CANNABINOID PRECURSOR PRODUCTION
20220154197 · 2022-05-19
Inventors
Cpc classification
C12Y121/03007
CHEMISTRY; METALLURGY
C12N15/8243
CHEMISTRY; METALLURGY
International classification
Abstract
A nucleic acid molecule is disclosed for transiently transforming a plant to produce Δ9-tetrahydrocannabinolic acid synthase, cannabidiolic acid synthase, and/or cannabichromenic acid synthase. The nucleic acid molecule corresponds to a nucleotide sequence comprising at least one of a nucleotide sequence fragment encoding a polypeptide having at least 78% sequence identity to SEQ ID NO: 4, SEQ ID NO:5 or SEQ ID NO:6 or comprising at least 15 contiguous nucleotides of the nucleotide sequence SEQ ID NO: 4, SEQ ID NO:5 or SEQ ID NO:6. The nucleotide sequence further comprises a KDEL or HDEL retrieval tag for targeting the nucleotide sequence to the endoplasmic reticulum. Further aspects relate to a viral vector comprising such nucleic acid molecule, and to methods for producing THCAS, CBDAS, CBCAS, THCA, CBDA, CBCA, THC, CBD and/or CBC based on transient expression of the nucleic acid sequence in a host plant.
Claims
1.-17. (canceled)
18. A nucleic acid molecule for transiently transforming a plant to produce Δ9-tetrahydrocannabinolic acid synthase, THCAS, and/or cannabidiolic acid synthase, CBDAS, and/or cannabichromenic acid synthase, CBCAS, the nucleic acid molecule corresponding to a nucleotide sequence comprising at least one of following: i) a nucleotide sequence fragment encoding a polypeptide having at least 78% sequence identity to SEQ ID NO: 4 or comprising at least 15 contiguous nucleotides of the nucleotide sequence SEQ ID NO: 4; and/or ii) a nucleotide sequence fragment encoding a polypeptide having at least 78% sequence identity to SEQ ID NO: 5 or comprising at least 15 contiguous nucleotides of the nucleotide sequence SEQ ID NO: 5; and/or iii) a nucleotide sequence fragment encoding a polypeptide having at least 78% sequence identity to SEQ ID NO: 6 or comprising at least 15 contiguous nucleotides of the nucleotide sequence SEQ ID NO: 6; said nucleotide sequence further comprising a KDEL or HDEL retrieval tag for targeting the nucleotide sequence to the endoplasmic reticulum.
19. The nucleic acid molecule of claim 18, wherein said nucleotide sequence further comprises a polyhistidine tag or other purification tag to facilitate purification.
20. The nucleic acid molecule of claim 18, wherein said nucleic acid molecule comprises at least one heterologous moiety and/or at least one linker and/or at least one signal sequence and/or at least one detection label.
21. The nucleic acid molecule of claim 20, wherein said nucleic acid molecule comprises said signal sequence corresponding to a PR-1a signal peptide, a pathogenesis-related protein 4 or a pathogenesis-related protein STH-2.
22. The nucleic acid molecule of claim 18, wherein said nucleic acid molecule corresponds to, or comprises, SEQ ID NO: 1 or SEQ ID NO:2 or SEQ ID NO:3.
23. A viral vector comprising the nucleic acid molecule of claim 18.
24. The viral vector of claim 23, wherein said viral vector further comprises a further nucleotide sequence for deglycosylation.
25. The viral vector of claim 24, wherein said further nucleotide sequence is codon-optimized for Nicotiana benthamiana or Nicotiana tabacum species.
26. A method for producing Δ9-tetrahydrocannabinolic acid synthase, THCAS, and/or cannabidiolic acid synthase, CBDAS, and/or cannabichromenic acid synthase, CBCAS, said method comprising: transiently transforming a plant with a nucleic acid molecule in accordance with claim 18, and extracting THCAS and/or CBDAS and/or CBCAS from plant biomass obtained from said transiently transformed plant.
27. The method of claim 26, wherein said plant is a Nicotiana benthamiana or Nicotiana tabacum plant.
28. The method of claim 26, further comprising filtrating and/or purifying the extracted THCAS and/or CBDAS and/or CBCAS.
29. The method of claim 28, wherein said filtrating and/or purifying comprises a chromatography process.
30. The method of claim 26, wherein said plant is also transiently transformed to co-express a deglycosylation sequence to obtain the expression of THCA and/or CBDAS and/or CBCAS without glycosylation.
31. The method of claim 26, comprising Introducing the nucleotide sequence, using the viral vector, into at least one Agrobacterium tumefaciens strain.
32. The method of claim 31, wherein said at least one Agrobacterium tumefaciens strain comprises a combination of a plurality of Agrobacterium tumefaciens strains comprising or consisting of GV3101, C58C1, and LBA4404 and wild-type strains A4, At06, At10, and At77.
33. A method for producing Δ9-tetrahydrocannabinolic acid (THCA), and/or cannabidiolic acid (CBDA), and/or cannabichromenic acid (CBCA), comprising a method in accordance with claim 26, and converting the THCAS to THCA, and/or the CBDAS to CBDA, and/or the CBCAS to CBCA, by cannabigerolic acid oxidocyclization without hydroxylation.
34. The method of claim 33, further comprising a decarboxylation performed on the obtained THCA, CBDA and/or CBCA to respectively yield THC, CBD and/or CBC.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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[0073] The drawings are schematic and not limiting. Elements in the drawings are not necessarily represented on scale. The present invention is not necessarily limited to the specific embodiments of the present invention as shown in the drawings.
DETAILED DESCRIPTION OF EMBODIMENTS
[0074] Notwithstanding the exemplary embodiments described hereinbelow, is the present invention only limited by the attached claims. The attached claims are hereby explicitly incorporated in this detailed description, in which each claim, and each combination of claims as allowed for by the dependency structure defined by the claims, forms a separate embodiment of the present invention.
[0075] Raw materials being used are not always described in detail. Such raw materials may be commercially available products. While process steps and/or preparation methods are not always described in detail, such process steps and/or preparation methods may be considered to be well-known by those skilled in the art.
[0076] The word “comprise,” as used in the claims, is not limited to the features, elements or steps as described thereafter, and does not exclude additional features, elements or steps. This therefore specifies the presence of the mentioned features without excluding a further presence or addition of one or more features.
[0077] In this detailed description, various specific details are presented. Embodiments of the present invention can be carried out without these specific details. Furthermore, well-known features, elements and/or steps are not necessarily described in detail for the sake of clarity and conciseness of the present disclosure.
[0078] Embodiments of the present invention are specifically described below with reference to the embodiments, so as to facilitate the understanding of the present invention by those skilled in the art. It should be noted that the embodiments are only used for further explanation of the present invention and cannot be understood to necessarily limit the protection scope of the present invention. The person skilled in the art will recognize that the protection scope of the present invention can be better understood by those skilled in the art.
[0079] In a first aspect, the present invention relates to a nucleic acid molecule for transiently transforming a plant to produce Δ9-tetrahydrocannabinolic acid synthase (THCAS) and/or cannabidiolic acid synthase (CBDAS) and/or cannabichromenic acid synthase (CBCAS). The nucleic acid molecule corresponds to a nucleotide sequence comprising at least one of following: [0080] i) a nucleotide sequence fragment encoding a polypeptide having at least 78% sequence identity to SEQ ID NO: 4 or comprising at least 15 contiguous nucleotides of the nucleotide sequence SEQ ID NO: 4; and/or [0081] ii) a nucleotide sequence fragment encoding a polypeptide having at least 78% sequence identity to SEQ ID NO: 5 or comprising at least 15 contiguous nucleotides of the nucleotide sequence SEQ ID NO: 5; and/or [0082] iii) a nucleotide sequence fragment encoding a polypeptide having at least 78% sequence identity to SEQ ID NO: 6 or comprising at least 15 contiguous nucleotides of the nucleotide sequence SEQ ID NO: 6.
[0083] The nucleotide sequence further comprises a KDEL or MEL retrieval tag for targeting the nucleotide sequence to the endoplasmic reticulum.
[0084] In a second aspect, the present invention relates to a viral vector comprising such nucleic acid molecule.
[0085] In a third aspect, the present invention relates to a method for producing Δ9-tetrahydrocannabinolic acid synthase (THCAS), and/or cannabidiolic acid synthase (CBDAS), and/or cannabichromenic acid synthase (CBCAS). The method comprises transiently transforming a plant with a nucleic acid molecule in accordance with embodiments of the first aspect of the present invention. The method comprises extracting THCAS and/or CBDAS and/or CBCAS from plant biomass obtained from the transiently transformed plant.
[0086] In a fourth aspect, the present invention relates to a method for producing Δ9-tetrahydrocannabinolic acid (THCA), and/or cannabidiolic acid (CBDA), and/or cannabichromenic acid (CBCA), comprising a method in accordance with embodiments of the third aspect of the present invention.
[0087] The method comprises: [0088] converting the (e.g. purified) THCAS to THCA through CBGA oxidocyclization without hydroxylation, e.g. by adding CBGA to the supernatant for 6 to 8 hours of incubation; [0089] converting the (e.g. purified) CBDAS to CBDA through CBGA oxidocyclization without hydroxylation, e.g. by adding CBGA to the supernatant for 6 to 8 hours of incubation; and/or [0090] converting the (e.g. purified) CBCAS to CBCA through CBGA oxidocyclization without hydroxylation, e.g. by adding CBGA to the supernatant for 6 to 8 hours of incubation.
[0091] Thus, the present invention provides engineered recombinant THCAS, CBDAS, and/or CBCAS fusion constructs, e.g. the nucleic acid molecule referred to hereinabove, as well as viral vectors comprising such construct and methods involving the use thereof.
[0092] Embodiments may provide or enable a more efficient and cost-effective process, capable of producing enzymes that are involved in cannabinoid biosynthesis by transient transformation of a plant, e.g. Nicotiana benthamiana. An illustrative method in accordance with embodiments may comprise inserting nucleic acid molecule, e.g. incorporating the genes of interest, in Agrobacterium tumefaciens and introducing the construct by agroinfiltration into the endoplasmic reticulum (ER) of plant cells, e.g. of 5 weeks old Nicotiana benthamiana plants.
[0093] Nicotiana benthamiana may be considered as a bioreactor of choice for the transient expression of recombinant protein in a manufacturing setting. The small ornamental plant has a high leaf to stem ratio and is very prolific in hydroponic culture. Nicotiana benthamiana tolerates the transfection vectors and delivers maximum synthesis of heterologous proteins in 5-7 days after transfection. Scale-up of this bioreactor is a matter of growing more plants not re-engineering processes. However, the skilled person may transpose the findings of the present disclosure to other host plant species, as he deems suitable, which are therefore also considered to be covered by embodiments of the present invention.
[0094] Plants have advantageously all the required eukaryotic cell machinery to accurately produce plant, human and animal proteins. Thus, the bioreactor may be an individual plant. Plants are well suited to express complex proteins, and minimize risk by not supporting growth of human or animal pathogens.
[0095] A benefit of this approach is that the production of the cannabinoid is fast and continuous, low cost and reliable, and only a specific cannabinoid is produced or a subset is produced. The extraction and purification process of the cannabinoid may be straightforward since there is only a single cannabinoid or a selected few cannabinoids present in the plant biomass. The process can be upscaled in a linear fashion, e.g. simply by growing more plants. Furthermore, it is a sustainable process which is more environmentally friendly than synthetic production and can also be purified to meet the requirements for pharmaceutical applications.
[0096] The acidic forms of the cannabinoids (THCA, CBDA, and CBCA) obtained through CBGA oxidocyclization without hydroxylation of THCAS, CBDAS, and CBCAS respectively, may be used as a pharmaceutical product or the acidic cannabinoids can be turned into their neutral form for use, for example THC, CBD, and CBC may be produced from THCA, CBDA, and CBCA respectively through decarboxylation. The resulting cannabinoid products may be used in the pharmaceutical/nutraceutical industry, e.g. to treat a wide range of health issues.
[0097] The contacting can for example be achieved by mixing the CBGA with recombinant THCAS, CBDAS, and CBCAS in a solution and/or in an immobilized state under conditions and for a length of time suitable to convert at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% of the CBGA to THCA, CBDA, and CBCA.
[0098] With regards to expressing the proteins in transiently transformed Nicotiana benthamiana, THCAS, CBDAS, and/or CBCAS encoding sequences may be fused to the endoplasmic reticulum (ER) retrieval tag, e.g. KDEL, and a poly-histidine tag, e.g. all fused to the C-terminus of the proteins (i.e. of the protein transcribed by the nucleic acid molecule). The KDEL tag allows proteins to accumulate in the ER, a strategy that may lead to higher accumulation and/or reduce in planta proteolytic degradation.
[0099] The THCAS construct comprises a catalytic nucleotide sequence (e.g. without the signal peptide) as represented by SEQ ID NO: 4, or a suitable analog or sufficient part thereof (i.e. sufficient to express the intended THCAS enzyme). Likewise, a CBDAS catalytic nucleotide sequence may be represented by SEQ ID NO: 5, and a CBCAS catalytic nucleotide sequence by SEQ ID NO: 6 (both considered with a tolerance for suitable analogs or substantial fragments thereof).
[0100] The nucleic acid molecule may comprise a bacterial PNGase F gene sequence for obtaining fully functional deglycosylated THCAS and/or CBDAS and/or CBCAS.
[0101] The nucleic acid molecule comprises an endoplasmic reticulum retrieval tag, e.g. a KDEL tag, e.g. located at the C-terminus of the protein.
[0102] The nucleic acid molecule may comprise a (nucleotide sequence fragment coding for a) signal peptide for the intended host plant, e.g. a Nicotiana tabacum PR-1a signal peptide.
[0103] The nucleic acid molecule may comprise a purification tag, such as a polyhistidine tag. For example, a tag peptide may be used, e.g. engineered into the primary structures of the engineered fusion enzyme, to facilitate purification of produced THCAS, CBDAS and/or CBCAS. Examples include a polyhistidine tag, a streptavidin (biotin-binding) tag, a flagellar antigen tag, a hemagglutinin tag, or a glutathionine S-transferase tag, among others.
[0104] For example, the nucleic acid molecule may comprise said catalytic nucleotide sequence for THCAS, CBDAS and/or CBCAS, a Nicotiana tabacum PR-1a signal peptide added to the N-terminus, followed by an endoplasmic reticulum (ER) retrieval tag KDEL and a polyhistidine tag, both fused to the C-terminus of the protein.
[0105] The nucleic acid molecule may be codon-optimized for expression in a specific host plant species, such as Nicotiana benthamiana species.
[0106] SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 disclose nucleotide sequences of such nucleic acid molecules for respectively transiently expressing THCAS, CBDAS and CBCAS in Nicotiana Benthamiana, i.e. nucleotide sequences of respectively PR-1A/THCAS/PNGASE-F/ER/6×HIS, PR-1A/CBDAS/PNGASE-F/ER/6×HIS and PR-1A/CBCAS/PNGASE-F/ER/6×HIS constructs, each codon-optimized for Nicotiana benthamiana.
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[0108] The above constructs can also be cloned into two separate vectors; one functioning as the construct containing the PNGase F gene sequence, the other functioning as the construct containing the THCAS, CBDAS and/or CBCAS gene. Embodiments of the present invention may relate to a combination of such separate vectors.
[0109] A short (346 bp) but strong constitutive cauliflower mosaic virus 35S promoter (P35S), a kozak translation initiation sequence, and a nopaline synthase polyadenylation terminator signal for regulation of gene expression may be utilized. The use of constructs for N- and C-terminally truncated and both N- and C-terminally truncated versions of both nucleotide sequences and all homologous coding sequences, with a minimum of 45% sequence identity, are also within the scope of this present invention.
[0110] Embodiments of the present invention may relate to a composition comprising the nucleic acid molecule, e.g. with no additional protein components are present in said composition.
[0111] For example, the percentage by weight of the recombinant THCAS, CBCAS and/or CBDAS fusion enzyme (i.e. the nucleic acid molecule) in such composition may be from about 0.00001% to 99.99999%, e.g. from about 0.00001% to 99.99999%, e.g. from about 0.0001% to 99.9999%, e.g. from about 0.001% to 99.999%, e.g. from about 0.001% to 99.999%, e.g. from about 0.01% to 99.99%, e.g. from about 0.1% to 99.9%, e.g. from about 1% to 99%.
[0112] The acquired THCAS, CBDAS, and CBCAS after expression and purification can be further biosynthesized by CBGA through oxidocyclization without hydroxylation to obtain THCA, CBDA, and CBCA respectively. The enzymaticaly synthesized THCA, CBDA, and CBCA can then be carboxylated, e.g. by heating at 120° C. to obtain THC, CBD, and CBC respectively. The obtained THCA, CBDA, and CBCA can also function as a basis for further modification to other cannabinoids for example via degradation or isomerization.
[0113] In the agroinfiltration experiments discussed hereinabove, 5- to 7-weeks-old N. benthamiana plants were used. Nicotiana benthamiana seeds were grown in a greenhouse. Seedling and germination of Nicotiana benthamiana plants were carried out under light emitting diode (LED) illumination 24 hours/day, 7 days/week. Red and blue diodes were selected that match the action spectrum of photosynthesis (25% blue and 75% red). Other wavelengths may be less or not productive. The LEDs were focused on the plants. Plants were grown to usable maturity 20% faster by this approach as compared to other commercial solutions. All seeds were germinated using identical soil and fertilizer at 26.6° C.
[0114] For the biosynthesis of the genes of interest, respectively THCAS (UniProtKB-Q8GTB6, entry version 71, sequence version 1, last sequence update Mar. 1, 2003), CBDAS (UniProtKB-A6P6V9, entry version 50, sequence version 1, last sequence update Aug. 21, 2007), and CBCAS (GenBank: LY658672.1, cf. KR 1020190025485-A/8 11 Mar. 2019) from Cannabis sativa (hemp, marijuana), were used in combination with the bacterial PNGase F gene sequence (UniprotKB-P21163, entry version 107, sequence version 2, last sequence update Nov. 1 1991), a Nicotiana tabacum PR-1a signal peptide (UniprotKB-Q40557, entry version 66, sequence version 1, last sequence update Nov. 1, 1996; only signal peptide part: positions 1-30 of sequence) added to the N-terminus, a KDEL endoplasmic retrieval tag, and a poly-histidine tag added to the C-terminus, forming a template to biosynthesize the novel engineered recombinant THCAS, CBDAS, and CBCAS enzymes. The restriction sites for EcoRI and BgIII were added to the 5′ and 3′ ends of the gene, respectively. Codon usage was optimized for Nicotiana benthamiana expression, and the gene synthesis was done by Genscript Inc. The THCAS 2436 bp-fragment and CBDAS 2613 bp-fragment were cloned into pUC57 vectors to facilitate gene subcloning into plant expression vector. (See SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 for the full nucleotide sequences of these illustrative THCAS, CBDAS, and CBCAS constructs respectively, and
[0115] The Cannabis genome sequence was analyzed for genes with high similarity to THCA synthase using BLAST analysis. This led to the identification of a gene with 96% nucleotide similarity to THCA synthase. Based on subsequent biochemical characterization, the authors named this gene Cannabis sativa cannabichromenic acid synthase (CBCAS) and was deposited as the entire sequence, including signaling peptide sequence, in the Genbank database under registration: LY658672.1. We've predicted the signaling peptide sequence (see
[0116] As the native THCAS, CBDAS, and CBCAS genes employs tandem rare codons, which could reduce the efficiency of translation or even disengage the translational machinery, the codon usage bias in Nicotiana benthamiana was used by upgrading the codon adoption index (CAI) from 0.72 to 0.86. The GC content and unfavorable peaks have been optimized to prolong the half-life of the mRNA. The Stem-Loop structures, which impact ribosomal binding and stability of mRNA, were broken. In addition, negative cis-acting sites were screened and successfully modified.
[0117] For the construction of a plant expression vector, the entire THCAS 2436 bp-fragmental-orf, CBDAS 2613 bp-fragmental-orf, and CBCAS 2616 bp-fragmental-orf were excised from pUC57-THCAS, pUC57-CBDAS, and pUC57-CBCAS by digesting with both EcoRI and BglII and subcloned into pPRP[Exp]-CaMV35S binary vectors in the corresponding sites and under the control of 35S-promoter. The transformed colonies were confirmed by restriction digestion. The recombinant plasmids pCambia-THCAS, pCambia-CBDAS, and pCambia-CBCAS, were extracted from the selected colony and consequently was transformed into Agrobacterium to carry out agroinfilteration experiments.
[0118] The pPRP[Exp]-CaMV35S-THCAS, pPRP[Exp]-CaMV35S-CBDAS, and pPRP[Exp]-CaMV35S-CBCAS constructs were transformed into Agrobacterium tumefaciens strains GV3101 C58C1 and LBA4404 and wild-type strains A4, At06, At10, and At77 using electroporation technique at 2.5 kV, 25 mF and 400Ω. The transformed cells were plated on LB agar medium containing 50 mg/ml Ampicillin (Sigma Aldrich).
[0119] Agroinfiltration was used for transient expression in Nicotiana benthamiana with Agrobacterium tumefaciens strains as previously described. 100 μl of transformed Agrobacterium frozen cells stock was inoculated in 5 ml LB broth (Thermo Fisher Scientific) and supplemented with 50 mg/ml Ampicillin for THCAS, CBDAS, and CBCAS respectively. Overnight, the cultures were incubated at 28° C., shaking at 220 rmp. 2×500 μl was used to inoculate 2× 50 ml of LB medium. The cultural cells were incubated at 28° C. shaking at 220 rpm until the culture had reached an O.D.600=0.6. The cells were harvested by centrifugation at 6000 rpm and resuspended in 2×50 ml IVIES buffer (10 mM IVIES; pH 5.5, 10 mM MgCl2). These mixtures were incubated for 2.5 hours at room temperature with 120 μM acetosyringone and was added to the Agrobacterium suspension in infiltration buffer (1×MS, 10 mM MES, 2.5% glucose) for THCAS, CBDAS, and CBCAS respectively. For the effect of monosaccharide on induction of vir gene, different percentages of glucose (0, 1, 2 or 4%) were added to the Agrobacterium suspension in the infiltration buffer (lx MS, 10 mM IVIES, 200 μM acetosyringone). 5- to 7-weeks old N. benthamiana plants were infiltrated in a vacuum chamber by submerging N. benthamiana plant aerial tissues in Agrobacterium suspension and applying a 50-400 mbar vacuum for 30, 45 or 60 seconds.
[0120] The most optimal infiltration was routinely applied at 50-100 mbar for 60 sec. Once the vacuum was broken, infiltrated N. benthamiana plants were removed from the vacuum chamber, thoroughly rinsed in water, and grown for 5-7 days under the same growth conditions used for pre-infiltration growth. To avoid any variability, the leaves and location on the leaf, comparably-sized leaves for each plant of similar age were agro-infiltrated for each experiment.
[0121] For the Southern Blot Analysis, individual agroinfiltrated leaves with the pPRP[Exp]-CaMV35S-THCAS, pPRP[Exp]-CaMV35S-CBDAS, and pPRP[Exp]-CaMV35S-CBCAS constructs were harvested at different time-intervals; 4, 6, 8 and 10 days post-infiltration, in addition to un-infiltrated plants were used as control. DNA of infiltrated leaves was extracted by DNeasy Plant DNA mini kit (QIAGEN) and fragmented by endonuclease enzyme; EcoRI. Both recombinant THCAS 2436 bp-fragmental-orf, CBDAS 2613 bp-fragmental-orf, and CBCAS 2616 bp-fragmental-orf released from pUC57-THCAS, pUC57-CBDAS, and pUC57-CBCAS respectively were used as a probe. Labeling and detection were carried out using Biotin Deca Label DNA Labeling Kit (Thermo Fisher Scientific) and Biotin chromogenic Detection kit (Thermo Fisher Scientific) respectively.
[0122] For the Western Blot Analysis, individual agroinfiltrated Nicotiana bentamiana leaves were harvested and grinded in liquid nitrogen. Total proteins were extracted using SDS-extraction buffer (2% SDS, 0.2% bromopheol blue, 10% glycerol), and the extracts were clarified by centrifugation at 14,500 g for 20 min at 4° C. The, supernatants were transferred to fresh tubes, and the protein content of THCAS, CBDAS, and CBCAS were determined (Bradford assay—1976). Total proteins (40 μg each) were separated by SDS-PAGE and then transferred onto polyvinylidene difluoride (PVDF) membranes. Polyvinylidene difluoride membranes were blocked for at least 2 hours and then probed with rabbit anti-THCAS, rabbit anti-CBDAS, and rabbit anti-CBCAS in a 1:500 dilution. After extensive washing, the membrane was incubated with the appropriate secondary antibody in a 1:5000 dilution and then was conjugated to alkaline phosphatase. BCIP/NBT (Amresco) was used for immunodetection.
[0123] For the detection of chimeric gene by real-time polymerase chain reaction (RT-PCR) an illustra RNAspin mini kit (GE healthcare) was used to extract, total RNA from the agroinfiltrated leaves. Oligonucleotides pair at the core region was designed to detect the presence of the THCAS, CBDAS, and CBCAS genes at the core region; using THCAS-specific forward primer: 5′-CTCGTATACACTCAACACGACC-3 ‘ (SEQ ID NO: 7) and reverse primer: GTAGGACATACCCTCAGCATCATG-3’ (SEQ ID NO: 8), CBDAS-specific forward primer 5′-GAGGCTATGGACCATTGA (SEQ ID NO: 9) and reverse primer: 5′-GGACAGCAACCAGTCTAA-3′ (SEQ ID NO: 10), and CBCAS-specific forward primer 5′-CGGATGTACTGTTATGCTCCAA-3′ (SEQ ID NO: 11) and reverse primer: 5′-AAGCTTTCATGGTACCCCATGATGATGCCGTGGAAGAG-3′ (SEQ ID NO: 12). PCR parameters have not been previously reported for the co-dominant DNA marker (Onofri et al. 2015; Pacifico et al. 2006) and these were optimised as follows: each reaction contained 1.5 mM MgCl2, 0.2 mM dNTPs, 0.4 μM for the forward primer and 0.2 μM for THCAS-specific, CBDAS-specific, and CBCAS-specific reverse primers, and 2 U Platinum® Taq DNA Polymerase (Life Technologies #10966-034). Thermocycling parameters were 94° C. for 2 min, then 25 cycles of 94° C. for 30 s, 58° C. for 30 s, 72° C. for 1 min 15 s. PCR reactions were performed in 0.2 mL 96 well PCR plates (Thermo Scientific #AB-0600) sealed with flat cap strips (Thermo Scientific #AB-0786) using a Gradient Palm-Cycler™ (Corbett Life Science) and occurred in a total volume of 50 μL. D589 and B1080/B1192 amplification products were separated by electrophoresis on a 1.5 and 1% SeaKem® LE agarose gel (Cambrex #50004) stained with GelRed™ (Biotium #41003) respectively. Amplification products were then visualized under UV illumination using the Bio-Rad Molecular Imager® Gel Doc™ XR+ system using Image Lab™ software.
[0124] To confirm the expression of the transgene after agroinfiltration, a direct ELISA protocol was carried out. ELISA-extraction buffer (2% PVP, 0.03 MNa2SO3) was used to extract total proteins for both TCHAS, CBDAS, and CBCAS. ELISA 96-well plates (Thermo Fisher Scientific) were coated with 250 μl antigen and total soluble protein for both THCAS, CBDAS, and CBCAS followed by overnight incubation at 4° C. Plates were washed three times with washing buffer the next day, three times, 5 minutes each. By adding 250 μl blocking buffer (PBS-Tween 20, 5% low-fat milk), the remaining protein-binding sites were blocked and incubated for 2.5 hrs at room temperature.
[0125] After washing with PBS and Tween 20, anti-TCHAS, anti-CBDAS, anti-CBCAS antibodies were diluted by 1:1000 in a blocking buffer and 250 μl was added to each well. The plates were incubated in a humid chamber at 37.5° C. for 3.5 hrs. The plates were decanted and washed three times, 5 minutes each. 250 μl of substrate buffer (0.3 g (NaN3), 96 ml diethanolamine, 600 ml H.sub.2O) was added to the plates followed by incubation at room temperature until the color developed. With an automated ELISA reader (BIOBASE 2000), absorbencies were finally read at 630 nm wavelength, 15 minutes each. ELISA values were expressed as the mean absorbance at wavelength (λ=640A°). Compared to negative control (C−), the S3, S5, S7 and S10 infiltrated samples showed positive results after 15 min and 30 min of read time. The highest expression level of THCAS, CBDAS, and CBCAS genes within Nicotiana benthamiana leaves were obtained at the fifth day post-infiltration followed by descending in expression level at 7th and 10th days post-infiltration.
[0126] The proteins were purified by immobilised metal affinity chelating chromatography (IMAC). To immobilise the metal ions on Chelating Sepharose Fast Flow, a solution of 200 mM NiSO4 (Sigma Aldrich) was passed through the column (GE Life Sciences). The column was washed with distilled water containing 0.02% azide to remove excess NiSO4. The column was then equilibrated with 10 column volumes of buffer ANiS (50 mM Tris-HCl, pH 7.4, 50 mM NaCl, 100 mM Imidazole, 10 mM β-Mercaptoethanol, 0.02% (w/v) Azide) with the flow rate of 3 ml/min. The crude extracts in buffer ANiS were applied to the column of Nickel Chelating Sepharose Fast Flow (column volume 10 ml) (GE Life Sciences). The column was washed with at least 10 column volumes of buffer ANiS, and was then switched to a linear gradient increasing the concentration of imidazole from 100 mM (buffer ANiS) to 500 mM of buffer BNiS (50 mM Tris-HCl, pH 7.4, 50 mM NaCl, 500 M Imidazole, 10 mM β-Mercaptoethanol, 0.02% (w/v) Azide.
[0127] To concentrate target proteins, ultrafiltration was used. The protein solutions were added to a stirred cell with a volume of either 10 ml or 50 ml (Amicon®, Millipore Sigma) and passed through an ultrafiltration cut disc regenerated cellulose membrane with a molecular weight cut off of 30 kDa (Ultracel®, Millipore Sigma). Using centrifugal filter devices (Microcon®, Millipore Sigma) according to the supplier's protocol, a concentration of small volumes of protein (300-600 μl) was achieved. To retain the target proteins, the exclusion limit of the membrane was selected.
[0128] To measure enzymatic activity of THCAS, CBDAS, and CBCAS, 150 mg of frozen plant material for each was homogenized in 500 ll of THCAS, CBDAS, and CBCAS reaction buffer (100 mM trisodium citrate, pH 5.5) and centrifuged (17,000×g, 15 min). Afterwards, the supernatants were incubated with CBGA (final concentration 0.05 mM, 1.9% (v/v) ACN) for 2 h at 37° C. To terminate the reactions, 275 μl of ice cold acetonitrile was added, followed by incubation on ice for 30 min. Finally, the supernatants were purified two times from solid particles by centrifugation (17,000×g, 30 min, 4° C.).
[0129] THCAS, CBDAS, and CBCAS assay extracts were analyzed by HPLC-MS using a Poroshell 120SB-C18 (3.0 9 150 mm, 2.7 μm) column. Detailed parameters of HPLC-MS analysis is described in the supplementary data. For confirmation of THCAS, CBDAS, and CBCAS mass spectra of compounds were juxtaposed with mass spectra of authentic standards and further confirmed by LC-ESI-MS/MS. Quantification of THCAS, CBDAS, and CBCAS was done by integration of peak areas of the UVchromatograms at 260 nm. The enzyme showed 137±14 fkat g.sub.FW.sup.−1 activity towards THCA production, while the activity towards CBDA was 132±11 fkat g.sub.FW.sup.−1 and the activity towards CBCA was 129±13 fkat g.sub.FW.sup.−1.
[0130] THCAS, CBDAS, and CBCAS were able to produce up to 2.11 g of THCA/kg leaf biomass, 2.03 g of CBDA/kg leaf biomass, and 1.48 g of CBCA/kg leaf biomass after CBGA feeding to the culture natant, demonstrating the capacity of transiently transformed Nicotiana benthamiana in the biosynthesis of (novel) cannabinoids with enhanced properties by the incorporation of tailoring enzymes. Furthermore, as aforementioned in the summary, the present invention enables the production of cannabinoid precursor enzymes on a continuous basis, week after week, which obviously is advantageous towards the long cultivation and harvesting times in traditional cultivation of Cannabis. These strategies will help to support the potential value of cannabinoids as pharmaceutical drugs.