high-level in vivo biosynthesis and isolation of water-soluble cannabinoids in plant systems

专利摘要:
the inventive technology refers to systems and methods for intensified in vivo production, accumulation and modification of cannabinoids. in one embodiment, the invention may include systems and methods for enhanced in vivo biosynthesis of chemically modified water-soluble cannabinoids in an integral plant or a cell suspension culture system.
公开号:BR112019019966A2
申请号:R112019019966
申请日:2018-03-26
公开日:2020-04-28
发明作者:Elton Carvalho Goncalves；Richard T Sayre；Tawanda Zidenga
申请人:Trait Biosciences Inc；
IPC主号:

专利说明:

DESCRIPTIVE REPORT
BHOSSÍNTESE UN WO GIVE HIGH LEVEL AND INSULATION GIVE CANABUNOUDES SOLUBE IN WATER IN PLANT SYSTEMS [001] This order claims the benefit and priority of Provisional Orders US 62 / 476,080, filed on March 24, 2017 and 62 / 588,662, deposited on November 20, 2017 and 62 / 621,166, filed on January 21, 2018. All descriptive reports and figures of the orders mentioned above are incorporated herein, in their entirety by reference.
SEQUENCE LISTING [002] This application contains a sequence listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety.
TECHNICAL FIELD [003] The field of the present invention generally relates to plant molecular biology and plant biotechnology. More specifically, the invention relates to new systems, methods and compositions for the in vivo production, modification and isolation of cannabinoid compounds from plant systems including whole plant and / or plant cell culture systems. In certain preferred embodiments, the inventive technology includes a new system of genetic modification of a plant or plant cell suspension culture to produce, modify and / or accumulate one or more cannabinoid targets in Cannabis and / or Nicotiana Benthamiana and / or Nicotiana tabacum
BACKGROUND [004] Cannabinoids are a class of specialized compounds synthesized by Cannabis. They are formed by condensation of precursors of terpenes and phenols. They include these most abundant forms: Delta-9-tetrahydrocannabinol (THC), cannabidiol (CBD), cannabichromene (CBC) and cannabigerol (CBG). Another cannabinoid, cannabinol (CBN), is formed from THC as a degradation product and can be detected in some plant strains. Typically,
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THC, CBD, CBC and CBG occur together in different proportions in the various plant strains.
[005] Cannabinoids are generally classified into two types, neutral cannabinoids and cannabinoid acids, based on whether they contain a carboxyl group or not. It is known that, in fresh plants, the concentrations of neutral cannabinoids are much lower than those of cannabinoid acids. One strain of Cannabis sativa contains approximately 61 compounds belonging to the general class of cannabinoids. These cannabinoids are generally lipophilic, nitrogen-free, mainly phenolic compounds, and are biogenetically derived from a monoterpene and phenol, cannabinoid acids from a monoterpene and phenol carboxylic acid and have a C21 for base material.
[006] Cannabinoids also find their corresponding carboxylic acids in plant products. In general, carboxylic acids serve as a biosynthetic precursor. For example, these compounds arise in vivo from the carboxylic acids THC by decarboxylation of the tetrahydrocannabinols Δ9- and A8-THC and CBD of the associated cannabidiol. As generally shown in Fig. 28, THC and CBD can be artificially derived from tetrahydrocannabinolic acid from its acid precursor (THCA) and cannabidiolic acid (CBDA) by non-enzymatic decarboxylation.
[007] Cannabinoids are widely consumed, in a variety of ways around the world. Cannabis-rich cannabis preparations, either in grass (ie, marijuana) or in resin form (ie, hash oil), are used by an estimated 2.6 to 5.0% of the world population (UNODC , 2012). Pharmaceutical products containing cannabinoids, which contain natural extracts of cannabis (Sativex®) or synthetic cannabinoids dronabinol or nabilone, are available for medical use in many countries.
[008] As noted above, Δ-9-tetrahydrocannabinol (also known as THC) is one of the main biologically active components in the Cannabis plant that has been approved by the Food and Drug Administration (FDA) for the control of
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3/114 nausea and vomiting associated with chemotherapy and, more recently, to stimulate the appetite of AIDS patients suffering from wasting syndrome. The drug, however, shows other biological activities that lend itself to possible therapeutic applications, such as in the treatment of glaucoma, headaches, migraines, spasticity, anxiety and as an analgesic.
[009] Indeed, it is well documented that agents, such as cannabinoids and endocannabinoids, that activate cannabinoid receptors in the body modulate appetite and relieve nausea, vomiting and pain (Martin BR and Wiley, J. L, Mechanism of action of cannabinoids : how it may lead to treatment of cachexia, emesis and pain, Journal of Supportive Oncology 2: 1-10, 2004), multiple sclerosis (Pertwee, RG, Cannabinoids and multiple sclerosis, Pharmacol. Ther. 95, 165-174, 2002 ) and epilepsy (Wallace, MJ, Blair, RE, Falenski, K. WW., Martin, BR, and DeLorenzo, RJ Journal Pharmacology and Experimental Therapeutics, 307: 129137, 2003). In addition, CB2 receptor agonists have been shown to be effective in treating pain (Clayton N., Marshall FH, Bountra C, O'Shaughnessy CT, 2002. CB1 and CB2 cannabinoid receptors are implicated in inflammatory pain. 96, 253-260; Malan TP, Ibrahim Μ. M., Vanderah TW, Makriyannis A., Porreca F., 2002. Inhibition of pain responses by activation of CB (2) cannabinoid receptors.Chemistry and Physics of Lipids 121,191 -200; Malan TP, Jr., Ibrahim Μ. M., Deng H., Liu Q., Mata Η. P., Vanderah T., Porreca F., Makriyannis A., 2001. CB2 cannabinoid receptor-mediated peripheral antinociception. 93, 239-245 .; A., Mata Η. P., Ibrahim Μ. M., Vanderah TW, Porreca F., Makriyannis A., Malan TP, Jr., 2003. Inhibition of inflammatory hyperalgesia by activation of peripheral CB2 cannabinoid receptors. Anesthesiology 99, 955 -960) and multiple sclerosis (Pertwee, RG, Cannabinoids and multiple sclerosis, Pharmacol. Ther. 95, 165-174, 2002) in animal models.
[0010] More recently, several states have approved the use of Cannabis and cannabinoid-infused products for recreational and medical use. As these new medical and commercial markets develop, the
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4/114 the need to develop more efficient production and isolation of cannabinoid compounds. Traditional cannabinoid production methods generally focus on the extraction and purification of cannabinoids from raw materials harvested from Cannabis. However, traditional cannabinoid extraction and purification methods present several technical and practical problems that limit their usefulness.
Limitations of Traditional Cannabinoid Production and Extraction Methods [0011] For example, in US Patent 6,403,126 (Webster et al.), Cannabinoids and other related compounds are isolated from raw materials harvested from Cannabis and treated with an organic solvent , typically a petroleum-derived hydrocarbon or a low molecular weight alcohol to solubilize cannabinoids for later isolation. This traditional method is limited in that it depends on naturally grown plant material that may have been exposed to various toxic pesticides, herbicides and the like. In addition, these traditional extraction methods are inaccurate, resulting in unreliable and varied concentrations of extracted THC. In addition, many strains of Cannabis are grown in hydroponic environments that are also unregulated and can result in widespread contamination of these strains with chemical compounds and other unwanted compounds.
[0012] In another example, US patent application 20160326130 (Lekhram et al.), Cannabinoids and other related compounds are isolated from raw materials harvested from Cannabis using, again, a series of organic solvents to convert cannabinoids into one salt and then return to its original carboxylic acid form. Similar to Webster, this traditional method is limited in that it depends on naturally grown plant material that may have been exposed to various toxic pesticides, herbicides and the like. In addition, several organic solvents used in this traditional process must be recovered and recycled and / or disposed of properly.
[0013] Another traditional method of cannabinoid extraction involves the generation of
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5/114 hash oil with the use of supercritical carbon dioxide (SCO2) Under this traditional method, again the dry vegetable matter is earth and subjected to an SCO2 extraction environment. The primary extract being obtained initially and further separated. For example, as generally described by CA2424356 (Muller et al.) Cannabinoids are extracted with the help of SCO2 under supercritical pressure and temperature conditions and by the addition of accessory solvents (modifiers) such as alcohols. In the process, this supercritical CO2 evaporates and dissolves in cannabinoids. However, this traditional process also has certain disadvantages. For example, due to the low solubility in supercritical SCO2, the recovery of cannabinoids of interest is inconsistent. In addition, all solvents used must be recycled and pumped back to the extractor, to minimize operating costs.
[0014] Another method uses butane to extract cannabinoids, in particularly high concentrations of THC, from raw materials harvested from Cannabis. Since butane is non-polar, this process does not extract water-soluble by-products such as chlorophyll and plant alkaloids. That said, this process can take up to 48 hours and is therefore limited in its ability to expand for maximum commercial viability. The other major disadvantage of traditional butane-based extraction processes is in relation to the potential dangers of using flammable solvents, as well as the need to ensure that all butane is completely removed from the extracted cannabinoids.
[0015] Another limiting factor in the viability of these traditional cannabinoid extraction methods is the inability to maintain the integrity of the Cannabis strain. For example, cannabanoids used in medical and research applications or which are subject to controlled clinical trials, are heavily regulated by various government agencies in the United States and elsewhere. These regulatory agencies require that strains of Cannabis remain chemically consistent over time. Unfortunately, the genetic / chemical compositions of Cannabis strains change over generations such that
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6/114 they cannot meet the regulatory mandates present in most clinical trials or certified for use in other pharmaceutical applications.
[0016] Several attempts have been made to resolve these issues. For example, efforts have been made to produce cannabinoids in genetically modified organisms. For example, in US Patent Application 14 / 795,816 (Poulos, et al.). In this case, the applicant claims to have generated a genetically modified yeast strain capable of producing a cannabinoid by inserting genes that produce the appropriate enzymes for this metabolic production. However, this application is limited in its ability to produce only a single or very limited number of cannabinoid compounds. This limitation is clinically significant. Recent clinical studies have found that the use of a single isolated cannabinoid as a therapeutic agent is not as effective as treatment with the naturally occurring "environment" of primary and secondary cannabinoids associated with several selected strains.
[0017] Additional attempts have been made to chemically synthesize cannabinoids, such as THC. However, the chemical synthesis of several cannabinoids is an expensive process when compared to the extraction of cannabinoids from naturally occurring plants. The chemical synthesis of cannabinoids also involves the use of chemicals that are not ecological, which can be considered as an additional cost to their production. In addition, the synthetic chemical production of several cannabinoids has been classified as less pharmacologically active than with those extracted from plants such as Cannabis sativa.
[0018] Efforts to generate large-scale Cannabis cell cultures have also raised a number of technical problems. Chief among them is the fact that cannabinoids are cytotoxic. Under natural conditions cannabinoids are generated and stored extracellularly in small glandular structures called trichomes. Trichomes can be seen as small hair or other growths on the epidermis of a Cannabis plant. As a result, in Cannabis cell cultures, the inability to store cannabinoids
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7/114 extracellularly means that any accumulation of cannabinoids would be toxic to cultured cells. Such limitations hamper the ability of Cannabis cell cultures to expand to industrial levels of production. Cannabinoid Biosynthesis Toxicity Limits In Vivo Production Systems [0019] Efforts to generate strains / cultures of Cannabis cells that produce or accumulate high levels of cannabinoids have raised several technical problems. Chief among them is the fact that cannabinoid synthesis produces toxic by-products. Notably, both CBDA and THCA synthases require molecular oxygen, in conjunction with a FAD molecule, to oxidize Cannabigerolic acid (CBGA). Specifically, as shown in Fig. 29, two electrons from the substrate are accepted by an enzyme-bound FAD and then transferred to molecular oxygen to reoxidate the FAD. CBDA and THCA are synthesized from ionic intermediates through stereoselective cyclization by enzymes. The hydride ion is transferred from the reduced flavin to molecular oxygen, resulting in the formation of hydrogen peroxide and the reactivation of the flavin for the next cycle. As a result, in addition to producing CBDA and THCA, respectively, this reaction produces hydrogen peroxide (H2O2) which is naturally toxic to the host cell. Due to this production of a toxic hydrogen peroxide by-product, cannabinoid synthesis generates a self-limited feedback loop that prevents high-level production and / or accumulation of cannabinoids in in vivo systems. One way that Cannabis plants deal with these cellular cytotoxic effects is through the use of trichomes for the production and accumulation of Cannabinoids.
[0020] Cannabis plants deal with this toxicity by sequestering cannabinoid biosynthesis and extracellular storage in small glandular structures called trichomes as noted above. For example, THCA synthase is a water-soluble enzyme that is responsible for producing THC. For example, THC biosynthesis occurs in glandular trichomes and begins with the condensation of geranyl pyrophosphate with olivetolic acid to produce acid
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8/114 cannabigerolic (CBGA); the reaction is catalyzed by an enzyme called geranylpyrophosphate: olivatolate geranotransferase. The CBGA then undergoes oxidative cyclization to generate tetrahydrocannabinolic acid (THCA) in the presence of THCA synthase. THCA is then transformed into THC by non-enzymatic decarboxylation. Subcellular localization studies using RT-PCR and analysis of enzyme activity demonstrate that THCA synthase is expressed in glandular trichome secretory cells, and then is translocated into the secretory cavity where the final THCA product accumulates. THCA synthase present in the secretory cavity is functional, indicating that the storage cavity is the site for THCA biosynthesis and storage. Thus, Cannabis is able to produce cannabinoids extracellularly and thus avoid the cytotoxic effects of these compounds. However, as a result, the ability to access and chemically alter cannabinoids in vivo is impeded by this cellular compartmentalization.
[0021] To address these issues, some have proposed to chemically modify cannabinoid compounds to reduce their cytotoxic effects. For example, Zipp et al. proposed the use of an in vitro method to produce cannabinoid glycosides. However, this application is limited to in vitro systems only. Specifically, as noted above, cannabinoid synthase enzymes, such as THCA synthase, are water-soluble proteins that are exported out of the cells of the basal trichome to the storage compartment where this is active and catalyze the synthesis of THCA. Specifically, in order to effectively mediate the cellular export of this cannabinoid synthase, this enzyme contains a 28-amino acid signal peptide that directs its export out of the cell and to the extracellular trichome, where cannabinoid synthesis occurs. As a result of this signal-dependent extracellular compartmentalization, for example, THCA synthase, this means that THCA is produced outside the cytoplasm and would not be accessible to genetically engineered glycosylation enzymes. As such, the simple expression of a UDP glycosyltransferase in plant cells, such as
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9/114 alluded to vaguely in Zipp, et al., Would not result in the effective glycosylation of cannabinoid molecules in the compartmentalized and extracellular trichome structure where cannabinoid synthesis occurs. Zipp's method also cannot generate acetylated cannabinoids, as well as cannabinoid molecules from O acetyl glycoside.
[0022] The previous problems related to the production, detoxification and isolation of cannabinoids may represent a long-felt need for an effective and economical solution for them. Although some elements of implementation are available, real attempts to meet this need may have failed to some extent. This may have been due to a failure of those with common skills in the technique of fully appreciating or understanding the nature of the problems and challenges involved. As a result of this lack of understanding, attempts to meet these felt needs may not have been able to effectively solve one or more of the problems or challenges identified here. These attempts may even have deviated from the technical guidelines adopted by the present inventive technology and may even result in the achievements of the present inventive technology being considered, to a certain extent, an unexpected result of the approach adopted by some in the field. [0023] As will be discussed in more detail below, the current inventive technology overcomes the limitations of traditional cannabinoid production systems, while meeting the objectives of a truly effective and scalable cannabis production, modification and isolation system.
SUMMARY OF THE INVENTION (S) [0024] The inventive technology can encompass systems, methods and compositions for the in vivo production, modification and isolation of cannabinoid compounds from Cannabis plants. In particular, the invention provides systems and methods for high-level in vivo biosynthesis of water-soluble cannabinoids.
[0025] Current inventive technology includes systems and methods to improve cannabinoid production and / or accumulation. In one embodiment, the invention can
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10/114 include systems and methods for increasing cannabinoid production and / or accumulation in an in vivo system, such as a plant or plant cell culture.
[0026] Another target of the present invention may include the generation of genetically modified plants that overexpress certain endogenous / exogenous genes that result in overproduction and / or accumulation of cannabinoids above wild-type levels. In a preferred embodiment, such transgenic plants may exhibit improved production and localized accumulation of cannabinoid precursor compounds, such as THCA (tetrahydrocannabinolic acid), CBCA (cannabichromic acid), and CBDA (cannabidiolic acid). These transgenic plants may additionally exhibit improved production and localized accumulation of cannabinoids, such as THCs, CBCs and CBDs. An additional object of the present invention may include the generation of genetically modified plants that express certain endogenous / exogenous ones that result in improved cannabinoid modification. In a preferred embodiment, such transgenic plants may exhibit improved cannabinoid modification, including hydroxylation and / or acetylation and / or glycosylation. In additional preferred embodiments, such transgenic plants may exhibit improved cannabinoid modification, including acetylation and glycosylation, as a form of O acetyl glycoside. For example, acetylation adds an acetyl group (-CH3OOH) to a cannabinoid such that the carboxylate group is acidic and charged at neutral pH making it highly soluble in water.
[0027] An objective of current inventive technology may be to generate a genetically modified or transgenic Cannabis plant that overexpresses one or more transcription factors, such as myb, which improves the flow of metabolites through the cannabinoid biosynthetic pathway. In a preferred embodiment, these transcription factors may include several analogues. In a certain preferred embodiment, one or more of these transgenes can be operationally linked to one or more promoters.
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11/114 [0028] Another objective of current inventive technology may be to generate a genetically modified or transgenic Cannabis cell culture that overexpresses one or more transcription factors that improve the flow of metabolites through the cannabinoid biosynthetic pathway. In a preferred embodiment, these transgenes can be operationally linked to one or more promoters.
[0029] Another objective of the current inventive technology may be to generate a genetically modified or transgenic Cannabis plant that expresses one or more exogenous / heterologous transcription factors that regulate the formation of trichomes to increase the accumulation of cannabinoids. In certain preferred embodiments, one or more of these exogenous transgenes may be operably linked to one or more promoters.
[0030] Yet another objective of current inventive technology may be to generate a genetically modified or transgenic Cannabis plant that expresses the enzyme that is configured to be able to reduce the levels of hydrogen peroxide (H2O2) that can be generated during the synthesis of cannabinoid. In a preferred embodiment, the current inventive technology may be to generate a genetically modified or transgenic Cannabis plant that expresses a chimeric protein. In one embodiment, this chimeric protein can include a first domain that can reduce the levels of hydrogen peroxide (H2O2) that can be generated during cannabinoid synthesis. Such a chimeric / fusion protein can further include a second domain that can comprise a trichome targeting domain that can allow the targeted localization of the chimeric protein to active cannabinoid synthesis sites. In some embodiments, a third domain may include a linker that can further separate the first domain from the second domain, such that said first domain and said second domain can each fold into their appropriate three-dimensional shape and retain their activity and said ligand bands and a length.
[0031] Another objective of current inventive technology may include the generation of one or more of the plants or cell cultures of genetically modified plants
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12/114 mentioned above using Agrobacterium Tiplasmid-mediated transformation.
[0032] Another objective of the present inventive technology relates methods and systems for in vivo cell localization of cannabinoid biosynthesis and modification. More specifically, the present inventive technology relates methods and systems for the in vivo cellular localization of hydroxylation, acetylation and / or glycosylation of cannabinoids. The inventive technology may include systems and methods for highly efficient localized chemical modification and isolation of cannabinoid compounds from suspension cultures. In this modality, several selected cannabinoid compounds can be chemically modified in soluble and non-toxic configurations.
[0033] Additional modalities of the inventive technology may include the transient modification of cannabinoid compounds to reduce and / or eliminate their cytotoxicity in plants or plant cell culture systems. In a preferred embodiment, such transiently modified cannabinoids can be accumulated at levels that would normally have a deleterious effect on the cell. Additional modalities may include the isolation of these transiently modified cannabinoids by conversion or enzymatic reconstitution to their original and / or partially modified structure.
[0034] Another objective of the invention may include the generation of a transgenic plant and / or plant cell cultures that can express heterologous genes that coupled the synthesis of cannabinoids and hydroxylation and / or glycosylation in the plant. Specifically, a goal of the technology may include the use of Nicotiana benthamiana to demonstrate the synthesis of CBDA from coupling and glycosylation in the plant. An additional objective of this modality may include additional modifications to the CBDA molecule, such as hydroxylation and acetylation. In yet another objective, this cannabinoid modification can be specifically localized, for example in the cytosol and / or trichome.
[0035] Another target of the invention may include the generation of a transgenic plant
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13/114 and / or plant cell cultures that can express endogenous genes that can be configured to modify cannabinoids. The additional objective may include the coexpression of heterologous transcription factors that can increase cannabinoid production. Another objective of the invention may include the coexpression of heterologous genes that detoxify the hydrogen peroxide by-products generated by cannabinoid biosynthesis. The coexpression of such genes can be additive with the coexpression of genes configured to modify and / or locate cannabinoid biomodifications.
BRIEF DESCRIPTION OF THE FIGURES [0036] Fig. 1 Chromatographic Elution Profile Representative of CBGA Glycosides found in In Vitro Assays. Chromatograms A, B and C represent the respective ion chromatograms extracted for each glycoside product. Chromatogram D is representative of the total ion chromatogram. Peak intensities are illustrated as abundance relative to the most abundant peak in each respective chromatogram.
[0037] Fig. 2. Chromatographic Elution profiles representative of functionalized CBGA and Glycosides found in in vitro assays. Chromatograms A, B and C represent ion chromatograms with respective extract classification for each product. Chromatogram D is representative of the total ion chromatogram. Peak intensities are illustrated as abundance relative to the most abundant peak in each respective chromatogram.
[0038] Fig. 3. Chromatographic Elution Profile Representative of CBDA Glycoside profiles found in Leaf Extracts. Chromatograms A, B, C, and D represent ion chromatograms with respective extract classification for each glycoside product. Chromatogram E is representative of the total ion chromatogram. Peak intensities are illustrated as abundance relative to the most abundant peak in each respective chromatogram.
[0039] Fig. 4. Chromatographic elution of functionalized CBDA and functionalized glycosides in leaf extracts. Chromatograms A, B, and C represent
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14/114 ion chromatograms with respective extract classification for each product. Chromatogram D is representative of the total ion chromatogram. Peak intensities are illustrated as abundance relative to the most abundant peak in each respective chromatogram.
[0040] Fig. 5. Gene construct for expression of the cytochrome P450 (CYP3A4) gene, (SEQ ID NO. 1) expressing the cytochrome P450 protein (CYP3A4) (SEQ ID NO. 2) and P450 oxidoreductase gene ( oxred) (SEQ ID NO. 3) expressing the protein P450 oxidoreductase (SEQ ID NO. 4) in plants. Both genes were driven by the constitutive 35S (35S) promoter and showed 5 'untranslated regions of Arabidopsis thaliana alcohol dehydrogenase (AtADH) as translation enhancers.
[0041] Fig. 6. Confirmation of CYP3A4 and P450 oxidoreductase expression in tobacco leaves. CB1-CB5, biological replicas of leaves infiltrated with CYP3A4 / P450 oxidoreductase; WT = wild-type tobacco leaves without infiltration. L = I kb plus scaling (Thermo Fisher Scientific, USA). The arrows show the expected band (500 bp) indicating the expression of the transgene.
[0042] Fig. 7. Enhanced glycosylation of cannabinoids in Nbenthamiana plants with overexpression of P450. CB1-CB5 are biological replicates that overexpress CYP3A4 + P450 oxidoreductase, the P control is the PI9 silencing suppressor ("empty vector control"). The vertical axis shows relative quantities expressed as the peak area per g of fresh weight.
[0043] Fig. 8 Gene construct for the cannabinoid production system of suspension culture and cytosol. 35S, cauliflower mosaic 35S promoter; HSPt, HSP terminator; 35PPDK, hybrid promoter that consists of a 35S enhancer of the cauliflower mosaic virus fused to the basal corn promoter C4PPDK (Yoo et al. 2007); 76G1, UDP Stevia rebaudiana glycosyltransferase; ABCG2, carrier of multiple human drugs.
[0044] Fig. 9. Demonstrates RT-PCR confirmation of the expression of CBDA synthase (a), UDP glycosyltransferase (b) and ABCG2 (c) in tobacco leaves. L is Ikb more
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11/15 scheduling (Thermo Fisher Scientific, USA). The numbers in the bands represent independent transgenic lines. The arrows point to the expected band that shows the expression of the transgene.
[0045] Fig. 10 Hydroxylation and glycosylation of cannabinoids in transgenic tobacco (SUS, numbered) overexpressing CBDA synthase, UDP glycosyltransferase and ABC transporter. WTS1 and 2 are wild type fed with substrate for endogenous reactions. There was some endogenous glycosylation of CBGA, as well as evidence of enhanced activity of transgenic glycosyltransferase (for example, SUS2, SUS3 and SUS4). The data were corrected for the peak area per g of fresh weight.
[0046] Fig. 11. Improved modification of cannabinoids in transgenic Nbentamian plants co-infected with constructs for glycosylation, P450-mediated functionalization (hydroxylation) and detoxification of hydrogen peroxide by catalase. SUS = construct for overexpression of CBDA synthase, UDP glycosyltransferase and ABC transporter; M3S = construct for overexpression of CBDA synthase, UDP glycosyltransferase and ABC transporter with Cannabis type MYB12 and Arabidopsis thaliana catalase.
[0047] Fig. 12 Increased glycosylation activity in transgenic N-Bentamian plants (TSA, TSB, TSC, SUS, SUS / P450) overexpress a glycosyltransferase compared to the wild type in 14-hour transient expression assays.
[0048] Fig. 13 Exemplary monooxygenase reaction, catalyzed by cytochrome P450.
[0049] Fig. 14 Gene 1 construct for the trichome cannabinoid production system. Cauliflower mosaic 35S promoter; AtADH 5'-UTR, translation enhancer element (Matsui et al. 2012); tsCBDAs, cannabidiolic acid synthase with its original trichome target sequence; HSP terminator; tsUGT76GI, UDP Stevia rebaudiana glycosyltransferase with CBDA trichome target sequence.
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16/114 [0050] Fig. 15 Gene 2 construct for the trichome cannabinoid production system, 35S promoter of the cauliflower mosaic; AtADH 5'-UTR, intensifier element; PM-UTR1, Arabidopsis thaliana UDP-glucose / galactose transporter directed to the plasma membrane; HSP terminator. [0051] Fig. 16 CBDA RT-PCR trichome target synthase (top), UDP trichome target glycosyltransferase (76G1) UGT RT-PCR (bottom). A, B, and C are biological replicas collected after 2DPI.
[0052] Fig. 17 PM-UTR1 RT-PCR. A, B and C are biological replicas collected after 2DPI.
[0053] Fig. 18 Gene construct for the cytosolic cannabinoid production system. 35S promoter of cauliflower mosaic; AtADH 5'-UTR, intensifier element; cytCBDAs, cannabidiolic acid synthase with the target sequence of the trichome removed; HSP terminator; cytUGT76GI, UDP Stevia rebaudiana glycosyltransferase.
[0054] Fig. 19 SUS-A to SUS-C are biological replicas for the transformation of cell suspension (201 -SUS) after 1 DPI.
[0055] Fig. 20. cytUGT RT-PCR (top), cytCBDAs RT-PCR (bottom). A, B, and C are biological replicas for cytosolic construct infiltration after 2DPI. [0056] Fig. 21. Detection of cannabinoids in leaves infiltrated with trichome or cell suspension constructs and fed with 2.7 mM CBGA. The color code refers to the target compartment for accumulation of CBDAs and UGT76G1 proteins, either trichome or cytostol in cell suspension. Y-axis: CBGA and CBDA expressed as parts per million (ppm). Primary, secondary, and acylated glycosides expressed as peak area.
[0057] Fig. 22. Detection of cannabinoids in leaves infiltrated with cell suspension or cytosolic construct and fed with 2.7 mM CBGA and 4 mM UDP-glucose. The color code refers to the target compartment for accumulation of CBDAs and UGT76G1 proteins. Y-axis: CBGA expressed as parts per million (ppm). All other cannabinoid derivatives expressed as peak area
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17/114 (no standards available).
[0058] Fig. 23. Ion chromatograms extracted from Functionalized R-OH 1 x Glycosylated CBDA Analog. (A) chromatographic trace, m / z ion, calculated elementary composition, confirming the presence of trace levels of the CBDA analog (B) Absence of CBDA analog in the control extract (C) Absence of CBDA analog in the duplicate control extract biological.
[0059] Fig. 24. Mass Spectrum of Direct Infusion of Cannabis sativa extract. The spectral inserts represent CBDA with a single glycosylation (519.2546 m / z), and CBDA functionalized with R-OH and a single glycosylation (535.2543 m / z). Peak intensities are illustrated as abundance relative to the most intense ion.
[0060] Fig. 25. Relative abundance of CBDA in extracts of various strains of Cannabis sativa infiltrated with Agrobacterium cultures containing combinations of CBDA synthase (CBDAs) and UGT plasmids. Normalized data of relative abundance are presented as the intensity of the ion of each compound divided by the intensity of the ion of the internal standard 7hydroxycoumarin (20 ppm).
[0061] Fig. 26. Relative abundance of modified CBDA (glycosylated and / or hydroxylated) in extracts of various strains of Cannabis sativa infiltrated with Agrobacterium cultures containing combinations of CBDAs and UGT plasmids. Normalized data of relative abundance are presented as the ion intensity of each compound divided by the intensity of ions of the internal standard 7-hydroxycoumarin (20 ppm).
[0062] Fig. 27. Gene construct used to enhance cannabinoid production and mitigate toxicity. CsMYBI 2, Cannabis sativa MYB transcription factor predicted to enhance flavonol biosynthesis; HSPt, efficient transcription terminator of the gene18,2 of the heat shock protein Arabidopsis thaliana; 35S, constitutive promoter of the cauliflower mosaic virus; Catalase, Arabidopsis thaliana catalase gene.
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18/114 [0063] Fig. 28. THC and CBD synthesis from the common CBGA precursor. [0064] Fig. 29. Generation of hydrogen peroxide during cannabinoid biosynthesis.
[0065] Fig. 30. Hydroxylation followed by THC oxidation by CYP2C9 / [0066] Fig. 31. Transfer of a glucuronic acid component to a cannabinoid substrate by UGT.
[0067] Fig. 32. Synthesis of olivetolic acid, a precursor to CBGA.
[0068] Fig. 33. Comparison of the amino acid sequence of the protein sequences of the exemplary Arabidopsis catalase.
[0069] Fig. 34. Schematic diagram of the increase in cannabinoid production associated with the reduced oxidative damage system in one modality. MODE (S) FOR CARRYING OUT THE INVENTION (S) [0070] The present invention includes a variety of aspects, which can be combined in different ways. The following descriptions are provided to list the elements and describe some of the embodiments of the present invention. These elements are listed with initial modalities, however it must be understood that they can be combined in any way and from any number to create additional modalities. The examples described in various preferred ways and modalities are not to be interpreted to limit the present invention to only the technical systems and applications explicitly described. In addition, this description must be understood to support and cover the descriptions and claims of all the various modalities, systems, techniques, methods, devices and applications with any number of the elements disclosed, with each element alone, and also with any and all various permutations and combinations of all elements in this or any subsequent application.
[0071] The inventive technology includes systems and methods for high-level production of cannabinoid compounds. As used here, the term “high level” in this modality can mean greater than biosynthesis or accumulation of the type
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19/114 wild of one or more cannabinoids in the plant or plant cell. In one embodiment, a suspension or hair root or cell suspension culture of one or more plant strains can be established. In a preferred embodiment, a suspension or hair root or cell suspension culture of one or more strains of Cannabis or tobacco plants can be established. It should be noted that the term strain can refer to a plant strain, as well as to a cell culture or cell line derived from a plant, such as Cannabis.
[0072] In a preferred embodiment, a suspension or hair root or cell suspension culture of Cannabis sativa or tobacco plant can be established in a fermenter or other similar apparatus. It should be noted that the use of C. sativa in this modality is only an example. For example, in certain other modalities, several strains of Cannabis, mixtures of strains, hybrids or clones of different strains, as well as different varieties can be used to generate a suspension or hair root culture. For example, strains such as C. sativa, C. indica and C. ruderalis can be used with the inventive technology. In still other modalities, other cannabinoid-producing or cannabinoid-type plants can be used. For example, in a certain embodiment, a cell suspension or hair root culture can be established for one or more of the following: Echinacea; Acmella Oleracea; Helichrysum Umbraculigerum; Radula Marginata (liver herb), Theobroma Cocoa or tobacco.
[0073] In certain modalities, such fermenters may include large industrial scale fermenters, allowing ample amounts of cannabinoid-producing C. sativa cells to be cultivated. In this modality, it may be possible to grow a large number of unadulterated cells from a single strain of, for example, tobacco or C. sativa, which can establish a cell culture with consistent production and / or modification of cannabinoid compounds in both of the quantity and type. Such cultivated growth can be continuously sustained by supplementing nutrients and other
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11/20 growth to culture. Such features can be automated or performed manually.
[0074] Another modality of the inventive technology may include systems and methods for high-level production of modified cannabinoid compounds. In one embodiment, a suspension or culture of hairy roots from one or more strains of tobacco plants can be established. It should be noted that the term strain can refer to a plant strain, as well as to a cell culture or cell line derived from a tobacco plant. In a preferred embodiment, a Nicotiana benthamiana plant hair suspension or culture can be established in a fermenter or other similar apparatus. It should be noted that the use of N. benthamiana in this modality is only an example. For example, in certain other modalities, various strains of Nicotiana, mixtures of strains, hybrids of different strains or clones, as well as different varieties can be used to generate a cell suspension or hair root culture.
[0075] In certain cases, these fermenters may include large industrial scale fermenters, allowing a large number of N. benthamiana cells to be cultivated. In this embodiment, the harvested cannabinoids can be introduced into this suspension culture and modified as generally described here. Similarly, such cultivated tobacco cell growth can be continuously sustained with the continuous addition of nutrients and other growth factors being added to the culture. Such features can be automated or performed manually.
[0076] Another embodiment of the invention may include the production of Cannabis and / or tobacco cells genetically modified to express exogenous and / or endogenous variable genes that can modify the chemical structure of cannabinoid compounds. Such transgenic strains can be configured to produce and / or modify large amounts of cannabinoid compounds in general, as well as increases targeted to the production of specific cannabinoids, such as THC, Cannabidiol (CBD) or Cannabinol (CBN) and the like.
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21/114 [0077] Another embodiment of the invention may include the production of genetically modified Cannabis cell cultures that express a mixture of cannabinoids that can be optimized for the treatment of specific medical conditions. For example, CBD is a non-psychoactive cannabinoid that can be used to treat seizures in those with epilepsy. However, decades of selective breeding resulted in the majority of Cannabis strains with low concentrations of CBD when compared to the psychoactive cannabinoid THC. Therefore, in certain embodiments, cell cultures specific to the disease or syndrome can be developed which express a calibrated mixture of cannabinoids for the treatment downstream of such conditions.
[0078] Additional modalities of the inventive technology may include new systems, methods and compositions for the production and in vivo modification of cannabinoid compounds in a plant system. In a certain way, these modifications in vivo can lead to the production of different forms of cannabinoids with special properties, for example, prodrugs or water-soluble cannabinoids, of slow release. In a preferred embodiment, the inventive technology may include new systems, methods and compositions for hydroxylation, acetylation and / or glycosylation. Modified cannabinoids can be soluble in water, for example, by glycosylation.
[0079] As noted above, the production and / or accumulation of high levels of cannabinoids would be toxic to a plant cell host. As such, a modality of the inventive technology may include systems and methods for transiently modifying cannabinoids in vivo. One of the objectives of the present invention may include the use of cytochrome P450 monooxygenases (CYP) to modify or functionally transiently the chemical structure of cannabinoids. CYPs are a family of major enzymes capable of catalyzing the oxidative biotransformation of many pharmacologically active chemical compounds and other lipophilic xenobiotics. For example, as shown in Fig. 13, the most common reaction catalyzed by cytochrome P450 is a mono reaction
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22/114 oxygenase, for example, insertion of an oxygen atom in the aliphatic position of an organic substrate (RH) while the other oxygen atom is reduced to water. [0080] Several cannabinoids, including THC, have been shown to serve as a substrate for human CYPs (CYP2C9 and CYP3A4). Likewise, CYPs that metabolize cannabidiol (CYPs 2C19, 3A4) have been identified; cannabinol (CYPs 2C9, 3A4); JWH-018 (CYPs 1A2, 2C9); and AM2201 (CYPs 1A2, 2C9). For example, as shown generally in Fig. 30, in an exemplifying system, CYP2C9 can "functionalize" or hydroxyl a resulting THC molecule into a hydroxyl form of THC. Additional oxidation of the hydroxyl form of THC by CYP2C9 can convert it to a form of carboxylic acid that loses its psychoactive capabilities, making it an inactive metabolite.
As such, another embodiment of the invention may include the creation of a cannabis strain or cell culture that can be transformed with artificially created gene constructs that encode one or more exogenous CYPs. In a preferred embodiment, genes encoding one or more isoforms and / or non-human analogues, as well as possibly other CYPs that can functionalize cannabinoids, can be expressed in transgenic Cannabis sativa or another plant. In another preferred embodiment, genes encoding one or more isoforms and / or non-human analogues, as well as possibly other CYPs that can functionalize cannabinoids, can be expressed in the transgenic or tobacco strains of Cannabis sativa in a suspension culture. Additional modalities may include elements of genetic control, such as promoters and / or enhancers, as well as post-transcriptional regulatory elements that can also be expressed in transgenic Cannabis strains such that the presence, quantity and activity of any CYP in the suspension or root culture hair can be modified and / or calibrated.
[0082] Another embodiment of the invention can include the creation of a tobacco strain or the cell culture can be transformed with artificially created gene constructs that encode one or more exogenous CYPs. In a modality
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23/114 preferred, genes encoding one or more non-human isoforms and / or analogues, as well as possibly other CYPs that can functionalize cannabinoids introduced into a suspension culture or transgenic Nbenthamiana plants. Additional modalities may include elements of genetic control, such as promoters and / or enhancers, as well as post-transcriptional regulatory elements that can also be expressed in transgenic Nbenthamiana strains such that the presence, quantity and activity of any CYP in the suspension or culture hair root can be modified and / or calibrated.
[0083] Another objective of the invention may be to further modify, in vivo, cannabinoids and / or cannabinoids already functionalized. In a preferred embodiment, glycosylation of cannabinoids and / or functionalized cannabinoids can convert them to a water-soluble form. In an exemplary embodiment shown in Fig. 31, the inventive technology can use one or more glycosyltransferase enzymes, such as UDP-glycosyltransferase (UGT), to catalyze, in vivo, glucuronosylation or glucuronidation of cannabinoids, such as primary cannabinoids (CBD, CBN) and secondary (THC, JWH-018, JWH-073). In one embodiment, glucuronidation may consist of the transfer of a glucuronic acid component from uridine diphosphate glucuronic acid to a cannabinoid substrate by any of several types of glycosyltransferases, as described here. Glucuronic acid is a sugar acid derived from glucose, with its sixth carbon atom oxidized to a carboxylic acid.
[0084] Yet another embodiment of the current invention may include the in vivo conversion of a functionalized cannabinoid, in this example, a form of cannabinoid acid carboxylic, to a glycosylated form of cannabinoid that can be both water-soluble and non-toxic to the cell hostess. These chemical modifications may allow higher levels of cannabinoid accumulation in a plant cell culture without the deleterious cytotoxic effects that could be seen with unmodified cannabinoids due to this solubility in water.
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24/114 [0085] Another embodiment of the invention may include the generation of transgenic or genetically modified strains of Cannabis, or other plants such as tobacco, having artificial genetic constructs that can express one or more genes that can increase the solubility of cannabinoids and / or decrease the cytotoxicity of cannabinoids. For example, inventive technology may include the generation of strains of transgenic plants or cell lines with artificial genetic constructs that can express one or more endogenous and / or exogenous glycosyltransferases or other enzymes capable of glycosylating cannabinoid compounds. For example, in one embodiment, one or more N-benthamian glycosyltransferases, or other non-cannabis plants, can be introduced into a cannabis plant or cell culture and configured for glycosylated cannabinoids in vivo. In another embodiment, endogenous N-benthamian glycosyltransferases can be overexpressed to increase cannabinoid glycosylation in vivo.
[0086] In an additional embodiment, the inventive technology may include the generation of artificial gene constructs with genes encoding one or more glycosyltransferases, including non-human analogs of those described here as well as other isoforms, which can be expressed in transgenic Cannabis sativa, N-benthamiana or another plant system that can still be grown in a suspension culture. Additional modalities may include elements of genetic control such as promoters and / or enhancers as well as post-transcriptional regulatory control elements that can also be expressed in a transgenic plant system so that the presence, quantity and activity of any glycosyltransferases present in suspension or hair root culture can be regulated.
The additional embodiment of the invention may include artificial gene constructs with one or more genes that encode one or more UDP- and / or ADPglycosyltransferases with localization sequences or domains that can assist in the movement of the protein to a certain portion of the cell, how cell sites that were cannabinoids and / or functionalized cannabinoids can be
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25/114 modified, produced, stored and / or excreted from the cell.
[0088] The additional embodiment of the invention may include artificial gene constructs having one or more genes that encode one or more UDP- and / or ADPglycosyltransferases being co-expressed with one or more exogenous genes that can assist in the movement of the protein to a certain portion of the cell, such as cell sites that were cannabinoids and / or functionalized cannabinoids can be stored, and / or excreted from the cell.
[0089] A preferred embodiment of the inventive technology may include the high-level in vivo production of water-soluble glycosylated cannabinoids, generally referred to as transiently modified cannabinoids that can be harvested from a plant or cell culture. In one embodiment, transiently modified cannabinoids can accumulate within the cell that is part of a suspension culture. In this example, the cell culture can be allowed to grow to a desired cell level or optical density or in other cases until a desired level of transiently modified cannabinoids has accumulated in the Cannabis cells. These exogenous genes can be located, for example, in the cytosol or trichome as generally described here, and can further be coexpressed with other exogenous genes that can reduce the toxicity of cannabinoid biosynthesis and / or facilitate cannabinoid transport through or outside the cell.
[0090] All or a portion of the Cannabis cells containing the transiently modified cannabinoids accumulated can be harvested from the culture, which in a preferred embodiment may be an industrial-scale fermenter or other apparatus suitable for large-scale cultivation of plant cells. Harvested cannabis cells can be lysed so that the accumulated transiently modified cannabinoids can be released into the surrounding lysate. Additional steps may include treating this lysate. Examples of such treatment may include filtering or screening this lysate to remove foreign plant material as well as chemical treatments to improve
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26/114 subsequent cannabinoid yields.
[0091] Another modality of the inventive technology may include the high-level in vivo generation of glycosylated and water-soluble cannabinoids, generally referred to as transiently modified cannabinoids that can be harvested from a plant or cell culture. In one embodiment, cannabinoids can be introduced into a culture of non-cannabinoid-producing cells, such as Nbenthamiana. In this preferred embodiment, the culture of non-cannabinoid-producing cells can be genetically modified to express one or more endogenous or exogenous genes that can modify cannabinoids, for example, through hydroxylation, acetylation and / or glycosylation. Such endogenous or exogenous genes can be located, for example, in the cytosol or trichome as generally described here, and can further be coexpressed with other exogenous genes that can reduce the toxicity of cannabinoid biosynthesis and / or facilitate cannabinoid transport through, or outside the cell.
This culture of non-cannabinoid-producing cells can be allowed to grow to a desired cell level or optical density, or in other cases until a desired level of transiently modified cannabinoids has accumulated in the cultured cells. All or a portion of the Nbenthamiana cells containing the accumulated cannabinoids can be harvested from the culture, which in a preferred embodiment may be an industrial-scale fermentor or other apparatus suitable for large-scale plant cell culture. The harvested N-benthamian cells can be lysed so that the accumulated transiently modified cannabinoids can be released into the surrounding lysate. Additional steps may include treating this lysate. Examples of such a treatment may include filtering or sieving that lysate to remove foreign plant material as well as chemical treatments to improve later cannabinoid yields.
[0093] Another objective of the inventive technology may include methods to isolate and purify transiently modified cannabinoids from a plant or
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27/114 suspension culture. In a preferred embodiment, a lysed Cannabis can be generated and processed using affinity chromatography or other purification methods. In this preferred embodiment, the affinity column with a protein ligand or receptor configured to bind to transiently modified cannabinoids, for example, through association with a functional group of glycosyl or glucuronic acid, among others, can be immobilized or coupled to a solid support. The lysate can then be passed over the column so that the transiently modified cannabinoids, having a specific binding affinity to the ligand, become bound and immobilized. In some embodiments, non-specific binding and non-binding proteins that may be present in the lysate can be removed. Finally, transiently modified cannabinoids can be eluted or displaced from the affinity column, for example, a corresponding sugar or other compound that can displace or interrupt cannabinoid-ligand binding. Eluted modified transient cannabinoids can be collected and further purified or processed.
[0094] A target of the invention may include the mode in which transiently modified cannabinoids are passively and / or actively excreted from a cell or into a cell wall. In an exemplary model, the exogenous ATP-binding cassette transporter (ABC transporters) or other similar molecular structure can recognize the glucuronic or glycosyl (conjugated) functional group in the transiently modified cannabinoid and actively transport it across the wall / membrane cell in the surrounding environment. In one embodiment, the cell culture can grow until an output parameter is reached. In one example, an output parameter may include letting the cell culture grow until a desired cell / optical density is reached or a desired transiently modified cannabinoid concentration is reached. In this modality, the culture medium containing the transiently modified cannabinoids can be harvested for
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11/284 later cannabinoid extraction. In some embodiments, this harvested medium can be treated in a similar manner to the lysate generally described above. In addition, the transiently modified cannabinoids present in the crude and / or treated medium can be isolated and purified, for example, by affinity chromatography in a manner similar to that described above.
[0095] In certain embodiments, this purified cannabinoid isolate may contain a mixture of primary and secondary glycosylated cannabanoids. As noted above, these purified glycosylated cannabinoids can be water soluble and metabolize more slowly than unmodified cannabinoids, providing a slow release capability that may be desirable in certain pharmaceutical applications, such as for use in specific tissue applications, or as a prodrug. Therefore, it is an objective of the invention to incorporate these purified glycosylated cannabinoids in a variety of pharmaceutical and / or nutraceutical applications.
[0096] For example, purified glycosylated cannabinoids can be incorporated into various solid and / or liquid release vectors for use in pharmaceutical applications. As noted above, these transiently modified cannabinoids may no longer have their psychoactive component, making their application in therapeutic research and pharmaceutical applications especially advantageous. For example, the treatment of children can be carried out by administering a therapeutic dose of transiently purified and isolated modified cannabinoids, without the unwanted psychoactive effect. Additional therapeutic applications may include harvesting and later administering a dose of an "environment" of transiently purified and isolated modified cannabinoids.
[0097] Another embodiment of the invention may include a system for converting or reconstituting transiently modified cannabinoids. In a preferred embodiment, glycosylated cannabinoids can be converted to non-glycosylated cannabinoids by treating them with one or more glycosidases
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Generalized or specific 29/114. The use and availability of glycosidase enzymes can be recognized by those skilled in the art without requiring undue experimentation. In this embodiment, these glycosidase enzymes can remove a portion of sugar. Specifically, these glycosidases can remove the portion of glycosyl or glucuronic acid by reconstituting the cannabinoid compound to a form exhibiting psychoactive activity. This reconstitution process can generate a highly purified “environment” of primary and secondary cannabinoids. These reconstituted cannabinoid compounds can also be incorporated into various solid and / or liquid release vectors for use in a variety of pharmaceutical and other commercial applications.
[0098] As noted above, in one embodiment of the invention, cannabis-producing strains of Cannabis, as well as other plants can be used with inventive technology. In certain preferred embodiments, instead of cultivating the target cannabinoid-producing plant in a cell culture, the raw plant material can be harvested and subjected to cannabinoid extraction using one or more of the methods described herein. These traditionally extracted cannabinoids can then be modified from their native forms through the in vitro application of one or more CYPs that can generate hydroxyl and carboxylic acid forms of these cannabinoids, respectively. These functionalized cannabinoids can be further modified by in vitro application of one or more glycosyltransferases, as generally described herein. In this modality, the new transiently modified cannabinoids can be isolated and purified through an affinity chromatography process or other extraction protocol, and then applied to various commercial and other therapeutic uses. In other modalities, transiently modified cannabinoids can be restored and reconstituted through the in vitro application of one or more glycosidase enzymes. These restored cannabinoids can also be applied to a variety of commercial and other therapeutic uses.
[0099] Another embodiment of the invention may include the use of other plants not
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30/114 cannabinoid producing plants, instead of growing a cannabinoid producing plant in a cell culture. Here, cannabinoids can be introduced into genetically modified plants or plant cell cultures that express one or more CYPs that can generate hydroxyl and carboxylic acid forms of these cannabinoids, respectively. These functionalized cannabinoids can also be modified through the action of one or more glycosidases that can also be expressed in the culture of plants or non-cannabinoid-producing cells. In a preferred embodiment, a culture of non-cannabinoid-producing cells can include tobacco plants or cell cultures.
[00100] One embodiment of the invention may include an in vivo method of accumulating and modifying cannabinoids targeting trichomes. A preferred embodiment of this in vivo system may include the creation of a recombinant protein that can allow the translocation of a CYP or glycosyltransferases to an extracellular cannabinoid synthesis site in an entire plant. More specifically, in this preferred embodiment, one or more CYPs or glycosyltransferases can be modified to express all or part of the N-terminal extracellular target sequence as present in the cannabinoid protein synthase, such as THCA synthase or CBDA synthase.
[00101] Another embodiment of the invention may include an in vivo method of biosynthesis, accumulation and / or modification of high-level cannabinoids targeting trichomes. A preferred embodiment of this in vivo system may include the creation of a recombinant protein that can allow the translocation of a catalase to an extracellular cannabinoid synthesis site in an entire plant. More specifically, in this preferred embodiment, one or more catalase enzymes can be engineered to express all or part of the N-terminal extracellular target sequence as present in the cannabinoid protein synthase, such as THCA synthase or CBDA synthase. In this modality, catalase can be directed to the cannabinoid biosynthesis site, allowing to neutralize
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11/114 hydrogen peroxide by-products more efficiently.
[00102] In this preferred embodiment, that N-terminal trichome targeting sequence or domain can generally include the first 28 amino acid residues of a generalized synthase. An exemplary trichome target sequence for THCA synthase is identified SEQ ID NO. 40, while the trichome target sequence for CBDA synthase is identified SEQ ID NO. 41. This extracellular target sequence can be recognized by the cell plant and cause the transport of cytoplasmic glycosyltransferase to the plant's trichrome, and In a particular embodiment the plant's trichrome storage compartment in which the cannabinoid extracellular glycosylation may occur. More specifically, in this preferred embodiment, one or more glycosyltransferases, such as UDP glycosyltransferase, can be engineered to express all or part of the N-terminal extracellular target sequence, as present in an exemplary synthase enzyme.
[00103] Another embodiment of the invention may include an in vivo method of production, accumulation and / or modification of cannabinoids targeting the cytosol. A preferred embodiment of this system in vivo may include the creation of a recombinant protein that can allow the localization of cannabinoid synthases and / or glycosyltransferases in the cytosol.
[00104] More specifically, in this preferred embodiment, one or more cannabinoid synthases can be modified to remove all or part of the N-terminal extracellular target sequence. An exemplary trichome target sequence for THCA synthase is identified SEQ ID NO. 40, while the trichome target sequence for CBDA synthase is identified SEQ ID NO. 41. Coexpression with this cytosolic target synthase with a cytosolic target CYP or glycosyltransferase can allow the localization of cannabinoid synthesis, accumulation and modification in the cytosol. Such cytosolic target enzymes can be coexpressed with catalase, ABC transporter or other genes that can reduce the toxicity of cannabinoid biosynthesis and or facilitate transport through, or
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32/114 outside the cell.
[00105] Another embodiment of the invention may include the generation of an expression vector comprising that polynucleotide, that is, an N-terminal extracellular target sequence of the cannabinoid synthase and glycosyltransferase genes, operably linked to a promoter. A genetically altered plant or parts of it and its progeny comprising this polynucleotide operationally linked to a promoter, in which said plant or parts of it and its progeny produce said chimeric protein, is yet another modality. For example, seeds and pollen contain this sequence of polynucleotides or a homologue thereof, a genetically altered plant cell comprising that polynucleotide operably linked to a promoter, so that said plant cell produces said chimeric protein. Another embodiment comprises a tissue culture comprising a plurality of genetically altered plant cells.
[00106] Another embodiment of the invention provides a genetically altered plant or cell that expresses a chimeric or fusion protein with an N-terminal cannabinoid synthase N-terminal extracellular target sequence (see, for example, SEO ID NO: 40-41; see also SEQ ID NO. 42 for the complete amino acid sequence of THCA synthase) coupled to a UDP glycosyltransferase gene, operably linked to a promoter. Another method provides a method for building a genetically altered plant or part of it with cannabinoid glycosylation in the extracellular storage compartment of the plant's trichome compared to a non-genetically altered plant or part of it, the method comprising the steps of: introducing a polynucleotide encoding the above protein in a plant or part of it to provide a genetically altered plant or part thereof, wherein said chimeric protein comprises a first domain, a second domain, and wherein said first domain comprises a target sequence N-terminal extracellular cannabinoid synthase and a second
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The 33/114 domain comprises a glycosyltransferase sequence. These domains can be separated by a third domain or linker. This ligand can be any sequence of nucleotides that can separate a first domain from a second domain, so that the first domain and the second domain can each fold into their appropriate three-dimensional shape and retain their activity.
[00107] A preferred embodiment of the invention may include a genetically altered plant or cell that expresses a cytosolic target cannabinoid protein synthase having an inactivated or removed N-terminal cannabinoid synthase extracellular target sequence (SEQ ID NOs. 40-41). In one embodiment, a cytosolic target THCA synthase (ctTHCAs) can be identified as SEQ ID NO. 46, while in another modality CBDA synthase of cytosolic target (cytCBDAs) is identified as SEQ ID NO. 22-23). This cannabinoid protein synthase of cytosolic target can be operationally linked to a promoter. Another modality provides a method to build a plant or part of it genetically altered with glycosylation of cannabinoids in the plant's cytosol compared to a plant or part of it not genetically altered, the method comprising the steps of: introducing a polynucleotide that encodes the protein above on a plant or part of it to provide a genetically altered plant or part thereof, wherein said cannabinoid synthase N-terminal extracellular target sequence has been disrupted or removed.
[00108] Yet another embodiment of the invention may include an in vivo method of glycosylating cannabinoids in a cannabis cell culture. In a preferred embodiment, to facilitate the glycosylation of cannabinoids in cannabis cell culture, which would lack an extracellular trichome structure, a cannabinoid synthase gene can be genetically modified to remove or interrupt, for example, through a targeted mutation, the extracellular N-terminal target domain that can be used to transform a Cannabis plant cell into a cell culture. In this modality, without that target domain, cannabinoid synthase, for example, THCA or CBDA synthase, can remain
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34/114 inside the plant cell, instead of being actively transported outside the cell, where it can be expressed with one or more glycosyltransferases, such as UDP glycosyltransferase in the cytoplasm.
[00109] Another modality of the inventive technology may include systems and methods for improved production and / or accumulation of cannabinoid compounds in an in vivo system. In a preferred embodiment, the invention may include the generation of a genetically modified or transgenic Cannabis plant that can produce and / or accumulate one or more cannabinoids at higher levels than those of the wild type. In one embodiment, a transgenic Cannabis plant can be generated to express one or more Cannabis sativa transcription factors that can improve the metabolic pathway (s) in cannabinoids. In a preferred embodiment, a polynucleotide that encodes one or more Cannabis sativa myb transcription factor genes and / or one or more exogenous ortholog genes that increase the flow of metabolites through the cannabinoid biosynthetic pathway can be generated.
[00110] In this preferred embodiment, a polynucleotide can be generated that encodes one or more genes for Cannabis sativa myb transcription factors, such as CAN833 and / or CAN738. As shown in Fig. 32, these transcription factors can boost the production of olivetolic acid, which is a precursor to CBGA, which in turn is a precursor in the biosynthetic pathway of THCs, CBDs and CBC. In an alternative embodiment, a polynucleotide can be generated that encodes one or more gene orthologs from Cannabis sativa myb transcription factors, specifically Cannabis Mybl2 (SEQ ID NOs. 11-12), Myb8 (SEQ ID NO. 43), AtMybl2 (SEQ ID N0.44), and / or MYB1 12 (SEQ ID NO. 45) which can also boost the production of olivetolic acid, which is a precursor to CBGA, which in turn is a precursor in the biosynthetic pathway of THCs, CBDs and CBC.
[00111] In a preferred embodiment, the invention may include methods for generating a polynucleotide that expresses one or more of the SEQ IDs related to the improved cannabinoid production identified herein. In certain modalities
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35/114 preferred, the proteins of the invention can be expressed using any of several systems to obtain the desired amounts of the protein. Typically, the polynucleotide that encodes the protein or its component is placed under the control of a promoter that is functional in the desired host cell. An extremely wide variety of promoters may be available and can be used in the expression vectors of the invention, depending on the specific application. Typically, the promoter selected depends on the cell in which the promoter must be active. Other expression control sequences, such as ribosome binding sites, transcription termination sites and the like, are also optionally included. Constructs that include one or more of these control sequences are called "expression cassettes" or "constructs". Therefore, the nucleic acids encoding the joined polypeptides are incorporated for high level expression in a desired host cell. [00112] Additional modalities of the invention may include the selection of a genetically altered plant or part of it that expresses the cannabinoid production transcription factor protein, wherein the expressed protein has increased cannabinoid biosynthesis capabilities. In certain embodiments, a polynucleotide encoding the cannabinoid production transcription factor protein is introduced by transforming said plant with an expression vector comprising said polynucleotide operably linked to a promoter. The cannabinoid-producing transcription factor protein may comprise a SEQ ID selected from the group consisting of SEQ ID NO: 11-2 or 43-45, or a homologue thereof.
[00113] As noted above, one embodiment of the invention can include systems and methods for general and / or localized detoxification of cannabinoid biosynthesis in an in vivo system. In a preferred embodiment, the invention may include the generation of a genetically modified or transgenic Cannabis or other plant that can be configured to be able to detoxify hydrogen peroxide by-products resulting from cannabinoid biosynthesis
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36/114 at higher levels than those of the wild type. In addition, this detoxification can be configured to be located in the cytosol and / or trichome structure of the Cannabis plant, where cannabinoids are being actively synthesized in an entire plant system. In this preferred embodiment of the invention, a transgenic plant, such as a cannabis or tobacco plant or cell, which expresses one or more genes can over regulate hydrogen peroxide detoxification. [00114] In a preferred embodiment, a polynucleotide can be generated that encodes one or more catalase genes for endogenous and / or exogenous transcription and / or orthologs that catalyze the reduction of hydrogen peroxide: Catalase
Ψ
H2O2 -> 2 H2O + O2 [00115] As such, in one embodiment, the invention comprises the generation of a polynucleotide that encodes an exogenous catalase protein that can be expressed within a transformed plant and / or cell culture. In a preferred embodiment, a configured catalase enzyme reduces the hydrogen peroxide (H202) generated during cannabinoid synthesis and can be used to transform a cannabis or other plant, such as a tobacco plant. Although several generic catalase enzymes can be included in this first domain, as merely an exemplary model, a first domain can include an exogenous catalase derived from Arabidopsis (SEQ ID NO. 13-14; see also Fig. 33), or Escherichia coli (SEQ ID NO. 15-16), or any suitable catalase ortholog, protein fragment or catalase with a homology between about 70% and approximately 100%, as defined herein.
[00116] Another embodiment of the present invention may include the location of the catalase enzyme in a trichome structure. As generally described above, in this embodiment, a trichome target sequence from a cannabinoid synthase can be coupled with one or more catalase enzymes in a fusion or chimera - terms that are generally interchangeable in the present
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37/114 request. This artificial trichome target catalase gene can be used to transform a plant with trichome structures, such as Cannabis or tobacco. In a preferred embodiment, a trichome target catalase from Arabidopsis thaliana with a THCA synthase trichome target domain is identified as SEQ ID NO. 47, while an Arabidopsis thaliana catalase targeting the trichome with a target domain of the CBDA synthase trichome is identified as SEQ ID NO. 48) In another embodiment, a collalvo Escherichia trichome target trichome catalase with a THCA synthase trichome target domain is identified as SEQ ID NO. 49, while an Escherichia catalase strikes the trichome target with a CBDA synthase trichome target domain is identified as SEQ ID NO. 50) [00117] Another embodiment of the invention comprises the generation of a polynucleotide from a nucleic acid sequence encoding the chimeric / fusion catalase protein. Another embodiment includes an expression vector comprising this polynucleotide operably linked to a promoter. A genetically modified plant or parts of it and its progeny comprising this polynucleotide operatively linked to a promoter, wherein said plant or parts of it and its progeny produce said fusion protein is yet another modality. For example, seeds and pollen contain this sequence of polynucleotides or a homologue thereof, a genetically modified plant cell comprising that polynucleotide operably linked to a promoter such that said plant cell produces said chimeric protein. Another embodiment comprises a tissue culture comprising a plurality of genetically modified plant cells.
[00118] In a preferred embodiment, a polynucleotide encoding a trichome target fusion protein can be operably linked to a promoter that may be appropriate for the expression of the protein in a Cannabis, tobacco or other plant. Exemplary promoters may include, but are not limited to: a non-constitutive promoter; an inducible promoter, a preferred tissue promoter; a tissue-specific promoter, a plant-specific promoter
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38/114 or a constitutive promoter. In a preferred embodiment, one or more selected genes can be operationally linked to a leaf-specific gene promoter, such as Cabl. Additional promoters and configurations operable for expression, as well as the coexpression of one or more of the selected genes are generally known in the art.
[00119] Another embodiment of the invention can provide a method for constructing a genetically modified plant or part of it with increased resistance to the cytotoxicity of hydrogen peroxide generated during cannabinoid synthesis compared to a non-genetically modified plant or part of it , the method comprising the steps of: introducing a polynucleotide that encodes a fusion protein into a plant or part of it to provide a genetically modified plant or part of it, wherein said fusion protein comprises a catalase and a target sequence trichome from a cannabinoid synthase.
[00120] In one embodiment, the invention can encompass a system to increase the production and general accumulation of cannabinoids in trichomes, while avoiding possible cytotoxic effects. As generally shown in Fig. 34, the system can include, in a preferred embodiment, the creation of a transgenic Cannabis, tobacco or other plant or plant suspension culture that overexpresses at least one Myb transcription factor to increase the total biosynthesis of cannabinoids. According to other preferred modalities, this transgenic plant can coexpress a catalase enzyme to reduce the oxidative damage resulting from the production of hydrogen peroxide associated with the synthesis of cannabinoids, reducing the toxicity of cells. In certain preferred embodiments, this catalase can be fused to a target domain of the N-terminal synthase trichome, for example, THCA and / or CBDA synthase, helping to locate the catalase in the trichome in the case of integral plant systems and reduce the potentially toxic levels of hydrogen peroxide produced by the activity of THCA, CBCA and / or CBDA synthase.
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39/114 [00121] Another embodiment of the invention may comprise a polynucleotide combining a nucleic acid sequence that encodes a combination of: 1) a cannabinoid-producing transcription factor protein, such as a myb gene; and / or a catalase protein, or any homologue thereof, which may further include a trichome target or localization signal. A genetically modified plant or parts of it and its progeny comprising this combination of polynucleotides operationally linked to a promoter, in which said plant or parts of it and its progeny produce said fusion protein is yet another modality. For example, seeds and pollen contain this sequence of polynucleotides or a homologue thereof, a genetically modified plant cell comprising that polynucleotide operably linked to a promoter such that said plant cell produces said protein. Another embodiment comprises a tissue culture comprising a plurality of genetically modified plant cells.
[00122] Another embodiment of the invention can provide a method for constructing a genetically modified plant or part of it with: 1) increasing the production of cannabinoids compared to a non-genetically modified plant or part of it; and / or 2) greater resistance to cytotoxicity of the hydrogen peroxide generated during the synthesis of cannabinoids compared to a non-genetically modified plant or part of it, the method comprising the steps of: introducing a combination polynucleotide into a plant or part of the to supply a genetically modified plant or part of it.
[00123] Additional modalities of the invention may include the selection of a genetically modified plant or part of it that expresses one or more of the proteins, wherein the expressed protein (s) can (1) increase the ability to produce cannabinoids, for example, through the overexpression of an endogenous myb gene; and 2) catalase with / or without localization capacity
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40/114 of trichome, or any combination thereof. In certain embodiments, a combination polynucleotide encoding proteins is introduced by transforming said plant with an expression vector comprising said combination polynucleotide operably linked to a promoter. The cannabinoid production transcription factor protein may comprise a SEQ ID selected from the sequences identified herein or homologues thereof. Naturally, these combinations and expression combination strategies, identified in Tables 7-8, 10 below and elsewhere, are exemplary, since various combinations of the elements as described herein are included in the invention.
[00124] In a preferred embodiment, the technology of the invention may include systems, methods and compositions of high levels of cannabinoid hydroxylation in vivo, acetylation and / or glycosylation and / or a combination of the three. In a preferred embodiment, hydroxylation of cannabinoids in vivo, acetylation and / or glycosylation and / or a combination of all three can occur in a plant system or culture of cannabinoid-producing cells. While in alternative modalities it may include a non-cannabinoid-producing plant or a cell culture system, such as a tobacco plant, such as N. benthamiana.
[00125] In one embodiment, the invention can include a system for the production, accumulation and modification of cannabinoids. In a preferred embodiment, a plant, such as cannabis or tobacco, can be genetically modified to express one or more heterologous cytochrome P450 genes. In this preferred embodiment, a heterologous human cytochrome P450 (CYP3A4) SEQ ID NO. 1 can be expressed in a cannabinoid-producing plant or cell culture system. While in alternative modalities a heterologous human cytochrome P450 (CYP3A4) can be expressed by a non-cannabinoid-producing plant or cell culture system, such as a tobacco plant, such as N. benthamiana. In this modality, the overexpression of a protein
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41/114 heterologous human cytochrome P450, identified as SEQ ID NO. 2, can functionalize endogenously created cannabinoids so that they can be more efficiently glycosylated and / or acetylated in vivo, making them soluble in water. [00126] In an alternative embodiment, the invention can include a system for the production, accumulation and modification of cannabinoids. In a preferred embodiment, a plant, such as cannabis or tobacco, can be genetically modified to express one or more heterologous cytochrome P450 oxidoreductase heterologous genes. In this preferred embodiment, a heterologous cytochrome P450 oxidoreductase (oxred) identified as SEQ ID NO. 3 can be expressed in a cannabinoid-producing plant or cell culture system. While in alternative modalities a heterologous cytochrome P450 oxidoreductase (oxred) can be expressed by a non-cannabinoid-producing plant or cell culture system, such as a tobacco plant, such as N. benthamiana. In this embodiment, the overexpression of a heterologous cytochrome P450 oxidoreductase protein (oxred) identified as SEQ ID NO. 4, can functionalize endogenously created cannabinoids so that they can be more efficiently glycosylated and / or acetylated in vivo, making them soluble in water.
[00127] In one embodiment, the invention can include a system of production, accumulation and modification of cannabinoids in a non-cannabinoid-producing plant. In a preferred embodiment, a plant, such as tobacco, can be genetically modified to express one or more heterologous cytochrome P450 oxidoreductase heterologous genes. In this preferred embodiment, a heterologous cytochrome P450 oxidoreductase (oxred) identified as SEQ ID NO. 3 can be expressed in a cannabinoid-producing plant or cell culture system. In alternative modalities, while in alternative modalities a heterologous cytochrome P450 oxidoreductase (oxred) can be expressed by a non-cannabinoid-producing plant or cell culture system, such as a tobacco plant, such as N. benthamiana. In this modality, the overexpression of a cytochrome P450 oxidoreductase protein
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42/114 heterologous (oxred) identified as SEQ ID NO. 4, can help to functionalize cannabinoids introduced into the culture system of plant cells or genetically modified plants, so that they can be more efficiently glycosylated and / or acetylated in vivo, making them soluble in water.
[00128] In a preferred embodiment, cytochrome 450 and P450 oxidoreductase are co-expressed.
[00129] In another embodiment, the invention may include the expression of one or more exogenous or heterologous genes, the terms being generally interchangeable, cannabinoid synthase gene in a non-cannabinoid-producing cell or plant culture system. In a preferred embodiment, this gene can include one or more of the CBG, THCA, CBDA or CBCA synthase genes. For example, in one embodiment, a cannabidiolic acid (CBDA) synthase, identified as SEQ ID NO. 5 (gene) or SEQ ID NO. 6 (protein) from Cannabis sativa can use castings in a non-cannabinoid-producing plant, such as suspension culture or N. benthamiana plant cell. In another preferred embodiment, a tetrahydrocannabinolic acid (THCA) synthase, identified as SEQ ID NO. 42 (gene) of Cannabis sativa can use castings in a non-cannabinoid-producing plant, such as suspension culture or N. benthamiana plant cell.
[00130] In another preferred embodiment, these cannabinoid synthase genes expressed in a culture of cannabinoid and / or non-cannabinoid plants or plant cell suspension can be targeted or located in certain parts of a cell. For example, in a preferred embodiment, cannabinoid production can be located in the cytosol, allowing cannabinoids to accumulate in the cytoplasm. In an exemplary embodiment, an artificially modified cannabinoid synthase protein synthase can be generated. In this exemplary embodiment, a CBDA synthase may have the trichome target sequence removed, forming a cytosolic CBDA synthase (cytCBDAs) identified as SEQ ID NO. 22, (gene) or 23 (protein). Alternative modalities would include the generation of other
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43/114 artificial cytosol target synthase genes, such as cytosolic THCA synthase (cyTHTHs) identified as SEQ ID NO. 46 (gene).
[00131] These preferred modalities may be particularly suitable for cannabinoid expression systems in cannabinoid cell suspension culture, since these culture systems lack the trichomes present in whole plants. As such, in a preferred embodiment, a cannabinoid-producing plant can be transformed into one or more cannabinoid synthase genes that are artificial cytosolic targets without a trichome target signal. In an alternative embodiment, these artificial cytosolic target cannabinoid synthase genes can be expressed in a suspension culture of cannabinoid-producing plants in which the corresponding endogenous wild-type synthase gene has been inhibited and / or eliminated.
[00132] In one embodiment, the invention can include a system of production, accumulation and modification of cannabinoids that can generate water-soluble cannabinoids. In a preferred embodiment, a plant, such as cannabis or tobacco, can be genetically modified to express one or more heterologous glycosyltransferase genes, such as UDP glycosyltransferase. In this preferred embodiment, UDP glycosyltransferase (76G1) (SEQ ID NO. 7) (gene) / SEQ ID NO. 8 (protein) of Stevia rebaudiana can be expressed in cannabinoid-producing plants or in cell suspension. In a preferred embodiment, the plant culture or suspension of cannabinoid-producing cells can be Cannabis. In another embodiment, one or more Nicotiana tabacum glycosyltransferase and / or a homologous Nicotiana benthamiana glycosyltransferase, can be expressed in a cannabinoid-producing plant, such as cannabis, or can be overexpressed in a plant and / or endogenous plant cell system of culture. In a preferred embodiment, a glycosyltransferase gene and / or protein can be selected from the exemplary plant, such as Nicotiana tabacum. This glycosyltransferase gene and / or protein may include, but is not limited to:
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44/114 (NtGT5a) Nicotiana tabacum (SEQ ID NO. 26) (amino acid); Glycosyltransferase (NtGT5a) Nicotiana tabacum (SEQ ID NO. 27) (DNA); Glycosyltransferase (NtGT5b) Nicotiana tabacum (SEQ ID NO. 28) (amino acid); Glycosyltransferase (NtGT5b) Nicotiana tabacum (SEQ ID NO. 29) (DNA); UDP-glycosyltransferase 73C3 (NtGT4) Nicotiana tabacum (SEQ ID NO. 30) (amino acid); UDPglycosyltransferase 73C3 (NtGT4) Nicotiana tabacum (SEQ ID NO. 31) (DNA); Glycosyltransferase (NtGTIb) Nicotiana tabacum (SEQ ID NO. 32) (amino acid); Glycosyltransferase (NtGTIb) Nicotiana tabacum (SEQ ID NO. 33) (DNA); Glycosyltransferase (NtGTIa) Nicotiana tabacum (SEQ ID NO. 34) (amino acid); Glycosyltransferase (NtGTIa) Nicotiana tabacum (SEQ ID NO. 35) (DNA); Glycosyltransferase (NtGT3) Nicotiana tabacum (SEQ ID NO. 36) (amino acid); Glycosyltransferase (NtGT3) Nicotiana tabacum (SEQ ID NO. 37) (DNA); Glycosyltransferase (NtGT2) Nicotiana tabacum (SEQ ID NO. 38) (amino acid); and / or glycosyltransferase (NtGT2) Nicotiana tabacum (SEQ ID NO. 39) (DNA). The Nicotiana tabacum sequences are exemplary only because other tobacco glycosyltransferase can be used.
[00133] As noted above, these glycosyltransferases can glycosylate cannabinoids and / or cannabinoids functionalized in a plant or plant cell suspension culture, as generally described here. Of course, other glycosyltransferase genes from alternative sources can be included in the present invention.
[00134] As noted above, in one embodiment, one or more glycosyltransferases can be targeted or located in a portion of the plant cell. For example, in this preferred embodiment, cannabinoid glycosylation can be localized to the trichomes allowing cannabinoids to accumulate higher than then the wild-type levels in that structure. In an exemplary embodiment, an artificially modified glycosyltransferase can be generated. In this example embodiment, a UDP glycosyltransferase (76G1) can be fused to a trichome target sequence on its N-terminal tail. It is
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45/114 trichome target sequence can be recognized by the cell and causes it to be transported to the trichomes. This artificial genetic construct is identified as SEQ ID NO. 19 (gene), or SEQ ID NO. 20 (protein). In one embodiment, a trichome target sequence or domain can be derived from any number of synthases. For example, in one embodiment, a THCA Synthase Trichome domain (SEQ ID NO. 40) can be coupled with a glycosyltransferase as generally described above. In addition, in another example, a CBDA Synthase Trichome target domain (SEQ ID NO. 41) can be coupled with a glycosyltransferase as generally described above.
[00135] In another embodiment, the invention may include a embodiment in which the transiently modified cannabinoids can be passively and / or actively excreted from a cell or into a cell wall. In an exemplary model, an exogenous ATP-binding cassette carrier (ABC or ABCt carriers) or other similar molecular structure can recognize the glycosyl or glucuronic acid or acetyl (conjugate) functional group in the transiently modified cannabinoid and actively transport it through the cell / membrane wall and in the surrounding environment.
[00136] In one embodiment, a plant can be transformed to express a heterologous ABC carrier. In this embodiment, an ABCt can facilitate the transport of cannabinoids out of cells in suspension cultures, such as a cannabis or tobacco cell suspension culture. In this preferred embodiment, a transported multi-drug human (ABCG2) can be expressed in a plant cell suspension culture of the same, respectively. ABCG2 is a plasma membrane target protein and can also be identified as SEQ ID NO. 9 (gene) or 10 (protein).
[00137] Generally, a trichome structure, as in Cannabis or tobacco, will have very little or no substrate for a glycosyltransferase enzyme to use to effect glycosylation. To solve this problem, in one embodiment, the invention can include systems, methods and compositions to increase
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46/114 substrates for glycosyltransferase, that is, sugars selected in a trichome. In a preferred embodiment, the invention may include the location or target of the sugar transport to the trichome. In this preferred embodiment, an exogenous or endogenous UDPglucose / UDP-galactose, transporter (UTR1) can be expressed in a trichome production unit, such as cannabis or tobacco and the like. In this embodiment, the UDP-glucose / UDP-galactose (UTR1) transporter can be modified to include a plasma membrane target sequence and / or domain.
[00138] With this target domain, the UDP-glucose / UDPgalactose (UTR1) transporter can allow the artificial fusion protein to be anchored to the plasma membrane. In this configuration, the sugar substrates of the cytosol can pass through the UDP-glucose / UDP-galactose transporter (PM-UTR1) attached to the plasma membrane to the trichome. In this modality, the substrates for the glycosyltransferase can be located in the trichome and allow it to accumulate even more, allowing an improved glycosylation of cannabinoids in the trichome. In one example, SEQ ID NO. 21 is identified as the sequence of the polynucleotide gene for a heterologous UDP-glucose / galactose (UTR1) transporter from Arabidopsis thaliana having a plasma membrane target sequence replacing a tonoplast target sequence. The target sequence of the plasma membrane of this exemplary fusion protein can include the following sequence (see SEQ ID NO 21)
TGCTCCATAATGAACTTAATGTGTGGGTCTACCTGCGCCGCT, or a sequence with 70-99% homology to the sequence.
[00139] It should be noted that various combinations and permutations of the genes / proteins described herein can be co-expressed and thus achieve one or more of the objectives of the present invention. Such combinations are examples of preferred modalities only, and not limiting in any way.
[00140] In one embodiment, a gene, such as a cannabinoid synthase, or a gene fragment corresponding to, for example, a signal domain
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47/114 can be inhibited, negatively regulated, interrupted or can even be knocked out. One skilled in the art will recognize the various processes that can be carried out without undue experimentation. In another embodiment, a knockout can mean overexpression of a modified endogenous or exogenous gene compared to the weight version.
[00141] For example, in one embodiment, high levels of cannabinoid glycosylation can be generated by coexpressing CYP3A4 and CYP oxidoreductase (cytochrome P450 with P450 oxidoreductase) and at least one endogenous glycosyltransferase in N. benthamiana. In another embodiment, one or more of the endogenous or exogenous genes can be expressed in plants or plant cell culture with the coexpression of myb and / or a catalase. In this configuration, there is an additive effect of overexpressing a Myb transcription factor and a catalase, one or more of which can be targeted or localized, in the synthesis of water-soluble cannabinoids (glycosylated and hydroxylated) in Cannabis sativa.
[00142] In certain embodiments, endocannabinoids can be functionalized and / or acetylated and / or glycosylated, as generally described here.
[00143] All sequences described herein include sequences of between 70-99% homology to the identified sequence [00144] The modified cannabinoid compounds of the present invention are useful for a variety of therapeutic applications. For example, the compounds are useful for treating or alleviating symptoms of diseases and disorders involving CB1 and CB2 receptors, including loss of appetite, nausea and vomiting, pain, multiple sclerosis and epilepsy. For example, they can be used to treat pain (that is, as pain relievers) in a variety of applications, including, but not limited to, pain management. In additional embodiments, these modified cannabinoid compounds can be used as an appetite suppressant. Additional modality may include the administration of modified cannabinoid compounds.
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48/114 [00145] By "treating" the inventors of the present invention say that the compound is administered in order to alleviate the symptoms of the disease or disorder being treated. Those skilled in the art will recognize that the symptoms of the disease or disorder that is treated can be completely eliminated or can simply be reduced. In addition, the compounds can be administered in combination with other medications or treatment modalities, such as chemotherapy or other cancer drugs.
[00146] The implementation can generally involve identifying patients suffering from the indicated disorders and administering the compounds of the present invention in an acceptable manner by an appropriate route. The exact dosage to be administered can vary depending on the age, sex, weight and general health of each patient, as well as the precise etiology of the disease. However, in general, for administration to mammals (e.g., humans), dosages in the range of about 0.1 to about 30 g of compound per kg of body weight per 24 η. More preferably, about 0.1 to about 10 mg of compound per kg of body weight for 24 hours are effective.
[00147] Administration can be oral or parenteral, including intravenous, intramuscular, subcutaneous, intradermal injection, intraperitoneal injection, etc., or by other routes (for example, transdermal, sublingual, oral, rectal and buccal delivery, inhalation of an aerosol , etc.). In a preferred embodiment of the invention, water-soluble cannabinoid analogs are provided orally or intravenously.
[00148] In particular, the phenolic esters of the present invention (Formula
1) are preferably administered systemically, in order to provide an opportunity for metabolic activation by in vivo ester divage. In addition, water-soluble compounds with azole moieties on the pentyl side chain (Formula 2, for example, with imidazole moieties) do not require in vivo activation and may be suitable for direct administration (eg specific injection into the site).
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49/114 [00149] The compounds can be administered in pure form or in a pharmaceutically acceptable formulation including suitable elixirs, binders, and the like (generally referred to as a "carrier") or as pharmaceutically acceptable salts (for example, alkali metal salts, such as such as sodium, potassium, calcium, lithium salts, ammonium, etc.) or other complexes. It is to be understood that pharmaceutically acceptable formulations include liquid and solid materials conventionally used to prepare injectable dosage forms and solid dosage forms, such as tablets and capsules and aerosol dosage forms. In addition, the compounds can be formulated with aqueous or oil-based vehicles. Water can be used as a vehicle for the preparation of compositions (for example, injectable compositions), which can also include buffers and conventional agents to make the composition isotonic. Other potential additives and other materials (preferably those that are generally considered to be safe [GRAS]) include: dyes; aromas; surfactants (TWEEN, oleic acid, etc.); solvents, stabilizers, elixirs and binders or encapsulants (lactose, liposomes, etc.). Solid diluents and excipients include lactose, starch, conventional disintegrating agents, coatings and the like. Preservatives such as methyl paraben or benzalkonium chloride can also be used. Depending on the formulation, it is expected that the active composition will consist of about 1% to about 99% of the composition and the vehicle "vehicle" will constitute about 1% to about 99% of the composition. The pharmaceutical compositions of the present invention can include any suitable pharmaceutically acceptable additives or adjuvants, insofar as they do not impair or interfere with the therapeutic effect of the active compound.
[00150] The administration of the compounds of the present invention can be intermittent, bolus dose or at a gradual or continuous, constant or controlled rate for a patient. In addition, the time of day and the number of times per day that the pharmaceutical formulation is administered can vary and are best determined by a specialist, such as a doctor. In addition
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50/114 Effective dose can vary depending on factors such as delivery mode, sex, age and other conditions of the patient, as well as the extent or progression of the disease. The compounds can be supplied alone, in a mixture containing two or more of the compounds, or in combination with other medications or treatment modalities. The compounds can also be added to the blood ex vivo and then supplied to the patient.
[00151] The genes encoding a combination polynucleotide and / or a homologue thereof can be introduced into a plant and / or plant cell using various types of transformation approaches developed for the generation of transgenic plants. Standard transformation techniques, such as transformation mediated by Agro bacterium, Ti plasmid, particle bombardment, microinjection and electroporation can be used to build transgenically transformed plants in a stable manner.
[00152] As used here, a - "cannabinoid" is a chemical compound (such as cannabinol, THC or cannabidiol) that is found in Cannabis plant species, among others like Echinacea; Acmella Oleracea; Helichrysum Umbraculigerum; Radula Marginata (Liverwort) and Theobroma Cacao, and metabolites and synthetic analogues thereof that may or may not have psychoactive properties. Therefore, cannabinoids include (without limitation) compounds (such as THC) that have high affinity for the cannabinoid receptor (for example, Ki <250 nM) and compounds that do not have significant affinity for the cannabinoid receptor (such as cannabidiol, CBD). Cannabinoids also include compounds that have a characteristic dibenzopyran ring structure (of the type seen in THC) and cannabinoids that do not have a pyran ring (such as cannabidiol). Therefore, a partial list of cannabinoids includes THC, CBD, dimethyl heptypentyl cannabidiol (DMHP-CBD), 6,12dihydro-6-hydroxy-cannabidiol (described in US Patent 5,227,537, incorporated by reference); (3S, 4R) -7-hydroxy-A6-tetrahydrocannabinol homologues and derivatives described in US Patent 4,876,276, incorporated by reference; (+) - 4- [4-DMH-2,6diacetoxy-phenyl] -2-carboxy-6,6-dimethylbicyclo [3.1.1] hept-2-ene and other derivatives of
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51/114
4-phenylpinene disclosed in US Patent 5,434,295, which is incorporated by reference; and cannabidiol (-) (CBD) analogs such as monomethyl ether (-) CBD, dimethyl ether (-) CBD; (-) CBD diacetate; (-) 3'-acetyl-CBD monoacetate; and ± AF1 1, all disclosed in Consroe et al., J. Clin. Phannacol. 2 1: 428S-436S, 1981, which is also incorporated by reference. Many other cannabinoids are also disclosed in Agurell et al., Pharmacol. Rev. 38: 31-43, 1986, which is also incorporated by reference.
[00153] As claimed here, the term "cannabinoid" can also include different modified forms of a cannabinoid, such as a hydroxylated cannabinoid or cannabinoid carboxylic acid. For example, if a glycosyltransferase were able to glycosylate a cannabinoid, it would include the term cannabinoid as defined elsewhere, as well as the modified forms mentioned above. It can also include several portions of glycosylation.
[00154] Examples of cannabinoids are tetrahydrocannabinol, cannabidiol, cannabigerol, cannabicromene, cannabicycline, cannabivarin, cannabielsoin, cannabicitrane, cannabigerolic acid, cannabigeric acid monomethyl, cannabigeric acid, cannabigeric acid, cannabigerovinic acid, cannabigerovinic acid, cannabigerovinic acid cannabidolic acid, cannabidiol monomethyl ether, cannabidio-C4, cannabidivarinic acid, cannabidiorcol, delta-9-tetrahydrocannabinolic acid A, delta-9-tetrahydrocannabinolic acid B, delta-9-tetrahydrocannabinolic-C4 acid, delta-9-tetrahydrochloric acid , delta-9-tetrahydrocannabivarina, delta-9-tetrahydrocannabiorcolico acid, delta-9-tetrahydrocannabiorcol, delta-7-cis-iso-tetrahydrocannabiol, delta-8-tetrahydrotetrahydrocannabilic acid, delta-8-tetrahydrocannabino , cannabicyclic acid, cannabicilovarin, cannabielsoic acid A, cannabielsoic acid B, cannabinolic acid, cannabinol methyl ether, cannabinol-C4, cannabinolC2, cannabiorcol, 10-ethoxy-9-hydroxy-delta-6a-tetrahydrocannabinol, 8,9-dihydroxyidelta-6a-tetrahydrocannabinol, cannabitriolvarina, ethoxy-cannabitriolvarina, dehydrocannabifuran, cannabifurane, cannabicuran, 10 delta
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11/114
6a-tetrahydrocannabinol, delta-9-cis-tetrahydrocannabinol, 3, 4, 5, 6-tetrahydro-7hydroxy-alpha-alpha-2-trimethyl-9-n-propyl-2,6-methane- 2H-1-benzoxocin-5-methanolcanabiripsol, trihydroxy-delta-9-tetrahydrocannabinol and cannabinol. Examples of cannabinoids in the context of this disclosure include tetrahydrocannabinol and cannabidiol.
[00155] The term "endocannabinoid" refers to compounds, including arachidonoyl ethanolamide (anandamide, AEA), 2-arachidonoyl ethanolamide (2-AG), 1-arachidonoyl ethanolamide (1-AG), and docosahexaenoyl ethanolamide (DHEA, synaptamide), oleoyl ethanolamide (OEA), eicsapentaenoyl ethanolamide, prostaglandin ethanolamide, docosahexaenoyl ethanolamide, linolenoyl ethanolamide, acid 5 (Z), 8 (Z), 1 1 (Z) - eicosatrienoic ethanolamide (ethanolamide amide acid); , stearoyl ethanoylamide, docosaenoyl ethanolamide, nervonoyl ethanolamide, tricosanoyl ethanolamide, lignoceroyl ethanolamide, myristoyl ethanolamide, pentadecanoyl ethanolamide, palmitoleyl ethanolamide, docosahexaenoic acid (DHA). Particularly preferred endocannabinoids are AEA, 2AG, 1 -AG and DHEA.
[00156] Hydroxylation is a chemical process that introduces a hydroxyl group (-OH) into an organic compound. Acetylation is a chemical reaction that adds an acetyl chemical group. Glycosylation is the coupling of a glycosyl donor to a glycosyl acceptor forming a glycoside.
[00157] The term "prodrug" refers to a precursor to a biologically active pharmaceutical agent (drug). Prodrugs must undergo chemical or metabolic conversion to become a biologically active pharmaceutical agent. A prodrug can be converted ex vivo to the biologically active pharmaceutical agent by chemical transforming processes. In vivo, a prodrug is converted into the biologically active pharmaceutical agent by the action of a metabolic process, an enzymatic process or a degradative process that removes the prodrug fraction to form the biologically active pharmaceutical agent.
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53/114 [00158] As used herein, the term "homologous" in relation to a contiguous nucleic acid sequence, refers to contiguous nucleotide sequences that hybridize under appropriate conditions to the reference nucleic acid sequence. For example, homologous sequences can have about 70% to 100, or more generally 80% to 100% sequence identity, such as about 81%; about 82%; about 83%; about 84%; about 85%; about 86%; about 87%; about 88%; about 89%; about 90%; about 91%; about 92%; about 93%; about 94% about 95%; about 96%; about 97%; about 98%; about 98.5%; about 99%; about 99.5%; and about 100%. The property of substantial homology is closely related to specific hybridization. For example, a nucleic acid molecule is specifically hybridizable when there is a sufficient degree of complementarity to avoid non-, specific binding of the nucleic acid to non-target sequences under conditions where specific binding is desired, for example, under hybridization conditions strict.
[00159] The term 'Operationally linked', when used in reference to a regulatory sequence and a coding sequence, means that the regulatory sequence affects the expression of the linked coding sequence. "Regulatory sequences" or "control elements" refer to nucleotide sequences that influence the time and level / amount of RNA transcription, processing or stability or translation of the associated coding sequence. Regulatory strings can include promoters; strings of translation leaders; introns; improvers; rod-loop structures; binding sequences to the repressor; termination sequences; polyadenylation recognition sequences; etc. Particular regulatory sequences can be located upstream and / or downstream of a coding sequence operationally linked to it. In addition, particular regulatory sequences operably linked to a coding sequence may be located on the associated complementary strand of a double-stranded nucleic acid molecule.
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54/114 [00160] As used herein, the term "promoter" refers to a region of DNA that may be upstream of the start of transcription and that may be involved in the recognition and binding of RNA polymerase and other proteins to initiate transcription . A promoter can be operationally linked to a coding sequence for expression in a cell, or a promoter can be operationally linked to a nucleotide sequence that encodes a signal sequence that can be operationally linked to a coding sequence for expression in a cell . A "plant promoter" can be a promoter capable of initiating transcription in plant cells. Examples of promoters under development control include promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, seeds, fibers, xylem vessels, tracheids or sclerenchyma. Such promoters are referred to as "preferential tissue". Promoters that initiate transcription in certain tissues only are referred to as "tissue specific".
[00161] A "cell type specific" promoter primarily directs expression in certain cell types in one or more organs, for example, vascular cells in the roots or leaves. An "inducible" prosecutor may be a prosecutor who may be under environmental control. Examples of environmental conditions that can initiate transcription by inducible promoters include anaerobic conditions and the presence of light. Tissue-specific, tissue-preferred, cell-specific and inducible promoters constitute the “non-constitutive” class of promoters. A "constitutive" promoter is a promoter that can be active under most environmental conditions or in most types of cells or tissues.
[00162] Any inducible promoter can be used in some embodiments of the invention. See Ward et al. (1993) Plant Mol. Biol. 22: 361-366. With an inducible promoter, the rate of transcription increases in response to an inducing agent. Exemplary inducible promoters include, but are not limited to: ACEI system promoters that respond to copper; In2 gene of
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55/114 corn that responds to the protection agents of benzene sulfonamide herbicides; TIO repressor of TIO; and the promoter inducible from a steroid hormone gene, whose transcriptional activity may be induced by a glucocorticosteroid hormone are general examples (Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88: 0421).
[00163] As used herein, the term "transformation" or "genetically modified" refers to the transfer of one or more nucleic acid molecule (s) to a cell. A plant is "transformed" or "genetically modified" by a nucleic acid molecule transduced into the plant when the nucleic acid molecule becomes stably replicated by the plant. As used herein, the term "transformation" or "genetically modified" encompasses all techniques by which a nucleic acid molecule can be introduced, such as a plant. [00164] The term "vector" refers to some means by which DNA, RNA, a protein or polypeptide can be introduced into a host. Polynucleotides, proteins and polypeptides that are to be introduced into a host can be therapeutic or prophylactic in nature; it can code or be an antigen; may be of a regulatory nature, etc. There are several types of vectors, including viruses, plasmids, bacteriophages, cosmids, and bacteria.
[00165] As is known in the art, different organisms preferentially use different codons to generate polypeptides. Such "codon use" preferences can be used in the design of nucleic acid molecules that encode the proteins and chimeras of the present invention, in order to optimize expression in a particular host cell system.
[00166] An "expression vector" is a nucleic acid capable of replicating in a selected host cell or organism. An expression vector can replicate as an autonomous structure or, alternatively, it can integrate, in whole or in part, into the host cell's chromosomes or the nucleic acids of an organelle, or is used as a transport service for the delivery of foreign DNA to cells, and thus replicate along with the host cell genome.
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Thus, an expression vector is polynucleotides capable of replicating in a selected host cell, organelle or organism, for example, a plasmid, virus, artificial chromosome, nucleic acid fragment and for which certain genes in the expression vector (including genes of interest) are transcribed and translated into a polypeptide or protein within the cell, organelle or organism; or any suitable construct known in the art, which comprises an "expression cassette". In contrast, as described in the examples in this document, a "cassette" is a polynucleotide containing a section of an expression vector of this invention. The use of cassettes assists in the assembly of expression vectors. An expression vector is a replicon, such as plasmid, phage, virus, chimeric or cosmid virus, and which contains the desired polynucleotide sequence operationally linked to the expression control sequence (s).
[00167] A polynucleotide sequence is operably linked to an expression control sequence (for example, a promoter and, optionally, an enhancer) when the expression control sequence controls and regulates the transcription and / or translation of that polynucleotide sequence . [00168] Unless otherwise indicated, a specific nucleic acid sequence also implicitly includes conservatively modified variants (e.g., degenerate codon substitutions), the complementary sequence (or complement) and the reverse complement sequence, as well as the sequence explicitly indicated. Specifically, substitutions of degenerate codons can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced with mixed base and / or deoxy-inosine residues (see, for example, Batzer et al ., Nucleic Acid Res. 19: 5081 (1991); Ohtsuka et al., J. Biol. Chem. 260: 26052608 (1985); and Rossolini et al., Mol. Cell. Probes 8: 91-98 (1994) ). Due to the degeneration of the nucleic acid codons, several different polynucleotides can be used to encode identical polypeptides. Table 1,
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57/114 below, contains information on which nucleic acid codons encode which amino acids.
TABLE 4 Amino acid nucleic acid codons
Amino Acid Nucleic acid codons Wing / A GCT, GCC, GCA, GCG Arg / R CGT, CGC, CGA, CGG, AGA, AGG Asn / N AAT, AAC Asp / D GAt, GAC Cys / TGT, TGC Gln / Q CAA, CAG Glu / E GAA, GAG Gly / G GGT, GGC, GGA, GGG His / H CAT, CAC lle / l ATT, ATC, ATA Leu / L TTA, TTG, CTT, CTC, CTA, CTG Lys / K AAA, AAG Met / M ATG Phe / F TTT, TTC Pro / P CCT, CCC, CCA, CCG Ser / S TCT, TCC, TCA, TCG, AGT, AGC Thr / T ACT, ACC, ACA, ACG Trp / W TGG Tyr / Y TAT, TAC Val / V GTT, GTC, GTA, GTG
00169] The term “plant” or “plant system” includes whole plants, plant organs, offspring of whole plants or plant organs, embryos, somatic embryos, embryo-like structures, protocorms, protocorm-like bodies (PLBs) and culture and / or plant cell suspensions. The organs
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58/114 plants comprise, for example, vegetative sprouting organs / structures (eg leaves, stems and tubers), roots, flowers and floral organs / structures (eg bracts, sepals, petals, stamens, carpels, anthers and ova), seeds (including embryo, endosperm, and lining seed) and fruit (mature ovary), plant tissue (eg vascular tissue, soil tissue, and the like) and cells (eg guard cells, egg cells, trichomes and the like). The invention can also include Cannabaceae and other strains of Cannabis, such as C. sativa in general.
[00170] The term "expression" as used here, or "expression of a coding sequence" (for example, a gene or a transgene) refers to the process by which the encoded information of a nucleic acid transcription unit (including, for example, genomic DNA) or cDNA) is converted into an operational, non-operational or structural part of a cell, usually including the synthesis of a protein. Gene expression can be influenced by external signals; for example, exposure of a cell, tissue or organism to an agent that increases or decreases gene expression. The expression of a gene can also be regulated anywhere in the path, from DNA to RNA and protein. The regulation of gene expression occurs, for example, through controls that act on the transcription, translation, transport and processing of RNA, degradation of intermediate molecules, such as mRNA, or through the activation, inactivation, compartmentalization or degradation of specific protein molecules after that they are made, or by combinations of them. Gene expression can be measured at the RNA level or the protein level by any method known in the art, including, without limitation, Northern transfer, RT-PCR, Western blot, or in vitro, in situ, or in the activity assay in vivo protein (s) [00171] The term "nucleic acid" or "nucleic acid molecules" includes single and double stranded DNA forms; forms of single-stranded RNA; and double-stranded forms of RNA (dsRNA). The term “nucleotide sequence” or “sequence
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59/114 nucleic acid “refers to the sense and antisense strands of a nucleic acid as individual strands or in the duplex. The term “ribonucleic acid” (RNA) is included in RNAi (inhibitor RNA), RNAdc (double-stranded RNA), siRNA (small interfering RNA), mRNA (messenger RNA), miRNA (micro-RNA), RNAhp (hook RNA) hair), tRNA (transfer RNA), loaded or unloaded with a corresponding acylated amino acid) and cRNA (complementary RNA). The term "deoxyribonucleic acid" (DNA) is included in cDNA, genomic DNA, and DNA-RNA hybrids. The terms "nucleic acid segment" and "nucleotide sequence segment", or more generally, "segment", will be understood by those skilled in the art as a functional term that includes both genomic sequences, ribosomal RNA sequences, sequences of RNA transfer, messenger RNA sequences, operon sequences and smaller manipulated nucleotide sequences that have encoded or can be adapted to encode, peptides, polypeptides or proteins.
[00172] The term "gene" or "sequence" refers to a coding region operationally linked to appropriate regulatory sequences capable of regulating the expression of the gene product (for example, a polypeptide or a functional RNA) in some way. A gene includes untranslated regulatory regions of DNA (for example, promoters, enhancers, repressors, etc.) preceding (upstream) and following (downstream) the coding region (open reading frame, ORF), as well as, when applicable, intermediate sequences (ie, introns) between individual coding regions (ie, exons). The term "structural gene", as used herein, is intended to mean a DNA sequence that is transcribed into mRNA, which is then translated into an amino acid sequence characteristic of a specific polypeptide.
[00173] A nucleic acid molecule can include one or both naturally occurring and modified nucleotides linked together by naturally occurring and / or non-naturally occurring nucleotide bonds. The nucleic acid molecules can be modified chemically or biochemically or can contain bases
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60/114 unnatural or derived nucleotides, as will be readily appreciated by those skilled in the art. Such modifications include, for example, markers, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications (for example, unloaded bonds: for example, methyl phosphonates, phosphotrieters, phosphoramidates, carbamates, etc.). ; charged bonds: for example, phosphorothioates, phosphorodithioates, etc.; pending portions: for example, peptides; intercalators: for example, acridine, psoralen, etc.; chelators; alkylating agents; and modified bonds: for example, alpha anomeric nucleic acids, etc.). The term "nucleic acid molecule" also includes any topological conformation, including single ribbon, double ribbon, partially duplex, triplex, cracked, hair-pinned, circular and padlocked. [00174] As used here in relation to DNA, the term "coding sequence", "structural nucleotide sequence" or "structural nucleic acid molecule" refers to a nucleotide sequence that is finally translated into a polypeptide, via transcription and mRNA , when placed under the control of appropriate regulatory sequences. With regard to RNA, the term "coding sequence" refers to a sequence of nucleotides that is translated into a peptide, polypeptide or protein. The limits of a coding sequence are determined by a translation start codon at terminal 5 'and a translation stop codon at terminal 3'. Coding sequences include, but are not limited to: genomic DNA; cDNA; EST; and recombinant nucleotide sequences.
[00175] The term "sequence identity" or "identity", as used herein in the context of two nucleic acid or polypeptide sequences, refers to residues in the two sequences that are equal when aligned for maximum match in a specified comparison window .
[00176] The term "recombinant" when used with reference, for example, to a cell, or nucleic acid, protein or vector, indicates that the cell, organism, nucleic acid, protein or vector has been modified by the introduction of an acid
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61/114 heterologous nucleic or protein, or the modification of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells can express genes that are not found in the native (non-recombinant or wild-type) form of the cell or express native genes that are otherwise abnormally expressed, overexpressed, underexpressed or unexpressed.
[00177] The terms "approximately" and "about" refer to a quantity, level, value or quantity that varies up to 30%, or in another modality up to 20% and in a third modality up to 10% to a quantity, level, value or reference quantity. As used here, the singular form "one", "one" and "o, a" includes plural references, unless the context clearly indicates otherwise.
[00178] As used here, "heterologous" or "exogenous" in reference to a nucleic acid is a nucleic acid that originates from a foreign species, or is synthetically designed, or, if from the same species, is substantially modified in its way native in composition and / or genomic locus by deliberate human intervention. A heterologous protein can originate from a foreign species or, if it belongs to the same species, it is substantially modified from its original form by deliberate human intervention. By "host cell" means a cell that contains an introduced nucleic acid construct and supports replication and / or expression of the construct. Host cells can be prokaryotic cells such as E. coli or eukaryotic cells, such as fungi, yeasts, insects, amphibians, nematodes or mammals cells. Alternatively, host cells are monocotyledonous or dicotyledonous plant cells. An example of a monocotyledon host cell is a corn host cell
EXAMPLES
Example 1: Functionalization of cannabinoids by cytochrome P450s.
[00179] The present inventors have demonstrated that cannabinoids can
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62/114 be functionalized in an in vivo plant system. Specifically, the present inventors used cytochrome P450 monooxygenases (CYP) to modify or functionalize the chemical structure of cannabinoids. As shown below, CYPs do this by inserting an oxygen atom into the hydrophobic molecules to make them more reactive and hydrophilic. A representative reaction can include the generalized reaction in Fig. 13.
[00180] The P450 enzyme system involves several species of cytochrome P450 and nonspecific oxidoreductases of cytochrome P450. As shown in Fig. 5, the present inventors used a human cytochrome P450 (CYP3A4) in a double construct with an exemplary exemplary human cytochrome P450 oxidoreductase, both expressed under the control of the constitutive promoter of CaMV 35S with regions not translated in 5 ' to improve the translation. The protein and DNA sequences for cannabinoid functionalization (CYP3A4 and P450 oxidoreductase) are identified as SEQ ID NOs. 1 -4. Expression was confirmed using RT-PCR using the forward and reverse primers identified in Table 3 below. As noted above, the present inventors demonstrated that the overexpression of P450s generated functionalized cannabinoids that could then be glycosylated, making them soluble in water.
Example 2: Overexpression of P450 improves hydroxylation and glycosylation in vivo of cannabinoids in plant systems.
[00181] The present inventors demonstrated that overexpression increased the hydroxylation and glycosylation in vivo of CBDA in an exemplary plant system. Specifically, as generally shown in Fig. 6, the present inventors demonstrate that the infiltration of tobacco leaves with Agrobacterium carrying CYP3A4 and P450 oxidoreductase was carried out as described in the present document. Expression confirmation was performed using RT-PCR 23 days after infiltration (Fig. 6).
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63/114 [00182] As generally shown in Fig. 7, the present inventors demonstrated that the overexpression of the CYP3A4 + P450 oxidoreductase construct and subsequent feeding of at least one cannabinoid, in this case, CBDA, after confirmation of the expression resulted in the in vivo glycosylation of CBDA in tobacco leaves (Fig. 7). On average, glycosylation increased 3-fold in transgenic N. benthamiana plants compared to control, while hydroxylation increased up to 13-fold. As such, in a certain embodiment, tobacco glycosyltransferases can be used as main targets in the current inventive technology for cannabinoid glycosylation.
Example 3: Identification of water-soluble cannabinoids modified by mass spectrometry.
[00183] The present inventors demonstrated the biosynthesis of modified functionalized, as well as water-soluble cannabinoids, both in vitro and in vivo, as well as plant system. Specifically, the present inventors identified the cannabinoid biotransformations associated with the gene constructs in both in vitro assays and transient leaf expression. Through the use of accurate mass spectrometry measurements, the present inventors were able to identify and confirm the biosynthesis of modified water-soluble cannabinoids.
[00184] Specifically, as generally shown in Figs. 1-4, the present inventors were able to identify glycosylated water-soluble cannabinoids in chromatographic analysis and were able to produce extracted ion chromatograms for peak integration. For example, Fig. 1, panel B, illustrates the identification of multiple constitutional isomers of cannabinoids from a single glycosidic fraction, while in Fig. 2 panel B, an example of multiple constitutional isomers of cytochrome P450 oxidation is illustrated. The peak areas for each identified molecule were used for relative quantification between treatments. Based on these results,
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64/114 we confirmed the biosynthesis of modified cannabinoid molecules containing up to two portions of glycosides, acetyl glycoside, as well as hydroxylation biotransformations (R-OH).
[00185] Tables 1 and 2 are provided below demonstrating even more the production of the selected modified cannabinoid molecules. Generally referring to Tables 1-2 below, the present inventors have demonstrated that, based on reduced water retention time: the acetonitrile HPLC gradient, the glycosylated and hydroxylated cannabinoids, which eluted earlier than their unmodified forms , are shown to be more water soluble than their unmodified forms.
Example 4: Generation of heterologous cytosolic synthesis and glycosylation gene constructs for expression in tobacco leaves and cell suspensions.
[00186] As shown in Fig. 8, the present inventors generated a triple gene construct for expression of cannabidiolic acid (CBDA) synthase in which the trichome target sequence had been removed and Stevia rebaudiana glycosyltransferase 76G1. In this construct, the ABC multi-drug vehicle ABCG2 was also included.
[00187] In one embodiment of the present inventive technology, the genetic construct can be used to transform a plant cell that can still be configured to be grown into a suspension culture. In a preferred embodiment, a cannabis cell can be transformed with the construct generally outlined in Fig. 8. In this preferred embodiment, the cannabinoids produced by cannabis cells in cell culture can be functionalized by overexpressing CYP3A4 + P450 oxidoreductase, as described above, and still glycosylated by the expression and action of the heterologous UDP glycosyltransferase (76G1) from Stevia rebaudiana refer to above. In addition, as generally in a scheme here, cannabinoids can be modified to be functionalized and / or glycosylated, or in general, soluble
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65/114 in water, and can then be segregated to the cell wall area, in the case of an entire plant, or turn media into suspension cultures, with the aid of the ABC carrier. In one embodiment, this construct can be used for the synthesis and modification of cannabinoids in cell suspension cultures, using bright yellow tobacco cells or cannabis cells.
[00188] As generally shown in Fig. 9, the in vivo expression of CBDA synthase, UDP glycosyltransferase 76G1 and ABCG2 was confirmed. The reverse and forward primers used in RT-PCR reactions are provided below in Table 4 below.
[00189] The gene and protein sequence identifications for CBDA synthase are provided as SEQ ID NO's 5 and 6, respectively. It should be noted that a variety of cannabinoid synthase genes / proteins can be used with current inventive technology, with CBDA synthase being only exemplary. In fact, it is specifically contemplated that the enzyme synthase associated with any of the cannabinoids identified herein can be incorporated into the present invention without undue experimentation. In one embodiment, one or more of these exogenous or endogenous synthase enzymes may still have the trichome target sequence excised, again, a step that can be readily performed without undue experimentation. Example can be THCA synthase, CBG synthase, THCA synthase, CBDA synthase or CBCA synthase, which may, in this embodiment, have its trichome target sequence had been removed.
[00190] The gene and protein sequence identifications for Stevia rebaudiana glycosyltransferase 76G1 are provided as SEQ ID NOs. 7 and 8, respectively. The gene and protein sequence identifications for the ABC multi-drug carrier ABCG2 are provided as SEQ ID NO's 9 and 10, respectively.
Example 5: Cytosolic synthesis in vivo and glycosylation of cannabinoids in N. benthamiana leaves and cell suspensions.
[00191] As shown in Fig. 10, the inventors of the present invention
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66/114 demonstrate that, in plants, in this modality N. benthamiana, expressing the cytosolic construct mentioned above, glycosylation of CBGA occurred, as well as formation of modified or hydroxylated CBDA. CBGA glycosylation shows in vivo glycosylation of cannabinoids by overexpression of a glycosyltransferase in plants of N. benthamiana. The presence of glycosylated cannabinoids in wild type plants suggests the presence of a strong glycosyltransferase in tobacco. As such, in one embodiment, overexpression of a heterologous or homologous tobacco glycosyltransferase can be expressed or overexpressed, resulting in improved in vivo biosynthesis of water-soluble cannabinoids in whole plants, as well as in suspension cultures. For example, in one embodiment, a heterologous tobacco glycosyltransferase can be expressed in a culture of cannabis plants or cells resulting in the in vivo biosynthesis of water-soluble cannabinoids in the Cannabis plant and / or suspended cannabis cultures.
Example 6: Water-soluble cannabinoid production systems using MTB and / or catalase transcription factor.
[00192] The present inventors have developed a plurality of systems for the biosynthesis and modification of cannabinoids based on the location of cells using new protein targeting methods. As shown in Table 10, the present inventors designed these new systems and methods to improve cannabinoid production and modification (glycosylation, acetylation and functionalization), as well as to mitigate toxicity resulting from cannabinoid accumulation. Certain modalities included the expression of a MYB transcription factor and a catalase (Fig. 27) to degrade the hydrogen peroxide resulting from CBDA synthase activity. In a preferred embodiment, the present inventors used Arabidopsis thaliana or an E. coli catalase gene and a predicted transcription factor for Cannabis MYB involved in the elevation of genes involved in cannabinoid biosynthesis. DNA and protein sequences for the MYB predicted transcription factor MYB (SEQ ID NOs. 1-12,
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67/114 DNA sequences and amino acids respectively), SEQ ID NOs 13-14 of the catalidase of Arabidopsis thaliana., DNA sequences and amino acids respectively) and / or E. coli catalase (SEQ ID NO. 15-16, DNA sequences and amino acids).
Example 7: Enhanced cytosolic synthesis and in vivo glycosylation of cannabinoids in tobacco leaves and cell suspensions.
[00193] The present inventors demonstrated the increase in in vivo modification of cannabinoids in transgenic plants co-infected with glycosylation constructs, P450-mediated functionalization (hydroxylation) and detoxification of hydrogen peroxide by catalase. Also as shown in Fig. 1, of functionalization and glycosylation, it was observed mainly the CBGA substrate in transgenic tobacco plants that overexpress CBDA synthase, UDP glycosyltransferase and the ABC transporter, but increased when the overexpression of this construct was coupled with cytochrome P450, transcription factor MYB and catalase. As noted earlier, overexpression of a cytochrome P450 increased cannabinoid glycosylation. As such, the present inventor demonstrated the formation and glycosylation of CBDA in vivo in transiently transformed tobacco leaves fed with the precursor CBGA.
[00194] The present inventors also compared the activities of endogenous and transgenic glycosyltransferase in tobacco. Specifically, as shown in Fig. 12, the present inventor performed in vitro assays for UDP glycosyltransferase and CBDA synthase. Short 3-hour trials at 30 ° C revealed no difference in CBGA glycosylation between wild and transgenic N. benthamiana plants, suggesting endogenous glycosylation. In prolonged trials (14 hours), there was a significant difference in the detection of glycosylated CBGA in transgenic plants compared to the wild type, demonstrating greater glycosylation activity in transgenic plants.
[00195] In a certain embodiment, glycosyltransferases from tobacco or other plants can be used as described herein. In one embodiment, one or more
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68/114 heterologous or homologous glycosyltransferases can be expressed or overexpressed in a plant, such as tobacco or Cannabis. Gene and protein sequences for exemplary glycosyltransferases are identified below in the Table
9.
Example 8: Generation of cannabinoid synthesis targeted by trichomes and cannabidiolic acid glycosylation constructs (CBDA).
[00196] As shown in Figs. 14-15, the present inventors demonstrated a target synthesis, synthesis and glycosylation system of trichomes, of cannabinoid compounds, such as CBDA. When targeting CBDA synthase, a plasma-oriented UDP-glucose / UDP-galactose (PM-UTRI) transporter, and a 76G1 Stevia UDP-glycosyltransferase (tsUGT) for the trichomes, these genes can produce and accumulate, in this case , CBDA and its glycosylated derivatives (primary and secondary glycosides), as well as new CBDA derivatives, in the trichomes.
[00197] SEQ ID NO. 17 is identified as the polynucleotide gene sequence for a CBDA synthase with a trichome target sequence. SEQ ID NO. 18 is identified as the corresponding protein sequence for a CBDA synthase with a trichome target domain.
[00198] SEQ ID NO. 19 is identified as the polynucleotide gene sequence for a UDP-glycosyltransferase alvotricomas (76G1) coding sequence, in this example being optimized for Arabidopsis thaliana expression, although other codon-optimized versions fall within the scope of the present invention. SEQ ID NO. 20 is identified as the corresponding protein sequence for a UDP-glycosyltransferase (76G1) having a trichome target domain.
[00199] SEQ ID NO. 21 is identified as the polynucleotide gene sequence for a UDP-glucose / galactose transporter (UTR1) having a plasma membrane target sequence.
Example 9: Trichome target synthesis and cannabidiolic acid glycosylation (CBDAPetition 870190120782, of 11/21/2019, page 70/155
69/114 h
[00200] As shown in Figs. 16-17, the expression of CBDA synthase, tsUGT and PM-UTRI genes in leaves infiltrated with N. benthamiana was confirmed 2DPI (Days after the infiltration of the Agrobacterium Ti plasmid constructs) via RT-PCR (Figs. 19 and 20 ). As expected, the CBGA substrate was detected in all infiltrated leaves and in the wild type control (without Agrobacterium infiltration). The primary and secondary CBGA glycosides were also detected in all infiltrated leaves and wild type control, also demonstrating an endogenous glycosyltransferase activity acting on CBGA. In addition, CBGA's primary acetylated glycoside was detected in all samples, including the WT control, providing evidence of endogenous acetylation. CBDA was detected at marginal levels in samples infiltrated with trichome and cell suspension constructs, but not in wild type plants.
Example 10: Synthesis of Cytosolic target and glycosylation of cannabidiolic acid (CBDAV) [00201] The present inventors demonstrated a system of synthesis and glycosylation of cytosol target cannabinoids. By targeting or locating CBDA synthase (CBDAs) and UDP-glycosyltransferase 76G1 (UGT) for cytosol, the present inventors demonstrated that plants that express these heterologous genes produce and accumulate, in this modality, CBDA and the glycosylated derivatives of the same (glycoside primary, secondary), as well as other CBDA derivatives, in the cytosol. As shown in Fig. 18, a gene expression vector was generated for the cytosolic cannabinoid production system. This construct included a cauliflower mosaic 35S promoter; AtADH 5'-UTR, intensifier element; cytCBDAs, cannabidiolic acid synthase with the target sequence of the trichome removed; HSP terminator; cytUGT76GI, UDP Stevia rebaudiana glycosyltransferase.
[00202] SEQ ID NO. 22 is identified as the polynucleotide gene sequence for a cannabidiolic acid synthase with the target sequence of
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70/114 trichome removed (cytCBDAs). SEQ ID NO. 23 is identified as the corresponding protein sequence of cytCBDAs.
[00203] SEQ ID NO. 24 is identified as the polynucleotide gene sequence for a cytosol target UDP-glycosyl transferase (UGT76G1) coding sequence (optimized for Arabidopsis thaliana expression) (cytUGT76GI or cytUTG). SEQ ID NO. 25 is identified as the corresponding protein sequence of cytUGT76GI or cytUTG.
[00204] As an exemplary plant model, the N. benthamiana plants were grown from seeds and, after 4 weeks of vegetative growth, the leaves were co-filtered with Agrobacterium tumefaciens GV3101, with the following constructs: cytosolic CBDAs + cytosolic UGT in pRI201-AN or cell suspension construct, Myb / catalase in pRI201-AN and pi 9 silencing suppressor in pDGB3alpha2. Agrobacterium density was normalized to 2 at 600 nm absorbance using a spectrophotometer and co-filtered cultures in the same proportion (1: 1: 1). After 2 and 4 days after Agrobacterium (DPI) infiltration, 1 ml of CBGA (2.7 mM) dissolved in 0.1% Tween 20 (Sigma-Aldrich) or 0.1% Triton X-100 (Sigma-Aldrich) was infiltrated in each leaf. In a second modality using the cytosolic construct, 4 mM UDP-glucose was added to the CBGA medium before feeding. Three biological replications were used. RT-PCR primers are described in Table 5 below.
[00205] As shown in Figs. 19-20, the expression of the cytCBDAs and cytUGT gene was confirmed via RT-PCR after 1 and 2DPI. No expression of the ABC transporter (ABCt) was observed after the IDPI in the construct of the suspension of cells infiltrated in the leaves. This does not affect this experiment, as the role of ABCt was to facilitate the transport of cannabinoids out of cells in suspension cultures. As shown in Fig. 21, CBGA and its glycosylated and acylated derivatives were detected in concentrations higher than the leaves infiltrated into the trichome construct, except for secondary glycosides. Besides that,
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71/114 CBDA was detected at higher concentrations (up to 34 ppm) in leaves infiltrated with the cell suspension construct, compared to experiments with trichomes (up to 2.6 ppm). As shown in Fig. 22, when UDP-glucose 4mM (substrate for UGT) was supplied together with CBGA (substrate for CBDAs), the present inventors detected low levels of glycosylated and hydroxylated CBDA in infiltrated sheets with the cell suspension construct and cytosolic, but not in the WT control. This result demonstrates the novelty in plant synthesis, glycosylation and hydroxylation of CBDA in the substitute plant N. benthamiana, as demonstrated by the extracted ion chromatograms shown in Fig. 23.
Example 11: Hydroxylation and glycosylation of cannabinoids in Cannabis Sativa.
[00206] The present inventors demonstrate the glycosylation and hydroxylation of cannabinoids in Cannabis sativa. To further confirm our findings using N. benthamiana as a plant model, we infiltrated Agrobacterium from the same plasmid constructs described in the section above into several strains of Cannabis sativa (see Fig. 24 sample IDs). As shown in Figs. 24-26, the expression of the selected genetic constructs in C. sativa, as in N. benthamiana, demonstrate the synthesis and accumulation of hydroxylated and / or glycosylated cannabinoids, in this case CBDA. A comparison of the results using different Agrobacterium genetic constructs is shown in Table 8 below.
[00207] As the present inventors demonstrated, in a modality, in which the cytosolic construct was reconverted with the expression vector Myb / catalase (MYBCAT), it produced the greatest detection of glycoside CBDA and CBDA, demonstrating the role of these genes in the mitigation of effects of toxicity due to the accumulation of hydrogen peroxide (catalase) and general increase in cannabinoid synthesis (transcription factor Myb).
MATERIALS AND METHODS
Example 12: Use of tobacco as an exemplary plant system for the
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72/114 in vivo functionalization and glycosylation of cannabinoids.
[00208] The present inventors demonstrated in vivo functionalization and glycosylation of cannabinoids in a plant model system. Specifically, the present inventors used N. benthamiana (tobacco) as a model system to demonstrate the in vivo functionalization and glycosylation of cannabinoids. In this modality, the transient transformation through the infiltration of Agrobacterium was carried out in N. benthamiana. The present inventors demonstrated expression of heterologous genes that were expressed in transformed N. benthamiana using various heterologous gene expression vectors (described below). In this exemplary modality, after confirming the expression of the heterologous genes that would work and glycosylated cannabinoid molecules, the present inventors introduced selected cannabinoid compounds to plants. In this modality, the present inventors introduced cannabigerolic acid (CBGA) and / or cannabidiolic acid (CBDA) to transgenic N. benthamiana plants. The present inventors have also demonstrated in vivo functionalization and glycosylation of cannabinoids in a cell suspension culture. Specifically, the inventors used exemplary cells of bright yellow tobacco (BY2) as a cell suspension system for studies of cannabinoid production, functionalization and / or glycosylation.
Example 13: Transient transformation of the exemplary model of Nicotian benthamian plant.
[00209] The present inventors used the Ti-mediated transformation of Agrobacterium tumefaciens with the plant expression vector pRI201-AN (Takara Bio USA), a binary vector for high-level expression of a foreign gene in dicotyledonous plants bearing the promoter Constitutive 35S and an Arabidopsis thaliana Alcohol dehydrogenase (AtAdh) as a translation enhancer (Matsui et al. 2012). N. benthamiana was transiently transformed according to the method described by Sparkes et al. 2006) The
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73/114 overnight cultures of the Agrobacterium GV3101 strain were transferred to a 250 ml flask with 50 ml of LB medium supplemented with 50 mg / L of Kanamycin, 50 mg / L of Gentamycin and 10 mg / L of Rifampicin and grown for 4- 8 hours until the optical density at 600 nm (OD600) reached approximately between 0.75 and 1. The cells were pelleted in a centrifuge at room temperature and resuspended in 45mL of infiltration medium containing 5g / L of D-glucose, 10mM MES , 10mM MgC12 and 100 μΜ acetosyringone. 1 ml of the solution was used to infiltrate the leaves, using a 1ML syringe. The expression of the transgene (s) was confirmed 2-4 days after infiltration by RT-PCR. For the analysis of RTPCR, 100 mg of leaf tissue were frozen in liquid nitrogen and crushed in a TissueLyser (QIAGEN Inc, USA). The RNA was extracted after the RNA extraction kit from the EZNA plant (Omega Bio-tek Inc, USA). Up to one microgram of total RNA was used to synthesize cDNA using the superscript cDNA synthesis kit III (Thermo Fisher Scientific, USA). The cDNA was used to verify the expression of the transgene (s) by RT-PCR.
Example 14: Introduction of cannabinoid substrates selected for transgenic N. benthamiana strain.
[00210] Selected enzyme substrates were introduced into the transgenic or genetically modified N. benthamiana strain two days after Agrobacterium infiltration and after confirmation of the transgene expression by RT-PCR. In this example, approximately 277 μΜ of cannabigerolic acid (CBGA) and / or cannabidolic acid (CBDA) was dissolved in 1 ml of buffer containing 10 mM MES, 10 mM MgCl 2 and 0.1% Triton XI 00 or 0.1% of Tween 20 and applied to the processed leaves either by infiltration or by rubbing with a cotton applicator. The plants were collected after 1 -4 days, weighed for fresh weight and frozen at -80 ° C before performing analysis by LC-MS for the presence of modified cannabinoids.
Example 15: trials / 7 for CBDA synthase and qlycosyltransferase activity. [00211] CBDA synthase is generally active in the pH range 4-6 (Taura et al.
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1996), while glycosyltransferases are normally active in the pH range 5.0 to 7.0 (Rini and Esko, 2017). Based on this difference in the ideal pH for enzymatic activity, the present inventors generated a single extraction buffer for a combined assay of CBDA synthase and UDP glycosyltransferase at pH 6 and 30 ° C in in vitro assays (Priest et al., 2006) . The present inventors ground the leaf tissue transformed into liquid nitrogen. A grinding buffer consisting of 50 mM MES, pH 6, 1 mM EDTA, 5 mM β-mercaptoethanol and 0.1% Triton X-100 was added in a 5: 1 ratio of buffer to weight fresh from the plant using a gral and pestle. The extract was filtered on ice through 2 layers of gauze to remove debris and centrifuged at 21,000 g for 5 minutes at 4 ° C. The supernatant was used in subsequent tests. The protein concentration of the supernatant was quantified by the Bradford test, using bovine serum albumin as a standard. To initiate the reaction, 100-200 pg of total crude protein was used. The test was performed with and without UDPglucose to check the glycosylation of the cannabinoid substrate, preventing reactions downstream or transport of CBGA. Wild-type plants were used as controls to separate UDP overexpressed glycosyltransferase activity from UDP. The reaction was initiated by adding 100 pg of protein and glucose 8 mM uridine diphosphate (UDPG) as a sugar nucleotide donor to a reaction mixture consisting of approximately 277 pM CBGA, 0.1% Triton X-100 (p / v), 3 mM MgCb and 50 mM MES (pH 6.0). The reaction was incubated at 30 ° C for 3 h or overnight for 14 hours. The reaction was terminated by freezing in liquid nitrogen and the samples were stored at -80 ° C before analysis by LC-MS.
Example 16: Trichome target synthesis and glycosylation.
[00212] As an exemplary plant model, N-benhamiana plants were grown from seeds and, after 4 weeks of vegetative growth, the leaves were co-filtered with Agrobacterium tumefaciens GV3101, with the following constructs: trichome CBDAs + UGT from trichome in pRI201 -AN
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75/114 (trichome construct), PM-UTR1 in pRI201-AN and p9 silencing suppressor in pDGB3alpha2. In a second experiment, the leaves were also infiltrated with Agrobacterium expressing a T plasmid with the Myb / catalase genes. Agrobacterium density was normalized to 1 or 2 with an absorbance of 600 nm using a spectrophotometer and co-filtered cultures in the same proportion (1: 1: 1). After 1 and 4 days after infiltration by Agrobacterium (DPI), I CBGA (277 μΜ) dissolved in 0.1% Tween20 (Sigma-Aldrich) or 3% DMSO (Sigma-Aldrich) was infiltrated in each leaf. Three biological replications were used. The experiment was repeated twice. After preliminary results, Agrobacterium densities from 2 to ODeo were selected for all subsequent infiltration experiments. In addition, 0.1% Tween20 was chosen over 3% DMSO due to the better solubilization of the CBGA substrate.
[00213] In this modality, leaf samples were collected in 2DPI and immediately frozen in liquid nitrogen. RNA extraction was performed using the mini-kit of the RNA plant, as described by the manufacturer (Qiagen). The cDNA was synthesized using RNA for the Ecodry Premix cDNA as described by the manufacturer (Takara). The model cDNA was normalized to 50 ng of corresponding total RNA per reaction. Annealing temperature in Celsius: 60. Extension time: 15s. 35 cycles. Q5 DNA polymerase kit used as described by the manufacturer (New England Biolabs). RT-PCR primers are described in Table 5 below.
Example 17: Transient transformation of Cannabis sativa.
[00214] The present inventors carried out the transient transformation of Cannabis sativa, mediated by Agrobacterium tumefaciens. The experimental groups consisted of young leaves of high variety of CBD (-10% in dried flowers) and leaves of trichomes of high variety of THC (-20% of dried flowers). [00215] To transform leaves of varieties with a high content of CBD, the present inventors germinated 100 seeds three times; this was done to ensure that a sufficient number of plants were available for all 9
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76/114 independent transformation events. To transform trichome leaves, the present inventors used small leaves containing trichomes of various varieties known to be high THC varieties. The experimental setup consisted of 2 different strains of Agrobacterium tumefaciens. For the transient transformation of the EHA 105 strain of Agrobacterium, the present inventors cultured cells in 10 ml of LB medium supplemented with 100 mg / L of rifampicin and 50mg / L of kanamycin and for the GV3101 strain of Agrobacterium: 6000 cells were grown with 50mg / L Kanamycin, 25 mg / L gentamicin and 50 mg / L rifampicin. A single colony of Agrobacterium was used for inoculation and grown overnight. Then, 1 ml of this culture was inoculated in 500 ml of the LB medium mentioned above, supplemented with 20 μΜ acetosyringone. Agrobacteria were cultivated at ODeo of approximately between 1 and 1.5. The cells were pelleted in a centrifuge at room temperature and resuspended in an infiltration medium containing 10 mM MES, 10 mM MgCb and 200 μΜ acetosyringone at an ODeo of 0.5.
[00216] The bacterial culture was then used for three different types of Cannabis Sativa transformations. In all cases, the transformation was carried out in the form of cotransformation, mixing all the relevant strains (plasmids) in the same proportion as the number of cells. First, for the present inventors, young Cannabis sativa plants (two weeks old) were infiltrated, completely spent, using a 1 ml syringe. Before the transformation, the plants were kept under plastic cover, to ensure maximum softness of the leaves. The infiltration was performed from the abaxial side, ensuring that the entire leaf surface is infiltrated at 12 / h / 12h day / night at 22 ° C.
[00217] Secondly, the present inventors have infiltrated young, two-week-old leaves in a vacuum, completely poured out of Cannabis sativa. Before the transformation, the plants were kept under plastic cover, to ensure maximum softness of the leaves. The leaves were then placed on Murashige and Skoog (1962) agar (½ MS) supplemented with 6
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77/114 1.8 to 8 mM ammonium and incubated for 5 days at 12 / h / 12h day / night at 22 ° C.
[00218] Third, the leaves of the trichome were detached, placed in 50 ml Falcon tubes and vacuum infiltrated with the mentioned bacterial solution 2 x for 10 minutes each. The leaves were then placed on% MS agar supplemented with 61.8 mM ammonium nitrate and incubated for 5 days.
[00219] All experiments were performed in triplicates, with the fourth replication performed for DNA / RNA collection and X-gluc staining to measure beta-glucuronidase (GUS) activity after co-infiltration with the GUS-containing Agrobacterium gene. In all cases, the leaves were harvested after 5 days of transformation, frozen in liquid nitrogen and stored at -80 ° C.
Example 18: Extraction of water-soluble cannabinoids from N. benthamiana [00220] Fresh transformed plant material was collected from greenhouse experiments in 15 mL or 50 polypropylene centrifuge tubes and quickly frozen in liquid N2. The frozen plant material was enzymatically extinguished by submerging the plant material in boiling methanol for 2 min. The methanol-quenched material was homogenized using a P-10-35 homogenizer (Kinematica, Bohemia NY). The homogenate was extracted by brief stirring in a final volume of 10 mL or 30 mL of 70% (v / v) methanol, corresponding to the size of the tube. The resulting extracts were clarified by centrifugation at 2,500 rpm at 4 ° C for 15 minutes in a Beckman J-6B floor centrifuge (Beckman Coulter, Indianapolis IN). The supernatant was transferred to a polypropylene tube and evaporated under a stream of N2 at 45 ° C until dry. The extracts were reconstituted in methanol containing 20 g / mL of the internal standard 7-hydroxyoumarin (Sigma-Aldrich, H24003). The reconstituted extracts were placed in 1.5 ml microcentrifuge tubes and clarified in a microcentrifuge at 10,000 g for 15 min. 500 pL of the supernatant was transferred to a 2 mL automatic sampler flask and kept at 4 ° C until analysis. Sample preparation for in vitro assays: the samples were filtered with a 0.45pm syringe with PVDF membrane in an automatic sampler vial
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78/114 of 2 ml.
Example 19: Extraction of water-soluble cannabinoids from Cannabis sativa. [00221] Fresh plant material was harvested from plants grown in the chamber in 1.5 ml polypropylene centrifuge tubes and quickly frozen in liquid N2. The frozen plant material was homogenized using pestle and mortar and enzymatically quenched by submerging the plant material in boiling 100% ethanol for 2 min. The homogenized solution was diluted in 70% ethanol. The resulting extracts were clarified by centrifugation at 2,500 rpm at 4 ° C for 15 minutes in an Eppendorf centrifuge (Centrifuge 5415 R). The supernatant was transferred to a polypropylene tube and concentrated three times using a vacuum centrifuge (Speedvac SCI 10, Savant). 2 μΙ_ of 20 pg / mL of the internal standard Umbelliferone (Sigma-Aldrich, H24003) was added to 98 μΙ_ of concentrated extract and taken for analysis.
Example 20: Liquid chromatography mass spectrometry used to confirm cannabinoid functionalization and glycosylation.
[00222] The present inventor used liquid chromatography mass spectrometry to confirm cannabinoid functionalization and glycosylation in the exemplary plant systems described here. Specifically, mass spectrometry was performed on a quadrupole time-of-flight (QTOF) mass spectrometer (QTOF Micro, Waters, Manchester, United Kingdom) equipped with a lockspray ™ electrospray ion source coupled to a Waters Acquity UPLC system (Waters, Manchester, United Kingdom)) Mass spectra were collected in the negative electrospray (ESI-) ionization mode. The nebulizer gas was adjusted to 400 L / h at a temperature of 350 ° C, the cone gas was adjusted to 15 L / H and the source temperature was adjusted to 110 ° C. The capillary voltage and cone voltage were adjusted to 2500 and 35 V, respectively. The voltage of the MCP detector was set to 2500 V. The micro MS Q-TOF acquisition rate was set to 1.0 s with a 0.1 s inter-scan delay. The scan interval was 100
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79/114 at 1500 m / z. The data were collected in continuous mode. A 50 ppm raffinose (503.1612 m / z) lockmass solution in 50:50 water: methanol was delivered at 20 pL / min through an auxiliary pump and acquired every 10 s during the acquisition of DM. The separations were performed on a Waters HSS T3 Cl 8 column (2.1 x 100 mm, particle size 1.8 pm) using a Waters ACQUITY UPLC system, equipped with an ACQUITY binary solvent manager, an ACQUITY column manager and an ACQUITY sample manager (10 sample loop, partial loop injection mode, 5 pL injection volume, 4 ° C). The eluents A and B were water and acetonitrile, respectively, both containing 0.1% formic acid. Elution was performed isocratically for 0.5 min at 10% eluent B and then linear gradient of 100% eluent B in 14.5 min and isocratically for 3 min at 100% eluent B. The column was rebalanced for 6 min . The flow rate was adjusted to 250 pL / min and the column temperature was maintained at 30 ° C.
Example 21: Demonstrates materials and methods for data processing. [00223] The identification of individual cannabinoid analogs was carried out by the present inventors, for their corresponding precise mass changes by Metabolynx (Waters Corp., Milford, USA). The parameters of the method for data processing were defined as follows: retention time interval 0.1-18 min, mass interval 100-1500 Da, retention time tolerance 0.2 min, mass tolerance 0.05 Da, peak intensity threshold 14. The accurate mass measurement of the data was performed using the raffinose blocking mass. The raw chromatographic data was further processed to integrate the peak sand area of the extracted ion chromatogram using Masslynx 4.1 (Waters Corp., Milford, USA). The selected cannabinoids, CBGA and CBDA were identified and quantified using certified reference materials (Cerilliant, Round Rock, TX). All chemical structures and physico-chemical and constitutional properties were generated using ChemDoodle version 8.1.0 (IChemLabs ™, Chesterfield, VA).
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TABLES
Table 1 CBGA Biotransformed Products
RRT for Expected Mistake Mistake Molecular Formula [ Product Source m / z m / z (niDa) (ppm) HI-
Found
R-OH 1 x Glycoside 0.58 537.2700 537.2703 -0.30 0.6 C28H41O10 2 x Glycoside 0.59 683.3279 683.3258 2.10 -3.1 C34H51014 1 x 0 acetyl glycoside 563.2856 563.2844 1.20 -2.1 C30H43010 1 x Glycoside # 1 0.74 521.2751 521.2734 1.70 -3.3 C28H4109 R-OH # 1 0.80 375.2171 375.2224 -5.30 14.1 C22H3105 1 x Glycoside # 2 0.81 521.2751 521.2727 2.40 -4.6 C28H4109 R-OH # 2 0.81 375.2171 375.2237 -6.60 17.6 C22H3105 R-OH # 3 0.94 375.2171 375.2192 -2.10 5.6 C22H3105 CBGA 1.00 359.2222359,2245 -2.30 6.4 C22H3104
RRT Relative Retention Time for Originating Molecule
ROH Functionalized by adding O atom.
Table 2. CBDA Biotransformed Products
Product RRT panSource a Expectedm / z FindError Mistake(PPm) FormulaMolecular [H1- m / z (mDa) 2 x Glycoside 0.56 681.3122 681,3097 2.50 -3.7 C34H49014 R-OH 1 x Glycoside 0.61 535.2543 535.2599 -5.60 10.5 C28H39O10 1 x Glycoside 0.71 519.2601 519.2594 0.70 1.3 C28H3909 1 x Acetyl Glycoside 0.71 561.2700 561.2700 0.00 0 C30H41010 R-OH # I 0.84 373,2015 373.2074 -5.90 15.8 C22H2905
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R-OH # 2 0.87 373,2015 373.2034 -1.90 5.1 C22H2905 R-OH # 3 0.96 373,2015 373.2040 -2.50 -8 C22H2905 CBDA 1.00 357.2066 357.2122 -5.60 15.7 C22H2904
RRT Relative retention time for the source molecule
R-OH functionalized by the addition of O atom.
Table 3. Direct and reverse primers for CYP3A4 and P450 oxidoreductase RT-PCR
String | CYP3A4 P4S0 oxidoreductase Initiators for i RT-PCR i Direct TGCCTAATAAAGCTCCTCCTACTReverse GCTCCTG TO AÁCAGTTCCATCTC Direct GGA AG AGCTTTGGTTCCTATGTInverse GCTCCCAATTCAGCAACAÁTATC
Table 4. Direct and reverse primers for CBDA synthase, UGT76G1 and ABCG2
Sequence CBDA synthase UCT% Gi À8CG2 Initiators forRT-PCR Direct Initiator:ACATCACAATCACACA AAACTÀACAAAAGReverse Starter:GGCCATAGTTTCTCATÇAATGG Direct Initiator;gattggaagaacaagcttCAGGATTTCCReverse Starter:CCATCCTG AATGAGTCCA AAAAGCTÇ Direct Initiator:CCTTCAGGATTGTCAGGAGATGReverse Starter:GCAGGTCCATGAAACATCAATC
Table 5. CBDA synthase (CBDAs) trichome target, UGT trichome target and UTR1 PM target
Sequence CBDAs targeting trichome UGT trichome target UTRiplasma membrane target Initiators for RT-PCR Direct Initiator;AAAGATCAAAAUCAA GTTCTTCACTGTReverse Starter: CCATGCAGTTTGGCTA TGAACATCT Direct Initiator:AGTGCTCAACATtCTCCTT TTGGTTReverse Starter: TCTGAAGCCAACATCAAC AATTCCA Direct Initiator:TTGTTCCTTÀAàCCTCGC CTTTGACReverse Starter: TCATTATGGAGCACTCCACTCTCTG
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Table 6. CBDA cytosolic target synthase (cytCBDAs), cytosolic target UGT (cytUGT)
[Sequence CBDA synthase cytosolic target UGT synthase cytosolic target Initiators1 for RT-PCR Direct Initiator:AÁAGÁTCA.AAAGCAAGTTCTTCACTGTReverse Starter:ATAA ACTTCTCCA AGGGTAGCtCCG Direct Initiator:AG.AAÇTGQAAGAATCCGÁAÇTGGAAReverse Starter;AAATCATCGGGACACCTTCAGAAAC
Table 7. Summary of the results of the glycosylation and functionalization experiments on N-bentamian leaves.
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CBGA
ÇBGA
Constructs of
Agrobacteriuci cytosol i glycosyltransferase e | _: CBGA transporter ABC's plasmatic membrane a
CBDA
CBGA
CBGA ritochrome Ρ4: ’· 0 with P4:’ C · isxsdsreih; rase
TricomaCBDA synthase 4Tricoma glycosyltransferase + PM-UTR1) * ^ * PIS dVcatalase suppressor _i süençiamento
CBDA cytosolic synthase, glycosyltransferase and plasma membrane ABC transporter a - í
MvLVeat3iase + Pl & silencing suppressor__ ____J
2ΰ1-SUS {CBDA synthase I
CBDA cytosolicaglycosyltransferase cytosolic -r
Mv & cataiase * + 'Pl 9 silencing suppressor________
P45GCsfYBca & la-se / cito ss ólic a
CBDA synthase; glycosyltransferase and three plasma membrane ABC transporter a
Without agrobacterium (negative control)
CBGA. I CBGA <®GA CBDA
Glycoside substrate 'fed kipantity] (acetylated quantity | (quantity
Irelative) | _rative) . ^ relative) relative)
CBD.A glycoside (relative quantity)
ND
ND * Co-infiltration with and without construct was tested in different replicates
Table 8. Summary of the results of the glycosylation and functionalization experiments on Cannabis sativa leaves.
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| CBDA L ConstructsAgrobseteritti »^ quantity^ relative)1 CBDAglycoside(relative quantity) CBDAHydroxyl(relative quantity) 1 Trichome CBDA synthase4 Trie orna glycosyltransferase |+ target sugar carrier plasma membrane} 4 ·] traits traits CBDA cytosolic synthase _c ] cytosolic glycosyltransferase 4-L 4 * · + Myb / 'at8la $ .8 4 + 4-t- ΉI-4-44 pObSÜSf CBDA synthase |cytosolic, glycosyltransferase and | ; ABC conveyor 1[lasma membrane) ] 44 · +4
Table 9. Identification of exemplary glycosyltransferase sequence
SEQ ID NO. Name Body Type SEQ ID NO. 26 NtGT5a Nicotiana tabacum Amino Acid SEQ ID NO. 27 NtGT5a Nicotiana tabacum DNA SEQ ID NO. 28 NtGT5b Nicotiana tabacum Amino Acid SEQ ID NO. 29 NtGT5b Nicotiana tabacum DNA SEQ ID NO. 30 NtGT4 Nicotiana tabacum Amino Acid SEQ ID NO. 31 NtGT4 Nicotiana tabacum DNA SEQ ID NO. 32 NtGTIb Nicotiana tabacum Amino Acid SEQ ID NO. 33 NtGTIb Nicotiana tabacum DNA SEQ ID NO. 34 NtGTIa Nicotiana tabacum Amino Acid SEQ ID NO. 35 NtGTIa Nicotiana tabacum DNA
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SEQ ID NO. 36 NtGT3 Nicotiana tabacum Amino Acid SEQ ID NO. 37 NtGT3 Nicotiana tabacum DNA SEQ ID NO. 38 NtGT2 Nicotiana tabacum Amino Acid SEQ ID NO. 39 NtGT2 Nicotiana tabacum DNA
Table 10. Models of cell compartmentalization of cannabinoid production. Different columns and shaded lines correspond to the different expression constructs used.
Cannabinoid accumulation / production system
CBDA
Tmp glycosyl transferase synthase
Conveyor
ABC of cannabinoid
Cytoplasmic accumulation
Less Trichome Target Sequence
Required but no target change
No genes needed
Trichome synthesis (low pH)
No change
Adding Trichome Target Sequence
No Alvopara gene needed; plasma memhrana
No gene needed; UDP glucose transporter
Express
Express
Myb Transcription Factor for cannabinoids [Catalase to degrade
ΪΗ2Ο2 of
CBDA synthase
Express
Express
Cell suspension cultures
Less Trichome Target Sequence
Required but no target change
Plasma membrane (FM) target
No gene needed
Express
Express
REFERENCES
The following references are hereby incorporated in their entirety by reference:
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86/114 [1] I flies OssawskL M R Mulvey, P A Leco, A Boiys sad P C Loewen, X
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88/114 [10] Siritmga, D., and Sayre, RT (2003J. Generation of cyanngen-iree transgenic cassava. Planta. 217, 347-373. Pain: lG.lOO7 / sQG425-OO3-10O5-8 [111 Sparkes , IA, Runions, J., Kearns, A, & Hawes, C. (200> 5). Rapid, transient expression of Suorescent fexisn proteins in tobacco plants and geaerati <m of stably transformed plants. A / stov 7 (4) , 2019-202.5. Https: //doi.Qrg/I 0 .. 103 S.inpiot. 2004.236 [13] Taura, F., Morimoto, S., & Shoysma, Y. (1996). PariScatirm and characterization of cannabidiolic -acid synthase from Cannabis sativa L. Biochemical analysis of a novel enzyme that catalyzes fee oxidocydizaticin of. </ SiW & g & Yd GaBijôy, 277 (29), 1741 1-17416.
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Cannabis sativa. FeMs fetters, 2929-34. ΣΧ3Ι: 10,101 õ / í..febsIet.2gO7.O5 .043 [15] Yeo, SD, Ch.o, YH, & Sheen, J. (20Q7). AraMopsis mesophyll protoplasts: The cel versifete! system for transient gene expression analysis. ^T e / sre .Prstocnih ,. 2 (7), 1565-1572.
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3054.1962 feOSC62.x [18] Ztpp, et al., Camiifemoid glycosides: In y ito preíhtóion of a new class of cannabinoids with m ^ proved physicochemical properties, bioRxiv preprint dai: http: //dx..doi Jrg / 'l 0 ..1101 / 1 04349
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89/114 [19] Mohamed, E. A., T. Iwaki, I. Munir, M. Tamol, S. Shigeoka, and A. Wadano. 2003. Overexpresswn of bacterial catalase in tomato leaf chloroplasts enhances photo-oxidative stress tolerance. Plant Cell Environ. 26: 2037-2046.
[2D] Akhtar, MT, 2013 ,. Doctoral Thesis, Leiden University. Cannabmsids and zebrafish. 2013-05-22. ht ^: / & dl.handfe.aet / f 887/20899 [21] Sayed Farag. Cainsabinaids produrttoti m Cannabis sativa L .: An in vitro approach. Thesis - January 2014. DOI: 10.17S77; DE2.9 ^: 0R-16298 [21] K, Watanabe, et al., Cytcchrome P45Q enzymes involved in the metabolism of tetrahydrocannabinols and cannabinol by human hepatic microsomes. Life Sciences. Volume 80, Issue 15, 2D March 2007, Pages 1415-1419 [22] Flores-Sanchez Π. et al .. Elicitation. studies in cell suspension, cultures of Cannabis sativa L. J'Bir ^ echnoL 2009 Ang 20; 143 (2): 157-68. doi: 10.lOldfj.jbiotec.
[23] Stephen M. Stout & Nína M. Cimino (2013) Exogenous cannabinoids as substrates, inhibitors, and inducers of human drug metabolizing enzymes: a systematic review, Drug Metabolism Reviews, 46: 1, 86-95 ,. DOI: 10.3 109703602532.20 13.849268 [24] Andre CM, Hausman J-F, Guerriero G. Cannabis sativa: The Plant of the Thousand and One Molecules. Frontiers st Plant Science. 2016-7: 19. doi: 10.3389 / fpis.2016.00019.
[25] Mahlberg PI. et: a; .. Accumulation of Cannabinoids in Glandular Trichomes of Cannabis (Cannabaceae). Journal of Industrial Hemp 9 (1): 15-36 June 2504 with 273 Reads DOI: 10.1300 / J237 O9 »01_04.
[25] Katalin S., et al. Mini Rev Med diem. 2017; 17 (13): 1223-1291. doi: 10..2174 / 1389557516666161004162133.
[26] Sirikantaramas S., et al., Tetrahydrucannabinohe Acid Synthase, the Enzyme Contelihig Marijuana Psychoactivity., Is Secreted into the Stooge Cavity · of & and Glandular Trichomes. Plant and Cell Physiology, Volume 46, Issue 9, 1 September 2005, Pages 15781582, https.://doi.Org < ^f 10.1093 / pcp / pcil66.
[26] Schilmdler AL, Last RL, Piehersky E (2008) Harnessing plant trichome biochemistry for the production of usefid compounds. Plant Journal 54: 702-71 1.
Petition 870190120782, of 11/21/2019, p. 91/155
90/114 [27] Matias.-HeiuaEidez _s L. et al. AaMYBl and its rathologste AíMYtídl affect terpene metabolism asd tochsme development in rfefíãsií am »and
J. 2017; 90: 520-534
SEQUENCE LISTING
As mentioned above, this application contains a complete Sequence Listing that has been submitted electronically in ASCII Format and is hereby incorporated by reference in its entirety. The following strings are additionally provided with and are incorporated here in the specification in their entirety:
SEQ ID NO. 1 DNA
Human Cytochrome P450 (CYP3A4)
ATGGCTTTCArrCCTGATTTGGCTATGGAAAC.TAGATTGTTGTTGGCTGTTTCATTGGTTTTGT TGTATTTGTATGGAACTCATTCACATGGArEGTrrAAAAAATTGGGAATTC / CTGGACGTACTCC TTTGCCTTTrrTGGGAAATATrrTGTCATATCATAAAGGATTrTGCATGTTTGATATGGAATGC: CATAAAAAATATGGAAAAGTTTGGGGATrrTATGATGGACAACAACXTGTTTTGGCTATTACTG ATCCTGATATGATrAAAACTGTnTGGrrAAAGAATGCTATrCAGTTTTTACTAATAGAAGACX: TTrTGGACCTGTTGGATTTATG.AAATCAGCTATTTCAATrGCTGAAGATGAAGAATGGAAAAGA TTGAGATCATTGTTGTCACXTACTrrTACTrCAGGAAAATTGAAAGAAATGGTrCCTATTATTG CTCAATATGGAGATGTTTTGGTTAGAAATTTGAGAAGAGAAGCTGAAACTGGAAAACCTGTrAC • rTTGÀÀAGATGTTTTTGGAGCTrATTCAATGGATGTTATTÀCTTCÀACTTCATTTGGAGTTAAT ATTGATTCATTGAATAATCCTCAAGATCCTrTTGTTGAAAATACTAAAAAATTGTTGAGATTTG ATTTrTTGGATCCTTTTTTrrTGTCAATTACTGTTTTTCCTTTTITGATTCCTATTrrGGAAGT TTTGAATATTTGCGTTTTTCCTAGAGAAGTrACTAATTTTTTGAGAAAATCAGTTAAAAGAATG AAAGAATCAAGATTGGAAGATACTCAAAÀACATAGAGTTGATTITTTGCAATTGATGATrGATT CACAAAATTCAAAAGAAACTGAATC'ACATAAAGCTTTGTCAGATTTGGAATTGGTTGCTCAATC AATTATTITrATTrTrGCTGGATGCGAAACTACTTCATCAGTrrTGTCATTTATrATGTATGAA TrGGCTACTCATCCTGATG rrCAACAAAAATTGC: AAGAAGAAATTGATGC: iGTTTTGCCTXATA AAGCTCCTCCTACTTATGATACTGTTTTGCAAATGGAATATITGGATATGGTFGTTAATGAAAC TTTGAGATTGTTrCCTATTGCTATGAGATTGGAAAGAGTTrGCAlAAAAGÀTGTTGAÁATrAAT GGAATGTTTATTCCTAAAGGAGTTGTTGTTATGATI € X; iTCATATfXTITCCATAGAGATCCTA AATATTGGACTGAACCTGAAAAATrmGCCTGAAAGATnTCAAAAAAAAATAAAGATÁATAT TGATCCTTÀTATTTATACTC-CTTTTGGATCAGGACCTAGAÀATTGCATTGGÀATGAGATTTGCT TTGATGAATATGAAATTGGCTTTGATTAGAGTTTTGCAAAATTTTTCAITTÀAACCTTGCAAAG AAACTCAAATr € 'CTTrGAÀATTGTCATTGGGAGGA.rTGTTGCAACCTGAÀÀAACCTGrrGTTTT GAAAGTTGAATCAAGAGATGGAACTGTTTCAGGAGCT
SEQ ID NO. 2
Amino Acid
Petition 870190120782, of 11/21/2019, p. 92/155
91/114
Human Cytochrome P450 (CYP3A4)
MAIJP®l.AaiETKLIXAVSLVLLYL ¥ GTHSHGLFKKLGI PGPTPI ^ FWNJLSVHKC ^ CaíFDX-IEC
HKKY <^ T ¥ <^ TX ^ QPVLAI TDPDMI KTAT.VKECYX ¥ TTNRRPFGPVGFMKS ftl S JAEDEEWKR
LRSLESPTFESGSLKEMVPÍ LA.QYGm VR.JSlLRREAETGKFVELKD ^ ^ ^ TGAYSMimTSESFG WSE ¥ ^ PQBPfWXTKKEEREDFLDPEFLS ITVFPELI PILE L CVFPREVTNFLRKSVKRM KESREE II 'TQKHRTOFEQEMI' SQNSKETESHKAESBLELVAQS 11 FI FAGCETTSSVLSFIMYE LÁTHP 'Day ⁷ LQAÍE VQQKLQEEIDAATPNKAPPT'i ^r ¥ EIÍMV NETLRL.FPIAAÍRLEKVCKK ^7' WIN CHF IFKG.WVMI PSYALaRBFKTWTEPEKFLPERFSKKNKDNmPYnTTFGSGPRNCIG & IRrA EXE ^ ¥ IKLALIRVTQNFSFKPCKETQI PLKLSLGGLLQPEKPWLI ^ TSEIlCiTVSGA
SEQ ID NO. 3
DNA
P450 oxidoreductase gene (oxred)
Human
Petition 870190120782, of 11/21/2019, p. 93/155
92/114
ATGATTAATÀTGGGÀGATTCACATGTTGATACTTeATCAACTGTTTCAGAAGCTGTrGCilGAAG AAGTTTCATTGTTTTCAÀTCACTGATATGATTTTGTTTTCATrGATTGTTGGATTGTTGACTTÁ TTGGTTTTTGTTTÀGAAAAÀÀAAÁAGAAGAAGTTCGTGAATTTACrAAÀATTGÀÀACTrTGÀCT TCATCAGTTÀGAGÀATCATCATrTGTTGAAAAAATGAÀAAÀAACTGGAAGâÀATAITAITGTIT TTTATGGATCAeAAACTGGAAeTGCTGAAGAATrTGCJAÀTAGATTGTCAAAAGÂTGCTCATAG ATATGGAATGACACGAATGTCAGC / rGATCCTGAAGAATATGATTTGGCTGAmGTCATCATTC CCTGxAAATTGATAATGCTTTGGTTGTTTmG € ATGGCTAeTrATGGAGAAGGAGAT € GrACTG ATÀATCXTCAAGATTrTTATGATTGGTTGCAAGAAACTGATGTTGATTTGTCAGGAGTTAAATT · TCCTGTTTTTGGATTGCGAAATAÀÀACTTATGAACATTTTAATGürATGGGAÁAATATGTTGAT AAAAGATTGGAACAATTGGGAGCTCAAAGAATmTGAATTGGGATTGGGAGATGATGATGGAA ATTrGCAAGAAGATirrATTACTTGGAGAG.AAQAATrrTCGTTCGCTGTrTGCGAA € ATTTTGG AGTTGAAGCTACTGGAGAAGÀATCATCAATTAGACAATATGAATTGGTTGTTGATACTGATATr GATGCTGCTAAÁGTrTATATGGGAGAAATGGGxAÀGATTGAAATCLATATGÀÀAATCAAAAAGCTC CTTTTGATGCTAAAÀATCCTTTTnGGCTGCTGTTACTACTAATAGAAAATTGAATCAAGGAAG TGAAAGACATTTCATGCATTTGGAATTGGATATTTGAGATTCAAÁAATTAGATATGAATCAGGA G4TCATGTrGCTGTTTATCCTGCTA ATCATTGAG € "nTGGTT.AATCAATTGCGAÀAAATTTTGG GAGGTGATTTGGATGTIGTTATGTCATTGAATAATTTGGATCxAAGAATCAÀATAÀAAAACATCC TTTTCCTTGCCCTÀCTTCATÀTAGAACTGCTTFGACTTATrATrTCGATATTACTAATCCTCCT AGAACTAATGTTTTGTATGÀATTGGCTÜAATATG € TTCÂGA.A << TrüAGÀACAAG.A4TTGTTGA GAAAAATGGCn € ATCA.TCAGGÀGAÀGGAAAAG.4ATTGTÀTTTGTCATGGGTFGTrGAAGCTAG AÀGACATATTrTGGCTATnTGCAAaATTGCCXTTCATTGAGACCTCCTATTQÀTCATTrGTGC GAATTGTTGCCTÀGATTGGAAGCTAGATATTATTCÀATTGCTTCATGATCAAAAGTrCATCCTA ATTCAGTTCATATTTGCGCTGTTGTTGTTGAATATGAAACTAAAGCTGGxAAGAATTAATAAAGG AGTTGCTACTAATTGGTTGAGAGCTAAAGAACCTGTTGGAGAAAATGGAGCAAGAGCTTTGGTT CCTATGTrTGTTAGAAAATCACÀATTTAGÀTTGGCTTTTAAÀGCTÁCTACTCCTGTTATTATGG TTGGA €€ TGGAACTGGAGTTGCTCCTTTTATTGGAniATTCAÁGAÀAGAGC: TTGGTTGAGACA ACAAGGAÁAAGÀAGTTGGAGAAMTTTGTTGTATTATGGATGGAGAAGATCAGATGAAGATTAT TTGTÀTAGAGAAGAATTGGCTCAATTTeATAGAGATCGAGCTTTGACTeAATlGÀÀTGTTGCTT TTTCAAG4GA4CÀATCÀCATAAWTTTATGTTCAA € ATTTGTTGAÀACÀÀGATA; QÀGAA € ATTT & τ € αΑΑΑΤΤί ^ ΑΤΤΟΑΑ € © Α ^ ΟΑ €€ Τ << 4ΤΑΓΓΤΑτ & 'ΓΤΤ & </ <ίΟΑ ^ ΑΤ €€ ΤΑ. © ΑΑΑΤΑΤΟ € ΧΤΑ © Α GAIGTTtAAÀATACITTTTATGAIATTGTTGCTGAATTG & GAGCTATCGAACAT & CTCAAGCrTG TTGÀTTATATTAAAAAATTGÀTGACTAAAGGAAGATÀTTCATTGGATGTTTCGTGA
SEQ ID NO. 4
Amino Acid
P450 oxidoreductase
Human
Petition 870190120782, of 11/21/2019, p. 94/155
93/114
MINMGDSm'DTSSTCSEA ¥ AEEVSLF5MTDX £ I LFSUVGLL-TVWEI ^ RKKKEE ^ TEFTOQTLT SSVRZSSFVXiaíKSTGRM IVFYGgQTGTÀEEFA ^ RLSKBÀiffiTG & IRCXISADLY. FEmFiAEWFCiiAn GDFTDXAQESFYSY'LQETBTOESG ^ ^ ^ FAVFGEG KTYEHFNAAJGKYYD KRLEQLGAí ^ · ^ RIFELGLGDESSG LEESFreWKEQFWLAVCEHFGyEAFGEESS IKQYELWHTDI BÂÃKVY iGEMGRLK.SWSQKFFWAKNPFLÀAVTlWKLKQGTEKHIAffiEELBISDSKIRYE.SG DHV'A ^ ^: ^ D & AT.A AL; f'QLGKlLGABE V © ^{^:} ^ N-SL íKLOEES ^KKHFFPCT, T & KTALTY ¥ ¥ LDI TNFF RTX XlTIAQYA ^ ^ ^ SEQELLRKMASSSGEGKELYLS- tEARRHILAI LQD <P8LEFFI »HLC EEÍFELíQAEYYS · LASSSKVHPKSVHI CA ^ ^ YTEyETKAGEl GYAT TRÀKEFVGE ^ ^ ^ ^r GGRALV PW KKSQn FK4TTP ^ ^ € ™ n'GPGTGVAlTX IQERA líR.QQGKE ^ ¥ ^ .TLL ¥¥ GCRR8BEBY L ^ KEEEAQ ^ EBGALTQE-M.'AFSEiEQSHE ^ 'YVQm.LKQDREKL ^^ L ^ .CGAHmOGSARM ^ AR S VQNTFYDIYAELGAMEHÀQ A VWIKKLAÍTKORYS LDWS
SEQ ID NO. 5 Cannabidiolic DNA (CBDA) synthase
Cannabis sativa
ATGAATCCTCGAGA4AA 7rTCCTTAAATGCTTCTCGCAATATATTCC € '€ AATAATG .4ACAAATC TÀAAACTCGTATAGACTGÀAàACAACCCATTGTATATGTCTGTCCTAAÀTTCCACAATACAGAA TCTTAGATTCACCTCTGACACAA C £ ££ €€ AAA4 ACTTGTTATCGTCACTCCTTCACATGTCTCT CATAECCAAGGCACTATTCTAEGCTCCAAGAAAGTTGGCTTGCAGATTCGAACTCGAAGTGGTG GTCATGATTCTG.AGGGCATGTCCTACATATCTCAAGFCCCATTTGTTATxAGTAGACTTGAGÂAA CATGCGTTCAATC.AAAÁTAGATGTTCATAíXGAAACTGCATGGGTTGAAGCCGGAGCTACCCTT GGAGAAGTTTATTATTGGGTTAATGAGAAAAATGAGAATCTTAGTTEGGCGGCTGGGTATTGCC CTACTGTTTCCGCAGGTGGAC ACTTTGGTG GAGGAGGCTATGGACCATTGATGAGAAACT 4TGG CCTCGGG GCTGATÀATATGATTGÀTGCACACTTAGTC .AACGTTCATGGAAAACTGCTAGATC GA AAATCTATGGGGGAAGAFCT TTrEGGG € € € TTTxACGTG TGGAG T ^ £ Λ · ÀGAAAGCTTCGG4ATt TTGTAGCATGG. AAAATTAGACTGGTTGCTGTCC € AAAGT £ TACTATGTETAGTCTTAAAAAGAT CATGGAGATACATGAGCTTGTCAAGTFAGTTAACAAATGCGAAAATATTGCTTACAAGTATOAG A4AGATTTATTACT € ATGA TCACTF £ £ £ _{ATA4CTAGGAACATTACAGÀTAAT;} 4A & GG.AAGAATA AGACAGCAATACACAeTTACTTCTCTTCAGTTTTCCTrGGTGGAGTGGÁTAGTCEÂGTCGÀCTI GATGAA AAGAGTmceTGAGTTGGGTATTAAÀAA4A € € € GCATTGeAGACAÁTTGAG TGGATr GATACTATCATCTTCTATAGTGGTG'TTGTxAAATTACGACACTGATAATTTTAACAAGGAAATTT TGGTrGATAGATCCGCTGGGCAGAACGGTGCTTTCAAGÀTTAAGTTAGAGTACGTTAAGÀÀACC AATTCXAGAATCTGTATTTGTGCAAATTTTGGAAAÂATTATATGAAGÀACATATAGGAGCTGGG ATGTATGCGTTGTACGGTTACGGTGGTATAATGGATGAGATTTCAGAATCAGCAATTCCATTCC CTCATCGAGCTGGAATCTTCTATGAGTrATGGTAGATATGTAGTTGGGAGAACCAAGAAGATAA CGAAAÀG AT € € € TA4ACTGGAETAGÀÀÀTATTTATÁACTT ÂTGACTCCTTATGTGTC <AAAAAT TCAAGÀTTCQeATATCTCAATTATAGACAGGUGATÀEAGGAATAAÀTGATCCCAAGAATCCAA ATAAErACACACAAGCACGTATTTGGGGTGAGAAGTATriTGGTAAAAATTTTGACAGGCTAGT AAÀAGTGAAAACCGTGGTTGATCCCAATAACTmTEAGAAACGAACAAAGGATCCCACCTCAA CCACGGCATCGTCATEAA
SEQ ID NO. 6
Amino Acid
Cannabidiolic acid (CBDA) synthase
Cannabis sativa
Petition 870190120782, of 11/21/2019, p. 95/155
94/114 wpri v
GaT <
IVDLRRKRSI KIDVHSQTAKVKAGaTL
OKSYGLAASN I IDAIfííVNVHSKVLDR
KSSO · '<i A > x WSVKKI. ^ EIHELWLWOQ ^ TÂ ¥ KYD
KDLL; > DSLVDLMÍXSFPEmiKKTOCK.gLGXI
3TIIS WKKP ^ FESywgiLSKLYE ^ IG & S
MSffi »> EKS} WNKKH Jí» .1 RR ITOWmSKS
SRLA xx _W Akx »^ .. A» ~ &WDRLVO'KTL VDW WWIPPS
SEQ ID NO. 7 DNA
UDP glycosyltransferase 76G1
Stevia rebaudiana
^vrr x “A 'X ^ AA J ^ TOCO ^ rCCTCAA-C:
~ * „ ^T -“ '' v A ”'”''. ”* X * ' ^T AiT ^ si ^ Jk ^ PTC ^ uA.QT ⁵ * ^' ^
'. — J. X .4. .4. · - · ._, x and-ΐ. <4. -x j. x. <. S „jr:>. Vxè-à.x- .4. x x 'X ·: ‘. 4C4-j x x x .1 .i. '-4> J ^' 5-> 4-il_i
SEQ ID NO. 8
Amino Acid
UPD gyrosyltransferase 76G1
Stevia rebaudiana í / 3 Ui
ΜΕΗΚΤΕΤίνΚδΚΕΙΙΙΙΕΚΡνΡΡ ^ ΗΣ ^ ΡΣ ^ Ι ^ νΐίΎδΚδ ^ ΙΤΙΡΉΤ ^ ΡίίΚΡΟΕΝΪΡΗΡΓ ^ Κ RXLíUÍí} I} ^: .Í ^ U'àSíi..X AJ .. A li '' 1 y '· ..' .. j .. :. £ 1
FAQSVimSÍJSLI®LVlWSSLF ^ FHAirVSLPQFDSLG ¥ WPWKTRLEEQaSGFP ^ LK.VKDIK ΑΥδΝϊί ^ ΙΙίΚΕΙΙ ^ ϊ ^ ΙΚςΤΠΪΑΕΕΟνΣ ^ ΝδΡΚΕ ^ ΕΕδΕΣ ^ Σνί ^ ΕΙΡΑΡΕΕΙΊΡδδΡΚΗΙίί
Petition 870190120782, of 11/21/2019, p. 96/155
95/114
LU> HBRTVTQWLDQQÍPSSlTinWGSTSE T> £ KBFlFlARGLV © SKQS FLVWIÍPGFVKGSTW ATPL SGELGESGRB'K ^ ATQQEA'LASGAIGAIA ^ ^: TSSG ^ 'N & TLES ¥ CEGVE'i £ í FSDFGLSM ASYXiSDVLK'i ^ GWLEKG ^ ERGEIA ^ AlEEA'MVDEEGEYIEQXAS ^ TKQKABVSL & IKGGSSEESL E.SLVS11 SSL
SEQ ID NO. 9
DNA
ABC ABCG2 conveyor
Human
ATGTC'ATCATCAAATGTTGAACTTTTTATTCCTGTTTCACAAGGAAATACTAATGGATFTeCTG CTACTGCTTC AAATGA'TTTGAAAGCTITIACTGAAGGAGGTGTTITGTC ATTTCAT AATÂTTTG CTATAGÀGTTAAATTGÀAATGAGGÂTTTTTGeCTTCCAGAAAACCTGTTGAAAAAGÂÀATTTTC TGÀAATÁTTAATCGAATTATCAAAGGTGGATTGAATGCTArrTIGGGACCTACTGGAGGAGGAA XATCATCATrCTTGGATGTTTTGGCTGCTAGAAAAGATCiCTTGAGGATTGTCAGGÀCATGTTTT GÀTrAATGGAGCTüCTACACCTGCTAATmAAATGCAATTCAGGATATGTrGTTCAAGATGAT CTTGTTATGGGAACTTTGACTGTTAGAGAÀAATTTGCAATTrECAGCTGCTTTGAGATTGGCJA CTACTATCACTAATCATQAAAAÂAATGÀAAGAATTAATAGAGTTATTGAAGAATTGGGATTGGA TAAAGTTGCTGATTCAAAAGTTG-GAACTCAATTTArTAGAGGAGTTTCAGGAGGAGAAAGAAAA ACAACTrCAATTGGAATGGÁATTCATTACTGATCGTTCÀATTTTGTTTTTGGATGAACCTAGTA CTGGAETGQATICATCAAeTGCTAATQ £: ^ TGTTTTGTTGTTGTTGAÁAAGAÀTGTeAAAACAACG AAGAACTATTATTTTTTC ATTCATÜAACCTAGATÀTTCÀATTrTTÀÀATTGTTTGAITCATTG The TFTGTTGG € € €€ TTCAGGAACATTGATGTTTCATGGAC'CT TCAAGAAGCTTTGGGATATTTTG ÀATCAGCTGGATATC: ATTG-CGÀAG € 'TTATAATAATCC-TGCTQATTTTTTETTGGATA.TTATTAÀ. AA TGGAGÀFT € € € TGCTGTTGCTTTG4ATAQAGAAGÀÀQATTTTAAACCTACTGÀAATrATTGAA CGTTCAAÀACAAGATAAACCTITQATTGAÀAAAETGGCTGAAATTIATGTTAÁUCAECATTTT ATAAÀGAAA: TAAAGCTGAATTG ATCAATTGTCAGGA © € GAGAAAÀAAAAÀÀAAAAATTACTGT TmAAÀGÁAATTTCATÀTACTACTTCATnTGCGATCAATTGAGATGGGTTTCÀAÀAAGATCA TTTÂAAÂÁTTTGTTGGGÂAATCCTCAAGCTTCAATTGCTCAAATTATTGTTACTGTTGTTTTGG GATTGGTTATTC ^ ^ CT AGCTATITATTTTGGATTGAiÀAATGATTC.AÀCTGGAATTCAÀAÀTAGACC AGTETTGTTITTTrrGACTACTÀATCAATCCTFITCÀTCAGTTTCAGCTGTTGAATTGTrT GTTGTTGAAAAAAAATTGTTrATTCATGAATATATTTCÀGGATATTATAGAGTTTCATCATATT TTTTGGGAAÀATTGTTGTCÀGATTTGTr <XCTATGAGÁATGTTGC-CTTCÂATTATITTTAeiTG CATTGITTAITTTATGTTGGGATTGAAAGCTAAAGCTGATCCTTTTTTTGTTATGATGTTTACT TTGATGATGGTTG TTATTCAGCTTCATCAATGGCTTTGG € € € TATTGCTG TGGA € AATCAG TirCAGTIGCTACTITGTTGATCÀCTÀTTrGCTrTGTTTTTATCÀTGATTTTTTCAGGATTGTT TTG <X TTAATTTGACTACTATTG € € Tr ÀIGGTTCTCATGCTTCCAATÀTTrTTCAATTC-CTAGATAT GGATrTACTGCTTTGCAACATAATGAÀTrrTTGGGACAAAATTTTTGCCCTGGATTGAATGCTA CTGGAAATAATCeTTGCAATTATGCTACTTGTGAGTGA TGGÀAAAATCÀTGTTGCTTTGGCTTGCATGATTGTTÀTTTrr TTGAGTATrGeTTATFTGAÀATTGTTGTTTTTT & AAAAÀATATTCA
SEQ ID NO. 10
Amino Acid
ABC ABCG2 conveyor
Petition 870190120782, of 11/21/2019, p. 97/155
96/114
Human
M & SS3SVEVR Ci ¥ RVKI ^ SCFLM.'KKPVEKlI L
SKJNGatKFeLWAI LGFTCGGK. $. SLLm ^ AÂgX »FS.GLSGma * ING.« »LA ÍTKCNSQTO'Q» D ® 0ΤΣ _ί ΤνΚ3Ν12Ρ3ΑΑΙ _} ΧΙΑ.ΤΜΤΜΙΕΟΕΕΙ ^ Νΐ2ΕΣ.01 _! ΠΟΑβ5θαΤ2: ΡΣΡ0Χ '· 5α0ΕΚΚ ^ Ζ8ΤΟΜΒΙ> ι · ΤΏΡΞΙ ^ ΡΣ.ΟΕ ΤΤΟ ^ 58Τ & Ν £ ν ^ ΙΑ _! ΚΚΜδΚ '·;' · '''·· ^, ··· ^Γ ' ”~ 2HQPOSIFKLFDSL TXJ'A.SGRIJi FRGPÃQEAX> G ¥ FES ^ YHCKAYN PADFFX _í DI 'χχ”' AUHSgSSFOTSIIg PSKQDKPLX EEKYKIKSKKKKKYKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKXKXKXKXKXKXXXXXXXXXXXXXXXXXXXXXXXXXXXXX rv ^ TTSFCRQLKWSKSS FOLLG PQASIAQIlVTWLG IGÂIYroLKNDSTGXQNRAGVLFFLTT ^ ^ ¥ I CFSgVGÃVELF WEKKLF XHE SG WSS ¥¥ ¥ ^ Κ ΕΙ & Ε8ΕΜ. _{ΕΜΡ.ΜΙ ί Ρ} £ · 11 ^ FTC XVTFHLGLOKADAFFVW4FT Χ _{ν ί ΑΥ3Α35ΚΑ1Α1} '.02δ νν5νΑΤ · ^ 1' ^ ΙΟΡνΜ4Ι _{5ΟΙίΙ ί ν3ϊΣ} > _! ^8 ^ 1 _! 5 ^ Χ42'ϊ% ·· 5JPRY GFTALQI® EFLSQ ^ FCPGLSATGraPC ^ YATCTGEK'Sb VKQGIDAS WGAWCTHVAL & raiVX F ΙΖΓΙΑΥΑΚΑΙιΈΕΚΟΕ
SEQ ID NO. 11
DNA
Similar to MYB12
Cannabis λ -_ '-. -i. ~ u. -_- L · .. · x, -, 'j, .ΐχΑ .; AA j. Α.-4ί4ΆΑΑ.- ± Ι1 · '.. Α ^ · / χ.31.χ5: < : x jí <jl c,,> ΐ. X is ^O,! ... iJ - : '- LJ- ^ k' ./ J
ACATOSTATTATCATCAT ·: x x'x .CAACANACATCATCAACAA.CTACASCAACATCATÜATT
& όΛΑδ δΛΛ "Α ν'χ%. ¥ 7> Α Ax '* · ^ AAA ΓΤ SAACTTrAÃTAÃTAATAATAATAÂTÃATAAÍ ATGGTSM'UAA ·' XX 'ΎΑ Λ ΤΑ ATTATCATCATCA.TAÃAOAAOAGGAÂGAAQAAGAA-I3A AG.A TGAÃQCATCTGCATCAOTAGCAGCTGTTOATGÃA.GGGATGTTOTTOTGCTTTGATGA.C âTAATÃGATAGC ^^ ^^ ^ TG AAÃTaC & AATG & GGTTTTGàCTTTA ^ SÃGÂÍ ^ ATAGGSAiaATG. · .Xa ^ 3 $ a ^ yx..A'.J '.. j.xy.A.L'GAt & A.3. . . ik ' ¹ . .t, ';.' · .yL Λ / Λ. . <'A' · .. ^:: . THE.·. ./ A'x. ; .ΑΑ..ΐ '* · όΑ.
TGA. * 3 'XA'i.'AACAA.'iAA'C ^and i'T ^l GA ^ GA ^ 3-'3 ^ TGA. $ JOTGí-2-AATAA'rAAC ^ 33JíAGÂ ^, .rT ^t A1 [ 'ü''i ^r rÂT ^p rAGTGAT ^t QÃTCÃTGAIGATCAGTAeTCGATAQA0GACGTa3Tr®lAGTIG.ACTTTTaSÃGTIGGGA ^ GTT xAíAk. I.. · '// .. à .4- .. i <ic i.;. £ s. i j. /. x.á-Xat-iAii-s.CA / jTAA. / Yi / li-i ^ TATGTGG TAATG & GAA & GASAAÃereTTGTCTFTGCTAimSATMTAGTGATAACAGCÃGC AGTTGGSAGTTAOAÃSÃTÂÃAAOCAATAATAATAATAÃTAA.TAÂTGTTCCTAACAÃATG ^^ ^ AÃ.O AGATTACCTCTSATAAAOÃAAATGCTATG PTGCATGGCTTCTCTCCTGA
SEQ ID NO. 12
Amino Acid
MYB12
Cannabis
Petition 870190120782, of 11/21/2019, p. 98/155
97/114
MONKSTS J®8N8SffiI ® I) I VS SSSSTTTTSSTTTATSSWNSKVTVSTDHI SlifLSDKQKR ξ5ΕΟΕϋδΑΕΚΕΕΕΕΕδ®0α € ΟΕΤ '' Εβ5 ®ΡΑΑΑΖΑΑΑΑΟΏ835σβΕϋΚΕί3βΗΣ1ΟΕΖΑΕΟΟ3Ε KK.TTEISSVWFM ¥ ^ WNKTS SNGDSVSGPYn-H ^ _i EEEEEEEEDEASASVAAVDgGXLLCFDD IlDSBI LNF EVLTLRED5®iECK2AADQIDKTTCIWTTTSÍDDWffiM ^ ^ ^ L3C NNGS ¥¥ ISD SW.DqWIDD ^ SVDFWS Í £ STrTVITQEQEQgQSQ ¥ QEqK53WDNEKEKLLSLL5iOíSM £ SS WSLQDKS KIMW PWXQE ITS DKEX ^ MV.WLLS
SEQ ID NO. 13
DNA
Catalase
Arabidopsis thaliana
ATGGATCGTTATAAATATAGAGCTGCTTCATCATATAATIGACCTTTTTTTACTACTAATTCAG
GAGCTC-CTGTTTGGAATAAIAATTCATCAATGAXTGTTGGACCT.AGAGG.ATTGATTTTDTTGGÂ AGATTATCAnTGGTTGAÂAAATTGGCTAATmGATAGAGAAAGAATTtXTGAAAGAGTrGTT CA.TGCTA.GAGGAÍj <'TICAÍj<TAAAGGÁTrTTTIGAÂGTTACTÜATtjATÀTTTeAAÀTrTGACTr GCX ^ CTGATTTTTTGAGAGCKX'TGGAGTK'AAACTCCTGTTATTGTTAGATITTCAACTGTTAT TCATGCTAGAGfâATCACCTGAAAdTTGAGAGATCCTACi.AGGATTTGCTGTT.AAATTTTATACT AGAGAÁ <K <A4.4TTTTGATTIGGTTGGÀAAIAATTTTCCTGrTTTTTTTAITÁGA.GATGGAATGA AAITTGGTGATATTGTrCATGCTTTGAAACCTAATCCrAAATCACATATTCAAGAAAATTGeAG ^{AAiTTTTGGATTTrTTTrC:} A.CATCATCCTGAATCATrGAATATGTTIA.CTTTTTrGTTTGÂTGÀT ATrGG.AATTCCrCÀA .GATTATÀGÁCATATGGATGGATC ^; AGGArTT'AATACTTATATGTTGÀ TTA ATAAAGCTGGxAAAAGCTCATTATGTTÂAATnüATIGGÂÀA ^ I K * CTTGCGGAGTTA.AATCATT GTTGGAÀGÂAGATGCTATTAGATrGGGAQGAACTAATCATR TGCTACTCÀAGATTTGTAT GATT0AÂTTGCTGeTGGAAA-TTATC '^ CTGAATGGÁÂA.TTGTTT 77s..AAATTA.TTGATC'CTGCTG
ÀIGÀAGATAA ^ TTreATmGATCC ^^ TGGATGTTÂCTAAAACTTGGCCTGÁAGATATTTTGCO
TTTGCAAiXTGTTeGAÂGAATGGTrTTGAATAAAAATATTGATAATTTTTTTGCrGÀAAATÍjAA
CAÁTFGGCITrrrGCCCIGCTATTAITGTTCCTGGAATrCAITATTC ^ JATGÁTAÀATTGTTGC AAACTAGAGTTmTCATATGCTGATACTCAAÂGACATAGÀTrGGGACCrAATTATTTGCAATT GGCTSTTAATGCiTCeTAAABSCGCTCATCATAAIAÁTCATCATGAAGGATTTATGAATTTTATG CATAGAGATGAAGAAGTTAATTATTITCCTTCAAGÂTATGATCAAGTTAGACATGCTCAAÁAAT ATCCT.ACTCC-TÜCTGCTGTTTGCTCAGGAAAAAGAGAAAGATGU.ATTATTGAAAAAG.AAAATAA TTTTAÀAGAAOCTGGAGAÂAGATATAGAACTTTTACTCGTGAAAGACAAGAAAGATTTATTCAA AGATGG.ATTGATGCTTTGTCÀGATCCTÀGAATTACTCATGÀAÀTTÂGATCAÁTTTGGATTTCAT ATrGGTCACAAGCTGÃTÀAATCATTGGGACAAAAATTGGGTTCAÀGATTGAATGTTAGACCTTC
AATT
SEQ ID NO. 14
Amino acid Catalase Arabidopsis thaiiana
WPY £ YRPASS ¥ NSFFFTT ^ GAPVWISr> ^ SSMTVGPRGLILLED ¥ HLVEKLÂNFDRERIFERVV
HARGASÁKGFFE ¥ THD1: LTCADFIJLAPGVQTPVIVEP5TVIHARGSPETLRDPRGFAV ^ £ ^ PEGN1 FYT) LVGJ PDIVHALKPI ^ ^^ ^^ PVFHRDG SHIQENWRILDFFSHHPES: LNMFTFLEDD ICSPQDYRH LDG ^ ^ ^ ini VNTll GKAHYVS ^ ^ ^ ^ I JKPTCGW DÀIRLGGI SHATQDLY DSIAAGK ¥ ^ ^ PEWLFIQS3PADEDKFDFDFLD TRTWPESILFLQFVGWVLKENmNFFAENE QLAFCPAn ^ Ti ± HySDDKI QT WS ^ ¥ ADTQRHkLGPN ^{TQIJ> WÂPKCAHHN,} NHHEGíMNE HKDEE ^ í n ^ SRYDQWHAEK PIPPA ¥ ¥ ¥ CSGKREPCIIEKENNFKEPGEE RTFTPERQERFlQ O®.ÂI5D TtnHSRSIV®WSQADKSL <3QKLASI ^ ^ 2WRP
Petition 870190120782, of 11/21/2019, p. 99/155
98/114
SEQ ID NO. 15
DNA
Catalase HPII (KatE)
Escherichia coli
ATGTCGCAA £ ATAAC GAAAAGAA € CCACATCAGCACCAG ^; TGACCA £; TACACGATTCCAG € GAAG OGAAACCGGGGATGGACTCACTGGCACXTGAGGACGGCTCICxATCGTÍXAGÜGGCTGAACCAAG ACCGCCAGGIGCACAACCTACCGCÜCCÂGGGAGCCTGAAAGCCCCIGATACGCGTAACGAAAAA <€ ^ AATTCTCTGGÁAGACGTACGCAAAGÜCAGIGAAAÁTTAIGCGCTGAC ACTAAirAGGGCG TGCGCATCGCCGACGÀTCAÀAAÜTCACT <KGIGCÜGGTAG <XGIGGTCCAACGCTG TGGAAGA TTTTAIT € € KK GKGAGAAA Thy ^ £ ^ mACITTGACCATGAGCGCATraXÍGAA CGTAITGTTCAT GCACGCGGATCAGCTKSIAGGGTTATrrCCAÍKX TATAAAÁGGTLAA & CGATÁrrAOCAAÁG CGGÃTnCCTGTCÂGAKWAACAAAÁT ACCCCAGTA.TTTGTÀCGTnCTCTÃ € <'^ CGTTCA.GGG TGGTGCTGGCTCTSTOATACWTGCGlGATATCCGTC TrTGCCACC.AA & ITCTATÁCGGÁA GAGGGTAIITrTGACCTÜGTFGGCAATAACACGCCAATüTTCTTTA.TCCAGGÀTGCGCÃTAAAT TCCCCGATTTTGTTCATGCGGTAAAACCAGAACCGCACTGGGCAATTCCACAAGGGCAAAGTGC CCACGATAC'TTK'TGGGAITATGTTTC'TCTGCAACCTGA.AACTCTGCACAACGTGATGTGGGCG AWTüGGATCXSCGGCÀTCCCCCGCAGTTACCGCACCATWAAGGCTTCGGTATTCACÀCCTreC GCÜTGATTAAIGCCGAAGGGÀÀGGCAACGTTTGTACXàTTTCCÀCTGGÀAAÜCACTGGÜÂGGrAA AGCCTCACICGTTTGGGATGAAQCAOAAAAÀGTCACüíMACGTGÀCCCGGAeTTOCACCGCCGC GAGTrGTGGGAAGCCATTGÂAGCAGGCGATTTTCCGGAATÀCGAACTOGOCTTCCAGTTGÁT TC CTGAAGAAGATGAATTGAAGTTCGÁÜTTCGATCTrCTCGATCÜAACCAAACTTATCCCGGAAGA ACTGGTGC C <GTTCAGCGIGTCGGCAAAATGGTGCTCAAlGGCAACCOsGATAAGTTCTTTGCT GAAAACGÀACAGtKX ^ CTITCCAI ^ CTGíX ^ ATATeGTGCCGGGACTGGACrirACCAACGATC CGCTGTTGCAGGGÀCGITrGTTCXCCTATACCGATACACAAÁTCAGTCGTCrrGGTGGGCüGAA TTT (XAIGAGATTWGÀ1TAÂ € CGTÍXX1ACCTGCCCTTACCATAATTWCAGCGTGACGGCAIG CATCG £ AT © CKX3AKGACACTAACCCGGCGAÂTTA £ OAÂCCGAAOCGAITAACGATÂÂCTGGC CGCÈXíGAÂACAaGCCOG ^ XGAAACGCGGaXSITITGAATCATÁCÜÁGGAGCGCGTGGAÀGG CÂATAAÀGTD: GCGAGCGQAC <mATCGITTGG £ GAATATTATTOXATCCGCGTCrGTTCTGG CTAAGTCAGACGC ^ AmGAGCAGCGCCATATFGTaSAT ^ iTITCAGTTTTGÀGTTAAGCAAAG TCGTTCGTCCOTATÂTTCGIWlGCGCGTmTTGACCAGCTGGCGCATATTGATCTCACTÜTGGC CCAGC <GGTGGCGAAAAATCT € GGTAT € OAACTGACTGACGACCAGCTGAATATCACCCCACCT CCGGADGTCAACmTCTGAAAAAGGÂTCCATCCTTÀAGTrTGTÁCGCÜÂTTCCIGAC <€ ^ rGATG TGAÂAGGTCGCGIGGTAGCGATmA TTAATOAIGAAGTGAQATCG (XÁGACCTrCTGGCCAT TCTCAAGGCGCTGAÁGGCCAAAGGCGITCATGCCAÀACTGCTCTACTCCCGAATGGGTGAÂGTG ACTGCGGATGÁÜGGTAaKsT & TTGCCTATAGCCG ^ ^ AC TTTGCCGGTGCACCTTCGCTGACGG TCGATGCGGT ATTGHXCnGCGGCAATAiaK € < TATCGCTGAGAACGC K ^ ^ ^ GATGCCAACTA CTAGCTGATGGAAGCCTAGAAACACCirAAACCGAnGCGÍTGGCGGGTGACGCGCGCiAÂGTrF AAÀGCÀACÁAKíAAGAKÍGC GACCAGGGTGÁAGAAGGGATrGTGGÀAGCTGACAGCGCTGACG GTAGTmATGGATGAAGTGCXAACGCTGATGGCAGCACACCGCG'TGTGGTCACGCArKiCTAA QAITGÀCAAAATTCCTGCCTCA
SEQ ID NO. 16
Amino Acid
Catalase HPII (KatE)
Petition 870190120782, of 11/21/2019, p. 100/155
99/114
Escherichia coli
MSt ^^ KJTPHQHQSPLHDSSEASOOsLAPEDGSERPAAEPIPPGAQPTAPGSIKAPDIENEK
L ^ LED 7i: <GSENWlTWG ^ AADDQNSLFAGSRGFIlAEDFIL ^ 3aTHFDHERI PEFSVH
ARGSAAKGITQP ¥ KSLSDnÈáDn.SDI ^ ÍKI TPVFVRS ^ WQGGAGSÂDTVSDtR ^ AlKFYTE
BGI EDLVGISkSiTPÍEFIQDAHKFEDP HAV ^ ^ ^ SFEPHWAI PQGQSÁHDTFWWSLQPEIEHISiVWiA ¥ ^ 3ΒΕΟΣΡΕ8 ΑΤ ^ ΊΕΟΕδΙΗΤΕΚ ΣΚΑΕδΚΑΤΕνΕΕΗΚΚΡ1Αί3ΚΆδΕ 'βΕΑ ^ ^ EL _{ΚΙ ί Τ355εΕη5ΉΚΚ} AIEAGDFFEYELGFQLjí PEEDEEKTOFDIIDPTKII PEEnWQEVGKWLNENFJWFA and QAAFHPGH ^ ^ ^ P GinFESDHAQCMLFSTEDTQI SRLGGFNFHEI PTCPYHNFQRDGM HWGWTNPANyEPNS INDN TREIP ^ ^ The RG®ES ¥ ¥ EG QER ^ KVRERSPS FGEYY3HFRLFW LSQFPFEQSinVDGFSFELSKWPYIREWDQLAHmmAQAyÀKNLGI ELTDEQLNITPF
PDWGLKKDPSLWFAI FDGDAIÍGR.WAI WWEVRSADÍUU I ^ LKAKGVHAKLLYS ^ ÍGEV
ΤΑΒΒΟΤΛ · ΤΡ1Α4ΙΕΛΟΑΡ «ΕΏΤ3ΑΑΏΤ <« Μ4δΕ4ΒΝ € ΦΑ ^ ΈΜΑνΈ3Ξ..ΚΡΕ41Α £ ·; &. ΑΒΕΕ
KWKLWQGEEGnT.WS.W € 4SI ^ fflELLTOlA4HRWSRIPKIBKI PA
SEQ ID NO. 17
DNA
CBDA synthase target of trichome
Cannabis
AZGAAGTCXTCA4CATTCT <.GTTrTGCTETrTrEGCAAGATAATATTTTTriTrTTCTCATTCA ATATC AÁACTT € <XAITG TAAT £ 'x E <C vAl-AAAU.TK:CTTAA4TKTT<T<«AATÀTATTCC AATAATGCAACAAATCTAAAAf T' <T r IT VJÂCTCAAAAC AACCXLATTGTATATGTCrGTCCTA .A4ITC £ & A XATACACX4TClTAGArrCAvLICBMCACAACiCXXÁAÀACCACTTGTTATCGTCA CTCCTTCÀCATGTeTC: TCATATCCA4GGCÀC: TATICTATGC: TCCAí & AAAGTIGGCTTG € AGAT TCCAÂCTCCAAGTCGTGGTCATGATTCTGÁCGGCÂTGTCGTÂCATÀTCTCAÀCTCCCATrrGTT ATACTAGA £ TFGACAA ^ AFG € <mAAICAAAAIAGATC ^ Catac 'CAA ^ TGCATGGGTrG N- GC € GGAG € TACCCTTGGAGA'; iTTArTATrGGGTTAWG4QAAAAiT € 4AGAATCTTAGTTr · C <XWCTGGGTATTÍXCCTA € TGTTT € XC "A <X; T <XACACTITGGTGGAGCAGGCTATGGACCA TTGAT & A <xAAACTATCAX: <TCGC : GGCTGATAATATCATTGATGCA <ACTTAGTCAiCGTTCÁTG GAAAACTGCTÀGATCXÍ ^ AAAATCTATGGGGGAAGATCTCTTTTGGGCTTTACGTGGTGGTGCAGC AGAAACá. TTCGG ATTGTA Aat I-Ai ^ .The GGAAA TTAGAC GGTTí.A TGTCC C 1 AAAGT1..TAC ATG T ^ TITAGTGIIAAAAAGATCATG GAEAGATGACXTTGTCAAGTTAGTTAACAAÂTGGCAÀÂAIA TrGCrTA € € AAGTAIGACAA4GATITA'nACTCATGACI ACTrCATAACTAGGA4CAITACAGÀ TAATCAAGGGAAGAATAAGALxAGCAATàCãCACTEàCTTCTCTTCàGTTTTCG-TTGGTGGAGEG gatAGECTAgT C GAC11GATGAACAáGAG.1 iii CC i Ai -gag1 iGGGT ÍÁAAAAAACGGAí IGCA GA € 'AATTGAGC: TGGATIGÀTACTATCATCTT TAEAGTGGTGTEGTAAATEA € €' € GAeAeTGATAA TTTTAACAAGGAAAITrTG TTGATA 'AK t TG £ € GCTGGGC AGAaCG TTTCAAGATTA4GTTA GACTÀCGTTAAG AAACCAATTCGAG LATI TCTÀTITGTC .4.A C' TnGGAÀAAATTATATGAAG AAGATATAGGAGCTGGGATGTATGC oTTGT ACCCTTAC GGT <5GTATAAIX TGAGATrTCAGA AICAGCXATT £ ^ £ rati <£ t £ ATCGAG TGGAATCTFGTATGAGTTATGGTACATATGTAGTrGG GAGAAGCAAGAAGATAACGAAAAGEAECTÁÀAtTGGATTAGAAAIAETTATA £ ^ € CTICATGACTC CTIAEGEGTGCÀAAAÀTCCAAGATIGGCAIATCTCAATrATAGAGACCTEGArATAGGAAIAÂA TGATCCC4AGAAI CAAATAATrAC4C £ X ^ 4A ACGTATn: GGI a4G4AGTATrTTGGTAAA AATnTGACAGGCEAGTAAAÁGTG.WLiC ^ € € XT 'Tm <£ TCCCAATAA TnTnAGAAACGA.AC AAA <^ ATCWACCrCTACCA <XSGC4TCGT ÀTrAA €
SEQ ID NO. 18
Petition 870190120782, of 11/21/2019, p. 101/155
100/114
Amino Acid
CBDA synthase target of trichome
Cannabis
MKCSTFSWFVCKH HTFSFMQTS IAIWENFLKCFSQY1Í ^ sn ^ TRFIBSTI KP »T ^ TPSm ^ HIQCITIL <^ KkTGLQníIIÍS-GCvHBSEGMm SQVPFVV
U DL W1RS liSDVHSQTAm-’EAGATLGEVn T T> EK ^^ lJEAAGYCFn’CAGGSFGGGGl’GP
LM ^ YC ^ A ^ iroAHL.Vm'SGSCV'lDKKSStiGEDLnVAI ^ OGGAE ^ GlIvAwKlSLVAX.'l ^ lM ^ ΈΚίΆΜΗΕΕΛΊα: Λ Ϋ́ £ ^ ^ Ί ^) ΚΒΕΕΕΧίΤΗΗ: Τ ^ ΙΤΏ ^ 0ΟΚΝΚΓΑΒΤΐΡ ^ ΎΈΙΧΧ ^ IK KID FSSC ^ TWDIIWNKH UDRSACQNGÁFSKL
DYVK13 »!. SESVEVQl EEEZYEEIMGAGM ^ 'AIYPY-GGBWEI. SESAl PTTEKAGI LYEOfYlCSW
EKQED LoLi ^ ^ ^ lE TCÍFMIPlYSK AlXNY ^ _í ^ IU DLDT <^ l> ^ f ^ l lTQAI3WGEK. £ ¥ EGL
NFDRL'fc’KVKTLWFJiNFfE ^ QS Ϊ FHPRHRH
SEQ ID NO. 19
DNA
Trichome target UDP glycosyltransterase 76G1
Stevia rebaudiana
& ATGAÂGTGGTCAACATTCTCCTTTFGGTTTGTTrGCAA ATAATAFTrTFCTFTTTCTCA.TTCA ATAKCAAACTTCCATTGCTAÍTCGTCGAGAAAATAAAACTGAÀACTACTGETAGAáGAAGAáG AAGAÂITATrriGTTTGCTGTTCCTTTTCAAGGACATATTAATCriÀTTTTGCAATTCGCTAAT GTTTTCTATTC:. AAAAGGÀTTTTCAATTACTÁTTTTTCATA € TAArrTTAATAAACCTAAÀÂ £ TT ^ CAÀATTATCGTCATTTTACTrFIÀGATTIATTrrGGATAATGATCCTCAAGATGÀÂAGAÀTTTC AAATTPGCCTACTCATGGACGTFTGGCTGQAATGAGAATTCCTATTATrAATGAACATGGAGCT GATGAATTGAGAAC GAATTGGÂÀTTGTTGATGrTGGCTTCAGAAGAAGATGAAGAAGT-rTCAT GCTTGATTAC'TGATGC'TTTGTGGTATTTTGCTCAATCAGTTGCTGATTCAnGAATTTGAGAAG ATTGGTTrTGATGACTTC.:ÀTCA.TTGTTTAATTTTCAT&CTOATGTTT£ATTGCCTCAATTTGAT GAATTGGGATATTTGGATCCTGATGATAAAACTAGATTGCLAAGAACAAGCTTCAGGATTTCCTA TGTTGAÀAGTT.4AAGATATTAÀÀTCAGClTATTeAAATrGGCÀÀATTTTGlÀ.AGAAATTTrGGG .AAAAATCxATTAAACAAACTAGACXTTCATCAGGAGTTATTTGGAATTCATTTAAAGAATTGGAA GxAATCAGAÂTTGGAAACTGTTATTxAGAGÂÂÂTTCCTGCTCCTTCArTTETGATTCCTTTGCCTxA ÀÂCATTTGACTGCTTCATCATCATCATTGTTGGATCATGATAGAACTGTTrTTCAATCGTTQGA TCAACAACCTGCTTCATCAGTm'GTATGTTTCATTTCGATCAACTTCAGAAGTTGATGAAAAA GATTTTTTGG AAATrGCTACAGGATTGGTTGATTCAAAACAATCATTTTTCTGGGTTGTrAGAC CTGGATTTGTTÀAAGGATCAACTIGOGTTGAACCTrTGCCTGATGGATTTTTGGGAGAAÂGAGG AAGAATTGTTAAAT <X ^ TrCCTCAACAAGAAGTTTTGGOTCATGGAGOTATTCGÀGCTrTTTG ACTCATT € € € AGGÀTGGAATrCAACTTTGGÀATCAGTTTGCGAA GAGTTC ATCAAC € ^ ^ TATGATinTrCAG ATTTlWATTC lTTGAÁTGCTAGÀTATÀTGTCAGATGTTTTGAAAGTTGGAGTrTA TTTGGAAAATGGATCGGAÀAGAGGAGA4ATTGCTÀATGCTATTAGAÀGAGTTATGGTTGATGAA GAA <Î € GAATÀEÀ1TAGÀCAAAATCCTAGÂGTrTTGAAÀCAAÁÁAG TGÀTGTTTCAITC.ÀTCA AAGGAGGATCATCATATGAATCATTGGAATCATFGGnTCATATATTTCATCATTGTÀA
SEQ ID NO. 20
Petition 870190120782, of 11/21/2019, p. 102/155
101/114
Amino Acid
Trichome target UDP glycosyltransferase 76G1
Stevia rebaudiana & ataCCSrF nrn'CKl I FFFF & FNI chitosan LV ^ ^ I ^ NKTETTt ERRRI IISSVFFQGHEOTLQLAX VL.YS3Í.GFS ΪΓΙ ΓΗΤ5Ϊ 5Κ5ΚΤ§ · Π> ΗΤΓΙ3ΪΚ LDND'P'QDEEI SM ^ ^ THGJLAGa.fRl PII2ÍEHG4 DELKFELE MLASEEBEEVSCIJ TBÁLWlTÂQSV.W ^ & SsllH Yian ^ LlSíyHAHV.SLÍQFI »ELGyLDPSDKTELEEQASGFPMLKVXDI KSAYSWQl LKEXLGKàSKQFRASSCTI WsFKELE ESELEIV3RS. RT & FLPKHLTASgSSLWHDE'nTQV.T.BQQPPS FÀFSFLI ^ ^ '^ l SFGSTSEWEK DFLE XÀKGLVSS TLWyn®GFVKSSTm PO-Z & GFLGEEGRI naVVS EWiBGAlGAW TH ^ ^ ^ GW TLESVCEGWMI FSDFGLBQFLNARy ^ ^ XSmyK XíVVTE CTORGEIASAERR ^ ^^ ^ Iffi ECSYW ÀSVLKQSG ^ ^ ySlAiKGC SYESLE.gLVSYI ísSL
SEQ ID NO. 21
DNA
PM-UTR1
Arabidopsis thaliana
AT <^ GGTCCATG <^ COGGÁTIC'CGT € GÂÃTrCKiTT & TIWCGTrGTGTAICTCCGGG ^ TCT CtGTQGGC-CTACA λ C TA..CCAAGCtL GTTC T ^ CAAgAGACTÜTGjTÜGACÁGAAGAGGGACH TTGC-.ATTCTTGAAL-TTaGC-TLjAAAGTljTAGTL.TGC- ZTGAZCTGCf TCTTÂTATÀATGAJTCAAGC ^ CTOGTCAÂAI ^ T <KyTAAC '<XjTGGA <^ A €' CÂTGGTGGACGTATT G * * TGl ACCX. ATTACT A AT ALAATTGGTCCTGCCATí ^ GGAATTGAâGLCTTGAAGTATà à LAG TTATC CAGCTC AGGTTTí ^ jGCAàAATCGTCAAAAt.TGâTílA AGT í. ATGCTA4TGGGAAC TTTA GTTTÂCGí € ^ ÁATA4GÀTÀCACTirTCCCTGAAT.Á-CATGTG AC'C'TTTCTrGTCGCTGíGÀGGAGTAT CLATLTTTCSL-TCTTVTTàAGALAâGL - ^ CTÀAGÂC-AATA AGC.AA.GCrr.AGC-ALArL <GAAATGGTW C -CTL GGTTÀCGG ACTTTG TTCCTTAÀAC L TCGC -OTTTG AÜGGATTC .ÀCAAÀTGCCAC AC. AAG AC TCiCATTCA ^GTCâAGG: T.áCCCAAAuAàLCGAà.CX GTGI G.AC'ATAATCA ^ '1 LjGiAAATGAALTZ'ATCXrG al GC-AATATACAALvATTATCTAC.aA.í.GTTTGGC TTGC.Cà, ^{CAÂ'GGGATGGAT:} TOGAãGCAâ.TTÜAG TiCTCAAAGXTAC, CAB-4GcCGGAAGCGGCATgC ATTCTAAAGTATTGTàTATGCájGTGCCgTC -G GALjAàAAGTTCATC · TTCATG. AC AÀ.TAAGT AALT'TCGGGS CACTAGCTAAC ACGAC ATAAL C AC C: GÂCCAGGAAGTTTGITÀGCATrGTreTATCATÜAGTAAIGAGGGGAAATGCATrGTCGTTGAAG CAATGGGCCATGTGTTTCGAICTGTGTlTC TGGTrrGGC'ATAIX'AAATTTATCTTAAATGGAACvA ^ $ AC AATTGCA & AG.ÁGTGG.ÁGTGCTCCÀ.TAArG.AACTTAATGTGTGGGTLTACCTGCGCLGJT.
SEQ ID NO. 22
DNA
Cytostolic CBDA synthase (cytCBDAs)
Cannabis sativa
Petition 870190120782, of 11/21/2019, p. 103/155
102/114
ATGÀATWICGAGÂAÃÂCTTWTTÂÂÁIGCTTCTCGCAATATATTGCCAÁTAÂTGCÂACAAATC
TÂÁÂÂCTaJTATÂCárTCÂÂÂÂCÂACCrATIGTATATGTCTGTCGTÂÂÂTrCGACÀÂTâCACAA TCTTAGÁTTCAttTCTGACÁÜAACCCCAAAACCACFrGTTATCGTCACTWriCACATGT CATAKC TCT £ ^ & AGGEACTÂTTCTATGCTGCAÁGÀAÁGTTGGCTTGCAGATrCGAÂCrCGAAGTGGTG GTCÀTGATTCTGAGGGCATGTCCTÂCATATCrCAAGTCCCATTTGTTATÂGTAGACTrGÃGAAÃ CÀTGvGITCÂAIÇAââATAGAT TKATAGCCâáACTGCATGGGTTGAAGGCGGAGCTÂCCCTT GGAGAAGTnATTATTGGGTTAATGAGAAAAATGAGAATCTTAGTTrG ^ GCOGGGTATTGGC:
CTACTGTTTGCGCAGGTGGACACTFTSGTGGÂGGAGGCTATGGACCATTGÂTGAGÂÀACTATGG EC ^ SGGeiSATAÀTATCÁTrGATGCÀCAGTTÂGTCiÀACGTTGÃTGGAÂAAGTGCTAGATCGÀ AÂATCFAlGGGGG-AAGÂTCrCTTTTGGGCTTTACGTGGTGGTGGAGCAGÂÀAGCITCGGÂATCÀ HGTAGCATGG ^ AAATTAGACTGGTTGCTGTCCCAAAGI € TACTATGTTTAGTGTTAAAAAGAT CATGGAGATÂCÂTGAGCnGTCAÂGTTAGTTÀACAÂATGGGAÂAÂTATTGCTTÂCAÂGTATGAC .AAAGATTTATTÀCTCATGACTCâCrTGATÃACTAGGAACÁTTACAGATAATCAAGGGAAGAATA AGÃCAGCÃÃTACACAClTACTFCrClTCAGTTTrcCTTGGTGGAGlGGATAGTCTÃGTCGACrr GATOAACAAGAGTTrTCCTGAGTTGGGFATrAAAAAAACGGATTGCAGACAATTGAGCTGGATT GATÀCEÀTCATCTTCTATÂGTGGTGTTGTAAATTACGACACTGATAATnTÂACÂAGGÂAATTT · TGCTTGATÂGATCC TG ^ ^ ^ XAGAACGGTCXmTCÂÁGÂTTAAGTTÂGACTACGTTAÂGAAÂCC AAT CAGÂATCTGTATTrGTCCAAAITTTGGAAAAATTATATGÁAGAAGATATAGGÂGCTGGG ATGTATGCGTT & TACCCTTACGGIGGTÂTAATGGÁTGAGATTTCAGÂATCAGCAÂTTCCATTCC
CICAIGGMKTOGÃATCTTGTATGAGTTATGGrÁCATATGTAGITGGGAGÁAGCAAGÀAGATAÀ CGAAAAGCATCTÀAAÍ TTA ^ ^ ^ GÍ AATAITrATAACTrCATGAGTCCTrÂTGimCCÂAAAAT CCAAGÂTTGGGATATCTCAÁTGATAGÂGACCTTGATATAGGAATAAATGATCCCAÂGAATCCÀÂ ATAATTACACACAAGCACGTATTTKAAÜTGAGAAGTArmWTAÂAAATATTGACA ACTAGT & AA4ÀGTGÁAÀACCCTGGTrGÂTCWAÀTÀÁCTITTTTAGÂÀACGÀÀCÁAAGCATCCCÁCCIürA CCACGGCATCGTCÁTTAÁ
SEQ ID NO. 23
Amino Acid
CBDA cytostolic synthase (cytCBDAs) Cannabis sativa
HK mLCSKKVC ^ ^ ^ Qa SGGHDSEGMSY QWFVITOI ^ I ^^ íSSaaDVHSQTAWVEÂGATL GEVTYWVNEKJSSNLSLAAGTCHVCAeGHFGGGGTGPLMSNyGLAADNI dAHLVNVHGKVLDR EDLFWALSGGGAESFGIIVÂWKISLVAYTXST KA ^ ^ ^ ffSVKKIMEIHELVKLVNK QNIÁAXYD KmiMrHFn ® ^ ^ N ^^ ^^ AJEM LGG ^ IS ^ ®aWU GIKKI3X 35QLSWI
DTS J ΡΑΈ-GWYDIDNFNKE xliDRSAGQNGAFXI KLDYVKKPI PESWVQS LEKL '¥ m> lGAG
MTALlT ¥ G <HMEEISESAIPFPHR4GiL ^ WYICSU ^ QED O2niTOIRNn N ^ fIP ¥ VSKN
FWLlTNYFJ> mO ^ I> KNTNN ¥ IK} ARWQ ^ T <^ 2rôm ^ ’Kn.X ^ Sa®FHySsEQSim.
PRHRH
SEQ ID NO. 24 DNA
UDP glycosyltransferase 76G1 with cytostolic target (cytUTG)
Stevia rebaudiana
Petition 870190120782, of 11/21/2019, p. 104/155
103/114
ATGGAAAAT ^ AMXGAÀACGWCGTCCG ^
I £ i ££ AGGGCCÂ GWATI £ € € ÂThe TGCAACTG (X2GAA € GIGCTGTÀIITOAAÂGGTrTCAGCAT CAC ATCTIWATAQjâACTI £ £ € AACAAGC GÀAGACCAGCAATTACCCGCACTTTACGTTCCGT TTTATTCIWATAACGA ro £ £ £ i £ CAGGAIGAACGQAI TAATCIGCCGA £££ ACGGCCCGCTGG GCKKHATGCGTATTWGATFÂTCAACXLAÀCACGGCGCAGÁIGAACTGCGTCGCGAACTGGAACT GCÍGÁTGCTGGCCAGCGAAGAAGATGAÀGAAGITTCnGCCTGATCACCaACGCACTGIGGTAT ITFGOXAGTCTGTIX ^ ^ AGATAGKTGÀACGTGCGiaKXnGGTCGTGAIGAíXAGCAGWIGT TCAATTITCATGCCGACGrTAGTCrGCCGCAGITCGATGAACrGGGTTATCTGGACCCGGATGA CAAAACCCGCC5GGAAGÁACAGGCX GCGGCTnGCGATGCT (^ AAGTCAAGGxATATTAAGTCA GCGTACTCGAACTGGCAGÂTTCTGAÀAGÀAÂTCCTGGGTÀAAAIGÂTTAAGCAAACCAAAGCAA GTTGCGGCGTCATCTGGAATAGTTTCAAAGAACIGGAAGAAI CGAACTGGAAACGGTGATTCG TGAAATCCCGGC ^ £ ^ CCGAGTTrTCTGATICCGCTGCCGAAGCATmGACCGOGAGCAGCAGCAGC CTGGTC ATCAWACWCACGGTGTTIÜAGTGGCTGGATCAWAÀCCGCCQAGrrarGTGCIGT ATGTFAWTI GGTAGTACCT £ £ € GGAAGIGGATGAAAAGGÁCTn IGGAAATCGCTCGIGGCCT GGTTGATAGCAAAGÂATümCCTGTQ ^ £ Q TGGTrCGCCCGGGTniGTGAAGGGÜTCTACGTGG GTIGÀAC TGCCGQACGGC £ ^ £ € OGGGIGAACXHWCCGCArrGTCAAÀTGGGTGC GCAGC AAGAAGTGCTGQ'GCAT <XKWGATrGGCXK; GTmGGACCCAETCC £ KJT1XX} AACICAACCK: T QGAÁiaKmTGTGAA <K ^ I £ CCGATOAITnriCAGATmGGCCTGG ^ CAGCCGCTGAAT GCACGTFATATGTCWATGTI £ TGAAAGTtWTGTGTGGGGGGGGGGGGGGGGGGGGGA
SEQ ID NO. 25
Amino Acid
UDP glycosyltransferase 76G1 with cytostolic target (cytUTG)
Stevia rebaudiana
MENETETTVRRlíSKSILFFWFQSHISPIigLSWLYSEGFSXTIFgTSFNOETSNYPgFTFE ΡΣ1ΏΪΠ) Ρβ333Σ3ϊί1 ΣΉ®Ρ1Α <^ ®: ΣΡΣ INEESADSISSSIELLMLA ^ ^ SSDSSVSCIXTSALWY FA.03VADãlJs''LSSLATL5ÍTSSLWFSASVSlPQFDE38YLDPDSKTRS33Q 3GFPSHKWDIKS .ΑΣ · 3 ™ ^ Ι1ΕΕ: Ι13ΚΜΣΚςΤΚΑ338 ¥ ^ Σξ 33ΕΕ13: Ε3Ε13Τνΐ33ΣΡΑΡ331ΙΡ1ΡΕ31ΤΑ8383 ΙΙϋΗδΚΤνΡα Σ ^ ^ ^ βΡΡβδνΐΥνΒΡδ ΤδΒνΠΕΚΒίΙΕΣΑΕδΙΥΒδΚςδΡΙΚΚΊΪΜΡνΚΘδΤ® ν3: Ρ1Ρϋ ®31δ3333ΣνΚ5ϊνΡ® _; 3νΐΑΗ8ΑΣδΑΓη · 33δΜ8 ^: Τ1Ε3νΰ3 «ΥΡΚΙΕ®Ώ3δ1Β ^ Ρ1Κ ΑΡ: Υ5ί3ΣινΐΚναν ¥ 1ΕΚ <^ 33 ^ ΕΣΑΜΑΣΡΡν ^ 1Ε3δΕΣ ^Γ ΣΚ ^^ Α®Λ ^ 13:. '3ΚΑϋν81ΜΕΟΟ33 ¥ ΕL1 SSIV
SEQ ID NO. 26
Amino Acid Glycosyltransferase (NtGT5a) Nicotiana tabacum
Petition 870190120782, of 11/21/2019, p. 105/155
104/114
ΜΟΡΒΡΕΤΙΡΒ ^^ ΡΡΟΕ & Π & ^ βΙΡΟΙίσΕ ^ ΤΤΪΕΡΟΣ ^ ΡΚΒΟΛΧΙ ^ ΕίΤΚΤδΚνΡΡνοαίνδΒα
FTLAAAQELeVPEVLFKTTSAeGFLGYWTCE V ^ ^^ ^ ¥ Μ EKGTAPLEDAEDLTNGyiiErTtDFIPG _{ΕΣ ί Εβ} & 8Ρ ^ Ε ^ ¥ ^ ΕΤΤ®Ρ3ΕΕΜΣΚΕ _{ΕΤΕΕΑΕΕΑΕΑΙΙΣ ί} Ε§ΣίΕ _{ΤΡΕΤΕΕΑΕνΐ ί} ^ ^ _{Σ ί ΕΡΡνΎΡ} IGPLHFL VKEVBDEJTLK.GIiE.SS LWKEEPECIQWLDTKEFKS WWFGG ITWTP QLTEFAKG LANSQ ^ ^ ^ I TFL ΣΕ .Ρ0 IVSGDAE i: LPPEFVEETraEGMLdASWC®QEEVLEHPaiVGFLTHSGWS
TLBSIESGVPKIíWíFFAEgQT ^ a ^ FSVTKWWGaffiinSDVXSZÍWgSLWSXiMVWKGKKMRi: Κ »Μ ^ Κ2Χ * Ε» ^ & 1 ^ 8α30ΥΧΪΕΣΒ81 _ί νΗϋΙΣ _ί Ι _ι 85ΚΗ
SEQ ID NO. 27 DNA
Glycosyltransferase (NtGT5a)
Nicotiana tabacum
ATGGGTTCCATTGGTGCTGAATTAACSAASCCACÀTSCAGTTTSCATACCATATCCCGCCCAAS GeeATATTAACCCCATSTTAAAGCTA.GCaAMA.TCCTTCA.TCACAAAGGCTTTeAQATCAeTTT TGTCAATACTÍ ^ ATTTAACCACCGACGTCTCCTTAAATCTCsSTGGCCCTGATTCTfTTCAAGGGT CTTTCTTCTrTCC®TTTTGAGA.CCATTGCTGATGGAeTTee®CCATGTGAGGCAGATGCeACA.C MSA.TATACCTTCTTTGTGrGAATOTACAACeAATACTTGe'TTGGCTCCTTTTAGGGATCTTCT TGQGAAÀCTCAATGATAGTMCA.GATCTAASGTGCGA ': © GTG ^C'GTTT; CA.TCGTCTCGGATSGT GTeATGAGCTTCACCTTA.GCCGCTGCACAAGAA.TTGQGAGTCCCTGAAGTTCTGTTTTGGACeA CTA@TGCTTGTQ®PTTCTTAGGTTA.CATGCATTACTSCAAGGTTATTGAAAAAGGATATgCTCC ACTTAAASSATQCGA.GTGACTTGACAAATs3 <3ATA € CTAGAGACAACATTíSSATTTTATACCAGGC ATSAAAGACSTACGTTTAAGG ^ TCTTCCAAGTTTCTTGAGAACTAQAAATCCAGATSAATTCA TGA.TÜAÃATTTGTCCTCCAAGAAACAGAGAÍ3AGCÃA.GAAAQGCTTCTGCAA.TTATCCTCAACAC
ATTTGAAACACTAGAGGCTGAAiHTGTrGAATCGCTCCGAAATCITCTrCCTCCAGTCTACCCC HVAC 4ÁGÀ ^ ^ ^ GWCTTGCATTrTCTAGTGAAACATGTTGATGAIGÀGAATTTGlAGGGACTTAGATCeA GCCirrGG AACCAGAGTCTATACAAT <TTGATACCAAAGAACCAAATTCTGTTGT TrATGTTAACTTTGGAAC ^ «€ ATTACTGTTATGA TCCTAATCAGCTTATTGAGTTrGeTTGGCGA GTTCCAAAGAGCCAGCAAACATTCTTATGGATGATAAGAGGTGATATTGTTTCAGGTGATGCAT GGATTCTrCCACCCGAATTCGTGG.AAGAÀACGAAGA.ACAGAGGTATGCITGeTAGTTGGTGrrC ACAAGAAGAAGTACTrâGTCACCCTGCAATAGTAGGATTCTTGAC-TCACAGTGGATGGAATTCG The ACTCXÍAÀAGTATAAÍ € € ^ AGTGGGCTG CrATGATrTGCTGGÜCATTrTTCGCTGÀACAGCAÀA GAAATTGTrGGTTTTCCGTCACTAAATGGGATGTTGGAATGGAGATTGACAGTGATGTGAAGAG AGATGAAGTGGAAAGCCTTGTAAGGGAATTGATGGTrGGGGGAAAÀGGCAAíAAG4TGxAAGAAÀ N- GGCAATGCAATGGAÁGGAATTGGCTGAAGCÀTCTGCTxWlGAACATTCAGGGTCATCTTATG TCAA ATTGÂÀAAGTTGGTCAATGATATTCTr € € € TTT IATC £ AAACATTAA
SEQ ID NO. 28
Amino Acid Glycosyltransferase (NtGT5b)
Nicotiana tabacum
Petition 870190120782, of 11/21/2019, p. 106/155
105/114
MGS IGAEFTOBAVCI Pl ^ AQG ^ DSFMLKLAEILHHKGFai TR ^ 'TFFKSKKLAKSRGPB.SLKG LSAFRIETI DC £ FPCBABATQ & I TSlXSSTTCTCTGPFRD’LIAKXJt’BTN’TSM ^ WSGI ISDC
VMS FTL.VV4QEL-GyPE ^ TJWTTSACGFLGYX: ffiYT £ ^, sTEKG ¥ AP'IJ £ DA§BLTNGYLT.TTLDFl PC ^ ÍKDyRMSI ^ SFLRTINFaEnS. KnTQETERAEKMAI SJM ^ ^ TLIAE lZSLKKIXPmT IGfLHn. ^ ^ IanWEJi GLRSSX ^ '^ C ^ KEE RilJJTKEThWTSWGS ITVMWfQLffiFAWG LANSQQSH.-W1 XRTDIVSGOAS EJTEnTEIKKRGAH.ASWSQEE SHFAIGGfL.THSG ^ S ^ I TLEs SSGVPMI TFFÀEQQTT% € <C ^ TSSTKRí®V'GW3I3CBVKl®ES 'ESIA ^T REIA £ VGGlíG13a-HÍK KAMEWKSLAEÀS AKESS GS STVMIEKW j®I LL.S SKH
SEQ ID NO. 29 DNA
Glycosyltransferase (NtGT5b)
Nicotiana tabacum
ATGGCTTCCATTGGTGCTGÀATTTACAAAGCXACATGCÀGTrrGCÀTACCATATCCCGCCCAAG GGCATATTAÀCCCCATGTTAAAGGTAGCCAAAATCCTTÜATGACAAAGGCTrTCACATCACTTT TGTCáATACTGÀATTTAACCACAGAGGTCTGCTTÂÀATCTCGTCGCCCTGATTCTCTCAAGGGT CTTTCTTCTrTCCGTTrTGAGACX TTC ^ ^^ ^ TGGACTTCCGCCATGTGATGCAGATGCCAGAC AAGATATACGTTCTTTC GTGAATCTACAACCAATAGTrCXTTGGGTCCTrTTAGGGATCTTCT € TG 'ÂAACTÜAATGATACTÀACA ATCTAA € € € GTG GACC GTTTCGTGCATC'AT € € TCAGATGGT GTCATGÀGCTTGAGCTTAQCCGCTGCACAAGAATTGCGAGTCCCTGAAGTIGTGTTTTGGACeA GTAGTGCTrGTGGTTTCrTTAGGTTACATGCATTATTACAAGGTTATTGAAAAAGGATACGCTCC ACTTAAAGATGCGAGTGACTTGACAAATGGATACCTÀGAGÀCAACAITGGAnTrATACGATGC ATGÀÀÀGAGGTACGTTrAAGGGÁTCTICCAAGTTTCTrGAGÀACTACAÀATCCAGÀTGAATTCA TGATCAÀATTTGTUCTCCAAfâAÁCAGAGÀGAGC' AWAAAGGCTTCTGCAATTATC GTCAACAC ATATGAAACACTÀGAGGCTGAAGTTCTTGAATCGCTCCGAAATCTTCTTCCTCCAGTCTACCCC ATTGGGCCCTT € XATTTrCTACTG4ÀACATCTTGATGÀTGACAATTTC; AAGGGACTTAGATCC, the GCUTTTGGAAAGÀGGÀA 'AGAGTCTATACAÀTGGCTTGATACCÀAAGAACC: AAA .TTCTGTTeT TTATGTTAACTTTQ <^ AACXATTAeTGTTATGACTCCT.AATCAACTTÀTTGAATTTGC: TTGGGGA € TrGCA ^ À € AGC € AAÜA ATCÀTTCTTÀTGGAT ATAAGA € € € CTGATATTGTTT AGCTGATGCAT CGATTCTTCCCCCCGAATTCGTGGAAGAAACGÀAGAAGAGAGGTATGCTTGCTAGTTGGTGTTC ACAACAA & AAGTACTTÂGTCAGGCTGCAATÂGG4CGATTCTTGACTCACÂGTGGATGG.AATTCG ACACTCGAAAGTATAÁGCAGTGGGCTQOCTATCATrTGCTGGCCATTTrTCGCTGAACAGCAAÀ CAAATTGTTGGTTTTCCGTCACTAAATGGGiTGTTGGÀATGGAGATTGACTGTGATGTGAAGAG CGATGAÀGTGGAAAGCrCTTGTAAGGGÀATTGATGGTrGGGGCAAAÀGGCAAÀAAGAIGXAGÀÀA AAGGCÀATGGAATGGÀAGGÀATTGGCTGAÀGCATÜTGCTAAAGAACATTCAGGGTCATCTFATG TGAACATTGÀGAAGGTGGTCAATGATATTCTTCTrrCGTCXAAACATTAA
SEQ ID NO. 30
Amino Acid
UDP-glycosyltransferase 73C3 (NtGT4)
Nicotiana tabacum
Petition 870190120782, of 11/21/2019, p. 107/155
106/114
MATQVHKLIHflL ^ LMATGHMI ΡΜΙΏΙΑΚΕΕΑΝΕ.ΕΛΤΤΏ ΠΤΒΤΜΚ STI TRAI KSCLRI QILlIJi SWA'GIiEGCEKIBXS ^ QQ ^ ¥ £ SLBLASKFFAAI SML E ÍLLEGWS ESCVI SDMGT PWETQIAQNFNI PRBTHGTCCTSLLCSYKI lASNTLOS TSDSEYFWDI TJ ^ ^ ^ BRI'ELTKAQVS GSTKNTTSVSAS VTEQnaAEESSVGVIVNSEEIXE ^ '^ YEKEySKAROKSCVWCVGPVS .NKEIEBLVTRGÍíKTAIBNQBeLKWLSNEETES TASLGSLSRLTELQAnELGLGLEESNREn ^ / ton. GCGma ^ & LEKWHZNG ^ £ QRIKERGV £ J RGWARQVLILSIffAlGGVLTHCC-WSTLEGI S AGLPIWT ^ TirAEQrCNEKL ^ TQTXKIG ^ LGI'K ^ 'T ^' KWCTEEJ ^ 'G ^ XVKKDBVKKKGGQKKKGGQQKKGQQKKGQQKKGG IEQQNHKEK
SEQ ID NO. 31
DNA
UDP-glycosyltransferase 73C3 (NtGT4)
Nicotiana tabacum
ATGCCA4CTCAACTGCACÀÀAGTTCATTTCATACTATTCCCTTT.AATGGCTCCAGGCCACATGA TTCETATGATAGACATAGCTAAACTTCTAGCAAATCGCGGTGTCATTAGCACTATCATCACCAC TCCAGTAA4CGCCAATCGTTTCAGTTCAACA, 4TTACTCGTGCCATAAAATCCGGT € TA4GAATC CAAATTCTTACACTCAAATTTCeAAGTGTAGAAGTAGGATTACCAGAAGCTTGCGAAAATATTC ACATGGTTCCTTCTCTTGACTTGGCTTCAAAGTTTnTGCTGCAATTAGTATGCTGAAACAACA AGTTGAAAATCTCITAGAAGGAATAAATCCAAGTCCAACTTGTGTTATTTCAGATATGGGATTT CCTTGGÀCTACTCAAATTGCACAAAATmAATATÜCCAAGAATTCITTTTCATGGTACTTGTT GTTT-CTCACTTTTATGTTCCTAT.4AAATACTTrCXTCCAACATT € TTGAAAATATAACCTCACA TTCxAGAGTATTTTGTTGTTCCTGATFrACCCGATAGAGTTCAACTAACGAAAGeTCAGGTTTCA GGATCGACGAAAAATACTACTTCTGTTAGTTCTTCTGTATTGAAAGAAGTEACTGAGCAAATCA GATTAGCCGAQGAATCATeATATGGTCTAATTGTTAATAGTmGAGGAGTTGGAGCAAGTGTA TCAGAAAGAATATACaAAAGCTAGAGGGAAAAAAGTTTGGTGTGTrGGTCCTGTTTCTTTGTGT AATAAGGAA4TTGÀAGATTTGGTTÀCÀAGGGGTÂATAAAACTGCAATTGATAÀTCAAGATTGCT TGAÀATGGTTAGATÀATTrTGxAAACÀGAATCTGTGGTTTATGCAAGTCTTGGAAGTTTATÜTCG TTTGACATTATrGCAAATGGTGGAACTTGG-TCTTGGTTTAGAAQAGTCAAATAGGCCTTTTCTA TGGGTATTAGGAGGAGGTGATAÀ ÀTTÀÀATGATTTAGAGAAATGGATTCTTGACAATGGÀTrTG AGC AAAG ^ TTAtóaAAAGAG-GÀGTTTTGATTAGA-GGATGGGCTCCTCAAGTGCTTATACTTTC ACACCCTGCAATTGGTGGAGTATTGACTCATTGCGGATGGAATTCTACATTGGAAGGTATTTCA GCAGCATTACCAATGCTAACATGGGCACTATTTGCTQAGCAATTFTGCAATGAGAAGTTAGTAG
TCGAAGTGCTAAAAATTGGAGTGAGCCTAGGTGTGAAGGTGCCTGTCAAATGGGGAGATGAGGA AAATCTTGGAGTTTEGGTAAAiAAGGATGATGTTAAGAAAGCATTAGACAAAGTAGA
TTGGAGAAGGTGGTTCTTGTTATGTTAACTTAACATCTCTGATTGAAGACATCATTGAGCAACA AAATCAC AAGGAAAAÀTAG
SEQ ID NO. 32
Amino acid glycosyltransferase (NtGTIb) Nicotiana tabacum
Petition 870190120782, of 11/21/2019, p. 108/155
107/114
MOAEl TiyAI ^ ^ ^ MGSLWn VAlLQLV'BKHEQLSrn ^ wati ΡΕΕΤΑΙ PSYTKSLSSDTSSRI TLLPLSQFETSVTMSSFNAIííFEEYI SSTKCSEKDAYSETS ESSgXSVKLÂGF TISSSFCTAMm VA ^ PS ^ EFGl lT ¥ ¥ ^r T.SSAASaGLQLlS QSL ΙΕ € δΡΚΑΉΝΤΛΈΡΕ8Ε ^ ΊΑ bTY5f> P P ^ I LA KCLPGE DESSTMn-WÀKSFSElKGimTaTTELESHÀLKALSBOEKl ΪΤΓΏΛ GPIL.YLENGNED WQElTJÀanaRXBEKF ^ ^ & SWFLCTGSKGSFEEDQllíESÀNALESSGYHH iLKRPPPKSKLQ:. rT: 8EFEisFEEVLFE®FTQKTKGKG £ £ F3GWAPQ AIL: ^{SKF§VGGFVSaCG1S:} The NSFl.E5yKSGW3A TK7L ¥ £ ^ QQS AFQL11O> LGMA TIKMI ^> K ¥ ^> f TRWI <LVKAEEIEI>GIRKLMI> SENKIKAKV TEMIA> K ^ AAE £ ECC§SWALGHFVETVMKN
SEQ ID NO. 33
DNA
Glycosyltransferase (NtGTIb)
Nicotiana tabacum
ATCAAGACAGCAGAGTTAGTATTCATTCCTGCTCCTGGGATGGGTCACGTEGTACCAACTGTGG AGGTGGCAAAGCAAC / T TAGTCGACAGACACGAGCAGCMTTCGATCACAGTieTAATCATGACAAT TrTGGAAACAAATATTCCATCATATACTAAATCA €€ € € TGTC TCAGACTACAGTTCTCGTATA ACGeTGCTTCCACTCTCTCAACCTGAGACCTCrCTrACTATCAGCAGTTTrAATGCCATeAATr TTTTTGAGTACATCTOCAGCTACAAGGGTCGTGTCAAAGATGCTGTTAGTCAAACCTCCTTTAG TTCGTCAAATTCTGTGAAACTTGCÁGGATTTGTAATAGACATGTTCTGCACTGCGATGÁTTGÁT GTÁGCGÀÀCGAGTrTGGAATGGGÀAGTTATGTGTrCTACAGTTCTAGTGCAGCTATGCTTGGÀC TACAACTGCATTrTCAAAGTCTTÀGCATTGAATGCAGTCeGAAAGTTCATAACrACGTFGAACC TGAATCAGAAGTTGTGATGTCAAGTIACATGAATCXGGTTCCAGTCAAATGTTTGCCCGGAATT ATACTAGTAAATGATGÀÀAGTAGCACCATGTTTGTCÁATCATGCACGxAAGATTGAGGGAGACGA AAGGAATrATGCTCÀAQACGTTQACTGAGGTrGAÀTCACAGGCTTTGAAAGCCCTTTCCGATGA TGA4AÀAAFCCCACCAATCTACCCAGTTGGÀCCTATAC: & TTAACCTTGAAAATGGGAAFGAAGAT CACAATCAAGAATATGAT l ^ ^ GATTAFGAÀGTGGCTTGACGAGAÀGCCTAATTCATCAGTGGTGT TCTTAIGCTTTGGAAGCAAGGGGTCTTTCGAAGAAGATCAGGTCÀAGGAAÁTAGCAAATGCTCT AC GAGCAGTGGCTACCACTrCTTGTGGTCGeTAAGCCGACCGCGACCAAAAGACAAGCTÀGAA TTCGCAAGCGAATTeGAGAAT CGAGAGGAAGTCTTACCAQAGGGATTCTTTCAAAGGACTÀAAG GÀAGAGGAAAGGTGATAGGATGGGCÀCCCCAGTTGGCTATTTTGTCTCATCCTTCAGTAGGAGG ATTCGTCTCGCÀTTCTGGGTGGAATTCAACTCTGGAGAGCGTTCGAAGTGGAGTGCCGATAGCA ACATGGCCATTGTÁTCCAGAGCAACAGAG € AATGCATTTCÀA € TGGTG4AGGATTTGGGTATGG GAGTAGAGATrAAGATGGATTÀGÀGGGAÀGATTTrAATACGACAAATCQACCACTGGTTAAAGC TGAGGAGATAGAAGATGGAATTAGGAÀGCTGATGGATTCAGAGAATÀÂÀATCÀGCGCTAAGGTG
ACGGAGÁTGAAGGACAAAAGTÁGA <^: AGCAOTGeTGOAGGG € GGATCAICATATGTAGCTCTrG GGCATTTTGTTGACACTGTCATGAAAATA
SEQ ID NO. 34
Amino acid glycosyltransferase (NtGTIa) Nicotiana tabacum
Petition 870190120782, of 11/21/2019, p. 109/155
108/114
ΜΚΠΕΙΛΤΓ PAPCMGHLATTl ^ AKQL ^ DRDEQLSmOMTLFLETNI PSVIKSLSSBYSS®!
'TLLQEAQPETSVSXISSF AINFFEEI S8 ^ ¥ K & K ri ⁷ »The ^^ TFSSgSSVKLKGFVSaiF tar £ € ^ FFIV ANEFGI PSWF SNAÀMLGLQLHFQSO lETSFKVEEíYLDFEKEVAI STV1KEI FVKCLPCI X ^ LD K§GTMF 7®AKRFKET ¥ ^ min ^ ^ TFAELESHALKALS & SEKI PPHWGPUJaGDG ESH SQEYSMlMKWLSE IHSS ^ ^ ^ TLCFGSKGSFEESQ' KE L ^ UZESCNSTLWSLRRPPPKBILQF, FSElTOTEE ¥ XWGEFQRTKGgGA ^ GA ¥ APQLAI L8HFAA'GGn. S.HCG ^ STLE8 ¥ 'R8GVHAT WPEVAEQQSW.O'QL ^ lOiLGMAXTIOffilliiESFNKEhTSLAlL ^ EEEEaQIRKLMBgENKIEAK ^' Ai EXEKBKSRAÂEEEGG8 ALGHEVETVMKN
SEQ ID NO. 35
DNA
Glycosyltransferase (NtGTIa)
Nicotiana tabacum
ATGAAGACAACAGAGTTAGTATTCATTCCTGCECCTGGCATGGGTCAGCTTGIAC-CCACTGTGG ÀGGEGGCAAAGCAACTAGTCGACA £ yiGACGAACAGC: TTT AAT € € ACAGTTCTCÀTCÀTGACGCT TCemGGAAACAAATÀTTGCÀTCATATACTAAATCACTGTCCTCAGACTACAGTTCTCGTATA ACGCTGCTTCAACTrTCTCÁAC "TGAGA € 'CTCTGTrAGTATGÀGC.'AGTTTTAATCCCATCAATT TrmGAGTACATCTeCAGCTACAAGGATCGTGTCAAAGAEGCTGTTAATGAAAECTTTAGTTe GTCAAGTTGTCTGAAACTCAAAGGATTTCTAÀTAGACATGTTCTGCACXGCGATGATTCATGTG GCGAACCiAGTTTGGAATCCCAAGTTATGTC / ETC.TACACTTCTAATGCAGCTATGCTEGGAeTCC AACTCCATTTTQAAAGTCTrACTÀTTGAATACAGTCCGÁAAGTTCATAATTACCTAGÀCCCTGA ATCAGAACTÀGCGATGTCAACTTACAETAATCCGATTCCAGTCAAATGTTTGCCCGGGATTATA CTAGACAATGÀTAAAACTGGCACCÀTGTTCGTCAATCATCXACGAAGATTCAGG
GAGACGxAAAGGAATTATGGTCAÀCACATTCGCTGACCXrTGAATCÀCACGCTTTGAAAGCCCTTr CCGATGÀTGAGAAAATCCGAC CAATCTAC CCAGTTGGGCCTÀTACTTAAC CTTGGAGATGGGAA. TGAAGATCACAATCAAGAATATGATATGATTATGAAGTGCCTCGACGAGCACCCTCATTCATCA GEGGTGTTCCTATGCTTTGGAAGCAAGGGATCTTTCGÀAGAAGAECAAGTGAÀGGAÀATAQCAA ATGCT ^€: TAGAGAGAAGTGG ¥ AACGGGTTE3TCTGGTCGC; TAAGACGACCGCCÂC € AAAAGACAe GCTACAATTC << AAGCGAATT € GAGAATC € AGACGAAGTCTTGCCGGTGGGAITCTTTCAÀAGG ACTÀAAGCAÀGACGAAACGTGÀTAGGATGGCCACCCCÁGTTGQCTÁTTTTGTeTCÁTCÜTGCAG TAGGAGGÀTE GTGTCGCATTGTQGGTGGAArrCAACTTTGG.AGAG.TGTT € € € GTAGTGGAGTACC GATAGCAA 'ATGGCCATTGTATG € ACAGCAACAGACCAATGCATrTCAACTCGTGAAGGATTTG GGGATGGCAGTQGAGATTXAGATCGATTAGAGGGAA.GATTTTAATAAGAC.AÀATCCACCACTGG ^{Tr.AAAGCTGAGGAGATAGAAGATGGÂATrAGGÀAGCTGATGGATTC: AGAGAATAAAATCAGGGC:} TÀÀGGTGATGGÀGÀTGÀAGGACAAÂAGTÀGAGCAGCGTTAITAGAAGGCGGATCATCATATGTÂ GCECTCGGGCATTTTGTTGAGACTGECATGÁAÀÀACTAA
SEQ ID NO. 36
Amino acid glycosyltransferase (NtGT3) Nicotiana tabacum
Petition 870190120782, of 11/21/2019, p. 110/155
11/114
MKEIKKSELVTI PGSOELVST K ^ l ^^ IQ TMAKLLIAREEQIA ITVLI> OXBSllQSV.tòTS & RLSJ3KLFQI® · »^ I HÍQLLKS FITHASHSSAVRSÁVÀBI LKSES ^ ^ TLAGJVTOIi'CTSStHBV .Α ΕΓΕ 'ΤΎ ^' ^^ ΓνΤ8βΑ7ΜΧίΕΗΪΉΙΟ ΣΤΝ1ΦΙΤΚΏίΙΪΣΤΈΕΏχ5 AU lATVUSITPAKCLFSV> KEGG & WnJ KRfKETK ^ ^^ ^ n LEÍZS ¥ ALNSI ^ KB13aJf I1TVG> VO LN ^ TGB »€« ^ 0ΚΤΜ, »1.ΒΒ0ΡΑϊ5 · νθΈ € Ε« § € Ο®ΙΈΚΗί2ΐ'ΚΣΙΑ ¥ ΑΕΕ§ SG € ®JXW: £ Lí®PPTEDÀK
FPSNYZXLEEILJEGri.ERIKGÍGKVtGWAPQLAIL5HKgTGGFA’SHCGW <STLESTYFGVFIÁ
T% l ^ AE <^ ÀNAFQL ¥ Kl »^ £ GVSKMm'EKBMKn _: iGKEA ^; n ^: KÀEEiEKA £ lEaíBSESEÍRl'K VKEi £ KSKSSÂA ^ fEGGSS ¥ T®IGGF £ Ql B £ ENSQ
SEQ ID NO. 37
DNA
Glycosyltransferase (NtGT3)
Nicotiana tabacum
ATGAAÁGAAACCÀÀGÀÀÀÀTAGAGTTAGTCTTCATTCGTTCACCAGGAATTGGCCATTTAGTÀT CCACAGTTGAAATGGCAAAGCTTCTTATAGCTAGÀGAAGAGCAGCTATCTATCACAGTCÍCTCAT CATCCAATGGCCTAACGAClAAGAAGCTCGATTCTTATATCCAATCAGTCGCCAATTTCAGCTCG CGTnG.AAATTCATTGGACTCCCTCAGGATGATrCCATTÀTGCAGCTAGTCAAÁÀGCÀÀCÀTTT TCACCACGTTTATTGCCAGTGATAAGCCTGCAGTrAGÀGATGCTGTTGCTGATATFeTCAAGTO AGAATCAÀATAATACGCTIAGCAGGTATTGTTATCGACTTGTTCTGCACCTCÀATGATAGACGTG GCCAATGA € TTC'CAGCTACCAACCTATCTTTTCTACACGTCTGGTGCAG € AACCÜTTGCTCTTC ATTATCATAT A "Ac ÀATCTCAGGGÀTGAATTTÀACAAAGATATTACCAAGTACAAÁGÀC GAACC TGÃÁGAAÁ AC TCTCTATÀCCAACATATCTCA4TCCATrT AGCAAÀATGTTTG € €€ C GTCTGTÁ GCCTTAGÀv.A4Av.AAGGTGCFreÁACÂATGTTTC: TTGATCTCG € AAÀÀÀGGTTTCGAGÀAACCA AAGGTATTÀTGATAAACACATrTCTAGAGCTCGAATeeTATGGATTAAACTCCCTCTCAeGAGA CAAGAATCTTCCACeTATATACCCTGTCGGACeAGTATTGAACCTTAACAATGTTGAAGGTGAC AACTTAGGTTCÁTCTGÀCCAGAATACTATGAAATGGTTAGATGATCAGCCCGCTTCÁTCTGTAG TGTTCCTTTGTrTTGGTAGTGGTGGAAGCTITGAAAAACATCAAGTTAAGGAAATAGCCTATGC TCTGGAGAGCAGTGGGTGTCGGTTTTTGTGGTCGITAAGGCGACCACCÁACCGAAGATGCAAGA TTTCCAAGe AACTATGAAAATCTTGAAGAAATrTTGeeACAAGGATTCTTGGAAAGAAeAAAAG GGATTGGAAAAGTGATAGGATCGGOÁeGTCAGTTGGCGATTrrGTGACATÀAÀTCGACGOGGGG ATTTGTGTCGCACIGTGGATGGÁATTCGACTTIGGAAAGTAGATATTWGGÀGTGCGAATAGCA AeeTGGCCAATGTAeGCGGAGCAACÂÂGCGAATGCATrrCÂÂTTGGTTAAGGATTTGAGAATGG GAGTrGAGATrAAGATGGATTATAGGAAGGATATGAAAGTGATGGGGAAAGAAGTTÁTAGTGAA AGCTGAGGÁGATTGAGAAÀGCÀÀTÀÂGAGAÁATTATGGATTCGGAGAGTGAAATTCGGGTGAAG GTaAAAGACATGAÀGGAGÀÀGAGOAGAGCAGCACAAATGGÀAGGTGGOTCTTCrTACACTTCTÀ TTGGAGGTTTCATCCAÁÁTTATCATGGAGAATECTGAÀTAA
SEQ ID NO. 38
Amino acid glycosyltransferase (NtGT2) Nicotiana tabacum
Petition 870190120782, of 11/21/2019, p. 111/155
110/114 .m'QEm LYTETAQGH ^ SC & TCLQFAKRLISSiGI EVTFATSTyAHRRJkiAKTTTSTLSKGLíSrÀAFS ¥ © € D TSABEHDSQHYMSEÍ KSKGSKTLKSI 'II ^ S ^ S & GRFiTSLA IXI ^ ^ ^ AAK' AKEfHl CALLWlQFATVLfíiy WNG ¥¥ ¥ £ »^ LF ^ al KCSl VCIQLEBLfLIJCSQBLPSFLLSSSNE SFALÍITKEQIJilWVEE ^ y ^ ^ SXX TFBAIZPKELKAI EKL ^ ^^ XIGiGFU PSTTLBGKBPLSSS FGGS1FQKSXSYI EWLNSEAKSS SFGSLEÍ SKNQEEEÍAKGLI E1 ^ & KOFLST.TR QENC KGBEKEEKISC € ^ SIEKQGKRTWCSQLEVLIHPS Ig: TDi n'8HCGWNSTLESESSGySWÀFPSW TXAKLXEDVWKTG ^ ^ '^ RLKKSEDG SSEE JERCI EIA-IÜDGCOlC EMRRK ^ ^ ^ QKWKELAREA XEGGSSE ^ SÍLEAFVQEVGSGC
SEQ ID NO. 39
DNA
Glycosyltransferase (NtGT2)
Nicotiana tabacum
ATGGTGCAACCCCATGTCCTeTTGGTGACTrTTCGAGCACAAGGCCATATTAATCCATGTCTCC AATTTGCXAAGAGGCTAATTAGAATGGGCATTGAGGTAACTTTT'GCCACGAGCGTTTTCCCCCA TCGTCXjTATGG £: AAAAACTAEGÁ TTOOACTCTATC £ £ € AÀGGG TTAAATTTTGOGGCÀTTCTüT GATGGGTAGGACCÁTGCTTTCAAGGCCCATGÁGCAECATTGTCAACATTACATGTCGGAGATAA AAAGTCGCGGTTCTAAAACCX ÀAÀÀGATATCATrTTGAAGAGCTGAGACGAGCGACGTCC ^ / EGT GACATCCCTCCTCTATTCTCTTTTGCTTCCATGGG € TGC.4ÀAGGTAGCGCGTCAATTTCACA.TA CCGTGCGCiGTTACTÀTGGATTCÀÀCeiGCAACTGTGCTAGÀCATÀTÀTTATTATTAGTTCJLATG GCTATGAGGATGCCATAAiAGGTAGCACCAATGATCCAiATTGGTGTATTeAATTGCCTAGGCT TCCACTACT.4.4AAAGCXAAGATCTT CTrCTTTTTTACTTTC © / TTCTAGTAATGAACAÀAAATAT ÂGCTITGCTCTACGAAGArrTAAAGÂGCÀÀCTTGÀCACATTAGATGTTGAAGÀÀÀATCCTAAAG TACTTGTQAACÂGATTrGATGOATTAGAGCGAAAGQÁÀGTCAAAGCTATTGAAÀAGTACXATTT AATrGGGATTGGAeCATTGATTCCTTCAACATTTTTGGAeGGAAAAGACCCTTTGGATTCTTCC TTTGGTGGTGATCTTTrTCAAAAGTCTAATGACTATÁTTGAATGGTTGAACTCAÀAGGCTAACT CATCTGTGGTTTATATCTGArETGCGAGTCTCTTGAATTTGTCÀÀÀÀÀATCÀAÀAGGAGGAGÁT TCCAAA.4GGGTTGATAGÀGATTA.4AA4GC: CATTCErGTGGGTAATAAGAGATeAAGAAAATGGT AAGGGAGATGA .AAÂAGA € ^ GAGAAATTA4GTTGTATGATGGAGTTGGAAAAGCAAGGGAAAATAG TACCATGGTGTTCÁCAACTTCAAGT TrAACACATCCATCT.4TAGGATGTITCGTGTCACATTG IGGATGGAATTCCaCTC TGGAAAGTTTÀE € GTCAGGCGTGTCÀCTÀGTGGCÀTTTCCTCA-No GG ACGGATCÀÂGGOAlAAATGCTAÀACTÀÂTTGXAGÀTGTTTGGAACÍACAGGTGTAAGGTTGAAÀÂ AGAAIGAAGATGGTGTGGTTGAaAGTGAAGAGATAAAAAGGTGCATAGAAATGGTAAEGGATGG TGGÂGAGÀA4GGAGAAG.A4ÂTGÀGAAGÀAATGCEG.A4ÂÂÂTGGAAAGAATTGGCAWGGAÂGCT GTAAAAGAAGGCGGATCTTCGG.A4ATGAATCTAAA4GCTrFTGTTCÂAGAAGTTCG £ € L TGA AAAGGTT
SEQ ID NO. 40
Amino Acid
THCA Synthase Trichome target domain
Cannabis
NÍNCSÀFSFWWCKI I FFFLSFHI QI $ I. A
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SEQ ID NO. 41
Amino Acid
Target domain of CBDA Synthase Trichome
Cannabis
WCCSTFS WFVCK1IFFFFS FNIQTSIA
SEQ ID NO. 42
THCA Amino Acid Synthase Cannabis
SSTIC ^ RPISGTXProitVIVTPSWSKI ^ TILCSKKVG-LQIRTRSG »'i 7„ V l ^ RNYGLÃADSÍ IDARLWVSGKVT-DRKS ^ OSSLWAIRGCXsGRWGI IA & OTKLTOWEEST
IFgVEW ¥ ^ SIHGLVKLFfJKKQK ΙΑ Κ5®Κ2Ε.ν 'ΓΗΕ ITOITDRHGKNOTVRGYFSgX PEGG VDSLVDEiynSTKSFPELGIO'riXORS IIFFriRFSGVXRPlS'TANFKra.I ^ ^ ^ DRSAGOl'APSTK I.DVVKKPIFSTMVKILEKE VEEIjVQAG ryLVpyGaiKggTgE.gAXPFPHgAGTMYEl ^ ^ ^ E ^ YZAS EDJfEFHXWVRSVYWTTFWSgKPRLAYLRYRDW-LGCTâRÃSPMYT-Q & RIWEKYPG OFTOEVOKTKTOP Σ ^ PPEEEQS PPLPPERR
SEQ ID NO. 43
Amino Acid
MYB8 - orthologist for CAN738
Humulus lupulus
MGR & PCGEOGUGCGRm G '''' I ^'5' ¥ I QSNGBSCWESLE35HÃGLLR CGKECRLEW I EYlsRMJ LKROTISSEREDIlXOsHS ♦ kW XASHLFGRTSNEIKSTWSiiLSRSSIHTFSPCFKTTTH WHS.FELVTVTKWL PlPKh RLÃMKKSKS STSI ^ SSVXKNDVGSSSSTTTTSVHQRT
TTTTPT TDQQÍSQLSRCRi ^ ^ tte ^. ^ ^ N Q & gTGTÍ KLGQ ^^. ^ ^ N VGSgCDSiD ICÍQLSFLCCg TTEES E ^ ^ ^ RMJ EGDKEVSGPYXJTDHEYEKETSySE ^ ^ R.CPEGIIDSKLLEPEEVLTLSEE SLNLGGÁLAíDTTTSTTTXSS STYaSYiSíssNGDCVlSDDHDCpVaiJDVyGyDFWSWESSTrVTaEQ
SEQ ID NO. 44
AtMYB12 amino acid - ortholog for CAN739 Arabidopsis thaliana
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.iDíjíjLíJ ^ SíJI-OIíLí 17'0'1.'4 UjG-IJG
ΒΙ ^ ΝΧΚδ8Ε®ΜίβΒδΙ5Τ «® ^ ®Ι © ϋΣ ₁ > Ι ^ 8ΟΡ8νΕ8ΪΙ ₁ ΗΧΒ · ^ 3νϊ © 1 ^ Ώ58Ρ333« η € ν »^ Ε®8Σ ^
EtWHBEENPDSMVSMIJiDGnSSATXSWNCENFíSEP & SHDDESALVÃÍIIIIE
SEQ ID NO. 45
Amino Acid
MYB112 - orthologist for CAN833
Arabidopsis thaliana miSRTEFJtíiCKTLXl®l £ EEV ^ Vein ^ IEIRRSPWTVEEDMKLVSYXSIá®SES8H »Sí ₁ SRSJl
03Ν8Χ0 £: 8ϋ81Ε & ΚΤ & ΕΡΟΙ520ΕΪ812Ε2ΕΣΣ3Ε3Ή38 »335Μ8ΚΧΑ2Ε3Ρ05Τ3ΚΕΧ2Ν ™
8Τ5να £ ΗΑΚ.11 <εϋνΚ · ΒΚ28Κ3ΤΣΚΗ.ΙΛΡδ.1ΙΕ8ΙΑΑΤ2δ ¥ 32Τ8ΝΗΥ8ΡΕΚ88νΑΤΑΤ88Τ8
88ΕΑ ^ .838Τ003α ^ ΡδΓ3Ο »ΊΝ <ϊ3Μ! 23 © 33Τδ’ΕΤ108Ε3Β2Ν57 113
SEQ ID NO. 46
Amino Acid
Cytosolic target THCA synthase (ctTHCAs)
Cannabis
HP "® ^ 2> XCFSKSIP» ^ SVaMPKLVTS ®QI TÍ®XI _I 'TXSSl®FXEDTTPlLPL ^ ¥ ^ IVTP ®SK IgATlLCSKO'SLQIBTRSiSiSgDAEMSTISQVPFWiDLSMHSSXIBVSSOTAmfSfeSATLG ®mw $ 8 SHWBS FPGGyCFTVSVSGS FSOSSYOALMSS WIMEI XDL ^ Ε S3E VKVDGKVIi ©! ^ ^ ΕΟΙ ΑΣΚΟ®3 ^ »ΡσΐΙΑΑ» ΕΣΕ & 5ΠΜΓΡδ ^ ΤΙ 8νΚΧ ^ ®Σ®δΣ.νΕΙ ₁ Β8Κ »Ι2ΝΕ & ΥΚΧδ ΧΧ> Σ.νΐ _ί « ΪΉΡΧ ^ ΗΧΤΕ®ΒΙ3® ^ Τ ™ 3 ¥ Ρ33ΣΡΒδδνΒ3Σ _> νΣ> Ι3®Εδ »3T> ILLDE8 AGKK.TAES IKWÍVXXS ¹ IPETMWKX 8EE1TEEDV0AS Μϊν3ϊ'Ρϊ00ΙΜΕΕΙ3Ε3ΑΙΡ88Η8.ΑδΙΜΪΒ1 »ΥΤΑ8 ^ ΈΕαΕ33522ΗΙϊ0Κ Β.8 ¥¥» ΤΤΡ ¥¥ 82.Ν PRÍAYSKY ^ IiKSKY ^ IIKES
SEQ ID NO. 47
Amino Acid
Trichome target catalase with THCA synthase trichome target domain
Arabidopsis thaliana
Petition 870190120782, of 11/21/2019, p. 114/155
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WCSAFSWFVCKIIFFFLDFaZ ^ IEXAWPYKYP.FASS ^ SPFFTTS'SGAPWWNDSKTVSP
S.OÍ.ZLl> ®5ÍHI «VBKSASFSR2SZF ^ .WK & RSasaKSFF8VTSDISSÍi: TCM ^ LRXPGygTFFX VPFS TVXS & FSS SET J3® FRSFAVRF YTREGSFDL V9HF PVFFISEGSKF PD IVδΤΡΣΤΡΣPlPl® ®68δΠ®ΤΧΜΧ »ΣΗΟ.0Ο2Τ« ΧΡ®ίΚΡ TSGVKSLLEEn & ZRLGGTW.SSÃTQDLZDSIAAGtTXPE ^ D.FZQXXDPADEDKFDFDPLDYTKT ^ ΕΕΙΙίΡΙ ^ Ρν ^ ΚΜνΕ ^ ΝΙΒΪΐΡΡΑΕΒΕδΣΛΡ ^ ΤΚ ϊϊΣ VN ¥ WSR ¥ DQWHÃEKyPTPPA ¥ CSGOSSC ΙΙΕΚΒ »ΡΚΕ ®ΕβΥβΤΈΤΡ®Κ0ΕΡΡΣ0Ρ» ΙϋΑΕ _ί §ΞΡΡΙΤΙ®: ΣΡ§ϊ «! Ι8 ™ 8> 3Α33Κ8Σ _! ® · 2ΧΈΑΒ S & WRPSI
SEQ ID NO. 48
Amino Acid
Catalase Trichome target with CBDA Synthase trichome target domain
Arabidopsis thaliana
MCCSTFSFSFVCSlIXFFFFSFKZaTSIMaDPYKTRFASSXKSPFFTX ^ ÍSGaPVSKSBÍS ^ fWSP RSfcSZ & BDXHLVSKLaSFDSEaiPERWSM ^ SASSFFSVTWXSF_SFSFP_SFSFP_SFSFJSJSJSJSJSJSJSJSJJSJSJSJSJSJSJSJSJSJSJSJSJJJSJSJSJSJSJSJSJSJSJSJSJSJSJSJSJSJSJSJSJSJSJSJSJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ
HIQSS5®XSDFFSH ^ SSS ^ FTFLFE © ISXP®PX8ffiS3GSSWTXMI> X ^ »GiaHXVKFS5®P
ΧΟ νΧδΖ.ΏΚΕΠΑΙΡΖβδΤΝδ ^ ^ Ε ^ & .Τ ΖΪΒδΣδΛαΝΪΡΣΜ.ΏΡΙβΙΣϋΡΆ ΜϋΚΡϋΡΩΡΜίνΤΚΤ ^ _{'ΡΚ33ΧΧΡΕ ί 2Ρ} <ΪΕΜΏ3ΪΚΪ ΣΪ ΡΡΑΕΚΕ ^ ¥ ^ Ι ₁ ΑΡαΡΑΙΣ Ρ®ΧΗΤ5 & _{Ι0Κ ί Σ ί} ΟΤΚνΡ§'Σ3'> ₁ Τ2ΕΚΚΣ
GPK'fXQLPraÃ.FXCAHBKNHHE®FmMgPDSSV ^r N ¥ EPSaZBgZTO6.EKTPTPPAZ ^ SGKRE8C
II ^ EWFS ^ t ^ YETFTPERSSR ^ ISWXDÃLSSFEITSSISSIWISWSQÃDÍKS & G ^ KLaS
RítôVPPSl
SEQ ID NO. 49
Amino Acid
HPII Catalase (KatE) with THCA Synthase Trichome target domain
Escherichia coli
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WCSAFS IWTCH IFFFLg PEI £ IS
SPAAEPTPPtSAQFTAFQSLKAPDTRKEKIjNSLEDYEKGSWEAXTT ^ QGVKIÃDD ^ SLSAGSE ®ΡΤ'ΧΧΕΕΕΣΣ _ί ®ΕΚΙΤΗΡΏΗΕβΣΡΕΕΣνΗΑΕΘ8ΑΑ2σΥΡ0ΡΪΚ®Ε§βΙΤΚΑβΡΙ ₁ 8ΒΡδΚΙΤΡνΕ7
ΕΕ3ΤναΘ®Α © ®ΑΓΤνΡ »ΣΡαΡΑΤΕΕΥΤΕΕ8ΧΕϋΕνδΜΤΡΙΕΕΙΏ · 0.ΑΗ ^ ΡΡΠΡνΉΑνΕΡΕΡΙ« Α
ISÍMlSA ^ WmyySLíSFXTI ^ VMWMfâSRGXF ^ YRraBSFGIHTFSIi ^ a ^ KATSWFH EEPXtAGKA8 & V $ E ^ AQ3 ^ tT @ RDPDF8RR £ LWEAXSAQK> FPEYEZtC ^^ LXPE ^ EPKPDFDLLX ^ ISaiP ^ NIR ^ WSKMVXJmtPDSFFASÍgÇl & aS ^^ JVPGlSFTSDSLl ^ RÍ.FSYTDTSI S® & GGFKFMEXPIHRPTCF ¥ HNFaSS5GMH5aegim ^ PMnrSP ' SXfiB3SWPRSTPPGFO.GG »8S
ΥαΕΕνΕΏΚΚνΕΕΕΕΕ ^ ΕδΕΥΥδΕΡΡΧΡ ^ ΕδδΤΪΤΕδΧΗΐνΒΏΕΡΡΕΧΕΟ ^ ΡΥΤΕΕΡΜί ^ ΕΑ HWLTI _t à ^ VM! SLGI ^ TDD2I ^ T> PI ^ nnSGLJ [KD SLS £ TAXPDS5 ¥ KGRWÍKXLMroiEVR.
SWLIAXLgALKAKS ¥ KAKLL ¥ S5aSKSVTMS3CTVI ₁ PSAA.TFAGftS'SLT ¥ DAVXVPCSKXÀDIA ΕΕΘΟΜηΠΓΣ38Ι »1ΚΗΣ ^ ΧΜΑαΐ» ^ ΚΡ8 »ΤΣΚΣΑΒ0αΕΒδΧνΕΑ» 3Ι ^ 8ΡΙΦΕ && Τί «ΑΑΗΒ
WSSIPEIDKXFA
SEQ ID NO. 50
Amino Acid
Catalase HPII (KatE) with CBDA Synthase Trichome target domain
Escherichia coli
Ι ^ ΧΡΤΡΕΕΪ ^ ΓϋΚΧΧΡΡΕΕΕΕϊη ^ δΣΑίΚςϊΒ ^ ΚΡΗΜδΡΧάϊΠδΕΒΜΕΡδϊΕίδΧΛΡΕββδϊϊ
ΕΡ ^: ΑΑΕΡΤΡΡβΑ ^ Ρ ^ ΑΡβΕβΚΑΡΠΤΚΪ ΕΚΕϊ ΕΕ® ^ Κ3®Ε ^ 'ΑΕΤΤΝς «νΕΣΜ> · δ ^ ίϊ®Ι _£ £ ΑδΡ®« ΡΤΖΖΕΒΕΙΣΕΕΕΣΤΚ ^ βΗΕΡΙΪΈΕΙΥΪ ^ ΕΐΕΕΑΑΗδΥ ^ ΡΥΚΕΕΕϋΣΤΚΑϋΡΕΕΕΡΝΕΣΤΡνΕν ΡΕ8Τνςΐ®®Αδ®ΑβΤΛ ΕβΧΕ®ΡΆΤΚΕ ¥ ΤΕΕ3: ΣΕΕΕ _ί ν®ϊ3®Τ'ΡΣΕΕΣ2ΠΑΪ3: ΡΡβΕνΉΑ ¥ Κ: ΡΕΡΗ · ».
I Re ^ SD®DTR® ¥ VSXígPETSS8Vl®®a ^ DRGI PES ¥ ΕΤΜΕ @ ΕΏΙΗΤΡΡΕΣΙ · ΑΕβΕ & ΤΕνΡΕΕ
WKPLÃGU ^: LV «Dia.QKXi.TSRD5ÍDSSRgEI.W ^ XEÃGDF G ¥ E & ® ^ 2I» XPEBDSSXFDFBI.WP
ΤΚΕΣΡΕΕΧίΥΡν ^ ΕναΚΚ’ίΣΜΝΡΒΝΕΕΑΕϊίΕςΑΑΕΗΡ ^ ΗΙΥΡβΕηΕΤΚδΡΕΕΟΕΕΕΕδΥΤβΤ ^ Σ
ΕΕΕΟ © Ρ8Ρ®ΕΣΡΙΝΕ.ΡΤ £! Ρ'Ώ0ίΡαΕΕ ·> 8®ΕΕΜΘΣβΤΪ® ^! ΑΜΕΡ »ΕΙϊ®Ν5ίΡΕΕΤΡΡ'3βΚΕί3 <3ΕΕΕ
Y ^ EP.VES ^ VSERSPSFGEyySHPREWL.SgTPFSQEHXWQESFELSKWSPYZKEP ^ SgLA
ΗΧβΣ _ι ΤΣΛ®ινΑΚΚΙ _ί ®ΧΕΕ> ΤϋδδΣ®ΣΤΡΡΡθνΚ «Ι _Ι ΕΚΣ Ρ313Ι _ί ΥΑΙΡ © ®Σ3ί ¥ Κ® Ϊ ΑΣΣ» £ ^ Εν®
SADLLAXLKM, KAKG ^ rEAEX <LYS: R ^ GEVTÃDE> GTVLPIAÂTFAGA.PSLTVI3A ¥ XVFe®riADIÃ
Σ2ί ^ 3ΑΗΥΧΣ ^ Α.ΥΚ »Σ« ΡΧΑΙΑ®ϊ & ΚΧΕΚΑΤΧΚΧΛΒ®®ΕΕ3Σν ^ Β3ΑΙΚ3δ ^ ®2Χ> Χ> Τ ^ ϊΑ «Η8 VSÍEKXPKXDKXPA

权利要求:
Claims (160)
[1]
1. Intensified in vivo method for the production of high-level water-soluble cannabinoids in a Cannabis suspension cell culture, characterized by the fact that it comprises the steps of:
- expressing in a genetically modified Cannabis cell a nucleotide sequence encoding a heterologous cytochrome P450;
- expressing in a genetically modified Cannabis cell a nucleotide sequence encoding a heterologous P450 oxidoreductase;
- expressing a nucleotide sequence encoding a glycosyltransferase in a genetically modified Cannabis cell;
- expressing in a genetically modified Cannabis cell a nucleotide sequence encoding a heterologous ABC transporter;
- expressing in a genetically modified Cannabis cell a nucleotide sequence encoding a myb transcription factor; and
- express in a genetically modified Cannabis cell a nucleotide sequence encoding a heterologous catalase.
[2]
2. Method according to claim 1, characterized in that said culture of Cannabis suspension cells comprises a culture of Cannabis sativa suspension cells.
[3]
Method according to claim 1, characterized in that said heterologous cytochrome P450 hydroxyls a cannabinoid to form a hydroxylated cannabinoid and / or oxidizes a hydroxylated cannabinoid to form a cannabinoid carboxylic acid.
[4]
4. Method according to claim 3, characterized by the fact that said heterologous cytochrome P450 is identified as SEQ ID NO. 1, or a sequence at least 80% identical to SEQ ID NO. 1
[5]
5. Method, according to claim 4, characterized by the fact that said heterologous P450 oxidoreductase facilitates the transfer of
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2/27 electrons of a nicotinamide adenine dinucleotide phosphate (NADPH) for said cytochrome P450.
[6]
6. Method according to claim 5, characterized by the fact that said heterologous P450 oxidoreductase is identified as SEQ ID NO. 3, or a sequence at least 80% identical to SEQ ID NO. 3.
[7]
Method according to claim 6, characterized by the fetus of which said glycosyltransferase is a heterologous glycosyltransferase.
[8]
8. Method according to claim 7, characterized by the fact that said heterologous glycosyltransferase is identified as SEQ ID NO. 7, or a sequence at least 80% identical to SEQ ID NO. 7.
[9]
9. Method according to claim 7, characterized by the fact that said heterologous glycosyltransferase is a glycosyltransferase of Nicotiana tabacum or Nicotiana benthamiana.
[10]
10. Method according to claim 9, characterized by the fact that said Nicotiana tabacum glycosyltransferase is selected from the group consisting of: SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39, or a sequence at least 80% identical to any of the listed sequences or a homologous sequence in Nicotiana benthamiana.
[11]
Method according to claim 1, characterized by the fetus of which said heterologous ABC carrier is identified as SEQ ID NO. 9, or a sequence at least 80% identical to SEQ ID NO. 9.
[12]
12. Method, according to claim 1, characterized by the fact that the said myb transcription factor is an endogenous myb12 transcription factor of Cannabis or an orthologist of the same.
[13]
13. Method according to claim 12, characterized by the fact that said endogenous myb transcription factor of Cannabis is selected from the group consisting of: SEQ ID NO. 11, SEQ ID NO. 42, SEQ ID NO. 43, SEQ
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ID NO. 44, or a sequence at least 80% identical to any of the listed sequences.
[14]
14. Method according to claim 1, characterized by the fact that said heterologous catalase is selected from the group consisting of: SEQ ID NO. 13, or SEQ ID NO. 15, or a sequence at least 80% identical to any of the listed sequences.
[15]
15. Method according to claim 1, characterized in that said water-soluble cannabinoids comprise glycosylated cannabinoids having one or more glycoside fractions.
[16]
16. Method according to claim 1, characterized by the fact that said water-soluble cannabinoids comprise acetylated cannabinoids.
[17]
17. Method according to claim 16, characterized in that said acetylated cannabinoids comprise a cannabinoid form of O acetyl glycoside.
[18]
18. Method according to claim 1, characterized by the fact that said water-soluble cannabinoids are isolated.
[19]
19. In vivo method of producing water-soluble cannabinoids in a Cannabis suspension cell culture, characterized by the fact that it comprises the steps of:
- expressing in a genetically modified Cannabis cell a nucleotide sequence encoding a heterologous cytochrome P450 enzyme;
- expressing a nucleotide sequence encoding a heterologous P450 oxidoreductase in a genetically modified Cannabis cell; and
- express a nucleotide sequence encoding a glycosyltransferase in a genetically modified Cannabis cell.
[20]
20. Method according to claim 19, characterized in that said Cannabis suspension cell culture comprises a Cannabis sativa suspension cell culture.
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[21]
21. Method according to claim 19, characterized in that said cytochrome P450 heterologous hydroxyl uni cannabinoid to form a hydroxylated cannabinoid and / or oxidizes a hydroxylated cannabinoid to form a cannabinoid carboxylic acid.
[22]
22. Method according to claim 21, characterized in that said heterologous cytochrome P450 is identified as SEQ ID NO. 1, or a sequence at least 80% identical to SEQ ID NO. 1.
[23]
23. Method according to claim 22, characterized in that said heterologous P450 oxidoreductase facilitates the transfer of electrons from a nicotinamide adenine dinucleotide phosphate (NADPH) to said cytochrome P450.
[24]
24. Method according to claim 23, characterized in that said heterologous P450 oxidoreductase is identified as SEQ ID NO. 3, or a sequence at least 80% identical to SEQ ID NO. 3.
[25]
25. Method according to claim 24, characterized in that said glycosyltransferase is a heterologous glycosyltransferase.
[26]
26. Method according to claim 25, characterized in that said heterologous glycosyltransferase is identified as SEQ ID NO. 7, or a sequence at least 80% identical to SEQ ID NO. 7.
[27]
27. Method according to claim 25, characterized in that said heterologous glycosyltransferase is a glycosyltransferase of Nicotiana tabacum or Nicotiana benthamiana.
[28]
28. Method according to claim 27, characterized by the fact that said Nicotiana tabacum glycosyltransferase is selected from the group consisting of: SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39, or a sequence at least 80% identical to any of the listed sequences or a homologous sequence in Nicotiana benthamiana.
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[29]
29. Method according to claim 19, characterized in that it further expresses in a genetically modified Cannabis cell a nucleotide sequence encoding an ABC transporter identified as SEQ ID NO. 9, or a sequence at least 80% identical to SEQ ID NO.
9.
[30]
30. Method according to claim 19, characterized in that it further expresses in a genetically modified Cannabis cell a nucleotide sequence encoding a Cannabis myb transcription factor selected from the group consisting of: SEQ ID NO. 11, SEQ ID NO. 42, SEQ ID NO. 43, SEQ ID NO. 44, or a sequence at least 80%> identical to any of the listed sequences.
[31]
31. Method according to claim 19, characterized in that it further expresses in a genetically modified Cannabis cell a nucleotide sequence encoding a heterologous catalase selected from the group consisting of: SEQ ID NO. 13, or SEQ ID NO. 15, or a sequence at least 80% identical to any of the listed sequences.
[32]
32. Method according to claim 19, characterized in that said water-soluble cannabinoids comprise glycosylated cannabinoids having one or more glycoside fractions.
[33]
33. The method of claim 19, characterized in that said water-soluble cannabinoids comprise acetylated cannabinoids.
[34]
34. Method according to claim 19, characterized in that said acetylated cannabinoids comprise a cannabinoid form of O acetyl glycoside.
[35]
35. Method according to claim 19, characterized by the fact that said water-soluble cannabinoids are isolated.
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[36]
36. In vivo method of producing hydroxylated and glycosylated cannabinoids in a Cannabis suspension cell culture, characterized by the fact that it comprises the steps of:
- express a nucleotide sequence encoding a heterologous cytochrome P450 enzyme:
- expressing a nucleotide sequence encoding a heterologous P450 oxidoreductase; and
- expressing a nucleotide sequence encoding a heterologous glycosyltransferase; and · expressing in a nucleotide sequence encoding a heterologous ABC transporter.
[37]
37. Intensified in vivo method for high-level production and accumulation of water-soluble cannabinoids in a Cannabis trichome, characterized by the fact that it comprises:
- a Cannabis plant:
- expressing a nucleotide sequence encoding a heterologous cytochrome P450;
- expressing a nucleotide sequence encoding a heterologous P450 oxidoreductase;
expressing a nucleotide sequence encoding a glycosyltransferase having a trichome target sequence;
- expressing a nucleotide sequence encoding a heterologous UDP-galactose / UDP-glucose transporter having a plasma membrane target sequence;
- expressing a nucleotide sequence encoding a myb transcription factor; and
- expressing a nucleotide sequence encoding a heterologous catalase.
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[38]
38. Method according to claim 37, characterized in that said Cannabis plant comprises Cannabis sativa.
[39]
39. The method of claim 37, wherein said heterologous cytochrome P450 hydroxyls a cannabinoid to form a hydroxylated cannabinoid and / or oxidizes a hydroxylated cannabinoid to form a cannabinoid carboxylic acid.
[40]
40. Method according to claim 39, characterized in that said heterologous cytochrome P450 is identified as SEQ ID NO. 1, or a sequence at least 80% identical to SEQ ID NO. 1.
[41]
41. Method according to claim 37, characterized by the fact that said heterologous P450 oxidoreductase facilitates the transfer of electrons from a NADPH to said cytochrome P450.
[42]
42. Method according to claim 41, characterized in that said heterologous P450 oxidoreductase is identified as SEQ ID NO. 3, or a sequence at least 80% identical to SEQ ID NO. 3.
[43]
43. The method of claim 42, characterized in that said glycosyltransferase having a trichome target sequence is a heterologous glycosyltransferase having a trichome target sequence.
[44]
44. method according to claim 43, characterized in that said heterologous glycosyltransferase having a trichome target sequence is identified as SEQ ID NO. 19, or a sequence at least 80% identical to SEQ ID NO. 19.
[45]
45. Method according to claim 43, characterized in that said heterologous glycosyltransferase having a trichome target sequence is a Nicotiana tabacum or Nicotiana benthamiana glycosyltransferase having a trichome target sequence.
[46]
46. Method according to claim 37, characterized by the fact that said myb transcription factor is an endogenous myb transcription factor 12 of Cannabis, or an orthologist thereof.
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[47]
47. Method according to claim 46, characterized by the fact that the said endogenous myb transcription factor of Cannabis is selected from the group consisting of: SEQ ID NO. 11, SEQ ID NO. 42, SEQ ID NO. 43, SEQ ID NO. 44, or a sequence at least 80% identical to any of the listed sequences.
[48]
48. Method according to claim 37, characterized in that said heterologous catalase is selected from the group consisting of: SEQ ID NO. 13, or SEQ ID NO. 15, or a sequence at least 80% identical to any of the listed sequences.
[49]
49. Method according to claim 37, characterized in that said heterologous catalase comprises a heterologous catalase having a trichome target sequence.
[50]
50. Method according to claim 49, characterized in that said heterologous catalase having a trichome target sequence is selected from the group consisting of: SEQ ID NO. 47, SEQ ID NO. 48, SEQ ID NO. 49, or a sequence at least 80% identical to any of the listed sequences.
[51]
51. The method of claim 37, characterized in that said water-soluble cannabinoids comprise glycosylated cannabinoids having one or more glycoside fractions.
[52]
52. The method of claim 37, characterized in that said water-soluble cannabinoids comprise acetylated cannabinoids.
[53]
53. Method according to claim 52, characterized in that said acetylated cannabinoids comprise a cannabinoid form of O acetyl glycoside.
[54]
54. Method according to claim 37, characterized in that said water-soluble cannabinoids are isolated.
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[55]
55. The method of claim 37, characterized in that said UDP-galactose / UDP-gucose transporter having a plasma membrane target sequence transports sugar substrate to said glycosyltransferase having a trichome target sequence in said Cannabis trichome.
[56]
56. Method according to claim 55, characterized in that said UDP-galactose / UDP-glucose transporter having a plasma membrane target sequence is identified as SEQ ID NO. 21, or a sequence at least 80% identical to SEQ ID NO. 21.
[57]
57. In vivo method of producing hydroxylated and glycosylated cannabinoids in a culture of Cannabis suspension cells, characterized by the fact that it comprises the steps of:
- express a nucleotide sequence encoding a heterologous cytochrome P450 enzyme:
- expressing a nucleotide sequence encoding a heterologous P450 oxidoreductase; and
- expressing a nucleotide sequence encoding a heterologous glycosyltransferase.
[58]
58. In vivo method of producing hydroxylated cannabinoids in a Cannabis plant, characterized by the fact that it comprises the steps of:
- a genetically modified Cannabis plant expressing:
- a nucleotide sequence encoding a heterologous cytochrome P450 enzyme; and
- a nucleotide sequence encoding a heterologous P450 oxidoreductase.
[59]
59. Method according to claim 58, characterized in that said heterologous cytochrome P450 hydroxyls a cannabinoid to form a hydroxylated cannabinoid and / or oxidizes a hydroxylated cannabinoid to form a cannabinoid carboxylic acid.
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[60]
60. Method according to claim 59, characterized in that said heterologous cytochrome P450 is identified as SEQ ID NO. 1, or a sequence at least 80% identical to SEQ ID NO. 1.
[61]
61. Method according to claim 58, characterized in that said heterologous P450 oxidoreductase facilitates the transfer of electrons from a NADPH to said cytochrome P450.
[62]
62. The method of claim 61, characterized in that said heterologous P450 oxidoreductase is identified as SEQ ID NO. 3, or a sequence at least 80% identical to SEQ ID NO. 3.
[63]
63. In vivo method for high-level production and accumulation of water-soluble cannabinoids in a Cannabis trichome, characterized by the fact that it comprises:
- expressing a nucleotide sequence encoding a heterologous cytochrome P450;
- expressing a nucleotide sequence encoding a heterologous P450 oxidoreductase;
- expressing a nucleotide sequence encoding a glycosyltransferase having a trichome target sequence; and · expressing a nucleotide sequence encoding a UDP-galactose / UDP-glucose transporter having a high plasma membrane sequence.
[64]
64. The method of claim 63, characterized by the fetus of which said Cannabis comprises Cannabis sativa.
[65]
65. Method according to claim 63, characterized in that said cytochrome P450 heterologous hydroxyl uni cannabinoid to form a hydroxylated cannabinoid and / or oxidizes a hydroxylated cannabinoid to form a cannabinoid carboxylic acid.
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[66]
66. Method according to claim 65, characterized in that said heterologous cytochrome P450 is identified as SEQ ID NO. 1, or a sequence at least 80% identical to SEQ ID NO. 1.
[67]
67. Method according to claim 63, characterized in that said heterologous P450 oxidoreductase facilitates the transfer of electrons from NADPH to cytochrome P450.
[68]
68. Method according to claim 67, characterized in that said heterologous P450 oxidoreductase is identified as SEQ ID NO. 3, or a sequence at least 80% identical to SEQ ID NO. 3.
[69]
69. Method according to claim 68, characterized in that said glycosyltransferase having a trichome target sequence is a heterologous glycosyltransferase having a trichome target sequence.
[70]
70. The method of claim 69, characterized in that said heterologous glycosyltransferase having a trichome target sequence is identified as SEQ ID NO. 19, or a sequence at least 80% identical to SEQ ID NO. 7.
[71]
71. Method according to claim 69, characterized in that said heterologous glycosyltransferase having a trichome target sequence is a Nicotiana tabacum or Nicotiana benthamiana glycosyltransferase having a trichome target sequence.
[72]
72. Method according to claim 63, characterized in that it further expresses a nucleotide sequence encoding a Cannabis myb transcription factor selected from the group consisting of: SEQ ID NO. 11, SEQ ID NO. 42, SEQ ID NO. 43, SEQ ID NO. 44, or a sequence at least 80% identical to any of the listed sequences.
[73]
73. Method according to claim 63, characterized in that it further expresses a nucleotide sequence encoding a heterologous catalase selected from the group consisting of: SEQ ID NO. 13, or SEQ ID
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AT THE. 15, or a sequence at least 80% identical to any of the listed sequences.
[74]
74. The method of claim 63, characterized in that said water-soluble cannabinoids comprise glycosylated cannabinoids having one or more glycoside fractions.
[75]
75. The method of claim 63, characterized in that said water-soluble cannabinoids comprise acetylated cannabinoids.
[76]
76. The method of claim 75, characterized in that said acetylated cannabinoids comprise a cannabinoid form of O acetyl glycoside.
[77]
77. Method according to claim 63, characterized in that said water-soluble cannabinoids are isolated.
[78]
78. The method of claim 63, characterized in that said UDP-galactose / UDP-glucose transporter having a plasma membrane target sequence is identified as SEQ ID NO. 21, or a sequence at least 80% identical to SEQ ID NO. 21.
[79]
79. Intensified in vivo method for high-level production and accumulation of water-soluble cannabinoids in a Cannabis cell cytosol, characterized by the fact that it comprises:
- generate a cannabis strain in which one or more cannabinoid synthase genes have been disrupted and / or eliminated;
- expressing in said cannabis strain one or more cannabinoid synthases that correspond to the deleted gene and in which said one or more cannabinoid synthases having their trichome target signal interrupted and / or removed;
- expressing a nucleotide sequence encoding a heterologous cytochrome P450;
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13/27 · expressing a nucleotide sequence encoding a heterologous P450 oxidoreductase;
- expressing a nucleotide sequence encoding a glycosyltransferase; and
- expressing a nucleotide sequence encoding a myb transcription factor; and
- expressing a nucleotide sequence encoding a heterologous catalase.
[80]
80. Method according to claim 79, characterized in that said one or more cannabinoid synthase genes comprise a cannabinoid synthase gene selected from the group consisting of: a CBG synthase gene, a THCA synthase gene, a CBDA synthase gene or a CBCA synthase gene.
[81]
81. Method according to claim 80, characterized in that said one or more cannabinoid synthases having their trichome target signal interrupted and / or removed are selected from the group consisting of: SEQ ID NO. 22 or SEQ ID NO. 46, or a sequence at least 80% identical to any sequence.
[82]
82. The method of claim 79, characterized in that said cytochrome P450 heterologous hydroxyl uni cannabinoid to form a hydroxylated cannabinoid and / or oxidizes a hydroxylated cannabinoid to form a cannabinoid carboxylic acid.
[83]
83. The method of claim 82, characterized in that said heterologous cytochrome P450 is identified as SEQ ID NO. 1, or a sequence at least 80% identical to SEQ ID NO. 1.
[84]
84. Method according to claim 83, characterized in that said heterologous P450 oxidoreductase facilitates the transfer of electrons from NADPH to cytochrome P450.
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[85]
85. Method according to claim 84, characterized in that said heterologous P450 oxidoreductase is identified as SEQ ID NO. 3, or a sequence at least 80% identical to SEQ ID NO. 3.
[86]
86. Method according to claim 85, characterized in that said glycosyltransferase is a heterologous glycosyltransferase.
[87]
87. Method according to claim 86, characterized in that said heterologous glycosyltransferase is identified as SEQ ID NO. 7, or a sequence at least 80% identical to SEQ ID NO. 7.
[88]
88. Method according to claim 86, characterized in that said heterologous glycosyltransferase is a glycosyltransferase of Nicotiana tabacum or Nicotiana benthamiana.
[89]
89. Method according to claim 88, characterized by the fact that said Nicotiana tabacum glycosyltransferase is selected from the group consisting of: SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39, or a sequence of at least 80% identical to any of the listed sequences of a homologous Nicotiana benthamian sequence.
[90]
90. Method according to claim 79, characterized in that it further expresses a nucleotide sequence encoding an ABC transporter identified as SEQ ID NO. 9, or a sequence at least 80%> identical to SEQ ID NO. 9.
[91]
91. Method according to claim 79, characterized in that it further expresses a nucleotide sequence encoding a Cannabis myb transcription factor selected from the group consisting of: SEQ ID NO. 11, SEQ ID NO. 42, SEQ ID NO. 43, SEQ ID NO. 44, or a sequence at least 80% identical to any of the listed sequences.
[92]
92. Method according to claim 79, characterized in that it further expresses a nucleotide sequence encoding a heterologous catalase selected from the group consisting of: SEQ ID NO. 13, or SEQ ID
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AT THE. 15, or a sequence at least 80%> identical to any of the listed sequences.
[93]
93. The method of claim 79, characterized in that said water-soluble cannabinoids comprise glycosylated cannabinoids having one or more glycoside fractions.
[94]
94. The method of claim 79, characterized in that said water-soluble cannabinoids comprise acetylated cannabinoids.
[95]
95. Method according to claim 94, characterized in that said acetylated cannabinoids comprise a form of cannabinoid acetyl glycoside.
[96]
99. The method of claim 79, characterized in that said water-soluble cannabinoids comprise hydroxylated cannabinoids.
[97]
100. In vivo method for high-level production and accumulation of water-soluble cannabinoids in a Cannabis cell cytosol, characterized by the fact that it comprises:
- generate a cannabis strain in which one or more cannabinoid synthase genes have been disrupted and / or eliminated;
- express in said cannabis strain one or more cannabinoid synthases that correspond to the deleted gene and in which said one or more cannabinoid synthases have their trichome target signal interrupted and / or eliminated:
- expressing a nucleotide sequence encoding a heterologous cytochrome P450;
- expressing a nucleotide sequence encoding a heterologous P450 oxidoreductase; and · expressing a nucleotide sequence encoding a glycosyltransferase.
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[98]
101. Method according to claim 100, characterized in that said one or more cannabinoid synthase genes comprise a cannabinoid synthase gene selected from the group consisting of: a CBG synthase gene, a THCA synthase gene, a CBDA synthase gene or a CBCA synthase gene.
[99]
102. The method of claim 101, characterized in that said one or more cannabinoid synthases having their trichome target signal interrupted and / or eliminated comprise SEQ ID NO. 22 or SEQ ID NO. 46, or a sequence at least 80% identical to any sequence.
[100]
103. Method according to claim 100, characterized in that said heterologous cytochrome P450 hydroxyls a cannabinoid to form a hydroxylated cannabinoid and / or oxidizes a hydroxylated cannabinoid to form a cannabinoid carboxylic acid.
[101]
104. Method according to claim 103, characterized in that said heterologous cytochrome P450 is identified as SEQ ID NO. 1, or a sequence at least 80% identical to SEQ ID NO. 1.
[102]
105. Method according to claim 104, characterized in that said heterologous P450 oxidoreductase facilitates the transfer of electrons from NADPH to cytochrome P450.
[103]
106. Method according to claim 105, characterized in that said heterologous P450 oxidoreductase is identified as SEQ ID NO. 3, or a sequence at least 80% identical to SEQ ID NO. 3.
[104]
107. Method according to claim 106, characterized in that said glycosyltransferase is a heterologous glycosyltransferase.
[105]
108. Method according to claim 107, characterized in that said heterologous glycosyltransferase is identified as SEQ ID NO. 7, or a sequence at least 80% identical to SEQ ID NO. 7.
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[106]
109. Method according to claim 107, characterized in that said heterologous glycosyltransferase is a Nicotiana tabacum or Nicotiana benthamiana glycosyltransferase.
[107]
110. Method according to claim 109, characterized by the fact that said Nicotiana tabacum glycosyltransferase is selected from the group consisting of: SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39, or a sequence at least 80% identical to any of the listed sequences or a homologous sequence of Nicotiana benthamiana.
110. Method according to claim 100, characterized in that it further expresses a nucleotide sequence encoding a Cannabis myb transcription factor selected from the group consisting of: SEQ ID NO. 11, SEQ ID NO. 42, SEQ ID NO. 43, SEQ ID NO. 44, or a sequence at least 80% identical to any of the listed sequences.
[108]
111. Method according to claim 100, characterized in that it further expresses a nucleotide sequence encoding a heterologous catalase selected from the group consisting of: SEQ ID NO. 13, or SEQ ID NO. 15, or a sequence at least 80% identical to any of the sequences listed above.
[109]
112. Method according to claim 100, characterized in that said water-soluble cannabinoids comprise glycosylated cannabinoids having one or more glycoside fractions.
[110]
113. The method of claim 100, characterized in that said water-soluble cannabinoids comprise acetylated cannabinoids.
[111]
114. Method according to claim 113, characterized in that said acetylated cannabinoids comprise a cannabinoid form of O acetyl glycoside.
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[112]
115. Method according to claim 100, characterized in that said water-soluble cannabinoids comprise hydroxylated cannabinoids.
[113]
116. Intensified in vivo method for high-level production and accumulation of water-soluble cannabinoids in a cytosol cell of a non-cannabinoid-producing plant, characterized by the fact that it comprises:
- expressing a nucleotide sequence encoding a heterologous cannabinoid synthase enzyme in which the trichome target sequence has been inactivated and / or removed;
· Express a nucleotide sequence encoding a heterologous cytochrome P450;
- expressing a nucleotide sequence encoding a heterologous P450 oxidoreductase;
- expressing a nucleotide sequence encoding a glycosyltransferase;
- expressing a nucleotide sequence encoding a heterologous catalase; and
- introduce a quantity of cannabinoids in the said non-cannabinoid producing plant.
[114]
117. Method, according to claim 116, characterized by the fact that said non-cannabinoid producing plant is Nicotiana benthamiana.
[115]
118. The method of claim 116, characterized by the photo that said one or more heterologous cannabinoid synthase enzymes, wherein the trichome target sequence has been inactivated and / or removed comprises a cannabinoid synthase gene selected from the group which consists of: a CBG synthase gene, a THCA synthase gene, a CBDA synthase gene or a CBCA synthase gene.
[116]
119. Method according to claim 118, characterized in that said nucleotide sequence encoding an enzyme from
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19/27 heterologous cannabinoid synthase in which the trichome target sequence has been inactivated and / or removed comprises a heterologous cannabinoid synthase enzyme in which the trichome target sequence has been inactivated and / or removed identified as SEQ ID NO. 46, or a sequence at least 80% identical to SEQ ID NO. 46, or SEQ ID NO. 22, or a sequence at least 80% identical to SEQ ID NO. 22.
[117]
120. Method according to claim 116, characterized in that said heterologous cytochrome P450 hydroxyls a cannabinoid to form a hydroxylated cannabinoid and / or oxidizes a hydroxylated cannabinoid to form a cannabinoid carboxylic acid.
[118]
121. Method according to claim 120, characterized in that said heterologous cytochrome P450 is identified as SEQ ID NO. 1, or a sequence at least 80% identical to SEQ ID NO. 1.
[119]
122. Method according to claim 121, characterized by the fact that said heterologous P450 oxidoreductase facilitates the transfer of electrons from NADPH to cytochrome P450.
[120]
123. Method according to claim 122, characterized in that said heterologous P450 oxidoreductase is identified as SEQ ID NO. 3, or a sequence at least 80% identical to SEQ ID NO. 3.
[121]
124. Method according to claim 123, characterized in that said glycosyltransferase is a heterologous glycosyltransferase.
[122]
125. Method according to claim 124, characterized in that said heterologous glycosyltransferase is identified as SEQ ID NO. 7, or a sequence at least 80% identical to SEQ ID NO. 7.
[123]
126. Method, according to claim VCIOO, characterized by the fact that said glycosyltransferase is a glycosyltransferase of Nicotiana tabacum Nicotiana benthamiana.
[124]
127. Method according to claim 126, characterized by the fact that the said glycosyltransferase from Nicotiana tabacum is selected from the group
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20/27 consisting of: SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39, or a sequence of at least 80% identical to any of the listed sequences or a homologous sequence of Nicotiana benthamiana.
[125]
128. Method according to claim 116, characterized in that it further expresses a nucleotide sequence encoding a Cannabis myb transcription factor selected from the group consisting of: SEQ ID NO. 11, SEQ ID NO. 42, SEQ ID NO. 43, SEQ ID NO. 44, or a sequence at least 80% identical to any of the listed sequences.
[126]
129. Method according to claim 116, characterized in that it further expresses a nucleotide sequence encoding a heterologous catalase selected from the group consisting of: SEQ ID NO. 13, or SEQ ID NO. 15, or a sequence at least 80%> identical to any of the listed sequences.
[127]
130. The method of claim 116, characterized in that said water-soluble cannabinoids comprise glycosylated cannabinoids having one or more glycoside fractions.
[128]
131. The method of claim 116, characterized in that said water-soluble cannabinoids comprise acetylated cannabinoids.
[129]
132. Method according to claim 131, characterized in that said acetylated cannabinoids comprise a cannabinoid form of O acetyl glycoside.
[130]
133. Method according to claim 116, characterized in that said water-soluble cannabinoids comprise hydroxylated cannabinoids.
[131]
134. In vivo method for high-level production and accumulation of water-soluble cannabinoids in a cell cytosol of a non-cannabinoid-producing plant, characterized by the fact that it comprises:
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Expressing a nucleotide sequence encoding a heterologous cannabinoid synthase enzyme in which the trichome target sequence has been inactivated and / or removed;
- expressing a nucleotide sequence encoding a heterologous cytochrome P450;
- expressing a nucleotide sequence encoding a heterologous P450 oxidoreductase;
- expressing a glycosyltransferase having a trichome target sequence; and · introducing a quantity of cannabinoids in the said non-cannabinoid producing plant.
[132]
135. In vivo method for high-level production and accumulation of water-soluble cannabinoids in the trichome of a non-cannabinoid-producing plant, characterized by the fact that it comprises:
- expressing a nucleotide sequence encoding one or more heterologous cababinoid synthase enzymes;
- expressing a nucleotide sequence encoding a heterologous cytochrome P450;
· Express a nucleotide sequence encoding a heterologous P450 oxidorreudtase;
- expressing a nucleotide sequence encoding a heterologous glycosyltransferase having a trichome target sequence;
- expresses a nucleotide sequence encoding a UDP-galactose / UDP-glucose transporter having a plasma membrane target sequence;
- expressing a nucleotide sequence encoding a heterologous catalase; and · introducing a quantity of cannabinoids in said non-cannabinoid producer.
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[133]
136. Method according to claim 135, characterized by the fact that the said non-cannabinoid-producing plant is Nicotiana benthamiana.
[134]
137. Method according to claim 135, characterized in that said one or more heterologous cannabinoid synthase enzymes, wherein the trichome target sequence has been inactivated and / or removed comprises a cannabinoid synthase gene selected from the group which consists of: a CBG synthase gene, a THCA synthase gene, a CBDA synthase gene or a CBCA synthase gene.
[135]
138. Method according to claim 137, characterized in that said nucleotide sequence encoding a heterologous cannabinoid synthase enzyme in which the trichome target sequence has been inactivated and / or removed comprises a heterologous cannabinoid synthase enzyme in that the trichome target sequence has been inactivated and / or removed identified as SEQ ID NO. 46, or a sequence at least 80% identical to SEQ ID NO. 46, or SEQ ID NO. 22, or a sequence at least 80% identical to SEQ ID NO. 22.
[136]
139. Method according to claim 135, characterized in that said heterologous cytochrome P450 hydroxyls a cannabinoid to form a hydroxylated cannabinoid and / or oxidizes a hydroxylated cannabinoid to form a cannabinoid carboxylic acid.
[137]
140. Method according to claim 139, characterized in that said heterologous cytochrome P450 is identified as SEQ ID NO. 1, or a sequence at least 80% identical to SEQ ID NO. 1.
[138]
141. Method according to claim 140, characterized by the fact that said heterologous P450 oxidoreductase facilitates the transfer of electrons from NADPH to cytochrome P450.
[139]
142. Method according to claim 141, characterized in that said heterologous P450 oxidoreductase is identified as SEQ ID NO. 3, or a sequence at least 80% identical to SEQ ID NO. 3.
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[140]
143. Method according to claim 142, characterized in that said heterologous glycosyltransferase having a trichome target sequence is identified as SEQ ID NO. 19. or a sequence at least 80% identical to SEQ ID NO. 19.
[141]
144. Method according to claim 135, characterized in that said glycosyltransferase is a glycosyltransferase from Nicotiana tabacum Nicotiana benthamiana.
[142]
145. Method according to claim 144, characterized by the fact that said Nicotiana tabacum glycosyltransferase is selected from the group consisting of: SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39, or a sequence of at least 80% identical to any of the listed sequences, each having a trichome target sequence or a homologous Nicotiana benthamian sequence, each having a trichome target sequence.
[143]
146. Method according to claim 135, characterized in that it further expresses a nucleotide sequence encoding a Cannabis myb transcription factor selected from the group consisting of: SEQ ID NO. 11, SEQ ID NO. 42, SEQ ID NO. 43, SEQ ID NO. 44, or a sequence at least 80% identical to any of the listed sequences.
[144]
147. Method according to claim 135, characterized in that it further expresses a nucleotide sequence encoding a heterologous catalase selected from the group consisting of: SEQ ID NO. 13, or SEQ ID NO. 15, or a sequence at least 80%> identical to any of the listed sequences.
[145]
148. Method according to claim 135, characterized in that it further expresses a nucleotide sequence encoding a heterologous catalase having a trichome target domain selected from the group consisting of: SEQ ID NO. 47, or SEQ ID NO. 48, or SEQ ID NO. 49, or SEQ ID
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24/27
AT THE. 50, or a sequence at least 80% identical to any of the listed sequences.
[146]
149. Method according to claim 135, characterized in that said water-soluble cannabinoids comprise glycosylated cannabinoids having one or more glycoside fractions.
[147]
150. The method of claim 135, characterized in that said water-soluble cannabinoids comprise acetylated cannabinoids.
[148]
151. Method according to claim 150, characterized in that said acetylated cannabinoids comprise a cannabinoid form of O acetyl glycoside.
[149]
152. The method of claim 135, characterized by the fact that said water-soluble cannabinoids comprise hydroxylated cannabinoids.
[150]
153. Intensified in vivo method for high-level production and accumulation of water-soluble cannabinoids in the trichome of a non-cannabinoid-producing plant, characterized by the fact that it comprises:
- expressing a nucleotide sequence encoding a cannabinoid synthase enzyme;
- expressing a nucleotide sequence encoding a heterologous cytochrome P450;
- expressing a nucleotide sequence encoding a heterologous P450 oxidoreductase;
- expressing a nucleotide sequence encoding a heterologous glycosyltransferase having a trichome target sequence;
- expressing a nucleotide sequence encoding a UDP-galactose / UDP-glucose transporter having a plasma membrane target sequence;
Petition 870190095591, of 9/24/2019, p. 30/196
25/27 · introduce a quantity of cannabinoids in the said non-cannabinoid producing plant.
[151]
154. Intensified in vivo method of producing water-soluble cannabinoids in a cell suspension culture of Nicotiana benthamiana, characterized by the fact that it comprises the steps of:
- generate a culture of Nicotiana benthamiana suspension cells;
- expressing a nucleotide sequence encoding a heterologous cannabinoid synthase enzyme in which the trichome target sequence has been inactivated and / or removed;
· Express a nucleotide sequence encoding a heterologous cytochrome P450;
- expressing a nucleotide sequence encoding a heterologous P450 oxidoreductase;
- express an endogenous glycosyltransferase;
- expressing a nucleotide sequence encoding a heterologous ABC transporter;
- expressing a nucleotide sequence encoding a heterologous catalase; and · introducing a quantity of cannabinoids in the said non-cannabinoid producing plant.
[152]
155. Intensified in vivo method of glycosylating cannabinoids in Nicotiana benthamiana, characterized by the fact that it comprises the steps of:
- expressing a nucleotide sequence encoding a heterologous cannabinoid synthase enzyme;
- expressing a nucleotide sequence encoding a heterologous cytochrome P450;
- expressing a nucleotide sequence encoding a heterologous P450 oxidoreductase;
Petition 870190095591, of 9/24/2019, p. 31/196
26/27 · introduce a quantity of cannabinoids in the said non-cannabinoid producing plant.
[153]
156. Method for increasing cannabinoid production, characterized by the fact that it comprises the steps of:
- expressing a nucleotide sequence encoding a heterologous catalase wherein said catalase has a trichome target sequence in a cannabinoid producing plant.
[154]
157. Method according to claim 156, characterized by the fact that said cannabinoid-producing plant is Cannabis Sativa.
[155]
158. Method according to claim 157, characterized in that said heterologous catalase wherein said catalase has a trichome target sequence selected from the group consisting of: SEQ ID NO. 47, or SEQ ID NO. 48, or SEQ ID NO. 49, or SEQ ID NO. 50, or a sequence at least 80% identical to any of the listed sequences.
[156]
159. Method to increase cannabinoid production, characterized by the fact that it comprises the steps of:
expressing a nucleotide sequence encoding a heterologous catalase wherein said catalase has a trichome target sequence; and · expressing a nucleotide sequence encoding a myb transcription factor.
[157]
160. Method according to claim 159, characterized by the fact that said cannabinoid-producing plant is Cannabis Sativa.
[158]
161. Method according to claim 160, characterized in that said heterologous catalase wherein said catalase has a trichome target sequence selected from the group consisting of: SEQ ID NO. 47, or SEQ ID NO. 48, or SEQ ID NO. 49, or SEQ ID NO. 50, or a sequence at least 80% identical to any of the listed sequences.
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[159]
162. Method according to claim 159, characterized by the fact that said myb transcription factor is an endogenous myb12 transcription factor of Cannabis or an orthologist thereof.
[160]
163. Method according to claim 162, characterized by the fact that said endogenous myb transcription factor of Cannabis is selected from the group consisting of: SEQ ID NO. 11, SEQ ID NO. 42, SEQ ID NO. 43, SEQ ID NO. 44, or a sequence at least 80% identical to any of the listed sequences.

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同族专利:

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公开号 | 公开日

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CA3056929A1|2018-09-27|

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US20190153460A1|2019-05-23|

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CN111373045A|2020-07-03|

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US20190338301A1|2019-11-07|

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IL269649D0|2019-11-28|

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US20190085347A1|2019-03-21|

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MX2019011323A|2020-01-27|

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CO2019011733A2|2020-01-17|

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US20190153461A1|2019-05-23|

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WO2018176055A2|2018-09-27|

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US20190161763A1|2019-05-30|

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AU2018239671A1|2019-10-10|

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US10378020B2|2019-08-13|

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EP3600361A4|2021-01-06|

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WO2018176055A3|2018-11-29|

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EP3600361A2|2020-02-05|

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ZA201906976B|2020-08-26|

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法律状态:
2021-10-19| B350| Update of information on the portal [chapter 15.35 patent gazette]|

优先权:

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申请号 | 申请日 | 专利标题

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US201762476080P| true| 2017-03-24|2017-03-24|

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US201762588662P| true| 2017-11-20|2017-11-20|

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