Patent Application
20190106684
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.
Terpenes or terpenoids are a structurally diverse and a very large group of organic compounds commonly found in plants ranging from essential and universal primary metabolites such as sterols, carotenoids and hormones to more complex and unique secondary metabolites. Terpenes are hydrocarbons assembled of five carbon terpene or isoprene subunits providing the carbon skeleton. Terpenoids are modified terpenes which typically comprise also oxygen. Terpenoids are classified accordingly to the length of the isoprene units as for example hemiterpenoids consisting of one, monoterpenoids consisting of two, sesquiterpenoids consisting of three and diterpenoids consisting of four isoprene units.
Diterpenes form the basis for many biologically important compounds such as retinol, retinal, and phytol and some compounds have shown anti-microbial and anti-inflammatory properties. A large number of diterpenes have been isolated from plants belonging to the family of Euphorbiaceae. The Euphorbiaceae or spurge family is a large family of flowering plants found all over the world, with some synthesising compounds of considerable biological activity such as ingenol mebutate (Euphorbia peplus), resiniferatoxin (E. resinifera), prostratin (E. cornigera), jatrophanes and lathyranes (Jatropha sp. and Euphorbia sp.), jatropholones, (Jatropha sp.), rhamnofolanes (Jatropha sp.) and jatrophone (Jatropha sp.).
The Euphorbiaceae produce a diverse range of casbene derived diterpenoids1,2, many of which are providing interesting leads in the development of new pharmaceuticals. These include the lathyranes which are inhibitors of ABC transporters responsible for the efflux of chemotherapy drugs in multidrug-resistant (MDR) cancers3,4 as wells as fungal5 and protozoal6 pathogens. The lathyranes are also precursors of many other active diterpenoids including ingenol mebutate, a licenced pharmaceutical used for the treatment of actinic keratosis7, prostratin, a lead compound for the treatment of latent HIV infections8, and resiniferatoxin, an ultrapotent capsaicin analog which is currently in clinical trial for the treatment of cancer-related intractable pain9. Although the relationship between casbene and lathyrane structure was noted several decades ago10, the mechanism leading to the ring closure required to convert the 14:3 casbane ring into the 5:11:3 lathyrane ring system has not previously been reported
In co-pending PCT application WO2015/104553 is disclosed genes encoding enzymes involved in diterpenoid biosynthesis, including casbene-5-oxidases. This disclosure relates to the identification and characterisation of additional enzyme activities involved in the biosynthesis of diterpenes, such as lathyranes, from geranylgeranyl pyrophosphate via the 9-oxidation of the casbene skeleton, as occurs for example in the biosynthesis of jolkinol C and epi-jolkinol C. The conversion of casbene to a lathyrane skeleton involves a co-ordinated cytochrome P450 mediated intramolecular carbon-carbon ring closure.
According to an aspect of the invention there is provided an isolated cell transformed or transfected with an expression vector adapted to express a nucleic acid molecule comprising a nucleotide sequence as set forth in SEQ ID NO: 3, or a nucleotide sequence that has at least 50% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 3 and encodes a polypeptide that has casbene 9-oxidase activity.
Substrates for the casbene 9-oxidase according to the invention are varied, for example, casbene, 9-hydroxy casbene, 5-hydroxy-casbene, 5-keto-casbene and 6-hydroxy-5-keto-casbene.
In an embodiment of the invention said isolated cell is further transformed with an expression vector adapted to express a nucleic acid molecule comprising nucleotide sequence as set forth in SEQ ID NO: 6, or a nucleotide sequence that has at least 50% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 6 and encodes a polypeptide that has casbene synthase activity.
In an alternative embodiment of the invention said isolated cell is further transformed with an expression vector adapted to express a nucleic acid molecule comprising nucleotide sequence as set forth in SEQ ID NO: 4, or a nucleotide sequence that has at least 50% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 4 and encodes a polypeptide that has casbene-5, 6-oxidase activity
In a further alternative embodiment of the invention said isolated cell is further transformed with an expression vector adapted to express a nucleic acid molecule comprising nucleotide sequence as set forth in SEQ ID NO: 5 or a nucleotide sequence that has at least 50% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 5 and encodes a polypeptide that has casbene-5, 6-oxidase activity
In an embodiment of the invention there is provided an isolated cell transformed with at least one vector comprising a nucleotide sequence from the group consisting of:
In an alternative embodiment of the invention there is provided an isolated cell transformed with at least one vector comprising a nucleotide sequence from the group consisting of:
In a further alternative embodiment of the invention there is provided an isolated cell transformed with at least one vector comprising a nucleotide sequence from the group consisting of:
In an embodiment of the invention said isolated cell transformed or transfected with an expression vector adapted to express a nucleic acid molecule encoding a polypeptide comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 1 or 2, or an amino acid sequence that is at least 50% identical to the amino acid sequence set forth in SEQ ID NO: 1 or 2 and which has casbene-9-oxidase activity.
In an embodiment of the invention said nucleic acid molecule comprises or consists of a nucleotide sequence that has at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% nucleotide sequence identity to the nucleotide sequences set forth in SEQ ID NO: 3, 4, 5 or 6 over the full length sequence or over the full length sequence of the amino acid sequence set forth in SEQ ID NO: 1 or 2.
In an embodiment of the invention said isolated cell is transformed or transfected with an expression vector adapted to express a nucleic acid molecule encoding a polypeptide comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 1 or 2.
In an embodiment of the invention said isolated cell is a plant cell.
In an alternative embodiment of the invention said cell is a microbial cell.
In an embodiment of the invention said microbial cell is a bacterial cell.
In an embodiment of the invention said microbial cell is a fungal cell, for example a yeast cell.
In a further alternative embodiment of the invention said cell is an algal cell.
If microbial cells, for example bacterial or yeast cells are used as organisms in the process according to the invention they are grown or cultured in the manner with which the skilled worker is familiar, depending on the host organism. As a rule, microorganisms are grown in a liquid medium comprising a carbon source, usually in the form of sugars, a nitrogen source, usually in the form of organic nitrogen sources such as yeast extract or salts such as ammonium sulfate, trace elements such as salts of iron, manganese and magnesium and, if appropriate, vitamins, at temperatures of between 0° C. and 100° C., preferably between 10° C. and 60° C., while gassing in oxygen.
The pH of the liquid medium can either be kept constant, that is to say regulated during the culturing period, or not. The cultures can be grown batchwise, semi-batchwise or continuously. Nutrients can be provided at the beginning of the fermentation or fed in semi-continuously or continuously. The diterpenoids produced can be isolated from the organisms as described above by processes known to the skilled worker, for example by extraction, distillation, crystallization, if appropriate precipitation with salt, and/or chromatography. To this end, the organisms can advantageously be disrupted beforehand. In this process, the pH value is advantageously kept between pH 4 and 12, preferably between pH 6 and 9, especially preferably between pH 7 and 8.
The culture medium to be used must suitably meet the requirements of the strains in question. Descriptions of culture media for various microorganisms can be found in the textbook “Manual of Methods for General Bacteriology” of the American Society for Bacteriology (Washington D.C., USA, 1981).
As described above, these media which can be employed in accordance with the invention usually comprise one or more carbon sources, nitrogen sources, inorganic salts, vitamins and/or trace elements.
Preferred carbon sources are sugars, such as mono-, di- or polysaccharides. Examples of carbon sources are glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose. Sugars can also be added to the media via complex compounds such as molasses or other by-products from sugar refining. The addition of mixtures of a variety of carbon sources may also be advantageous. Other possible carbon sources are oils and fats such as, for example, soya oil, sunflower oil, peanut oil and/or coconut fat, fatty acids such as, for example, palmitic acid, stearic acid and/or linoleic acid, alcohols and/or polyalcohols such as, for example, glycerol, methanol and/or ethanol, and/or organic acids such as, for example, acetic acid and/or lactic acid.
Nitrogen sources are usually organic or inorganic nitrogen compounds or materials comprising these compounds. Examples of nitrogen sources comprise ammonia in liquid or gaseous form or ammonium salts such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate or ammonium nitrate, nitrates, urea, amino acids or complex nitrogen sources such as corn steep liquor, soya meal, soya protein, yeast extract, meat extract and others. The nitrogen sources can be used individually or as a mixture.
Inorganic salt compounds which may be present in the media comprise the chloride, phosphorus and sulfate salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron.
Inorganic sulfur-containing compounds such as, for example, sulfates, sulfites, dithionites, tetrathionates, thiosulfates, sulfides, or else organic sulfur compounds such as mercaptans and thiols may be used as sources of sulfur for the production of sulfur-containing fine chemicals, in particular of methionine.
Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts may be used as sources of phosphorus. Chelating agents may be added to the medium in order to keep the metal ions in solution. Particularly suitable chelating agents comprise dihydroxyphenols such as catechol or protocatechuate and organic acids such as citric acid.
The fermentation media used according to the invention for culturing microorganisms usually also comprise other growth factors such as vitamins or growth promoters, which include, for example, biotin, riboflavin, thiamine, folic acid, nicotinic acid, pantothenate and pyridoxine. Growth factors and salts are frequently derived from complex media components such as yeast extract, molasses, corn steep liquor and the like. It is moreover possible to add suitable precursors to the culture medium. The exact composition of the media compounds heavily depends on the particular experiment and is decided upon individually for each specific case. Information on the optimization of media can be found in the textbook “Applied Microbiol. Physiology, A Practical Approach” (Editors P. M. Rhodes, P. F. Stanbury, IRL Press (1997) pp. 53-73, ISBN 0 19 963577 3). Growth media can also be obtained from commercial suppliers, for example Standard 1 (Merck) or BHI (brain heart infusion, DIFCO) and the like.
All media components are sterilized, either by heat (20 min at 1.5 bar and 121° C.) or by filter sterilization. The components may be sterilized either together or, if required, separately. All media components may be present at the start of the cultivation or added continuously or batchwise, as desired.
The culture temperature is normally between 15° C. and 45° C., preferably at from 25° C. to 40° C., and may be kept constant or may be altered during the experiment. The pH of the medium should be in the range from 5 to 8.5, preferably around 7.0. The pH for cultivation can be controlled during cultivation by adding basic compounds such as sodium hydroxide, potassium hydroxide, ammonia and aqueous ammonia or acidic compounds such as phosphoric acid or sulfuric acid. Foaming can be controlled by employing antifoams such as, for example, fatty acid polyglycol esters. To maintain the stability of plasmids it is possible to add to the medium suitable substances having a selective effect, for example antibiotics. Aerobic conditions are maintained by introducing oxygen or oxygen-containing gas mixtures such as, for example, ambient air into the culture. The temperature of the culture is normally 20° C. to 45° C. and preferably 25° C. to 40° C. The culture is continued until formation of the desired product is at a maximum. This aim is normally achieved within 10 to 160 hours.
The fermentation broth can then be processed further. The biomass may, according to requirement, be removed completely or partially from the fermentation broth by separation methods such as, for example, centrifugation, filtration, decanting or a combination of these methods or be left completely in said broth. It is advantageous to process the biomass after its separation. However, the fermentation broth can also be thickened or concentrated without separating the cells, using known methods such as, for example, with the aid of a rotary evaporator, thin-film evaporator, falling-film evaporator, by reverse osmosis or by nanofiltration. Finally, this concentrated fermentation broth can be processed to obtain the diterpenoids present therein.
According to an aspect of the invention there is provided a plant transformed with a nucleic acid transcription cassette comprising a nucleotide sequence selected from the group consisting of:
In an embodiment of the invention said plant further comprises a transcription cassette comprising a nucleotide sequence selected from the group consisting of:
In an embodiment of the invention said plant further comprises a transcription cassette comprising a nucleotide sequence selected from the group consisting of:
In an embodiment of the invention said plant further comprises a transcription cassette comprising a nucleotide sequence selected from the group consisting of:
Plants according to the invention can be selected from: corn (Zea mays), canola (Brassica napus, Brassica rapa ssp.), alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cerale), sorghum (Sorghum bicolor, Sorghum vulgare), sunflower (helianthus annuas), wheat (Tritium aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium hirsutum), sweet potato (lopmoea batatus), cassava (Manihot esculenta), coffee (Cofea spp.), coconut (Cocos nucifera), pineapple (Anana comosus), citris tree (Citrus spp.) cocoa (Theobroma cacao), tea (Camellia senensis), banana (Musa spp.), avacado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifer indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia intergrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), oats, barley, vegetables and ornamentals.
Preferably, plants of the present invention are crop plants (for example, cereals and pulses, maize, wheat, potatoes, tapioca, rice, sorghum, millet, cassava, barley, pea, and other root, tuber or seed crops. Important seed crops are oil-seed rape, sugar beet, maize, sunflower, soybean, and sorghum). Horticultural plants to which the present invention may be applied may include lettuce, endive, and vegetable brassicas including cabbage, broccoli, and cauliflower, and carnations and geraniums. The present invention may be applied in tobacco, cucurbits, carrot, strawberry, sunflower, tomato, pepper, chrysanthemum.
Grain plants that provide seeds of interest include oil-seed plants and leguminous plants. Seeds of interest include grain seeds, such as corn, wheat, barley, rice, sorghum, rye, etc. Oil-seed plants include cotton, soybean, safflower, sunflower, Brassica, maize, alfalfa, palm, coconut, etc. Leguminous plants include beans and peas. Beans include guar, locust bean, fenugreek, soybean, garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea, etc.
In preferred embodiment of the invention said plant is from the Soanaceae family (e.g., Nicotiana tabacum or Nicotiana bethamiana).
In a preferred embodiment of the invention said transcription cassette is part of a vector adapted for expression in a plant cell.
According to an aspect of the invention there is provided a cell culture comprising a cell according to the invention.
According to a further aspect of the invention there is provided a process or the manufacture of a lathyrane diterpene, or intermediates thereof, comprising the steps:
i) providing a cell culture according to the invention wherein cells comprised in the culture express a casbene 9-oxidase [SEQ ID NO: 3], and cell culture medium supplemented with a compound selected from the group consisting of casbene, 6-hydroxy-5-keto-casbene, 5-keto-casbene, 5-hydroxycasbene or 9-hydroxy-casbene;
ii) culturing said cells in said culture; and optionally
iii) isolating or purifying synthesized compounds from the cells and/or cell culture medium.
According to an aspect of the invention there is provided a process for the manufacture of at least 9-keto casbene comprising the steps:
According to a further aspect of the invention there is provided a process or the manufacture of a lathyrane diterpene, or intermediates thereof, comprising the steps:
According to a further aspect of the invention there is provided a process or the manufacture of a lathyrane diterpene, or intermediates thereof, comprising the steps:
In a preferred method of the invention said compound is jolkinol C or epi-jolkinol C.
According to an aspect of the invention there is provided an isolated polypeptide comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 2, or an amino acid sequence that has greater than 96% amino acid sequence identity to SEQ ID NO: 2.
In an embodiment of the invention said polypeptide has at least 97%, 98% or 99% amino acid sequence identity to SEQ ID NO: 2.
According to an aspect of the invention there is provided a nucleic acid molecule encoding a polypeptide according to the invention.
In an embodiment of the invention said nucleic acid molecule is part of an expression vector adapted for expression of said nucleic acid molecule.
In an embodiment of the invention said vector is adapted for expression in a microbial host cell.
According to an aspect of the invention there is provided the use of a polypeptide encoded by a nucleic acid molecule comprising a nucleotide sequence set forth in SEQ ID NO 3, or a nucleic acid molecule comprising a nucleotide sequence that has at least 50% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 3 and encodes a polypeptide that has casbene 9-oxidase activity in the transformation of casbene diterpenes, such as 6-hydroxy-5-keto-casbene to lathyrane, lathyrane diterpene intermediates and lathyrane diterpenes.
In a preferred embodiment of the invention said nucleic acid molecule is expressed by an isolated cell.
In a preferred embodiment of the invention said isolated cell is selected from the group a plant cell, a microbial cell, a fungal cell or an algal cell.
Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of the words, for example “comprising” and “comprises”, means “including but not limited to”, and is not intended to (and does not) exclude other moieties, additives, components, integers or steps. “Consisting essentially” means having the essential integers but including integers which do not materially affect the function of the essential integers.
Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.
Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.
An embodiment of the invention will now be described by example only and with reference to the following figures:
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.
SEQ ID NO 1: The full length amino acid sequence of casbene-9-oxidase.
SEQ ID NO 2: The amino acid sequence of casbene-9-oxidase minus an amino terminal membrane associated domain.
SEQ ID NO 3: The cDNA nucleotide sequence encoding casbene-9-oxidase.
SEQ ID NO 4: The cDNA nucleotide sequence of cytochrome P450 JCGZ 2819.
SEQ ID NO 5: The cDNA nucleotide sequence of cytochrome P450 CYP726A20.
SEQ ID NO 6: The cDNA nucleotide sequence of casbene synthase.
SEQ ID NOS 7-66: Primer sequences.
Analysis of Gene Expression by qPCR
RNA extraction, DNase treatment and cDNA synthesis were performed as described previously using three biological and four technical replicates per tissue13. qPCR primers (Table 1) were designed using Primer3Plus23, 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 previously13 and expression levels normalised against an β-actin gene (Genbank accession XM_012232498) using the delta-delta CT method24 with correction for amplification efficiencies obtained using LinReg PCR25.
Gene Cloning and Transient Gene Expression in Nicotiana benthamiana.
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(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 method26. 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 600nm of 1.0 in infiltration buffer (10 mM MgCl2, 200 μM acetosynringone and 0.015% Silwet L-77). Five days after infiltration, ca. 2 cm2 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
In-silico prediction of plastidial transit peptides was performed using ChloroP27. 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.
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 previously13 to yield ca. 1 mg of metabolite.
Data for [4] 6-hydroxy-5-keto-casbene: 1H NMR (700 MHz, CDCl3): δ 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)); 13C NMR (175 MHz, CDCl3): δ 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]+ calcd. for C20H30O2, 303.2319; found, 303.2313.
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 μg of metabolite. Data for [6] 9-keto-casbene: 1H NMR (700 MHz, CDCl3): δ 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)); 13C NMR (175 MHz, CDCl3): δ 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]+ calcd. for C20H30O, 287.2369; found, 287.2368.
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 μg of jolkinol C (mixture of epimers) and 740 μg of 8-hydroxy-5,9-diketocasbene.
Data for [7] 9-hydroxy-5-keto-casbene: 1H NMR (700 MHz, CDCl3): δ 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)); 13C NMR (175 MHz, CDCl3): δ 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]+ calcd. for C20H30O2, 303.2319; found, 303.2313.
Data for [9] Jolkinol C: 1H NMR (700 MHz, CDCl3): δ 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)); 13C NMR (175 MHz, CDCl3): δ 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]+ calcd. for C20H28O3, 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.
Data for [10] epi-Jolkinol C (characterized as a ca 1:4 mixture with Jolkinol C): 1H NMR (700 MHz, CDCl3; 1H resonances for which no multiplicity is given were resolved from Jolkinol C in HSQC but not in 1 D-1H NMR; resonances which are not reported were not resolved from Jolkinol C in either HSQC or 1H NMR): δ 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)); 13C NMR (175 MHz, CDCl3): δ 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).
Data for [11] 8-hydroxy-5,9-diketocasbene: 1H NMR (700 MHz, CDCl3): δ 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)); 13C NMR (175 MHz, CDCl3): δ 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]+ calcd. for C20H28O3, 317.2111; found, 317.2107.
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 tiglianes11 (
Using qPCR, we analysed the expression of the genes present within this cluster
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 neocembrene13. 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 (
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-oxidation13. 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 (
J. curcas cDNA sequences into AgeI and XhoI
J. curcas
J. curcas
J. curcas
A. thaliana
A. thaliana
| Number | Date | Country | Kind |
|---|---|---|---|
| 1604427.3 | Mar 2016 | GB | national |
This is the U.S. National Stage of International Application No. PCT/GB2017/050679, filed Mar. 13, 2017, which was published in English under PCT Article 21(2), which in turn claims the benefit of Great Britain Application No. 1604427.3, filed Mar. 16, 2016.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/GB2017/050679 | 3/13/2017 | WO | 00 |
