will now be described by example only and with reference to the following figures:

[0088] FIGS. 1a-1d: Diterpenoids of the (a) lathyrane (b) jatropholane (c) rhamnofolane and (d) tigliane class which have been isolated from J. curcas. The red oxygen atom highlighted on each of the molecules corresponds to the 5-position of casbene, whereas the blue oxygen atom corresponds to the 9-position of casbene. The carbon-carbon bond highlighted in green corresponds to the 6, 10-positions of casbene.

[0089] FIG. 2: A diterpenoid biosynthesis gene cluster. The diagram corresponds to a 300 kbp region present on scaffold 123 of the J. curcas genome (Genbank accession NW_012124159). Different classes of enzymes have been colour-coded, e.g., cytochrome P450 genes are shown in blue.

[0090] FIGS. 3a-3d: (a) GC and LC chromatographs of casbene and casbene metabolites produced by transient expression of casbene synthase and casbene synthase with a single cytochrome P450 from the J. curcas gene cluster in N. benthamiana. The structures of the metabolites denoted by [n] are shown in FIG. 3b. The corresponding mass spectra are provided in FIG. 5(b) Summary of enzyme activities the P450s encoded by JCGZ_2819, CYP726A20 and JCGZ_2811 (c) LC chromatographs obtained from co-expression of casbene synthase with two cytochrome P450s from the J. curcas gene cluster. The lower panels show the results with co-expression of the J. curcas genes with 1-deoxy-D-xylulose 5-phosphate synthase (DXS) and a plastidial geranylgeranyl pyrophosphate synthase (GGPPS) from Arabidopsis thaliana (d) Presumed facile enolization at the 5-keto group is the key step for the .sup.7,8.fwdarw..sup.6,7 double bond isomerization in 6-hydroxy-5,9-keto-casbene, which leads to a tri-keto precursor that spontaneously converts to jolkinol C via an intramolecular aldol reaction

[0091] FIG. 4 Analysis of expression of the J. curcas cluster genes shown in FIG. 2 by qPCR in leaf, stem and root. The bars have been colour coded to match FIG. 2. The error bars represent the standard deviations from three biological replicates. Expression levels are relative to -actin. Genes for which no expression was detected are not shown;

[0092] FIGS. 5a-5b: Determination of molecular weights of diterpenoids by high resolution mass spectrometry; and

[0093] FIG. 6: Transient expression of eGFP fusion proteins in the epidermis of N. benthamiana. The first 72 amino acids from casbene synthase, the first 93 amino acids from JCGZ_2819, and the first 80 amino acids of CYP726A20 were fused to eGFP. The upper panel is a control experiment where N. benthamiana plants were infiltrated with an empty vector control. The left hand column shows the chlorophyll autofluorescence. The middle column shows the eGFP fluorescence. The right had column shows the two fluorescent merges with the bright field image showing epidermal (pavement) cells. The yellow bar in each picture corresponds to a distance of 50 W. NB, when N. benthamiana plants are infiltrated using syringes, transgene expression is typically confined to the epidermal pavement cells. The diffuse red background fluorescence that appears in some images corresponds to mesophyll cells which are out of the focal plane;

[0094] FIG. 7a is the full length amino acid sequence of casbene-9-oxidase [SEQ ID NO: 1]; FIG. 7b is the sequence of casbene-9-oxidase amino acid sequence minus an amino terminal membrane associated domain [SEQ ID NO: 2];

[0095] FIG. 8 is the cDNA nucleotide sequence encoding casbene-9-oxidase [SEQ ID NO: 3];

[0096] FIG. 9 is the cDNA nucleotide sequence of cytochrome P450 JCGZ 2819 [SEQ ID NO: 4];

[0097] FIG. 10 is the cDNA nucleotide sequence of cytochrome P450 CYP726A20 [SEQ ID NO: 5]; and

[0098] FIG. 11 is the cDNA nucleotide sequence of casbene synthase [SEQ ID NO: 6].

SEQUENCE LISTING

[0099] The nucleic and amino acid sequences are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. The sequence listing submitted herewith, generated on Aug. 28, 2018, 32 Kb is herein incorporated by reference.

[0100] SEQ ID NO 1: The full length amino acid sequence of casbene-9-oxidase.

[0101] SEQ ID NO 2: The amino acid sequence of casbene-9-oxidase minus an amino terminal membrane associated domain.

[0102] SEQ ID NO 3: The cDNA nucleotide sequence encoding casbene-9-oxidase.

[0103] SEQ ID NO 4: The cDNA nucleotide sequence of cytochrome P450 JCGZ 2819.

[0104] SEQ ID NO 5: The cDNA nucleotide sequence of cytochrome P450 CYP726A20.

[0105] SEQ ID NO 6: The cDNA nucleotide sequence of casbene synthase.

[0106] SEQ ID NOS 7-66: Primer sequences.

Materials and Methods

[0107] Analysis of Gene Expression by qPCR

[0108] RNA extraction, DNase treatment and cDNA synthesis were performed as described previously using three biological and four technical replicates per tissue.sup.13. qPCR primers (Table 1) were designed using Primer3Plus.sup.23, and their specificity verified by a blastN search against the J. curcas genome. Optimal annealing temperatures were determined empirically by gradient PCR. qPCR reactions were then performed as described previously.sup.13 and expression levels normalised against an -actin gene (Genbank accession XM_012232498) using the delta-delta CT method.sup.24 with correction for amplification efficiencies obtained using LinReg PCR.sup.25.

Gene Cloning and Transient Gene Expression in Nicotiana benthamiana.

[0109] cDNA was synthesised using total RNA from J. curcas roots or A. thaliana seedlings using g Superscript II reverse transcriptase (Invitrogen, Carlsbad, Calif.) and a 5-T.sub.(18)VN-3 primer. The open reading frame for each gene was then amplified and inserted into the pEAQ-HT expression vector via conventional restriction enzyme or Gibson cloning using the primers detailed in Table 3. In each instance, a 5-AAAA-3 Kozak sequence was included immediately upstream of the start codon. DNA assembly was then performed using NEB Gibson Assembly Mastermix (NEB, Ipswich, Mass.) according to the manufacturer's protocol. After confirming the presence of the correct inserts by Sanger sequencing, the expression vectors were transformed into Agrobacterium tumefaciens LBA4404 using the freeze-thaw method.sup.26. For initial experiments to detect the production of novel diterpenoids, leaves were infiltrated with syringes with equal mixtures A. tumefaciens cultures at a final OD 600.sub.nm of 1.0 in infiltration buffer (10 mM MgCl.sub.2, 200 M acetosynringone and 0.015% Silwet L-77). Five days after infiltration, ca. 2 cm.sup.2 of leaf material was extracted with 1 ml of ethyl acetate by grinding for 1 minute with a steel bead at 30 Hz for 2 minutes in a Retsch homogenizer. After centrifugation, the supernatant was used either directly for GC-MS, or for LC-MS analysis after removal of the ethyl acetate and redissolving the extract in methanol. For the preparation of compounds for NMR analysis, multiple plants were infiltrated by immersing in cultures resuspended in infiltration buffer and then applying a partial vacuum to a pressure of 100 mbar for 1 minute.

In-Silico Analysis of Plastidial Transit Peptides and Creation of eGFP Fusion Constructs and Visualization of Subcellular Localization

[0110] In-silico prediction of plastidial transit peptides was performed using ChloroP.sup.27. pEAQ-HT expression vectors containing the N-terminal portions of proteins and eGFP were created by Gibson assembly using the primers detailed in Table 2. Leaves from N. benthamiana plants were examined by confocal microscopy five days after infiltration. A 20 magnification was used. Chlorophyll auto-fluorescence was observed using an excitation wavelength of 561 nm and an emission wavelength of 633-735 nm. GFP fluorescence was observed using an excitation wavelength of 488 nm and an emission wavelength of 495-600 nm.

Preparation and Identification of [4] 6-Hydroxy-5-Keto-Casbene

[0111] 23.8 g of freeze-dried leaf material that had been infiltrated with casbene synthase and JCGZ_2819 was extracted once with 250 ml ethyl acetate and once with 100 ml of ethyl acetate. The ethyl acetate was removed by rotary evaporation to yield 1.30 g of a green oily residue which was taken up in 10 ml of n-hexane. The extract was then applied to a 40 g Grace Resolve silica column and fractions collected on a 0-50% ethyl acetate in hexane gradient. Fractions containing the desired product were pooled and then further purified using C30 reversed-phase HPLC as described previously.sup.13 to yield ca. 1 mg of metabolite.

[0112] Data for [4] 6-hydroxy-5-keto-casbene: .sup.1H NMR (700 MHz, CDCl.sub.3): 6.35 (d, J=11 Hz, 1H (H-3)), 5.25 (d, J=9 Hz, 1H (H-6)), 5.09 (d, J=9 Hz, 1H (H-7)), 4.84 (dd, J=9, 4 Hz, 1H (H-11)), 2.25 (m, 1H (H-10a)), 2.24 (m, 1H (H-13a)), 2.20 (m, 1H (H-9a)), 2.14 (m, 1H (H-9b)), 2.12 (m, 1H (H-14a)), 2.03 (m, 1H (H-10b)), 1.96 (s, 3H (H-18)), 1.77 (ddd, J=12, 10, 3 Hz (H-13b)), 1.70 (s, 3H (H-19)), 1.58 (s, 3H (H-20)), 1.56 (dd, J=11, 8 Hz, 1H (H-2)), 1.21 (ddd, J=12, 8, 2 Hz, 1H (H-1)), 1.18 (s, 3H (H-16)), 1.02 (s, 3H (H-17)), 0.84 (dddd, J=12, 12, 10, 3 Hz (H-14b)); .sup.13C NMR (175 MHz, CDCl.sub.3): 200.2 (C-5), 145.2 (C-3), 142.2 (C-8), 136.3 (C-12), 134.2 (C-4), 124.2 (C-7), 123.8 (C-11), 68.4 (C-6), 39.8 (C-13), 38.7 (C-9), 35.8 (C-1), 29.2 (C-16), 28.2 (C-2), 27.5 (C-15), 25.9 (C-14), 23.9 (C-10), 16.0 (C-17), 15.5 (C-19), 15.4 (C-20), 12.0 (C-18); HRMS (m/z): [M+H].sup.+ calcd. for C.sub.20H.sub.30O.sub.2, 303.2319; found, 303.2313.

Preparation and Identification of 9-Keto Casbene [6]

[0113] 19.32 g of freeze-dried leaf material that had been infiltrated with casbene synthase and casbene-9-oxidase was extracted with ethyl acetate as described above. The ethyl acetate was removed by rotary evaporation to yield 1.09 g of a green oily residue which was taken up in 10 ml of n-hexane. The extract was then subjected to normal-phase silica flash chromatography and C30 reversed-phase HLPC as described above to yield 770 g of metabolite. Data for [6] 9-keto-casbene: .sup.1H NMR (700 MHz, CDCl.sub.3): 6.55 (dd, J=7, 7 Hz, 1H (H-7)), 5.12 (dd, J=8, 6 Hz, 1H (H-11)), 4.80 (d, J=10 Hz, 1H (H-3)), 3.56 (dd, J=12, 8 Hz, 1H (H-10a)), 3.02 (dd, J=12, 6 Hz, 1H (H-10b)), 2.41 (m, 2H, (H-6a/6b)), 2.32 (m, 2H (H-5a and H-13a)), 2.11 (ddd, J=13, 7, 7 Hz, 1H (H-5b)), 1.93 (dd, J=12, 12 Hz, 1H (H-13b)), 1.85 (ddd, J=14, 5, 1 Hz, 1H (H-14a)), 1.77 (s, 3H (H-20)), 1.75 (s, 3H (H-19)), 1.74 (s, 3H (H-18)), 1.29 (dd, J=10, 9 Hz, 1H (H-2)), 1.12 (dddd, J=14, 12, 10, 3 Hz, 1H (H-14b)), 1.08 (s, 3H (H-16)), 0.87 (s, 3H (H-17)), 0.68 (ddd, J=10, 9, 1 Hz, 1H (H-1)); .sup.13C NMR (175 MHz, CDCl.sub.3): 202.0 (C-9), 144.5 (C-7), 138.1 (C-12), 135.6 (C-8), 132.6 (C-4), 123.1 (C-3), 119.7 (C-11), 40.4 (C-13), 40.1 (C-10), 38.9 (C-5), 31.5 (C-1), 29.2 (C-16), 26.5 (C-2), 26.0 (C-6), 24.2 (C-14), 20.8 (C-15), 17.7 (C-20), 15.8 (C-18), 15.5 (C-17), 11.0 (C-19); HRMS (m/z): [M+H].sup.+ calcd. for C.sub.20H.sub.30O, 287.2369; found, 287.2368.

Preparation and identification of [7] 9-hydroxy-5-keto-casbene, [9] jolkinol C, [10] epi-jolkinol C and [11] 8-hydroxy-5, 9-diketocasbene

[0114] 13.8 g of freeze-dried leaf material that had been infiltrated with deoxy-xylulose-5-phosphate synthase, geranylgeranyl pyrophosphate synthase, casbene synthase, casbene 5,6-oxidase (JCGZ_2819) and casbene-9-oxidase (JCGZ_2811) was extracted with ethyl acetate as described above. The ethyl acetate was removed by rotary evaporation to yield 600 mg of a green oily residue which was taken up in 10 ml of n-hexane. The extract was then subjected to normal-phase silica flash chromatography using a 10% to 100% ethyl acetate in hexane gradient. Fractions containing the desired metabolites were then further purified using preparative C18 reversed-phase HPLC to yield ca. 1.17 mg of 9-hydroxy-5-keto-casbene, 450 g of jolkinol C (mixture of epimers) and 740 g of 8-hydroxy-5,9-diketocasbene.

[0115] Data for [7] 9-hydroxy-5-keto-casbene: .sup.1H NMR (700 MHz, CDCl.sub.3): 6.33 (d, J=10 Hz, 1H (H-3)), 5.29 (dd, J=9, 4 Hz, 1H (H-7)), 4.69 (dd, J=9, 4 Hz, 1H (H-11)), 4.17 (dd, J=8, 6 Hz, 1H (H-9)), 3.69 (dd, J=14, 9 Hz, 1H (H-6a)), 2.95 (dd, J=14, 4 Hz, 1H, (H-6b)), 2.29 (m, 2H (H-10a/10b)), 2.18 (ddd, J=10, 10, 10 Hz, 1H (H-13a)), 2.10 (dddd, J=15, 12, 10, 3 Hz, 1H (H-14a)), 1.88 (s, 3H (H-18)), 1.74 (dd, J=11, 11 Hz, 1H (H-13b)), 1.62 (s, 3H (H-20)), 1.57 (s, 3H (H-19)), 1.50 (dd, J=10, 9 Hz, 1H (H-2)), 1.17 (s, 3H (H-16)), 1.16 (ddd, J=12, 9, 3 Hz, 1H (H-1)), 1.10 (s, 3H (H-17)), 0.81 (ddd, J=12, 12, 12 Hz, 1H (H-14b)); .sup.13C NMR (175 MHz, CDCl.sub.3): 199.4 (C-5), 143.3 (C-3), 139.0 (C-8), 138.3 (C-12), 137.1 (C-4), 120.7 (C-7), 119.4 (C-11), 76.9 (C-9), 40.1 (C-13), 38.6 (C-6), 35.0 (C-1), 31.6 (C-10), 29.0 (C-16), 27.6 (C-2), 26.2 (C-14), 25.9 (C-15), 15.9 (C-17), 15.3 (C-20), 11.7 (C-18), 11.6 (C-19); HRMS (m/z): [M+H].sup.+ calcd. for C.sub.20H.sub.30O.sub.2, 303.2319; found, 303.2313.

[0116] Data for [9] Jolkinol C: .sup.1H NMR (700 MHz, CDCl.sub.3): 7.36 (d, J=12 Hz, 1H (H-3)), 5.35 (d, J=10 Hz, 1H (H-11)), 3.51 (dd, J=14, 9 Hz, 1H (H-7a)), 3.03 (d, J=10 Hz, 1H (H-10)), 2.66 (br d, J=13 Hz, 1H (H-13a)), 2.58 (dq, J=9, 7 Hz, 1H, (H-8)), 2.19 (ddddd, J=14, 4, 4, 2, 2 Hz, 1H (H-14a)), 1.86 (s, 3H (H-18)), 1.69 (ddd, J=13, 12, 2 Hz, 1H (H-13b)), 1.59 (dd, J=14, 2 Hz, 1H (H-7b)), 1.57 (dddd, J=14, 12, 12, 2 Hz, 1H (H-14b)), 1.46 (dd, J=12, 8 Hz, 1H (H-2)) 1.38 (s, 3H (H-20)), 1.29 (d, J=7 Hz, 3H (H-19)), 1.19 (s, 3H (H-16)), 1.14 (ddd, J=12, 8, 3 Hz, 1H (H-1)), 1.09 (s, 3H (H-17)); .sup.13C NMR (175 MHz, CDCl.sub.3): 219.73 (C-9), 198.15 (C-5), 152.18 (C-3), 144.91 (C-12), 132.33 (C-4), 118.79 (C-11), 88.65 (C-6), 57.99 (C-10), 40.43 (C-7), 38.98 (C-8), 35.87 (C-13), 35.72 (C-1), 29.86 (C-2), 29.17 (C-16), 27.65 (C-14), 25.28 (C-15), 20.89 (C-20), 18.39 (C-19), 16.25 (C-17), 12.16 (C-18); HRMS (m/z): [M+H].sup.+ calcd. for C.sub.20H.sub.28O.sub.3, 317.2111; found, 317.2096. N.B. The lathyrane system is not used; the casbane numbering system has been retained to allow comparison with precursor molecules.

[0117] Data for [10] epi-Jolkinol C (characterized as a ca 1:4 mixture with Jolkinol C): .sup.1H NMR (700 MHz, CDCl.sub.3; .sup.1H resonances for which no multiplicity is given were resolved from Jolkinol C in HSQC but not in 1 D-.sup.1H NMR; resonances which are not reported were not resolved from Jolkinol C in either HSQC or 1H NMR): 7.34 (d, J=12 Hz, 1H (H-3)), 5.29 (d, J=12 Hz, 1H (H-11)), 2.86 (d, J=11 Hz, 1H (H-10)), 2.61 (1H (H-7a)), 2.61 (1H (H-8)), 2.17 (1H (H-7b)), 1.83 (3H (H-18)), 1.70 (1H (H-14b)), 1.59 (1H (H-13b)), 1.43 (s, 3H (H-20)), 1.21 (3H (H-19)); .sup.13C NMR (175 MHz, CDCl.sub.3): 219.11 (C-9), 198.38 (C-5), 151.82 (C-3), 145.10 (C-12), 132.32 (C-4), 118.83 (C-11), 86.77 (C-6), 56.93 (C-10), 41.47 (C-7), 40.30 (C-8), 36.00 (C-13), 35.44 (C-1), 29.83 (C-2), 29.18 (C-16), 27.85 (C-14), 25.23 (C-15), 20.74 (C-20), 16.25 (C-17), 14.61 (C-19), 12.09 (C-18).

[0118] Data for [11] 8-hydroxy-5,9-diketocasbene: .sup.1H NMR (700 MHz, CDCl.sub.3): 6.52 (d, J=17 Hz, 1H (H-6)), 6.49 (d, J=17 Hz, 1H (H-7)), 6.22 (d, J=9 Hz, 1H (H-3)), 5.21 (dd, J=8, 6 Hz, 1H (H-11)), 3.42 (dd, J=15, 6 Hz, 1H (H-10a)), 3.35 (br s, OH), 3.23 (dd, J=15, 8 Hz, 1H, (H-10b)), 2.37 (ddd, J=14, 8, 8 Hz, 1H (H-13a)), 2.15 (dddd, J=15, 8, 8, 3 Hz, 1H (H-14a)), 1.89 (s, 3H (H-18)), 1.88 (ddd, J=14, 9, 3 Hz (H-13b)), 1.70 (s, 3H (H-20)), 1.53 (s, 3H (H-19)), 1.48 (dd, J=10, 9 Hz, 1H (H-2)), 1.19 (s, 3H (H-17)), 1.14 (ddd, J=10, 8, 2 Hz, 1H (H-1)), 0.99 (s, 3H (H-16)), 0.93 (m, 1H (H-14b)); .sup.13C NMR (175 MHz, CDCl.sub.3): 209.2 (C-9), 194.3 (C-5), 144.0 (C-7), 142.8 (C-3), 140.9 (C-12), 138.5 (C-4), 128.9 (C-6), 116.5 (C-11), 79.2 (C-8), 39.2 (C-13), 39.1 (C-10), 32.9 (C-1), 29.0 (C-17), 27.4 (C-2), 25.6 (C-15), 24.8 (C-14), 23.7 (C-19), 16.2 (C-20), 16.1 (C-16), 12.4 (C-18); HRMS (m/z): [M+H].sup.+ calcd. for C.sub.20H.sub.28O.sub.3, 317.2111; found, 317.2107.

Example 1

[0119] Recently, we reported a diterpenoid biosynthetic gene cluster in the castor (Ricinus communis) which contained genes encoding diterpene synthases and several cytochrome P450, including casbene synthases and casbene-5-oxidases. We also demonstrated the existence of similar clusters in other Euphorbiaceae including Jatropha curcas, a plant that produces a variety of diterpenoids including lathyranes, jatropholanes, rhamnofolanes and tiglianes.sup.11 (FIG. 1). Using a recently released version of the Jatropha curcas genome.sup.12, we were able to perform further in silico analysis of this cluster, and found it contained a number of enzyme-encoding genes, including casbene synthases, cytochrome P450s, alcohol dehydrogenases and alkenal reductase-like genes (FIG. 2). The P450 genes were all members of the CYP71D tribe, and all except two were part of the CYP726A taxon-specific bloom found so far only in the Euphorbiaceae.sup.13,14.

Example 2

[0120] Using qPCR, we analysed the expression of the genes present within this cluster FIG. 4). The majority of the genes for which we were able to detect transcripts were most abundantly expressed within the roots. The exceptions to this was JCGZ_2811, which was most abundant in leaves, but still abundant in both stems and roots. This observation was consistent with the roots of J. curcas being rich in diterpenoids.sup.11.

Example 3

[0121] Phylogenetic analysis of the P450 genes suggested JCGZ_2819 was orthologous to CYP726A18 and CYP726A15 from castor. The former of these P450s is able to convert casbene in 5-ketocasbene via a hydroxyl intermediate, whereas the latter catalyses a similar reaction with neocembrene.sup.13. When JCGZ_2819 was transiently co-expressed with casbene synthase in Nicotiana benthamiana leaves, we were able to detect a metabolite with a molecular mass of 302.23 (FIG. 3A and FIG. 4). After vacuum-infiltration of multiple N. benthamiana plants, we were able to purify the metabolite which was identified as 6-hydroxy-5-keto casbene (FIG. 3b) by NMR in CDCl.sub.3 solution. This diterpenoid has previously be reported as a product of casbene oxidation by CYP726A14 from castor.sup.15. Interestingly, in our previous study, we only observed 5-keto-casbene production with CYP726A14, but we were able to obtain 6-hydroxy-5-keto casbene when using pEAQ-HT vectors conferring higher levels of transient gene expression in N. benthamiana.sup.16 (data not shown).

Example 4

[0122] In addition to JCGZ_2819, CYP726A20 was also able to convert casbene into 6-hydroxy-5-keto casbene. This observation was similar to castor, where we identified more than one P450 gene that was able to perform casbene-5-oxidation.sup.13. In silico analyses of JCGZ_2819, and CYP726A18 and CYP726A15 (neocembrene-5-oxidase) revealed the presence of a putative plastidial transit peptide. Fusion of N-terminal for GFP resulted in the import of transiently expressed GFP into the plastids of N. benthamiana (FIG. 6). CYP726A20 did not contain a predicted chloroplast transit peptide, and consistent with this, fusion of the first 80 amino acids of this protein to GFP did not result in import into plastids. Thus it would appear in both Jatropha and castor, enzymes catalysing casbene 5-oxidation are located in both the plastid and endoplasmic reticulum. Both Jatropha enzymes were also able to catalyse 6-hydroxlation. Interestingly, in castor.sup.13, Euphoriba peplus.sup.13 and J. curcas (FIG. 2A), the plastidial casbene-5-oxidases are adjacent to a casbene synthase, indicating the order of these genes may be conserved in the Euphorbiaceae.

TABLE-US-00001 TABLE1 SequencesofprimersusedforqPCRanalysis ofgeneexpressiononJ.curcasgenomescaffold123 Positionon Forward/Reverse Annealing GeneID Annotation scaffold123 (SEQIDNO) temperature 105629799 JCGZ_2803 2-alkenal (40398..41923) 5-CCCAGAAGGAAGTA 60 C. reductase TGCCCG-3 (7) like 5-CTTTGCAAGTTGCC CAACGA-3 (8) 105629800 JCGZ_2805 2-alkenal (70021..71581) 5-CTCCAAGTCCCAGA 65 C. reductase AGGAAGT-3 (9) like 5-CGGGAAAATCTAGG CTGAGTGT-3 (10) 105629801 JCGZ_2806 2-alkenal (84833..87073) 5-GCAGTGTTGCTGAA 65 C. reductase TATGAGGC-3 (11) like 5-TCCCGCAATGAATC TTGTCTGA-3 (12) 105629802 JCGZ_2807 CYP726A24 (104379..106016) 5-AGCTCGCAGGCTAC 65 C. CAATTT-3 (13) 5-CTTCTTTGGCCATT TCCGGC-3 (14) 105629803 JCGZ_2808 2-alkenal (109911..111959) 5-CTGGGCATCCTTTT 60 C. reductase GCACCA-3 (15) like 5-TCTTGAAGTCTGGC GGCG-3 (16) 105629805 JCGZ_2810 CYP726A23 (127836..129469) 5-TAACAGGAAGGCGG 63 C. CAGTTC-3 (17) 5-CTGCCAGCCCCAAA CATTTC-3 (18) 105629806 JCGZ_2811 CYP71D-like (134771..137289) 5-TGCTGGGATAAACA 57 C. GTAAGGAGG-3 (19) 5-ATGACGTGTCACTA CCAGCG-3 (20) 105629816 JCGZ_2812 CYP71D-like (149297..150867) 5-CAGCTCGGCGAAAT 65 C. TACCAC-3 (21) 5-GTGCGAGTGCGATA TCTGTG-3(22) 105629807 JCGZ_2813 Short-chain (152208..153156) 5-GGGTTTGAGCGAAC 65 C. alcohol AGCAAG-3 (23) dehydrogenase 5-AGCAAGGTACAAAG CAGCCT-3 (24) 105629814 JCGZ_2814 Monoterpene (174139..176533) 5-CTCAAACCCAGCTT 62 C. synthase TTGCCC-3 (25) 5-TCGTTGGGGTTATT GGCACA-3 (26) 105629808 JCGZ_2815 Monoterpene (191855..195188) 5-ATGGCGGGTTCGGA 65 C. synthase TCTTAC-3 (27) 5-GACATTGCTTGTTG AGCCGT-3 (28) 105629820 JCGZ_2816 Monoterpene (209488..211654) 5-GCTACTGCGTACCT 65 C. synthase GCTGAT-3 (29) 5-AGGGCCACTAAAAA CTCGGG-3 (30) 105629809 JCGZ_2819 Casbene5,6- (237179..240418) 5-AACATAAAGCCGAC 59 C. oxidase AGGGCA-3 (31) 5-CTGCCTGCGCCAAA TGTATC-3 (32) 105629810 JCGZ_2820 Casbene (246989..249381) 5-CCTAGTGGCAAGCT 65 C. synthase3 GAACGA-3 (33) 5-TGGACGAGTGTCTG TCTCTGA-3 (34) 105629821 JCGZ_2821 Casbene (252084..255566) 5-ACATGTTTAATGGC 55 C. synthase2 GGGGTT-3 (35) 5-TTCGCCTCCAGCTT GATTGA-3 (36) 105629811 JCGZ_2822 Casbene (259916..262727) 5-GGTCCACAGAAGTT 65 C. synthase1 GTGCCA-3 (37) 5-TCAGTTGTGAAGAG TCCGTGT-3 (38) 105629812 JCGZ_2823 CYP726A20 (284198..285983) 5-TTGGGATAGGAGCG 58 C. AAGCTG-3 (39) 5-TCGCTTCCAGCACC AAACAT-3 (40) 105629813 JCGZ_2824 CYP726A21 (297137..298771) 5-CTGATCGACCGCTT 58 C. GTCCTT-3 (41) 5-CTCCGTACAGCCCA AAACCT-3 (42) XM_012232498 Actin n/a 5-TGCCATCCAGGCCG 61 C. TTCTATCT-3 (43) 5-GGAGGATAGCATGT GGAAGAGCG-3 (44)

TABLE-US-00002 TABLE2 Primersusedforcreationof GFPfusionconstructsinpEAQ-HTvia GibsonAssembly Fragment Domain Forward/Reverse(SEQIDNO:) Casbenesynthaseplastidialtransitsequence. Fragment AA1-72 5-CTGCCCAAATTCGCGACCGGTAAAA 1 ATGGCAATGCAACCTGCA-3 (45) 5-TTGCTCACCCATACAGTAGGAGGAA AGTAG-3 (46) Fragment eGFP 5-CTGTATGGGTGAGCAAGGGCGAGGA 2 G-3 (47) 5-GAAACCAGAGTTAAAGGCCTTACTT GTACAGCTCGTCCATG-3 (48) JCGZ_2819plastidialtransitsequence. Fragment AA1-93 5-CTGCCCAAATTCGCGACCGGTAAAA 1 ATGTCGCTGCAACCAGCA-3 (49) 5-TTGCTCACGAATATTTTGGTAAGAC TTGTGGTAGTTG-3 (50) Fragment eGFP 5-CAAAATATTCGTGAGCAAGGGCGAG 2 GAG-3 (51) 5-GAAACCAGAGTTAAAGGCCTTACTT GTACAGCTCGTCCATG-3 (52) CYP726A20N-terminal Fragment AA1-80 5-CTGCCCAAATTCGCGACCGGTAAAA 1 ATGGAACACCAAATCCTC-3 (53) 5-TTGCTCACGAAAGGAACTTGCCCAA G-3 (54) Fragment eGFP 5-GTTCCTTTCGTGAGCAAGGGCGAGG AG-3 (55) 2 5-GAAACCAGAGTTAAAGGCCTTACTT GTACAGCTCGTCCATG-3 (56)

TABLE-US-00003 TABLE3 Sequencesofprimersusedinsertionof J.curcascDNAsequencesintoAgeIandXhoI sitesofpEAQ-HTvector Organism GeneID Annotation Forward/Reverse Conventionalcloningusingrestriction digestionwithBsaIandligationintoAgeI andXhoIsitesofpEAQ-HT J.curcas 105629806 JCGZ_2811 5-AAAAGGTCTCACCGGAAAAATGCTTTT CTTCATCACCGTACTC-3 (57) 5-AAAAGGTCTCATCGACTATCTTGAGAT TTTACCAACTGCTG-3 (58) Conventionalcloningusingrestriction digestionAgeIandXhoIintoAgeIandXhoI sitesofpEAQ-HT J.curcas 105629809 JCGZ_2819 5-AAAAACCGGTAAAAATGTCGCTGCAAC CAGCAATTTTAC-3 (59) 5-AAAACTCGAGTCATAATGCTTTTAAGT GTGGGCAC-3 (60) GibsoncloningintotheAgeIandXhoIsitesofpEAQ-HT J.curcas 105629812 CYP726A20 5-TATTCTGCCCAAATTCGCGAAAAAATG GAACACCAAATCCTCTCATTT-3 (61) 5-TGAAACCAGAGTTAAAGGCCTTAGGGA CGGAATGGAATGGGG-3 (62) A.thaliana At4g15560 DXS 5-TATTCTGCCCAAATTCGCGACCGGTAA AAATGGCTTCTTCTGCATTTG-3 (63) 5-TGAAACCAGAGTTAAAGGCCTCGAGTC AAAACAGAGCTTCCCTTG-3 (64) A.thaliana At4g36810 GGPPS11 5-TATTCTGCCCAAATTCGCGACCGGTAA AAATGGCTTCAGTGACTCTAG-3 (65) 5-TGAAACCAGAGTTAAAGGCCTCGAGTC AGTTCTGTCTATAGGCAATG-3 (66)

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