Plants for producing cannabinoids

Abstract

The present invention relates to polynucleotides for the production of genetically modified plants, algae or plastids thereof capable of producing cannabinoids. In one aspect, the invention also relates to methods of producing cannabinoids.

Description

Technical Field

[0001] The present invention relates to polynucleotides for producing genetically modified plants, algae or plastids thereof capable of producing cannabinoids. In one aspect, the present invention also relates to methods for producing cannabinoids. Background Art

[0002] The plant cannabis (Cannabis sativa) has been used as a medicine for many centuries. For example, cannabis was previously used in China to treat gout, malaria, digestive disorders, and menstrual pain, and was further used in Western medicine to treat rheumatism and epileptic seizures (Bostwick, 2012; Russo, 2016; Kinghorn et al., 2017; Baron, 2018). Since then, a growing number of cannabinoid drugs have been approved for use in humans, including nabilone in 1985, dronabinol in 1986, rimonabant in 2006 (in Europe; withdrawn in 2008), dapoxetine in 2010, and tamoxifen in 2011. and 2018 An increasing number of countries have also approved the use of cannabis to treat various medical conditions, with cannabidiol (CBD) oil being a popular mode of administration.

[0003] In cannabis, the cannabinoid biosynthesis pathway begins with the precursor molecules olivetolic acid (OA) and geranyl pyrophosphate (GPP), which combine to form cannabigerolic acid (CBGA) (Shoyama et al., 1975; Fellermeier and Zenk, 1998; Fellermetier et al., 2001; Gülck and 2020). CBGA serves as a precursor to most other cannabinoids and is converted into Δ9-tetrahydrocannabinolic acid (Δ9-THCA), cannabidiolic acid (CBDA), and cannabichromenic acid (CBCA) in cannabis ( Figure 1 ). Because CBGA acts as a precursor molecule for other cannabinoids, its content in cannabis is usually very low.

[0004] Enzymatically produced cannabinoids (including CBGA) are produced in their acidic form and then decarboxylated to produce the "active" form (i.e., THC (tetrahydrocannabinol), CBD (cannabidiol), CBC (cannabichromene), CBG (cannabigerol), etc.). With the recent deregulation of CBD and other cannabis-derived cannabinoids, there is increasing interest in cannabinoid pharmacology (www.usda.gov / farmbill). Specifically, despite having CBG as a common precursor, Δ9-THC, CBD, and CBC have significantly different physiological effects. For example, Δ9-THC is known to produce euphoria and appetite stimulation (Volkow et al., 2014), while CBD is non-euphoric and has been shown to have anti-epileptic effects (Jones et al., 2010) and anti-inflammatory effects (Carrier et al., 2006).

[0005] Different methods for producing cannabinoid compounds in plant hosts have been attempted. In WO 2021 / 081648, the authors use algae as a system for producing olive acid (OA), which includes the production of olive acid synthase (OAS) and olive acid cyclase (OAC) fusion proteins, but scaling up commercial production is costly and requires high energy levels. The authors of WO 2018 / 200888 overexpressed geranyl pyrophosphate: olivetate geranyl transferase polypeptides to catalyze the production of cannabinoid acid from geranyl pyrophosphate and olive acid in yeast. However, small-scale yeast scaling is unpredictable and is associated with a lack of certainty in actual recovery. Yeast also uses geranyl pyrophosphate (GPP) to prepare isoprenoids, so it is necessary to develop methods to block the consumption of substrates required for cannabinoid production. Yet another method involves producing components of the cannabinoid pathway in heterologous plants, but the authors of this work were unable to detect the production of CBGA, despite the formation of OA and OA-glucoside (Gulck et al., 2020). The authors speculated that the lack of CBGA may be due to glycosylation of OA, hindering the biosynthesis of downstream cannabinoid products, highlighting the difficulty in successfully producing cannabinoids from it.

[0006] In response to the growing demand for medical use of cannabis, many countries have attempted to legalize and decriminalize cannabis by relaxing laws related to drug control, however, cannabis cultivation remains strictly controlled by strict regulations (Alharbi, 2020; et al. 2021). For example, within the EU, regions control which varieties may be grown based on THC content (Hazekamp, ​​2018). While cannabis varieties with reduced activity of the three main synthases can accumulate higher levels of CBGA, the presence of the euphoric THCA remains a puzzling issue (Fellermeier and Zenk, 1998; Fellermeier et al., 2001). Because THC levels are difficult to control, it is not possible to breed or modify cannabis to produce only CBD. Furthermore, reliable and efficient transformation and regeneration protocols for modifying cannabis have not yet been established (Schachtsiek et al., 2018; Deguchi et al., 2020). In addition, the production of the main cannabinoids is mainly limited to one organ, the female flower; as a result, most of the biomass is wasted.

[0007] These and other complexities associated with cannabinoid metabolism lead to challenges in developing robust and cost-effective methods for producing cannabinoids.

[0008] Therefore, there is a need to develop alternative strategies for producing cannabinoids. Summary of the invention

[0009] The present inventors have demonstrated for the first time a functional cannabinoid biosynthetic pathway in a heterologous plant. Specifically, expression of components of the cannabinoid pathway in a heterologous plant successfully resulted in the synthesis of OA and CBGA. The present inventors showed that targeting components of the cannabinoid pathway to the plastids of the host plant increased the yields of OA, CBGA and their respective glycosylated forms. When components of the cannabinoid biosynthetic pathway were overexpressed using an optimized vector expressed in plastids, significant increases in the levels of all four cannabinoids were also obtained.

[0010] Thus, the inventors have demonstrated for the first time the reconstruction of a functional cannabinoid biosynthetic pathway in a heterologous plant, which results in the production of OA and CBGA. Such a system is suitable for the generation of a scalable production system, such as plant biomass, with industrial applicability.

[0011] Thus, the present inventors have developed polynucleotides that can be used to generate genetically modified plants, algae and plastids thereof to increase cannabinoid production.

[0012] Thus, in a first aspect, there is provided a genetically modified plastid of a plant, part thereof or algae, the plastid comprising at least one polypeptide selected from the group consisting of a polynucleotide encoding a polyketide synthase, a polyketide cyclase, an acyl-activating enzyme, a prenyl transferase, a plastidial acyl-lipid thioesterase, a plastidial lipase or a plastidial tomato 13-lipoxygenase,

[0013] wherein the at least one polypeptide increases the production of cannabigerolic acid (CBGA) in the presence of hexanoic acid (C6) and geranyl pyrophosphate (GPP) when compared to plastids of wild-type plants, parts thereof or algae.

[0014] In another aspect, the present invention provides a genetically modified plastid of a plant, part thereof or algae, the plastid comprising at least one polypeptide selected from the group consisting of acyl activating enzyme 1 (AAE1), olivetol synthase (OLS) and olivetate cyclase (OAC),

[0015] wherein the at least one polypeptide increases the production of cannabigerolic acid (CBGA) in the presence of hexanoic acid (C6) and geranyl pyrophosphate (GPP) when compared to plastids of wild-type plants, parts thereof or algae.

[0016] In another aspect, the present invention provides a genetically modified plastid of a plant or part thereof, the plastid comprising an acyl activating enzyme 1 (AAE1) polypeptide, an olivetol synthase (OLS) polypeptide and an olivetate cyclase (OAC) polypeptide, optionally further comprising a plastid-localized prenyltransferase polypeptide, preferably cannabigerolic acid synthase (CBGAS),

[0017] wherein the polypeptide increases the production of cannabigerolic acid (CBGA) in the presence of hexanoic acid (C6) and geranyl pyrophosphate (GPP) when compared to plastids of a wild-type plant or part thereof.

[0018] Optionally, the at least one polypeptide or plurality of polypeptides increases the production of olivetic acid (OA) in the presence of hexanoic acid (C6) when compared to plastids of wild-type plants, parts thereof or algae.

[0019] In one embodiment, the at least one polypeptide or multiple polypeptides selected from the group consisting of AAE1, OLS and OAC are encoded by one or more polynucleotides, wherein the one or more polynucleotides, optionally each polynucleotide encodes a fusion polypeptide comprising a plastid transport peptide, preferably a chloroplast transit peptide (CTP); and wherein the one or more polynucleotides are operably linked to a promoter capable of directing expression of the one or more polynucleotides in the plant or part thereof. In one embodiment, the at least one polypeptide or multiple polypeptides selected from the group consisting of AAE1, OLS and OAC are encoded by one or more polynucleotides, wherein the polynucleotides are integrated into the plastid genome, and wherein the polynucleotides are operably linked to a promoter capable of directing expression of the polynucleotides in the plant or part thereof.

[0020] In yet another embodiment, the at least one polypeptide or multiple polypeptides selected from the group consisting of AAE1, OLS and OAC are encoded by one or more polynucleotides, wherein the one or more polynucleotides are operably linked to a viral vector sequence capable of expressing the polynucleotides in a plant or a part thereof, or are included in a viral vector capable of expressing the polynucleotides in a plant or a part thereof; and optionally, wherein the polynucleotide encodes a fusion protein comprising a plastid transit peptide.

[0021] Optionally, each polynucleotide encoding the polypeptide is operably linked to a viral vector sequence capable of expressing the polynucleotide in a plant or part thereof, or is included in a viral vector capable of increasing expression of the polynucleotide; and the CBGA and / or OA production is increased.

[0022] In another embodiment, the plastid comprises the polypeptides AAE1, OLS and OAC, optionally further comprising a plastid-localized prenyltransferase, preferably cannabigerolic acid synthase (CBGAS). In this embodiment, at least two polypeptides, optionally each polypeptide comprises a plastid transport peptide, preferably a chloroplast transit peptide (CTP).

[0023] In another aspect, the present invention provides a genetically modified plastid of a plant, part thereof or algae, the plastid comprising at least one polynucleotide encoding acyl activating enzyme 1 (AAE1), olivetol synthase (OLS) and olivetate cyclase (OAC),

[0024] wherein the at least one polynucleotide is operably linked to a promoter capable of directing expression of the polynucleotide in the plastid;

[0025] wherein the at least one polypeptide encoded by the polynucleotide increases the production of cannabigerolic acid (CBGA) in the presence of caproic acid (C6) and geranyl pyrophosphate (GPP) when compared to the plastids of a wild-type plant, part thereof or algae. Optionally, the at least one polypeptide or multiple polypeptides encoded by the polynucleotide increase the production of olivetolic acid (OA) in the presence of caproic acid (C6) when compared to the plastids of a wild-type plant, part thereof or algae. In one embodiment, the plastids of the genetically modified plant, part thereof or algae include polynucleotides encoding AAE1, OLS and OAC, and further include polynucleotides encoding a plastid-localized prenyltransferase, preferably cannabigerolic acid synthase (CBGAS).

[0026] In one embodiment, the polynucleotide is contained in a nucleic acid construct comprising a sequence capable of integrating the polynucleotide into the genome of the plastid, and preferably not into the genome of the nucleus of the plant, part thereof or algae. Non-limiting examples of specific integration sites may include trnH / pbA, trnG / trnfM, ycf3 / trnS, rbcL / accD, petA / psbJ, 5'rps12 / clpP, petD / rpoA, ndhB / rps7, 3'rpsl2 / trnV, trnV / rrnl6, rrnl6 / trnl, trnl / trnA, trnN / trnR and rp32 / trnL.

[0027] In another aspect, a genetically modified plant, part thereof or algae is provided, comprising at least one polynucleotide selected from the group consisting of a polynucleotide encoding a polyketide synthase, a polyketide cyclase, an acyl activating enzyme, a prenyl transferase, a plastidial acyl-lipid thioesterase, a plastidial lipase or a plastidial tomato 13-lipoxygenase,

[0028] wherein said at least one polynucleotide is operably linked to a promoter capable of directing expression of said polynucleotide in said plant, part thereof or algae,

[0029] Optionally wherein the at least one polynucleotide is codon-optimized for expression in the plant, part thereof or algae, and

[0030] The polypeptide encoded by the at least one polynucleotide increases the production of cannabigerolic acid (CBGA) when expressed in the plant, part thereof or algae in the presence of caproic acid (C6) and geranyl pyrophosphate (GPP) when compared to wild-type plants, parts thereof or algae.

[0031] In another aspect, the present invention provides a genetically modified plant, part thereof or algae, comprising at least one polynucleotide selected from the group consisting of: a polynucleotide encoding acyl activating enzyme 1 (AAE1), a polynucleotide encoding olivetol synthase (OLS) and a polynucleotide encoding olivetate cyclase (OAC), optionally further comprising a polynucleotide encoding a plastid-localized prenyltransferase polypeptide, preferably a cannabigerolic acid synthase (CBGAS),

[0032] wherein said at least one polynucleotide is operably linked to a promoter capable of directing expression of said polynucleotide in said plant, part thereof or algae,

[0033] wherein said at least one polynucleotide is codon-optimized for expression in said plant, part thereof or algae, and

[0034] When expressed in the plant, part thereof or algae in the presence of caproic acid (C6) and geranyl pyrophosphate (GPP), the polypeptide encoded by the at least one polynucleotide increases the production of cannabigerolic acid (CBGA) when compared to wild-type plants, parts thereof or algae. Optionally, the at least one polypeptide increases the production of olivetolic acid (OA) in the presence of caproic acid (C6) when compared to wild-type plants, parts thereof or algae.

[0035] In another aspect, a genetically modified plant, part thereof or algae is provided, comprising at least one polynucleotide selected from the group consisting of a polynucleotide encoding acyl activating enzyme 1 (AAE1), a polynucleotide encoding olivetol synthase (OLS) and a polynucleotide encoding olivetate cyclase (OAC), optionally further comprising a polynucleotide encoding a plastid-localized prenyltransferase polypeptide, preferably cannabigerolic acid synthase (CBGAS),

[0036] wherein the at least one polynucleotide is operably linked to a promoter capable of directing expression of the polynucleotide in the plant, part thereof or algae, and

[0037] The polypeptide encoded by the at least one polynucleotide increases the production of cannabigerolic acid (CBGA) when expressed in the plant in the presence of hexanoic acid (C6) and geranyl pyrophosphate (GPP) when compared to wild-type plants, parts thereof or algae.

[0038] In another aspect, a genetically modified plant or part thereof is provided, comprising a polynucleotide encoding acyl activating enzyme 1 (AAE1), a polynucleotide encoding olivetol synthase (OLS) and a polynucleotide encoding olivetate cyclase (OAC), optionally further comprising a polynucleotide encoding a plastid-localized prenyltransferase polypeptide, preferably cannabigerolic acid synthase (CBGAS),

[0039] wherein each polynucleotide is operably linked to a promoter capable of directing expression of said polynucleotide in said plant or part thereof,

[0040] wherein the polynucleotide is codon-optimized for expression in the plant or part thereof, and

[0041] The polypeptide encoded by the polynucleotide increases the production of cannabigerolic acid (CBGA) when expressed in the plant or part thereof in the presence of caproic acid (C6) and geranyl pyrophosphate (GPP) when compared to wild-type plants or parts thereof.

[0042] In one embodiment, the at least one polynucleotide or multiple polynucleotides are included in a vector, preferably a viral vector, optionally wherein the at least one polynucleotide or multiple polynucleotides encode a fusion polypeptide comprising a plastid transit peptide; and / or the polypeptide encoded by the at least one polynucleotide or multiple polynucleotides is expressed in the presence of a plastid-localized prenyl transferase; wherein the polypeptide encoded by the at least one polynucleotide or multiple polynucleotides increases the production of cannabigerolic acid (CBGA) when expressed in the plant in the presence of hexanoic acid (C6) and geranyl pyrophosphate (GPP) compared to wild-type plants, parts thereof or algae. Optionally, the genetically modified plant, part thereof or algae further comprises a plastid-localized prenyl transferase, preferably cannabigerolic acid synthase (CBGAS).

[0043] In one embodiment, the genetically modified plant is a vegetative plant part, preferably a vegetative plant part of a vascular plant.

[0044] In another embodiment, the genetically modified plant or part thereof is a high biomass plant, preferably selected from the group consisting of: Acrocomia aculeata (macauba palm), Arabidopsis thaliana, Arachis hypogaea (peanut), Astrocaryum murumuru (murumuru), Astrocaryum vulgare (tucuma), Indaia-rateiro (Attalea geraensis), American oil palm (Attaleahumilis), Attalea oleifera (andaia), Attaleaphalerata (uricuri), Babassu palm (Attalea speciosa or babassu), Avena sativa (oats), Beta vulgaris (sugar beet), Brassica rapa (Brassica sp., such as Brassica carinata, Brassica juncea, Brassica napobrassica, Brassica napus (canola), Camelina sativa (falseflax), Cannabis sativa (hemp), Carthamus tinctorius (safflower), Caryocar brasiliense (pequi), Coconut (Cocos nucifera), Crambe abyssinica (Abyssinian kale), Cucumis melo (melon), Elaeis guineensis (African palm), Glycine max (soybean), Gossypium hirsutum (cotton), Helianthus sp.) such as sunflower (Helianthus annuus), barley (Hordeum vulgare), physic nut (Jatropha curcas), arara nut-tree (Joannesia princeps), duckweed (Lemna sp.) (duckweed) such as Lemna aequinoctialis, Lemna disperma, Lemna ecuadoriensis, Lemna gibba (swelling duckweed), Lemna japonica, Lemna minor, Lemna minuta, Lemna obscura, Lemna paucicostata, Lemna perpusilla, Lemna tenera, Lemna trisulca, Lemna turionifera), Valdivian duckweed (Lemna valdiviana), Yungas rock duckweed (Lemna yungensis), Oti tree (Licania rigida or oiticica), Flax (Linumusitatissimum or flax), Lupinus angustifolius or lupin, Mauritia flexuosa (buriti palm), Maximiliana maripa (inaja palm), Miscanthus sp. such as Miscanthus x giganteus and Miscanthus sinensis, Nicotiana sp. (tobacco) such as Nicotiana tabacum or Nicotiana benthamiana, Oenocarpus bacaba (bacaba-do-azeite), Oenocarpus bataua)(pataua), Oenocarpus distichus(bacaba-de-leque), Oryza sp.) (rice) such as Asian rice (Oryza sativa) and African cultivated rice (Oryza glaberrima), switchgrass (Panicum virgatum), Paraqueiba paraensis (mari), avocado (Persea amencana), Pongamiapinnata (Indian beech), Populus trichocarpa, Ricinus communis (castor), Saccharum sp. (sugarcane), sesame (Sesamumindicum), potato (Solanum tuberosum), Sorghum sp. such as Sorghum bicolor (Sorghum vulgare), Theobroma grandiforum (cupuassu), Trifolium sp., Brazilian needle palm (Trithrinax brasiliensis) palm), Triticum sp. (wheat) such as common wheat (Triticum aestivum), and corn (Zea mays or corn).

[0045] In another embodiment, the plant or part thereof is tobacco, preferably Nicotiana benthamiana or Nicotiana tabacum.

[0046] In another embodiment, the plant or part thereof is a non-Cannabis plant.Alternatively, the plastid is not from algae.

[0047] In one embodiment, at least one polypeptide or polynucleotide is active in the cytosol of said plant.

[0048] In another embodiment, when the plant, part thereof or algae comprises polynucleotides encoding AAE1, OLS and OAC, the polynucleotides are contained in the same or different nucleic acid constructs. Optionally, each polynucleotide is operably linked to a sequence capable of directing the polypeptide encoded by the polynucleotide to the plastid of the plant, algae or part thereof. Optionally, each polynucleotide encodes a fusion polypeptide comprising a plastid transit peptide. Optionally, one, more or all of the polynucleotides are integrated into the plastid genome. In one embodiment, the polynucleotides encoding the OLS and OAC are fused.

[0049] In one embodiment, the one or more polypeptides increase the production of CBGA in the vegetative part of the plant or part thereof. In another embodiment, the production of CBGA is increased by at least about 1.5 to about 2 times, about 2 to about 2.5 times, about 2.5 to about 3.0 times, about 3 to about 3.5 times, about 3.5 to about 4.0 times, about 4 to about 4.5 times, about 4.5 to about 5.0 times, or about 5 to about 5.5 times or more when compared to a wild-type plant, part thereof, algae, or plastid thereof.

[0050] In another embodiment, the production of CBGA is increased by at least about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, about 90% to about 100%, about 100% to about 120%, about 120% to about 140%, about 150% to about 160%, about 170% to about 180%, about 190% to about 200%, about 210% to about 220%, about 230% to about 240%, about 250% to about 260%, about 270% to about 280%, about 290% to about 300%, about 310% to about 310%, about 320% to about 330%, about 340% to about 350%, about 360% to about 370%, about 380% to about 390%, about 400% to about 410%, about 420% to about 430%, about 440% to about 450%, about 460% to about 470%, about 480% to about 490%, about 500% to about 50 ... 140% to about 160%, about 160% to about 180%, about 180% to about 200%, about 200% to about 220%, about 220% to about 240%, about 240% to about 260%, about 260% to about 280%, about 280% to about 300%, about 300% to about 320%, about 320% to about 340%, about 340% to about 360%, about 360% to about 380%, about 400% to about 420%, or more.

[0051] In one embodiment, the increase in the production of CBGA in the plastid, algae, plant or part thereof can be determined by chromatography.

[0052] In one embodiment, the plastid is a chloroplast. In another embodiment, the plastid transport peptide is a chloroplast targeting peptide, preferably a chloroplast transit peptide (CTP). In yet another embodiment, the CTP is a stroma targeting peptide, preferably wherein the stroma targeting peptide is a Rubisco small subunit or a Rubisco large subunit, preferably a Rubisco small subunit. Preferably, the Rubisco small subunit is located at the N-terminus of the polypeptide.

[0053] In one embodiment, the plant, part thereof or algae further comprises a polynucleotide encoding a prenyltransferase, preferably a cannabigerolic acid synthase (CBGAS), wherein the polynucleotide encodes a fusion polypeptide comprising a plastid transit peptide. In another embodiment, the plant, part thereof or algae further comprises at least one polynucleotide selected from the group consisting of a polynucleotide encoding an acyl-lipid thioesterase (ALT4), a polynucleotide encoding a plastidial lipase 1 (PLIP1) and a polynucleotide encoding a tomato 13-lipoxygenase (TomLoxC), optionally wherein the polynucleotide encodes a fusion polypeptide comprising a plastid transit peptide.

[0054] In another embodiment, the plastid further comprises a polypeptide encoded by a prenyltransferase, preferably cannabigerolic acid synthase (CBGAS). In another embodiment, the cannabigerolic acid synthase (CBGAS) is integrated into the plastid genome. In yet another embodiment, the cannabigerolic acid synthase (CBGAS) is expressed in a viral vector. In another embodiment, the plastid further comprises at least one polypeptide selected from the group consisting of: acyl-lipid thioesterase (ALT4), plastidial lipase 1 (PLIP1) and tomato 13-lipoxygenase (TomLoxC).

[0055] In another aspect, the present invention provides a nucleic acid construct encoding a polypeptide for expression in a plant, part thereof or algae, the nucleic acid construct comprising one or more or all of the polynucleotides encoding a polyketide synthase, a polyketide cyclase, an acyl activating enzyme, a prenyl transferase, a plastidial acyl-lipid thioesterase, a plastidial lipase and a plastidial tomato 13-lipoxygenase;

[0056] wherein the polynucleotide is operably linked to a promoter capable of directing expression of one or more or all of the polynucleotides in the plant, part thereof or algae,

[0057] Optionally, wherein the one or more or all of the polynucleotides are operably linked to a polynucleotide encoding a plastid transit peptide;

[0058] Optionally wherein the nucleic acid construct encodes a fusion polypeptide comprising the plastid transit peptide, and

[0059] When expressed, the polypeptide encoded by the polynucleotide increases the production of cannabigerolic acid (CBGA) compared to wild-type plants, parts thereof or algae. Optionally, the polynucleotide increases the production of olivetolic acid (OA) when compared to wild-type plants, parts thereof or algae.

[0060] In one embodiment, the nucleic acid construct is used for expression in the plastid of the plant, part thereof or algae. In another embodiment, the nucleic acid construct comprises a polynucleotide encoding a silencing suppressor polypeptide, preferably a p19 silencing suppressor polypeptide,

[0061] In yet another embodiment, the nucleic acid construct is included in a vector suitable for expression in plants, parts thereof or algae. In another embodiment, the nucleic acid construct is operably linked to a component of a viral vector suitable for expression in plants, parts thereof or algae. In one embodiment, the viral vector is a geminivirus, more preferably the viral vector is BeYDV. In an alternative embodiment, the nucleic acid construct is operably linked to a viral vector replication sequence. In a further embodiment, the viral vector replication sequence comprises a long intergenic region (LIR), a short intergenic sequence (SIR) and Rep / RepA, preferably wherein the viral vector replication sequence is from a geminivirus.

[0062] In one embodiment:

[0063] i) the polynucleotide encoding the polyketide synthase is olivetol synthase (OLS);

[0064] ii) the polynucleotide encoding the acyl-activating enzyme is acyl-activating enzyme 1 (AAE1);

[0065] iii) the polynucleotide encoding the polyketide cyclase is olivetate cyclase (OAC);

[0066] Optionally wherein the nucleic acid construct comprises a polynucleotide encoding a silencing suppression polypeptide, preferably a p19 silencing suppression polypeptide,

[0067] wherein one or more or all of the polynucleotides are operably linked to a promoter capable of directing expression of the polypeptide encoded by the polynucleotide in a plant, part thereof or algae;

[0068] Optionally, wherein the one or more or all of the polynucleotides are operably linked to a polynucleotide encoding a plastid transit peptide; and

[0069] Optionally wherein the nucleic acid construct encodes a fusion polypeptide comprising a plastid transit peptide.

[0070] In one embodiment, the nucleic acid construct is suitable for transient expression in plants, plant parts, algae or their cells, for example, by using an inducible promoter system as described herein or known in the art. In another embodiment, the nucleic acid construct is suitable for stable expression in plants, plant parts, algae or their cells.

[0071] In one embodiment, when the polynucleotide is expressed in a plant or a part thereof, the production of CBGA is increased by at least about 1.5 to about 2 times, about 2 to about 2.5 times, about 2.5 to about 3.0 times, about 3 to about 3.5 times, about 3.5 to about 4.0 times, about 4 to about 4.5 times, about 4.5 to about 5.0 times, or about 5 to about 5.5 times or more compared to a wild-type plant, part thereof, or algae.

[0072] In another embodiment, when the polynucleotide is expressed in a plant or part thereof, the production of CBGA is increased by at least about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, about 90% to about 100%, about 100% to about 120%, about 120% to about 140%, about 150% to about 160%, about 170% to about 180%, about 190% to about 200%, about 210% to about 220%, about 230% to about 240%, about 250% to about 260%, about 270% to about 280%, about 290% to about 300%, about 310% to about 310%, about 320% to about 330%, about 340% to about 350%, about 360% to about 370%, about 380% to about 390%, about 400% to about 410%, about 420% to about 430%, about 440% to about 450%, about 460% to about 470%, about 480% to about 490%, about 500% to about 50 ... About 140%, about 140%-about 160%, about 160%-about 180%, about 180%-about 200%, about 200%-about 220%, about 220%-about 240%, about 240%-about 260%, about 260%-about 280%, about 280%-about 300%, about 300%-about 320%, about 320%-about 340%, about 340%-about 360%, about 360%-about 380%, about 400%-about 420%, or more.

[0073] In another embodiment, the one or more polypeptides are preferably expressed in the vegetative parts and / or seeds of a vascular plant, preferably leaves, more preferably in said chloroplasts of said leaves.

[0074] In another aspect, the present invention provides a fusion polypeptide for expression in plants, parts thereof or algae, the fusion polypeptide comprising:

[0075] i) a polypeptide selected from the group consisting of a polyketide synthase, a polyketide cyclase, an acyl-activating enzyme, a prenyl transferase, a plastidial acyl-lipid thioesterase, a plastidial lipase or a plastidial tomato 13-lipoxygenase; and

[0076] ii) plastid transport sequences,

[0077] Wherein the polypeptide of (i) increases the production of cannabigerolic acid (CBGA) in the presence of caproic acid (C6) and geranyl pyrophosphate (GPP) when compared to wild-type plants or algae. Optionally, the fusion polypeptide increases the production of olivetolic acid (OA) when compared to wild-type plants, parts thereof or algae.

[0078] In one embodiment, i) the polypeptide selected from the group consisting of a polyketide synthase, a polyketide cyclase, an acyl-activating enzyme, a prenyl transferase, a plastidial acyl-lipid thioesterase, a plastidial lipase or a plastidial tomato 13-lipoxygenase is encoded by the polynucleotide, and ii) the plastid trafficking sequence is encoded by a polynucleotide, preferably a polynucleotide encoding a chloroplast transit peptide. In another embodiment, the fusion polypeptide is for expression in the plastid of the plant, its part or algae, preferably for expression in the plastid of the vegetative part and / or seed of a vascular plant, preferably the plastid of a leaf, more preferably the chloroplast of the leaf.

[0079] In one embodiment, the polyketide synthase is olivetol synthase (OLS), the polyketide cyclase is olivetate cyclase (OAC), the acyl activating enzyme is acyl activating enzyme 1 (AAE1), the plastidial acyl-lipid thioesterase is ALT4, the plastidial lipase is PLIP1, the plastidial tomato 13-lipoxygenase is TomLoxC, and the prenyltransferase is cannabigerolic acid synthase (CBGAS), preferably wherein the plastidial trafficking sequence is a chloroplast transit peptide (CTP).

[0080] In another embodiment, one or more or all of the following are true:

[0081] i) the polynucleotide encoding AAE1 comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8% or identical to the sequence shown in any one of SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7;

[0082] ii) the polynucleotide encoding OLS comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8% or identical to the sequence shown in any one of SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10;

[0083] iii) the polynucleotide encoding OAC comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8% or identical to the sequence shown in any one of SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13;

[0084] iv) the polynucleotide encoding the plastid transit peptide comprises a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8% or identical to the sequence shown in SEQ ID NO:15.

[0085] In another embodiment, the polynucleotide encoding the isoprenyl transferase comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8% or identical to the sequence shown in SEQ ID NO:14.

[0086] In another embodiment,

[0087] i) the polynucleotide encoding ALT4 comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8% or identical to the sequence shown in any one of SEQ ID NO: 24 or SEQ ID NO: 25;

[0088] ii) the polynucleotide encoding PLIP1 comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8% or identical to the sequence shown in SEQ ID NO: 26; and

[0089] iii) the polynucleotide encoding TomLoxC comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8% or identical to the sequence shown in SEQ ID NO:27.

[0090] In another aspect, the present invention provides a plant, part thereof, algae, plastid or cell thereof, comprising the polypeptide or fusion polypeptide of the present invention.

[0091] In another aspect, the invention provides a method for producing cannabigerolic acid (CBGA) in a plant, part thereof or algae, the method comprising culturing a plant, part thereof or algae comprising a nucleic acid construct of the invention under conditions sufficient to express the polypeptide in the plant, part thereof or algae, wherein the polypeptide increases the yield of cannabigerolic acid (CBGA) in the plant, part thereof or algae when expressed in the presence of caproic acid (C6) and geranyl pyrophosphate (GPP) when compared to a wild-type plant, part thereof or algae. Optionally, the method further comprises introducing the nucleic acid construct of the invention into the plant, part thereof or algae. Optionally, the method produces olivetonic acid (OA) when compared to a wild-type plant, part thereof or algae.

[0092] In another aspect, the invention provides a method for producing olivetic acid (OA) in a plant, part thereof or algae, the method comprising culturing a plant, part thereof or algae comprising a nucleic acid construct of the invention under conditions sufficient to express a polypeptide in the plant, part thereof or algae, wherein OA is produced in the plant, part thereof or algae when expressed in the presence of olivetol synthase (OAS) and / or olivetate cyclase (OAC) when compared to a wild-type plant, part thereof or algae.

[0093] In another aspect, the invention provides a method for producing cannabigerolic acid (CBGA) in a plant, part thereof or algae, the method comprising culturing a plant, part thereof or algae comprising a nucleic acid construct of the invention under conditions sufficient for expression of the polypeptide in the plant, part thereof or algae, wherein CBGA is produced in the plant, part thereof or algae when expressed in the presence of cannabigerolic acid synthase (CBGAS) when compared to a wild-type plant, part thereof or algae.

[0094] In another aspect, the invention provides a method for producing C6-CoA from C6 in a plant, part thereof or algae, the method comprising culturing a plant, part thereof or algae comprising a nucleic acid construct of the invention under conditions sufficient to express one or more polypeptides in the plant, part thereof or algae, wherein C6-CoA is produced in the plant, part thereof or algae when expressed in the presence of acyl activating enzyme 1 (AAE1) when compared to a wild-type plant, part thereof or algae.

[0095] In one embodiment, the method further comprises providing exogenous C6, malonyl-CoA and / or GPP to the plant, part thereof or algae, optionally in the form of a plant feed, an amino acid supplement or a C6 and / or GPP substrate.

[0096] In one embodiment, one or more polypeptides described herein are expressed in the plastids of the plant, part thereof or algae.

[0097] In one embodiment, the CBGA is produced in the vegetative parts of the plant and / or seed, preferably wherein the vegetative parts are aerial parts including leaves of the plant. In one embodiment, the increase in the yield of CBGA in the vegetative parts of the plant can be determined by chromatography.

[0098] In one embodiment, the method further comprises producing cannabinoids, the method comprising:

[0099] i) recovering the CBGA produced by the method of the present invention; and

[0100] ii) optionally feeding said CBGA to a modified microorganism, preferably a yeast or a bacterium; and

[0101] iii) optionally subjecting said CBGA to a chemical, physical or biochemical transformation using said CBGA as a starting material, thereby producing said cannabinoid.

[0102] In one embodiment, the method comprises producing at least one of cannabigerol (CBG), tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA) and cannabichromene acid (CBCA), preferably cannabidiolic acid (CBDA), optionally comprising producing tetrahydrocannabinol (THC), cannabidiol (CBD), cannabinol (CBN) and cannabichromene (CBC), preferably cannabigerol (CBG) or cannabidiol (CBD).

[0103] In another embodiment, the method comprises a CBGA decarboxylation step after recovering the CBGA-containing extract, preferably by incubating the CBGA with heat or light.

[0104] In another aspect, the invention provides cannabigerol (CBG) produced by the methods of the invention.

[0105] In another aspect, the present invention provides cannabigerolic acid (CBGA) produced by the methods of the present invention. In one embodiment, the CBGA is substantially glycosylated. In another embodiment, the CBGA is substantially free of glycosylation.

[0106] In another aspect, the present invention provides cannabidiolic acid (CBDA) or cannabidiol (CBD) produced by the methods of the present invention.

[0107] In another aspect, the present invention provides cannabichromene acid (CBCA) or cannabichromene (CBC) produced by the method of the present invention.

[0108] In another aspect, the present invention provides olivetic acid (OA) produced by the method of the present invention.

[0109] In another aspect, the present invention provides tetrahydrocannabinolic acid (THCA) or tetrahydrocannabinol (THC) produced by the method of the present invention.

[0110] In another aspect, the present invention provides a method for producing a genetically modified plant, the method comprising:

[0111] i) regenerating a plant from a plant cell transformed with the nucleic acid construct of the present invention;

[0112] ii) optionally collecting seeds from said plant; and / or

[0113] iii) optionally producing one or more progeny plants from the genetically modified plant, thereby producing the genetically modified plant.

[0114] In another aspect, the present invention provides a method for producing a genetically modified seed, the method comprising:

[0115] i) regenerating a plant from a plant cell transformed with the nucleic acid construct of the present invention; and

[0116] ii) collecting seeds from said plants, thereby producing genetically modified seeds.

[0117] In one embodiment, the method further comprises introducing the nucleic acid construct of the present invention into the plant or cell thereof.

[0118] In one embodiment, the encoded polypeptide is expressed in the vegetative parts, preferably aerial vegetative parts, more preferably the leaves of the plant. In another embodiment, the plant is a vascular plant, preferably a vascular plant of the genus Nicotiana, more preferably Nicotiana benthamiana or Nicotiana tabacum.

[0119] In another aspect, the present invention provides a method for producing a genetically modified plant or algae, the method comprising:

[0120] i) crossing two parental plants or algae, wherein at least one of the plants or algae comprises a genetic modification introduced by a nucleic acid construct of the invention;

[0121] ii) screening one or more progeny plants or algae from the cross in i) for the presence or absence of the genetic modification; and

[0122] iii) selecting a progeny plant or algae comprising said genetic modification, thereby producing said plant or algae.

[0123] In one embodiment, the method further comprises:

[0124] iv) crossing a first genetically modified parent plant with a second parent plant, wherein the first genetically modified parent plant is a plant comprising a genetic modification introduced by a nucleic acid construct as described herein; and

[0125] v) backcrossing the progeny plant of said cross of step (iv) with a plant of the same genotype as said second parent plant to produce a plant having most of the genotype of said second parent and including said genetic modification.

[0126] In one embodiment, said vegetative part or aerotrophic part is capable of photosynthesis, preferably wherein said leaves of said plant comprise said genetic modification.

[0127] On the other hand, the present invention provides a genetically modified and / or recombinant plant or algae cell for producing cannabigerolic acid (CBGA), comprising a nucleic acid construct of the present invention. Optionally, the genetically modified and / or recombinant plant or algae cell produces olive acid (OA).

[0128] In another aspect, the present invention provides a genetically modified plant, part thereof or algae for producing cannabigerolic acid (CBGA), comprising a nucleic acid construct of the present invention. Optionally, the genetically modified plant, part thereof or algae produces olive acid (OA).

[0129] In another aspect, the present invention provides a plant part of the genetically modified plant of the present invention. Optionally, the plant part is a seed comprising the genetic modification.

[0130] In another aspect, the present invention provides a method for producing a plant part from a genetically modified plant, the method comprising:

[0131] i) growing a plant according to the method of the present invention; and

[0132] ii) collecting said plant parts.

[0133] In another aspect, the present invention provides a use of a nucleic acid construct of the present invention for producing a recombinant plant or algal cell and / or a genetically modified plant and / or algae.

[0134] In another aspect, the present invention provides a crop or plant population or algae comprising:

[0135] (i) a plant or algae, a plastid, a nucleic acid construct or a fusion polypeptide of the invention; or

[0136] (ii) plant or algae, plastid, nucleic acid construct or fusion polypeptide and wild-type plant or algae of the present invention. In one embodiment, the crop or plant population comprises a vegetative plant part, preferably a vascular plant, more preferably Nicotiana, most preferably a vegetative plant part of Nicotiana benthamiana or a common tobacco plant. In a further embodiment, the crop or plant population further comprises a non-vascular plant.

[0137] In one embodiment, the plant or algae comprises a higher yield of CBGA, optionally OA, when compared to a wild-type plant or algae, wherein the CBGA and / or OA are optionally determinable by chromatography.

[0138] In another aspect, the present invention provides an industrial plant biomass obtained from leaves of a genetically modified plant of the present invention, wherein said leaves of said plant biomass comprise a higher yield of CBGA, optionally OA, optionally determinable by chromatography, when compared to leaves of a wild-type plant.

[0139] In another aspect, the present invention provides a composition comprising the plasmid, nucleic acid construct or plant cell of the present invention and one or more acceptable carriers.

[0140] In another aspect, a method for identifying a polynucleotide encoding a polypeptide for producing cannabigerolic acid (CBGA), preferably OA, in a plant, part thereof, algae, or plastid or cell thereof is provided, the method comprising:

[0141] i) obtaining the nucleic acid construct of the present invention;

[0142] ii) introducing the nucleic acid construct into a plant, part thereof, algae or plastid or cell thereof;

[0143] iii) determining whether the level of CBGA, preferably OA, is increased relative to a corresponding wild type plant, part thereof, algae or plastid or cell thereof lacking said nucleic acid construct; and

[0144] iv) Optionally, selecting a polynucleotide which, when expressed, produces a polypeptide suitable for producing CBGA, preferably OA. Preferably, said CBGA is produced in the plastids of said vegetative part of said plant, optionally as can be determined by chromatography.

[0145] In another aspect, there is provided a method for producing an industrial product, the method comprising the steps of:

[0146] (i) obtaining the plant, part thereof, algae or plastid of the present invention;

[0147] (ii) treating the plant, part thereof, algae or plastid;

[0148] (iii) converting at least some of the cannabinoids in the plant, part thereof, algae or plastid of step (i), or the treated plant, part thereof, algae or plastid obtained by step (ii), to in situ cannabinoids by applying thermal, chemical or enzymatic means or any combination thereof; and

[0149] (iv) recovering said cannabinoids, thereby producing said industrial product.

[0150] In a preferred embodiment, the industrial product is cannabigerolic acid (CBGA) or olive acid (OA). In a preferred embodiment, the industrial product is cannabigerolic acid (CBGA) or cannabigerol (CBG).

[0151] In another aspect, a method for producing extracted cannabigerolic acid (CBGA) is provided, the method comprising the steps of:

[0152] (i) obtaining the plant, part thereof, algae or plastid of the present invention;

[0153] (ii) extracting CBGA from the plant, part thereof, algae or plastid; and

[0154] (iii) recovering the extracted CBGA, thereby producing extracted CBGA.

[0155] Optionally, the extracted CBGA includes olivetic acid (OA).

[0156] In one embodiment, the extraction process of step (ii) includes one or more or all of the following: (a) grinding of the dried material; (b) solvent (e.g., ethanol, hydrocarbon) or supercritical CO2 extraction; (c) winterization and filtration; (d) drying; (e) distillation; and / or (f) chromatography.

[0157] In another aspect, a method for producing an extract enriched in cannabigerolic acid (CBGA) is provided, the method comprising the steps of:

[0158] (i) obtaining the plant, part thereof, algae or plastid of the present invention;

[0159] (ii) treating the plant, part thereof, algae or plastid of step (i);

[0160] (iii) centrifuging the treated plant, part thereof, algae or plastid from step (ii) to obtain an extract;

[0161] (iv) filtering the extract obtained by step (iii);

[0162] (v) drying the filtered extract of step (iv) and resuspending it in a buffer solution;

[0163] (vi) purifying the extract of step (v) using a solid phase extraction column; and

[0164] (vii) recovering an extract rich in CBGA,

[0165] Thereby producing the CBGA-enriched extract. Optionally, the extracted CBGA comprises olivetic acid (OA).

[0166] In another aspect, there is provided a method for producing a pharmaceutical composition, the method comprising:

[0167] (i) obtaining the extract or cannabinoid of the present invention; and

[0168] (ii) formulating the extract or cannabinoid with one or more pharmaceutically acceptable carriers,

[0169] A pharmaceutical composition is thereby produced.

[0170] In another aspect, a pharmaceutical composition obtained by the method of the present invention is provided.

[0171] In another aspect, a method of treating and / or preventing a condition or disease responsive to cannabinoid treatment in a subject is provided, the method comprising administering to the subject a therapeutically effective amount of a cannabinoid, an extract or a pharmaceutical composition of the invention, thereby treating and / or preventing a condition or disease responsive to cannabinoid treatment.

[0172] In another aspect, there is provided the use of a therapeutically effective amount of a cannabinoid of the invention, an extract of the invention or a pharmaceutical composition of the invention for the preparation of a medicament for treating a condition or disease responsive to cannabinoid treatment.

[0173] In another aspect, there is provided a therapeutically effective amount of a cannabinoid of the invention, an extract of the invention or a pharmaceutical composition of the invention for use in treating a condition or disease responsive to cannabinoid treatment.

[0174] In one embodiment, the condition or disease is selected from the group consisting of: chronic pain, neuropathic pain, cancer, nausea and / or vomiting associated with cancer chemotherapy, loss of appetite, multiple sclerosis, spasticity associated with multiple sclerosis or spinal cord injury epilepsy, Parkinson's disease, anorexia and / or weight loss, irritable bowel syndrome, Tourette syndrome, amyotrophic lateral sclerosis, Huntington's disease, dystonia, dementia, glaucoma, dermatitis, acne, microbial infection, traumatic brain injury and / or intracranial hemorrhage, addiction, anxiety, depression, sleep disorders, post-traumatic stress disorder, microbial infection including methicillin-resistant Staphylococcus aureus (MRSA) and biofilm-producing organisms, dermatitis, acne, schizophrenia and other psychiatric disorders.

[0175] In another aspect, there is provided a use of a plant, part thereof, algae or plastid of the invention for the preparation of an industrial product, preferably a cannabinoid, more preferably cannabigerolic acid (CBGA) and / or cannabigerol (CBG).

[0176] Unless explicitly stated otherwise, any embodiment herein should be considered applicable to any other embodiment mutatis mutandis.

[0177] The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for purposes of illustration only. Functionally equivalent products, compositions and methods, as described herein, are clearly within the scope of the present invention.

[0178] Throughout this specification, references to individual steps, compositions of matter, groups of steps, or groups of compositions of matter shall be deemed to cover both one and more (i.e., one or more) of such steps, compositions of matter, groups of steps, or groups of compositions of matter, unless expressly stated otherwise or the context requires otherwise.

[0179] The invention is hereinafter described by way of the following non-limiting examples and with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0180] Figure 1 .Cannabinoid biosynthetic pathway in cannabis trichomes.

[0181] Figure 2 OA glycosylation in infiltrated N. benthamiana leaves. A LC-MS chromatogram of OA standard. B LC-MS chromatogram of N. benthamiana leaf extract after infiltration with OA.

[0182] Figure 3CBGA glycosylation in infiltrated N. benthamiana leaves. A LC-MS chromatogram of CBGA standard. B LC-MS chromatogram of N. benthamiana leaves after infiltration with CBGA.

[0183] Figure 4 .Reconstruction of a functional CBGA biosynthetic pathway across both the cytosol and chloroplasts in Nicotiana benthamiana.

[0184] Figure 5 .Reconstitution of a functional CBGA biosynthetic pathway in Nicotiana benthamiana in chloroplasts.

[0185] Figure 6 OA biosynthesis in Nicotiana benthamiana leaves. CsAAE1, CsOLS, CsOAC, and CsCBGAS were co-infiltrated with C6 and GPP as substrates for the reconstituted cannabinoid biosynthetic pathway.

[0186] Figure 7 Biosynthesis of glycosylated OA in Nicotiana benthamiana leaves. CsAAE1, CsOLS, CsOAC, and CsCBGAS were co-permeated with C6 and GPP as substrates for the reconstituted cannabinoid biosynthetic pathway.

[0187] Figure 8 CBGA biosynthesis in Nicotiana benthamiana leaves. CsAAE1, CsOLS, CsOAC, and CsCBGAS were co-infiltrated with C6 and GPP as substrates for the reconstituted cannabinoid biosynthetic pathway.

[0188] Fig. 9 Biosynthesis of glycosylated CBGA in Nicotiana benthamiana leaves. CsAAE1, CsOLS, CsOAC, and CsCBGAS were co-permeated with C6 and GPP as substrates for the reconstituted cannabinoid biosynthetic pathway.

[0189] Fig.10 Subcellular localization of CsAAE1, CsOLS and CsOAC in Nicotiana benthamiana. The gene was fused to the coding sequence of mNeonGreen fluorescent protein at the 3' end and contained either the Rubisco SSU chloroplast targeting sequence ('tp') (left two columns) or the hemagglutinin epitope ('HA') (right two columns) as a fusion at the 5' end. For each construct (line), the left panel shows mNeonGreen fluorescence, and the right panel shows mNeonGreen fluorescence and chloroplast autofluorescence.

[0190] Fig.11.Levels of OA (A) and CBGA (B) in Nicotiana benthamiana leaves. CsAAE1, CsOLS, CsOAC and CsCBGAS were co-expressed in the cytosol using a standard binary expression vector (Cyto), targeted to the chloroplast using a standard binary expression vector (Plast), localized in the cytosol using a deconstructed viral expression vector (Cyto VV), or targeted to the chloroplast using a deconstructed viral vector (Plast VV). OA and CBGA biosynthetic genes were co-infiltrated with C6 and GPP as substrates for the recombinant cannabinoid biosynthetic pathway. Both free forms and glycosylated derivatives of OA and CBGA were detected. P19+GFP, negative control. The relative amounts of the products are shown as peak areas.

[0191] Fig.12 LC-MS chromatograms of Nicotiana benthamiana leaves infiltrated with BeYDV-adapted expression vectors containing the coding sequences of OA and CBGA biosynthetic genes. A, C Cytoplasmic localization of CsAAE1, CsOLS and CsOAC. B, D Chloroplast targeting of CsAAE1, CsOLS and CsOAC.

[0192] Fig.13 . Biosynthesis of OA in Nicotiana benthamiana leaves. Chloroplast targeting of CsAAE1, CsOLS and CsOAC was co-expressed with C6 substrate (Plast VV) or with AtALT4 (Plast VV+ALT), but not with exogenous C6. Both free OA forms and glycosylated derivative OA forms were detected. P19+GFP, negative control. Relative amounts of products are shown as peak areas and are based on four independent biological replicates.

[0193] Key to sequence listing

[0194] SEQ ID NO: 1—Amino acid sequence of the cannabinoid enzyme CsAAE1.

[0195] SEQ ID NO:2—Amino acid sequence of the cannabinoid enzyme CsOLS.

[0196] SEQ ID NO:3—Amino acid sequence of the cannabinoid enzyme CsOAC.

[0197] SEQ ID NO:4—Amino acid sequence of the cannabinoid enzyme CsCBGAS.

[0198] SEQ ID NO:5—Codon-optimized nucleotide sequence of the cannabinoid enzyme CsAAE1 designed as a GG class 0 module without N- or C-terminal fusions.

[0199] SEQ ID NO:6—Codon-optimized nucleotide sequence of the cannabinoid enzyme CsAAE1 designed as a GG class 0 module for N-terminal fusion.

[0200] SEQ ID NO:7—Codon-optimized nucleotide sequence of the cannabinoid enzyme CsAAE1 designed as a GG class 0 module for N- and C-terminal fusions.

[0201] SEQ ID NO:8—Codon-optimized nucleotide sequence of the cannabinoid enzyme CsOLS, designed as a GG class 0 module without N- or C-terminal fusions.

[0202] SEQ ID NO:9—Codon-optimized nucleotide sequence of the cannabinoid enzyme CsOLS, designed as a GG class 0 module for N-terminal fusion.

[0203] SEQ ID NO: 10—Codon-optimized nucleotide sequence of the cannabinoid enzyme CsOLS, designed as a GG class 0 module for N- and C-terminal fusions.

[0204] SEQ ID NO: 11 - Codon-optimized nucleotide sequence of the cannabinoid enzyme CsOAC, designed as a GG 0-level module without N- or C-terminal fusions.

[0205] SEQ ID NO: 12—Codon-optimized nucleotide sequence of the cannabinoid enzyme CsOAC, designed as a GG class 0 module for N-terminal fusion.

[0206] SEQ ID NO: 13 - Codon-optimized nucleotide sequence of the cannabinoid enzyme CsOAC, designed as a GG class 0 module for N- and C-terminal fusions.

[0207] SEQ ID NO: 14 - Codon-optimized nucleotide sequence of the cannabinoid enzyme CsCBGAS, designed as a GG class 0 module without N- or C-terminal fusions.

[0208] SEQ ID NO: 15—Nucleotide sequence encoding Rubisco small subunit targeting peptide; designed as a GG class 0 module as an N-terminal fusion.

[0209] SEQ ID NO: 16—Nucleotide sequence encoding mNeonGreen; designed as a GG level 0 module as a C-terminal fusion.

[0210] SEQ ID NO: 17—Nucleotide sequence encoding EN38510; designed as a GG class 0 promoter module, the long intergenic region (LIR) sequence is underlined.

[0211] SEQ ID NO: 18—Nucleotide sequence encoding EN38509; designed as a GG class 0 5'UTR module.

[0212] SEQ ID NO: 19—Nucleotide sequence encoding EN38511; designed as a GG class 0 terminator module, LIR sequence is underlined, SIR sequence is bold, Rep / RepA sequence is italicized.

[0213] SEQ ID NO:20—Amino acid sequence of the Arabidopsis thaliana enzyme AtALT4.1.

[0214] SEQ ID NO:21—Amino acid sequence of the Arabidopsis thaliana enzyme AtALT4.2.

[0215] SEQ ID NO:22—Amino acid sequence of the Arabidopsis enzyme AtPLIP1.

[0216] SEQ ID NO:23—Amino acid sequence of TomloxC.

[0217] SEQ ID NO:24—Codon-optimized nucleotide sequence of the Arabidopsis thaliana enzyme AtALT4.1 designed as a GG class 0 module without N- or C-terminal fusions.

[0218] SEQ ID NO:25—Codon-optimized nucleotide sequence of the Arabidopsis thaliana enzyme AtALT4.2, designed as a GG class 0 module without N- or C-terminal fusions.

[0219] SEQ ID NO:26—Codon-optimized nucleotide sequence of the Arabidopsis thaliana enzyme AtPLIP1 designed as a GG class 0 module without N- or C-terminal fusions.

[0220] SEQ ID NO:27 - Codon-optimized nucleotide sequence of TomloxC designed as a GG class 0 module without N- or C-terminal fusions.

[0221] SEQ ID NO:28—Nucleotide sequence encoding EN38113 hemagglutinin epitope; designed as a GG class 0 module as an N-terminal fusion. DETAILED DESCRIPTION

[0222] General techniques and definitions

[0223] Unless specifically defined otherwise, all technical and scientific terms used herein should be construed to have the same meanings as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, plant molecular biology, phytocannabinoid synthesis, protein chemistry, and biochemistry).

[0224] Unless otherwise indicated, the recombinant polynucleotide, polypeptide, cell and plant culture and immunological techniques utilized in the present invention are standard procedures well known to those skilled in the art. Such techniques are described and explained in the literature in sources such as J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984); J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989); T. A. Brown (ed.), Essential Molecular Biology: A Practical Approach), Vols. 1 and 2, IRL Press (1991); D. M. Glover and B. D. Hames (eds.), DNA Cloning: A Practical Approach, Vols. 1-4, IRL Press (1995 and 1996); and F. M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date) and J. E. Coligan et al. (eds.), Current Protocols in Molecular Biology. Protocols in Immunology, John Wiley & Sons (contains all updates to date).

[0225] The term "and / or", such as "X and / or Y" should be understood to mean "X and Y" or "X or Y", and should be considered to provide clear support for both meanings or either meaning.

[0226] Throughout this specification, the word "comprise" or variations such as "comprises" or "comprising" should be understood to imply the inclusion of stated elements, integers or steps or groups of elements, integers or steps, but not the exclusion of any other elements, integers or steps or groups of elements, integers or steps.

[0227] The general use of the term "about" and ranges, whether or not qualified by the term about, means that the quantities understood are not limited to the exact quantities shown herein, and are intended to refer to ranges substantially within the referenced ranges without departing from the scope of the invention. As used herein, "about" will be understood by one of ordinary skill in the art and will vary to some extent depending on the context in which it is used. If there is a use of a term that is unclear to one of ordinary skill in the art, "about" will mean up to ±10%, more preferably ±5%, more preferably 1% of the particular term, taking into account the context in which the term is used.

[0228] As used herein, "wild type" refers to a cell, plastid, tissue or plant that has not been modified according to the present invention. For example, in one embodiment, a genetically modified plant or part thereof is provided, which includes a polynucleotide encoding acyl activating enzyme 1 (AAE1), a polynucleotide encoding olive alcohol synthase (OLS) and a polynucleotide encoding olivetate cyclase (OAC), which, when expressed, can increase the production of cannabigerolic acid (CBGA) compared to a wild-type plant or part thereof. In this embodiment, the wild-type plant does not include a polynucleotide encoding acyl activating enzyme 1 (AAE1), a polynucleotide encoding olive alcohol synthase (OLS) and a polynucleotide encoding olivetate cyclase (OAC), but will be similar in other aspects in genotype and phenotype when compared to the genetically modified plant or part thereof. Wild-type cells, plastids, tissues or plants can be used as controls to compare the expression level of exogenous nucleic acids or the degree and nature of trait modification with modified cells, plastids, tissues or plants as described herein.

[0229] Cannabinoid biosynthetic pathway

[0230] In cannabis, cannabinoids are produced from the common metabolite precursors geranyl pyrophosphate (GPP) and caproyl-CoA through the action of three polypeptides. Specifically, caproyl-CoA and malonyl-CoA are combined by butenone synthase (TKS) (olive acid synthase; OAS) polypeptides to form a 12-carbon butenone intermediate. The butenone intermediate is then cyclized by the olivetate cyclase (OAC) polypeptide to produce olivetate (OA). OA is then isoprenylated with the common isoprenoid precursor GPP by a geranyl transferase polypeptide (e.g., CsPT4 polypeptide) to produce cannabigerolic acid (CBGA). Various synthase polypeptides then convert CBGA into other cannabinoids. For example, tetrahydrocannabinolic acid (THCA) synthase produces THCA and cannabidiolic acid (CBDA) synthase produces CBDA. In the presence of heat or light, the acidic cannabinoids undergo decarboxylation, thereby producing cannabigerol (CBG) from CBGA, tetrahydrocannabidiol (THC) from THCA and cannabichromene (CBD) from CBDA.

[0231] Geranyl pyrophosphate (GPP) and hexanoyl-CoA can be produced by several pathways, including through amino acid supplementation or through the mevalonate (MVA) pathway. The term "mevalonate pathway" or "MVA pathway" refers to a biosynthetic pathway that converts acetyl-CoA to isopentenyl pyrophosphate (IPP) and dimethyl allyl pyrophosphate (DMAPP). The mevalonate pathway includes polypeptides that catalyze the following steps: (a) condensing two acetyl-CoA molecules to produce acetoacetyl-CoA (e.g., by the action of an acetoacetyl-CoA thiolase polypeptide); (b) condensing acetoacetyl-CoA with acetyl-CoA to form hydroxymethylglutaryl-CoA (HMG-CoA) (e.g., by the action of an HMG-CoA synthase (HMGS) polypeptide); (c) converting HMG-CoA to mevalonate (e.g., by the action of an HMGCoA reductase (HMGR) polypeptide); (d) phosphorylating mevalonate to 5-hydroxymethylglutaryl-CoA (HMG-CoA). (e) converting 5-phosphomevalonate to 5-pyrophosphate mevalonate (e.g., by the action of a mevalonate kinase (MK) polypeptide); (e) converting 5-phosphomevalonate to 5-pyrophosphate mevalonate (e.g., by the action of a phosphomevalonate kinase (PMK) polypeptide); (f) converting 5-pyrophosphate mevalonate to isopentenyl pyrophosphate (e.g., by the action of a mevalonate pyrophosphate decarboxylase (MVD) polypeptide); and (g) converting isopentenyl pyrophosphate (IPP) to dimethylallyl pyrophosphate (DMAPP) (e.g., by the action of an isopentenyl pyrophosphate isomerase (IDI) polypeptide). A geranyl diphosphate synthase (GPPS) polypeptide then acts on IPP and / or DMAPP to produce GPP. Additionally, the polypeptide that produces GPP or is part of a biosynthetic pathway that produces GPP can be one or more polypeptides that have at least one activity of a polypeptide present in the deoxyxylulose-5-phosphate (DXP) pathway, but not at least one activity of a polypeptide present in the MVA pathway.

[0232] In addition to the MVA pathway located in the cytosol, ER, and peroxisomes, these intermediates can also originate from the recently identified methylerythritol 4-phosphate (MEP) pathway located in plastids (Hemmerlin et al., 2012).

[0233] Hexanoyl-CoA can be produced by polypeptides that produce acyl-CoA compounds or acyl-CoA compound derivatives (e.g., hexanoyl-CoA synthase (HCS) polypeptides, acyl activating enzyme polypeptides, fatty acyl-CoA synthase polypeptides, or fatty acyl-CoA ligase polypeptides). Hexanoyl-CoA can also be produced by pathways that include one or more malonyl-CoA-producing polypeptides, such as acetyl-CoA carboxylase (ACC) polypeptides. Additionally, hexanoyl-CoA can be produced by polypeptides that are part of a biosynthetic pathway that produces hexanoyl-CoA, including but not limited to: malonyl-CoA-acyl carrier protein transacylase (MCT1) polypeptides, PaaH1 polypeptides, Crt polypeptides, Ter polypeptides, and BktB polypeptides; MCT1 polypeptides, PhaB polypeptides, PhaJ polypeptides, Ter polypeptides, and BktB polypeptides; short chain fatty acyl-CoA thioesterase (SCFA-TE) polypeptides; or fatty acid synthase (FAS) polypeptides. Hexanoyl-CoA derivatives, acyl-CoA compounds, or acyl-CoA compound derivatives can also be formed via such pathways and polypeptides.

[0234] The malonyl-CoA intermediate is involved in many metabolic processes; it is a substrate in acylation and condensation reactions and is therefore found in plastids, cytosol and mitochondria. It is prepared by acetyl-CoA carboxylation in plastids and cytosol, and is mainly produced by the cytosolic ATP-dependent acetyl-CoA carboxylase (ACCase) malonyl-CoA synthase (Guan et al. (2016)).

[0235] GPP and hexanoyl-CoA can also be produced by a pathway including a polypeptide that condenses two acetyl-CoA molecules to produce acetoacetyl-CoA and a pyruvate dehydrogenase complex polypeptide that produces acetyl-CoA from pyruvate. Hexanoyl-CoA derivatives, acyl-CoA compounds, or acyl-CoA compound derivatives can also be formed by such pathways. Figure 1 An exemplary cannabinoid biosynthetic pathway from cannabis is shown.

[0236] The term "cannabinoid" refers to a group of closely related compounds that include cannabinol and the active ingredients of cannabis, and may include cannabinoid derivatives, cannabinoid precursors or cannabinoid precursor derivatives. Cannabinoids can be in acid form or non-acid form, the latter also known as decarboxylated form, because the non-acid form can be produced by decarboxylating the acid form.

[0237] Cannabinoids may include, but are not limited to, cannabichromene (CBC) types (e.g., cannabichromene acid), cannabigerol (CBG) types (e.g., cannabigerolic acid), cannabichromene (CBD) types (e.g., cannabigerolic acid), Δ9-trans-tetrahydrocannabinol (Δ9-THC) types (e.g., Δ9-tetrahydrocannabinolic acid), Δ8-trans-tetrahydrocannabinol (Δ8-THC) types, cannabicycline (CBL) types, cannabichromene (CBE) types, cannabinol (CBN) types, cannabidiol (CBND) types, cannabitriol (CBT) types, cannabigerolic acid (CBGA), cannabigerolic acid monomethyl ether (CBGAM), cannabigerol (CBG), cannabigerol monomethyl ether (CBGM), cannabigerolic acid (CBGV A), cannabigerol (CBGV), cannabichromene (CBCA), cannabichromene (CBC), cannabigerolic acid (CBCV A), cannabichromene (CBCV), cannabidiolic acid (CBDA), cannabichromene (CBD), cannabichromene monomethyl ether (CBDM), cannabichromene-C4 (CBD-C4), cannabidiolic acid (CBDV A), cannabichromene (CBDV), cannabidiolic acid (CBD-C1), Δ9-tetrahydrocannabinolic acid A (THCA-A), Δ9-tetrahydrocannabinolic acid B (THCA-B), Δ9-tetrahydrocannabinol (THC), Δ9-tetrahydrocannabinolic acid-C4 (THCA-C4), Δ9-tetrahydrocannabinol-C4 (THC-C4), Δ9-tetrahydrocannabinolic acid (THCVA), Δ9-tetrahydrocannabinol (THCV), Δ9-tetrahydrocannabinol (TH CA-C1), Δ9-tetrahydrocannabinol (THC-C1), Δ7-cis-isotetrahydrocannabinol, Δ8-tetrahydrocannabinolic acid (Δ8-THCA), Δ8-hydrocannabinol (Δ8-THC), cannabicyclic acid (CBLA), cannabicyclyl (CBL), cannabinol (CBLV), cannabielsoic acid A (CBEA-A), cannabielsoic acid B (CBEAB), cannabinoids (CBE), cannabielsoic acid (cannabielsoic acid acid), cannabidipyran cycloalkanoic acid (cannabicitranic acid), cannabinolic acid (CBNA), cannabinol (CBN), cannabinol methyl ether (CBNM), cannabinol-C4 (CBN-C4), cannabinol (CBV), cannabinol-C2 (CNB-C2), cannabidiol (CBN-C1), cannabidiol (CBND), cannabidiol (CBVD), cannabitriol (CBT), 10-ethoxy-9-hydroxy-delta-6a tetrahydrocannabinol, 8,9-dihydroxy-δ-6a-tetrahydrocannabinol, cannabidiol vesiculoyl (CBTVE), dehydrocannabidifuran (DCBF), cannabifuran (CBF), cannabichromone (CBCN), cannabidipyranocene (CBT), 10-oxo-δ-6a-tetrahydrocannabinol (OTHC), Δ9-cis-tetrahydrocannabinol (cis-THC), 3,4,5,6-tetrahydro-7-hydroxy-α-α-2-trimethyl-9-n-propyl-2,6-methylene-2H-1-benzoxazin-5-methanol (OH-iso-HHCV), cannabinoid resin (CBR) and trihydroxy-δ-9-tetrahydrocannabinol (triOH-THC).

[0238] In one embodiment, the present invention relates to the production of OA and / or CBGA, which can be processed into any of the cannabinoids described above, preferably CBG, THC, CBD and CBN, most preferably CBG and CBD, by suitable methods described herein or known in the art.

[0239] As disclosed herein, the increased cannabinoid production produced by genetically engineered plants, algae or plant cells may be the result of increased expression of one or more enzymes associated with the cannabinoid biosynthetic pathway. Increasing the expression of an enzyme in a plant, algae or plant cell may include, for example, introducing a polynucleotide sequence encoding the enzyme into a plant, algae, part thereof, plastid or plant cell. Increasing expression can be achieved by increasing the copy number of one, two or all of the genes in the pathway, thereby inducing a gene dosage effect, such as seen in plants expressing endogenous biosynthetic pathways, where the introduction of additional copies of the gene contributes to a greater accumulation of the desired product content in the plant. One example involves increasing the expression of a polynucleotide sequence encoding a polypeptide described herein in cannabis by inducing a sufficient gene dosage effect, such that expression produces, for example, greater accumulation of CBGA or OA in the plant. In one embodiment, the introduction of a polynucleotide sequence encoding an enzyme can be accomplished by transformation or other methods known in the art or described herein. The polynucleotide can be introduced under the control of an inducible or developmentally triggered promoter or a constitutively expressed promoter, the promoter targeting a plant part, such as a trichome, leaf or stem expression. The polynucleotide sequence may be introduced into the nuclear genome, the plastid genome or present in a vector such as a geminivirus vector or a plasmid containing a geminivirus sequence or replication machinery. Examples of suitable cannabinoid biosynthetic pathway enzymes include, but are not limited to, hexanoyl-CoA synthase, acetyl-CoA carboxylase, MVA type III polyketide synthase (e.g., butenone synthase (TKS), Steely 1 and Steely 2), OAC, geranyl pyrophosphate synthase (GPPS), aromatic prenyltransferase, geranyl pyrophosphate: olivetate geranyltransferase, cannabichromene synthase, tetrahydrocannabinolic acid synthase and cannabidiolic acid synthase.

[0240] Apart from Figure 1 In addition to the exemplary cannabinoid biosynthetic pathway from cannabis shown, alternative biosynthetic intermediates can be used in the cannabinoid biosynthetic pathway of genetically engineered plants or plant cells. For example, olive alcohol is an intermediate that lacks the carboxyl group of olive acid. The use of olive alcohol rather than olive acid in the cannabinoid biosynthetic pathway will produce cannabinoids that similarly lack a carboxyl group, such as cannabigerol (CBG), tetrahydrocannabinol (THC), or cannabichromene (CBD). In another example, butenone synthase (TKS) condenses butyryl-CoA and malonyl-CoA to form the intermediate trioxydecanoyl-CoA, and olivetate cyclase (OAC) catalyzes the intramolecular aldol condensation of trioxydecanoyl-CoA to produce divarinolic acid. Divarinolic acid is an intermediate that contains an n-propyl group instead of the n-pentyl group found in olive acid. The use of divarinol in the cannabinoid biosynthetic pathway instead of oleic acid will produce cannabinoids that similarly contain an n-propyl group, such as cannabigerolic acid (CBGVA), tetrahydrocannabinolic acid (THCVA), cannabidiolic acid (CBDVA) or cannabichromenic acid (CBCVA). In another example, divarinol is an intermediate that lacks the carboxyl group of divarinol and contains an n-propyl group in place of the n-pentyl group found in oleic alcohol. The use of divarinol in the cannabinoid biosynthetic pathway instead of divarinol will produce cannabinoids that similarly contain an n-propyl group and lack a carboxyl group, such as cannabigerolic acid (CBGV), tetrahydrocannabinol (THCV), cannabidiolic acid (CBDV) or cannabichromenic acid (CBCV). The skilled person will appreciate that these alternative biosynthetic intermediates can be used to produce OA and / or CBGA or other cannabinoids according to the present invention.

[0241] Apart from Figure 1In addition to the exemplary cannabinoid biosynthetic pathway from cannabis shown, alternative enzymes can be used in the cannabinoid biosynthetic pathway of genetically engineered plants or plant cells. For example, in addition to the enzymes found in cannabis, alternative enzymes of the cannabinoid biosynthetic pathway can be found in other plants (e.g., Humulus lupulus, Rhododendron sp. such as Rhododendron dauricum or Rhododendron adamsii or Radula sp. such as Radula perrottetii or Radua arginateata), bacteria (e.g., Streptomyces) or protists (e.g., Dictyostelium discoideum). It should be understood that in the cannabinoid biosynthetic pathway of genetically engineered plants or plant cells, enzymes with different structures but performing the same function can be used interchangeably.

[0242] In addition to the wild-type enzymes found in the organisms discussed herein (e.g., cannabis), modified variants of these enzymes can be used in the cannabinoid biosynthetic pathway in genetically engineered plants or plant cells. Variants of enzymes for cannabinoid biosynthetic pathways can be generated by altering the polynucleotide sequence encoding the enzyme, such as increasing / decreasing the activity of a domain, adding / removing a domain, adding / removing a signaling sequence, or otherwise altering the activity, abundance, or specificity of the enzyme.

[0243] Exemplary enzymes and / or products of the cannabinoid biosynthetic pathway include, but are not limited to, the following.

[0244] Cannabiyl activating enzyme (CsAAE1) is a cytosolic CsAAE1 enzyme present in trichomes. It is a trichome-specific acyl-CoA synthase that converts C6 to CoA derivatives. CsAAE1 is a member of the acyl-activating enzyme (AAE) superfamily that activates carboxylic acids via an adenylate intermediate (Stout et al., 2012). This family of enzymes all have a highly conserved 12 amino acid residue AMP binding motif (PROSITE PS00455) (Shockey and Browse, 2011). AAE1 belongs to the EC6.2.1.2 classification and CsAAE1 is known to accept FAs with different chain lengths (Stout et al., 2012). An exemplary sequence for use according to the present invention is AFN42527.1 or SEQ ID NO: 1.

[0245] Cannabis olive alcohol synthase (CsOLS) is one of two enzymes that work together to produce OA from C6-CoA and malonyl-CoA. Taura et al. (2009) identified the CsOLS gene as a polyketide synthase in cannabis trichomes that condenses C6-CoA and a variety of other short-chain fatty acids with malonyl-CoA. CsOLS can accept C4-C8 CoA substrates (Taura et al., 2009). CsOLS is a cytoplasmic protein (Stout et al., 2012) and its crystal structure has recently been resolved (Kearsey et al., 2020). Non-natural variant OLS mutants have recently been described in WO2020 / 214951. An exemplary sequence used according to the present invention is BAG14339.1 or SEQ ID NO: 2.

[0246] Cannabinol cyclase (CsOAC) is the second enzymatic step required for the production of OA. CsOAC is located in the cytosol (Gagne et al., 2012) and refers to 3,5,7-trioxododecanoyl-CoA cyclase or 3,5,7-trioxoundecanoyl-CoA cyclase (EC 4.4.1.26). This type of enzyme is capable of converting 3,5,7-trioxododecanoyl-CoA into OA or 3,5,7-trioxoundecanoyl-CoA into divalinoic acid. An exemplary sequence used according to the present invention is AFN42527.1 or SEQ ID NO: 3.

[0247] Cannabis CBGA synthase (CBGAS). In cannabis, OA is transported to chloroplasts by CsCBGAS (EC 2.5.1.102) for further conversion to CBGA. Recently, Luo et al. (2019) and Gulck et al. (2020) both identified CsPT4 as a functional CBGAS prenyltransferase in cannabis. Unlike CsOLS and CsOAC, CsCBGAS is a membrane-bound enzyme present in chloroplasts (Gulck et al., 2020). Valliere et al. (2019) identified an alternative prenyltransferase NphB from Streptomyces CL190 strain that is capable of producing CBGA. The group used structure-based protein design to enhance the nonspecific CBGAS activity of soluble NphB. CBGAS is able to convert GPP and oleic acid (OA) or GPP and divaleric acid (DVA) into cannabigerolic acid (CBGA) or cannabigerolic acid (CBGVA). An exemplary sequence for use in accordance with the present invention is DAC76710.1 or SEQ ID NO:4.

[0248] Cannabis THCA and CBDA synthases (THCAS; CBDAS). The final step of the cannabinoid biosynthetic pathway consists of the conversion of CBGA to THC or CBD as the main cannabinoid end products by CsTHCAS or CsCBDAS, respectively. In cannabis, these two enzymes are located in the apoplast, which explains the accumulation of major cannabinoids such as THC and CBD in extracellular storage globules at very high levels (Sirikantaramas et al., 2004; Taura et al., 2007). CBDA synthase (EC1.21.3.8) is able to convert CBGA or CBGVA to (CBDA) or CBDVA. CBDAS can accept CBGA (C5 tail) and CBGVA (C3 tail) in vitro (Valliere et al., 2019). THCA synthase (EC1.21.3.7) is able to convert CBGA or CBGVA to THCA or THCVA.

[0249] Peptides

[0250] The terms "peptide", "polypeptide" and "protein" may be used interchangeably herein and may refer to a polymeric form of amino acids of any length, which may include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, full-length polypeptides, fragments of polypeptides, or polypeptides with modified peptide backbones. The polypeptides disclosed herein may exist in modified or engineered forms, including truncated or fused forms, retaining the described activity. The polypeptides disclosed herein may also be variants that differ from a specifically described "reference" polypeptide (e.g., a wild-type polypeptide) by amino acid insertions, deletions, mutations and / or substitutions, but retain substantially similar activity to the reference polypeptide.

[0251] The genetically engineered plants, algae, plastids or parts thereof of the present invention can include exogenous polynucleotides encoding polypeptides as defined herein. In these cases, plants and cells produce recombinant polypeptides. In the context of polypeptides, the term "recombination" refers to polypeptides encoded by exogenous polynucleotides when produced by cells, which have been introduced into cells or progenitor cells by recombinant DNA or RNA technology, such as transformation. Typically, cells include non-endogenous genes that cause the polypeptides of the amount to be changed to be produced. In one embodiment, a "recombinant polypeptide" is a polypeptide prepared by expressing exogenous (recombinant) polynucleotides in plant or algae cells.

[0252] In one embodiment, the polypeptide of the present invention can be active in the cytosol and / or plastid of a plant. In this context, the term "active" should be understood to mean that the polypeptide is able to exert its normal biological activity, such as by catalyzing the synthesis of olivetate or CBGA, whether in the cytosol and / or plastid of a plant.

[0253] The % identity of the polypeptides is determined by GAP (Needleman and Wunsch, 1970) analysis (GCG program) with a gap creation penalty = 5 and a gap extension penalty = 0.3. The query sequence is at least 100 amino acids in length, and the GAP analysis aligns the two sequences over a region of at least 100 amino acids. More preferably, the query sequence is at least 300 amino acids in length, and the GAP analysis aligns the two sequences over a region of at least 300 amino acids. Alternatively, the query sequence is at least 500 amino acids in length, and the GAP analysis aligns the two sequences over a region of at least 500 amino acids. Even more preferably, the GAP analysis aligns the two sequences over the entire length of any amino acid sequence disclosed herein, including the amino acid sequences defined in SEQ ID NOs: 1-4 and 20-23.

[0254] With regard to defined polypeptides, it will be appreciated that higher % identity figures than those provided above will encompass preferred embodiments. Thus, where applicable, based on the minimum % identity figures, it is preferred that a polypeptide comprises an amino acid sequence that is preferably at least 50%, at least 60%, at least 70%, more preferably at least 75%, more preferably at least 76%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.8% and even more preferably at least 99.9% identical to the relevant designated SEQ ID NO.

[0255] In one embodiment, the polypeptide used and / or belonging to the present invention is in some cases a fusion polypeptide, including a "chloroplast transport peptide" (CTP), also known as a "chloroplast transit peptide". Examples of suitable CTPs include any signal fragment capable of directing secretion or localization of a polypeptide defined herein to a plastid such as a chloroplast. Non-limiting examples of plastid transport proteins include the plastid transport proteins described in AT1G61800, AT5G16150, AT5G33320, AT5G46110, AT4G15530, AT2G36580, AT3G52990, AT3G55650, AT3G55810, AT4G26390, AT5G08570, AT5G56350, AT5G63680, AT1G32440, AT3G22960, AT3G49160 and AT5G52920.

[0256] In one embodiment, the CTP is a Rubisco small subunit (Hirakawa and Ishida, 2010). In a preferred embodiment, the CTP is a Rubisco small subunit having an amino acid sequence at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the nucleotide sequence shown in SEQ ID NO: 15.

[0257] It will be appreciated that the use of a particular plastid targeting peptide depends on the targeting location of the protein in the chloroplast, e.g., matrix / inner membrane / outer membrane / thylakoid membrane, and whether the enzyme is membrane-bound. Suitable plastid targeting peptides may comprise any peptide disclosed in the art, including Dugdale et al., 2013; Dugdale et al., 2014; Lee et al., 2008; Jarvis 2008 and Bruce 2000. For the cannabinoid pathway, the biosynthetic genes of the cannabinoid pathway are generally soluble and thus can be targeted to the matrix using Rubisco targeting peptides. The predicted sequence motifs of suitable plastid targeting peptides may comprise any of the following predicted sequence motifs listed in Table 1.

[0258] Table 1: Predicted sequence motifs

[0259] LKETT FSNHL IKEQR

[0260] MAYSA FLHQS IICKA QSHED

[0261] AMKLP FPNAI TDSES SLGHR VNGSK LDSAA

[0262] CRLFR CNLLP LPKPL NRIAS PNGVP <h2 style=";text-align:left;direction:ltr">

[0263] <h2 style=";text-align:left;direction:ltr"> EERKK YQNGD DVYAD LDWDN YMYVM CSKDG<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0264] <h2 style=";text-align:left;direction:ltr"> AFATG PTFLG KISNT SVVSC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0265] <h2 style=";text-align:left;direction:ltr"> RQQTP QSLFG VTAMA KFITP<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0266] <h2 style=";text-align:left;direction:ltr"> MITLR NLSRN SHQST RSFHN STGGF SSSND<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0267] <h2 style=";text-align:left;direction:ltr"> RRLLQ NLPAL QQIPL SRGFI<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0268] <h2 style=";text-align:left;direction:ltr"> FSSSC FLHRR KLTAI EQTTL DNRFS GSDSD<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0269] <h2 style=";text-align:left;direction:ltr"> EALTT LFANF GLVSI LSTLG<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0270] <h2 style=";text-align:left;direction:ltr"> ASTIK ATVRI TAAVA GVAAV VLLGG<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0271] <h2 style=";text-align:left;direction:ltr"> VPIMS GISSR RFSIG FGFRF DVPFK RLRFV<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0272] <h2 style=";text-align:left;direction:ltr"> EESSM DFVAT KFPAE LRYFP<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0273] <h2 style=";text-align:left;direction:ltr"> MAAST SSPAF LSPAALGSGR TMRKT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0274] <h2 style=";text-align:left;direction:ltr"> KAVKL PAASE GPFSG PGDYG<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0275] <h2 style=";text-align:left;direction:ltr"> YSTQA QPQTN LITCA LAADS KSTFM GAAKP<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0276] <h2 style=";text-align:left;direction:ltr"> VYPLA GLVSF KKLFL RAMSE DKESS<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0277] <h2 style=";text-align:left;direction:ltr"> MAAAT GRLSL AQASF FVLQL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0278] <h2 style=";text-align:left;direction:ltr"> NAAVF SQWRP EDPRS KAPPY KGKFL DVNQA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0279] <h2 style=";text-align:left;direction:ltr"> HFLRN QPKVH VPLAL PKKVE PKSKV<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0280] <h2 style=";text-align:left;direction:ltr"> FATIA SAAVK GGSSL GAKLF GDKSV EDLGN<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0281] <h2 style=";text-align:left;direction:ltr">LQARP LAIAA SFSNG LSFNL RQLPT LTVSC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0282] <h2 style=";text-align:left;direction:ltr"> SLQAR PRQLV QVKSF LRQLP VRKQL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0283] <h2 style=";text-align:left;direction:ltr"> TFTAT SMVAP SSSSF RRQSV RRSSS IVCAA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0284] <h2 style=";text-align:left;direction:ltr"> NSISF RPLRL VVRAE SIVIS LSLFL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0285] <h2 style=";text-align:left;direction:ltr"> AAVTR ARRLG SAVVD ARGVI<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0286] <h2 style=";text-align:left;direction:ltr"> VGTSF LRSLP NTQSL KVKFI ELEVE VLDAA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0287] <h2 style=";text-align:left;direction:ltr"> QLLLS HSQIT PATKI VWRPK SSAVD PTKTL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0288] <h2 style=";text-align:left;direction:ltr"> AQATM FNGLK TRKAN RVNCM GKKKF LTDSE<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0289] <h2 style=";text-align:left;direction:ltr"> MAAAM SLNPS QTKSK LAVTS SLSYS<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0290] <h2 style=";text-align:left;direction:ltr"> MAAAM SPALS SSSSF RLRVS ASSVT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0291] <h2 style=";text-align:left;direction:ltr"> ATVTI LKLAV TLSTIPPKLA VIAVA ASIVL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0292] <h2 style=";text-align:left;direction:ltr"> HVGMK MLQIA SSVFF AISPA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0293] <h2 style=";text-align:left;direction:ltr"> AGFTV TAARG SGRKG<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0294] <h2 style=";text-align:left;direction:ltr"> AVIQT GLLSL LSFNG FQTFE GISIS FICKA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0295] <h2 style=";text-align:left;direction:ltr"> FSLTF SVLVA AAQTT LKDGF<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0296] <h2 style=";text-align:left;direction:ltr"> LNHKS KGSFF TCKAK SEASV PLGIN<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0297] <h2 style=";text-align:left;direction:ltr"> MAATA FPALG KISNF IHCMS KAYKR<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0298] <h2 style=";text-align:left;direction:ltr"> NFANL PINPG FLFKS TVQAR AEKTV<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0299] <h2 style=";text-align:left;direction:ltr">MASAN SSASV SSRQS KRTIR FSVRA VKEIA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0300] <h2 style=";text-align:left;direction:ltr"> MAATN GVSLN HRLQS FAVKA VVSAA KGTSD<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0301] <h2 style=";text-align:left;direction:ltr"> RVLVS LRHSG SNNNK TTVRF SLNEI GLDSS<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0302] <h2 style=";text-align:left;direction:ltr"> PPPPL QLSND<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0303] <h2 style=";text-align:left;direction:ltr"> RPALL PMGKK RCSME AVMSN PAMAL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0304] <h2 style=";text-align:left;direction:ltr"> SAVIG SGCAQ LRSKF SIRME ESSKT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0305] <h2 style=";text-align:left;direction:ltr"> INRAP FLGKK SFKVL VKEDK AYDTS<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0306] <h2 style=";text-align:left;direction:ltr"> SGIFG QARFG PAPPP NPPEW<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0307] <h2 style=";text-align:left;direction:ltr"> RKSLS RCSVE TVTER LLLRA VDRPP<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0308] <h2 style=";text-align:left;direction:ltr"> SACYI KSNNL PLAVE KELRS FVQTA VTGSL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0309] <h2 style=";text-align:left;direction:ltr"> LFSCC NISQK SHCPI SQSQE PLRSV SLFAL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0310] <h2 style=";text-align:left;direction:ltr"> IDSRA RLASA PSCIS TLPIQ EAPAT TTEAE<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0311] <h2 style=";text-align:left;direction:ltr"> RIKLS ESLPI AICAA SSVEE<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0312] <h2 style=";text-align:left;direction:ltr"> GTRVV DISSN KKAKT FPGAK DPFGL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0313] <h2 style=";text-align:left;direction:ltr"> SIGSC FKAPN VLISA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0314] <h2 style=";text-align:left;direction:ltr"> MDTLL NKLDF FHGFE SNNPY VKKRA GSAAL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0315] <h2 style=";text-align:left;direction:ltr"> LVLSE RPLPR IYTTP NFLSN SFGLN PLTTP<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0316] <h2 style=";text-align:left;direction:ltr"> MASVA LISSP PSVSF RSSLS STSRR SFAVK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0317] ETLIP MTTGT ALSFL LQLMR

[0318] TKPKP NTATP THHQK PPSLV VAFSE RDVTT

[0319] VAAVK GGSSL ANGVV GDKSV

[0320] GASVF SEFPI ESSKI LKKNA TRNVG AMQAG

[0321] GFSKS FGISS GPGSS ENPRP

[0322] QFHRS SLLGS LGISS GSDFS NLTAS IENPR

[0323] AMSLE LSRNH SSLLG RSFQN DFSSR NLNVT

[0324] VTFSV FTEFS GSSGG NGFGR

[0325] ASTSL FLSPS SSRLP IKCSK TETSS

[0326] ASIFR TGSSG FTSIG GSLAG

[0327] RAAIL ASIKL SNGGI RCNAV VTELA

[0328] LQPAK KSFGL TCSLH AGFAL SALVV

[0329] QPSIL FSNNG KRAPT CMAVA ETKTA

[0330] ASLKL ISKVG GLSFL RLIVT VVAPW AESKK

[0331] RRFST PPPPT KEIQE

[0332] PLCDS SQFVI NRLLS SSFWG GVSGC ASLGG

[0333] LCSST TTLSS QTSSS RRELS SSLVA SNDRR

[0334] GIFNS SGVKK FSTTH AIRAE

[0335] <h2 style=";text-align:left;direction:ltr">SNLTT VSSKP SSLSF RASSV<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0336] <h2 style=";text-align:left;direction:ltr"> AGLRG RVSVG RTGLV RSVIG<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0337] <h2 style=";text-align:left;direction:ltr"> ASLGV MLGTP LNFRAFSKKK TSDEL DGLLD<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0338] <h2 style=";text-align:left;direction:ltr"> SPMAS SASLS PKGIS VSPTK FTVRASDKTT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0339] <h2 style=";text-align:left;direction:ltr"> AFAFP SYIIT KGAST HLLFS VAINC AVGRR<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0340] <h2 style=";text-align:left;direction:ltr"> MASLL FGSKL KNRSR MNVAT EINST GKFDS<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0341] <h2 style=";text-align:left;direction:ltr"> KINGS LNGSG VAQRS AQQSE RSVIG VAAGL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0342] <h2 style=";text-align:left;direction:ltr"> AFSVR FKDSS ASITD QIKSE RNLAI<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0343] <h2 style=";text-align:left;direction:ltr"> MDVAS HPSYY GKPIC SLIRI FMGET<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0344] <h2 style=";text-align:left;direction:ltr"> RHAPR STDPK STQSASVKWS PDQKD<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0345] <h2 style=";text-align:left;direction:ltr"> AAAAA GIHPG PLWYP GSLVG<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0346] <h2 style=";text-align:left;direction:ltr"> MASNA LSSFT NPALS SASPS FSRKV VAAAT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0347] <h2 style=";text-align:left;direction:ltr"> VLTRQ SSRVN CSLGS VSAIG TAIIR<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0348] <h2 style=";text-align:left;direction:ltr"> ASVYA FPIQN VSFRL KLRFL VKAQS GASSN<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0349] <h2 style=";text-align:left;direction:ltr"> LNPDP RVLES NQRLN QIPTW<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0350] <h2 style=";text-align:left;direction:ltr"> PLFLL LYISR RAFPP PLVSL LIPTF NHQVR<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0351] <h2 style=";text-align:left;direction:ltr"> AQATM TRKAN VSCMK IGKKK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0352] <h2 style=";text-align:left;direction:ltr"> AQATM TRKAN VSCMK KKFET<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0353] <h2 style=";text-align:left;direction:ltr">DTVFL FAGAK HIRTK NSRFH SIIAS AGTDS<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0354] <h2 style=";text-align:left;direction:ltr"> QFTRN PFFSV IACAACACTL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0355] <h2 style=";text-align:left;direction:ltr"> FSVRK FKESS SLSEQ SKADF SSSLR KAIIR<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0356] <h2 style=";text-align:left;direction:ltr"> APRKG SIFAT AVRSN GDNAV<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0357] <h2 style=";text-align:left;direction:ltr"> SVLTC GSGSV VGLVN VGFGQ KQMIM QRVVG<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0358] <h2 style=";text-align:left;direction:ltr"> MTIAL FSGLP SSFSS SFRLK VVCSS SSSVM<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0359] <h2 style=";text-align:left;direction:ltr"> RSIGS SVNHS NVPRS PMKPV PALQF<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0360] <h2 style=";text-align:left;direction:ltr"> SSSSS LNSGS NLSSL ASPTS PILGV NKDPS<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0361] <h2 style=";text-align:left;direction:ltr"> MAVCN FLQAP SRRAT TLSAG YGRLK TVTFC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0362] <h2 style=";text-align:left;direction:ltr"> RLLSA HVTSI LSNRT NLRRF<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0363] <h2 style=";text-align:left;direction:ltr"> WTPLC PPERN PHCSP FRTAL PHLVA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0364] <h2 style=";text-align:left;direction:ltr"> PFSSH PNPST ETERP DDVTP<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0365] <h2 style=";text-align:left;direction:ltr"> FNGLR PMRRK IMVTS TTLML RFGLA ANRKA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0366] <h2 style=";text-align:left;direction:ltr"> AMLQT TSPTF SYRSQ RVNRS SSSTG VKEKS<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0367] <h2 style=";text-align:left;direction:ltr"> NVATM TGYSG YAPSF SIKCV SDTKK STNLI<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0368] <h2 style=";text-align:left;direction:ltr"> VIAVA IVSGI LSGIL LTGVR FSSIG DAQSS<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0369] <h2 style=";text-align:left;direction:ltr"> SAVKK HLFSS APAVK PQLDP NTPSP<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0370] <h2 style=";text-align:left;direction:ltr"> FLFSR KSLVR SELPE KSVAE PAVVP

[0371] FLQTN LEIIP SLTDH KKRYN LQKAG GLEFD

[0372] LGSTK VQISL QTRKN FQIQA

[0373] SFISQ AAPET SDNTP QTIKV

[0374] FILTA GGGSS TTIVS DTPPAAKVPA

[0375] FSGLR SPKLD FHRVN VVAMA

[0376] LVNFH DVSPL ISSNL SSGKS STTDA

[0377] LQSPG VSRLL TIFSV CISKG YSPLL

[0378] LHKAS ALSLL VQAAK KSLTG EVKKE TPFVS

[0379] ASMTM SLEVK RDLMF VCSLA EPKRG

[0380] KSQSK KIPFL HNPLR VAAPP VPTSD EEKRI

[0381] FSLLN DISPL SSGKS TLSGV PFEEV

[0382] LSPAT ALMAM SGLFV TKMNH ISCQA LGALS

[0383] MAMTT PANVT ASSSR SSVSF VVRAA ASSSS

[0384] FELSR PVSPL RSLVP TPNSE PVYVP

[0385] GLAIR PPSDG IPADS HPISATADFP

[0386] MAMQS PVPKL DKNFF YCKSK

[0387] FLQTN SLTDH KKRYN QKAGS

[0388] FRTAS KPIAH IPARH SCINP YPPSN

[0389] YKSNL LTLDS RCCKI LLVAT EEGDY

[0390] ASSFT PISLN FLLKK AVSPI

[0391] NSLMS AVYPS LLSSS KSKFV IRMAA

[0392] AQATM TRKTN WSCMK KKKFE DLSDV

[0393] ETALL VNFSG MASGL TALAF LAVVK QGLLA

[0394] ASPVL LKPNAKNRVS RIVVK AAASG ESKEE

[0395] ATKIF VISAT GTAAS AANYG YGGNS

[0396] LQLIL PPRPR QELSS TSPPI LPKLI PLTTP

[0397] VSAIF VTVAALRQFQ IECSS

[0398] RSYLT SVKPL SVQVT RHLLS SGARR

[0399] MATCI SRFCC NFFRP SVNRK KSDVK ASITS

[0400] LSLKL PLFPF ISSEF SPDAD

[0401] ERRLD EQTKE LEKVE VSAID WVAMV LFPQC

[0402] HTARH PSRLF RFSDR RSLLS VRDSR QRSWE

[0403] STATM KGQSL LTVRA LVKTA SNATC

[0404] NPTSH WLKPP HSLPI

[0405] LSLRL PTTGG CRIPY SGVPT CSLDS DSSVV

[0406] ATTVH SLFIQ LTSQG FDFKL YMIRC AESVN<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0407] <h2 style=";text-align:left;direction:ltr"> ALLSD RSPPP PLTTP SLVEV SDPPP SSGSG<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0408] <h2 style=";text-align:left;direction:ltr"> NPKSL PLCVS VLPPS PAEYP FVRNV VSSDF<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0409] <h2 style=";text-align:left;direction:ltr"> GFPVI DQNPS KRRHI SGSSQ EKISP<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0410] <h2 style=";text-align:left;direction:ltr"> LTISG PLNVAKSRSG IHCSK LPLIH<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0411] <h2 style=";text-align:left;direction:ltr"> AFTCT FAVLP LTSIH VVCEA QETTT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0412] <h2 style=";text-align:left;direction:ltr"> ATVPL QFPCK IGVSV FKIRA GQDGV ASSST<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0413] <h2 style=";text-align:left;direction:ltr"> QPTSD KSPKG HPKPE NPTRP DVGID SGTST<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0414] <h2 style=";text-align:left;direction:ltr"> PQLLL QFRFT NYVAS CQRAS VIEGL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0415] <h2 style=";text-align:left;direction:ltr"> STDSC TDLPS NNSHS YSNRP<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0416] <h2 style=";text-align:left;direction:ltr"> ITTMN AISDT LRGQF QRIHF LQHTR FGHVS<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0417] <h2 style=";text-align:left;direction:ltr"> AAAMS KISTG SSYGD<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0418] <h2 style=";text-align:left;direction:ltr"> MASLM GSTSL NKDKL KLGTS FLKAK TMTVA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0419] <h2 style=";text-align:left;direction:ltr"> MTKLL LCFAK GYCSN TCYAT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0420] <h2 style=";text-align:left;direction:ltr"> TNSFL NTQIP TTQVQ SQERV FAAGP<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0421] <h2 style=";text-align:left;direction:ltr"> GQSVL LTVRA IASPG SNATC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0422] <h2 style=";text-align:left;direction:ltr"> LSLIC NTPNK CARVG EIPAN RARQI<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0423] <h2 style=";text-align:left;direction:ltr"> SMISI FVISS SFSRK VSTLP SVPVV<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0424] <h2 style=";text-align:left;direction:ltr"> HSRSP LSAAM EKSRQ SKVTV KLIAS NALKL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0425] <h2 style=";text-align:left;direction:ltr"> GSETP PIVGP<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0426] <h2 style=";text-align:left;direction:ltr"> SRTPP QAIDP PCNPL LSRFL ATPDP<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0427] <h2 style=";text-align:left;direction:ltr"> EFTVN NLRSL LRHGF SSQSW LKLAR LAMYS<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0428] <h2 style=";text-align:left;direction:ltr"> TPHTI RLRSL SILPD GDDFI VFEDP<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0429] <h2 style=";text-align:left;direction:ltr"> CLLPQ CPPDS FFQPQ SFDTD DSLIS<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0430] <h2 style=";text-align:left;direction:ltr"> RTYIF SPLCL PRGDS LRPRV ESYGS LLRRP<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0431] <h2 style=";text-align:left;direction:ltr"> QSQFL ANNRV CQITR SFKVE KFQLQ VKNGI<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0432] <h2 style=";text-align:left;direction:ltr"> MGVSL TSHNN LRHLS LLQRK FQHSV<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0433] <h2 style=";text-align:left;direction:ltr"> AVIGL KKASN<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0434] <h2 style=";text-align:left;direction:ltr"> TELNF HLLPI LLSFT SIRME ATGAG<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0435] <h2 style=";text-align:left;direction:ltr"> KSKKE KKRGV IRNHR TLPEL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0436] <h2 style=";text-align:left;direction:ltr"> YAIPR SFVKM SMGIK KLALA VTCMA MEVEA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0437] <h2 style=";text-align:left;direction:ltr"> HLNLR SRIGN KNLRN KLPPD RKPKI<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0438] <h2 style=";text-align:left;direction:ltr"> KLTVK SRQSD EKQRF GDSSS SQNAE<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0439] <h2 style=";text-align:left;direction:ltr"> SKIKL TLLLS FTRLC KAMDS CCISN<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0440] <h2 style=";text-align:left;direction:ltr"> EDEYIDYEKE GLEDK EEVME EQMGA VLILM<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0441] <h2 style=";text-align:left;direction:ltr"> MAYLA SMTMN TTAAE KQKKR ESKGF EEMRF<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0442] <h2 style=";text-align:left;direction:ltr"> PSENV RFSVA RKVLA PGKLE REPVE<h2 style=";text-align:left;direction:ltr">

[0443] FQTKT SVVML ASVLS

[0444] REILR AYQAF DFDPS ERSGL RLTKN

[0445] VLTCA TKKLT TELSI RCSGN VLLEG

[0446] MNIVS AFGGK

[0447] MGSKQ GAYYG ILSLI VAVIL ILWLI

[0448] ASSSL SLRPR SFINP SLGRR AAKKK

[0449] SRPRI TNSML PHPLI LLRSD IPMTS

[0450] SSSTF LTTTS VSRKS KTTST

[0451] SMAMC TGTGK RLSVD IATRF RAEII SDKIQ

[0452] SSFTI MGLKL LKVTC ASTMT

[0453] MTSMI VKTTP SRSNR RFPRT RLSIA LSPVS

[0454] FSQAS NRTLC STPRN REAFC SCRFH NGAPS

[0455] MELTL SSSSL AKSSR QESSS AKMKP KTFTP

[0456] TQESA SRNRS SNALY FNGFR LSFNT

[0457] LCSAS FHALK SRKKH SMRCS SMKSS KSSKP

[0458] TILTV FSSSR VSLKS STSSP KAGVS

[0459] GQAFS WSSAS SSGVF ERCGC

[0460] DVPFF KDKVV

[0461] VMALA TSKSA VSVTS HRIKS AERTK

[0462] ALQRL PRRPP PPPSS IVGYP ELKFA

[0463] LTSVA TTITT RLCFS RFRIA KSSVV

[0464] CLLPR SDSVN CKSKI SSVKR QSKHE EPDDD

[0465] LLSRT NPSPN NFGSQ YTINP SAIAS

[0466] ALNSS AFFVP LSSAR TRSTI SRRAF SPQKS

[0467] In a specific embodiment, the fusion protein or polypeptide for expression in a plant or algae comprises: i) a polypeptide selected from the group consisting of a polyketide synthase, a polyketide cyclase, an acyl-activating enzyme, a prenyl transferase, a plastidial acyl-lipid thioesterase, a plastidial lipase or a plastidial tomato 13-lipoxygenase; and ii) wherein the polypeptide of (i) increases the production of cannabigerolic acid (CBGA) in the presence of hexanoic acid (C6) and geranyl pyrophosphate (GPP) as compared to a wild-type plant or algae.

[0468] It will be appreciated that polyketide synthases (PKS) are a family of multidomain enzymes or enzyme complexes that produce polyketides, a large class of secondary metabolites that are particularly relevant to plants, and may include type I polyketide synthases, type II polyketide synthases, and type III polyketide synthases, such as oliverate synthase (OAS). It will be appreciated that polyketide cyclases represent a number of cyclases involved in polyketide synthesis in many actinomycete species, and may include oliverate cyclase (OAC).

[0469] Acyl activating enzyme

[0470] Acyl-activating enzymes are a group of different proteins that catalyze the activation of many different carboxylic acids, primarily through the formation of thioester bonds. This group of enzymes is found in all living organisms and includes acyl-CoA synthases, 4-coumaric acid: CoA ligases, luciferases, and non-ribosomal peptide synthases. The members of this superfamily share little overall sequence identity, but contain 12 amino acid motifs common to all enzymes, which activate their acidic substrates using ATP via enzyme-bound adenylate intermediates. A non-limiting example of this enzyme is acyl-activating enzyme (CsAAE1).

[0471] Prenyltransferases are a class of enzymes that transfer an allylic prenyl group to an acceptor molecule. Prenyltransferases are generally referred to as prenyl diphosphate synthesis. Prenyltransferases are generally divided into two classes, cis and trans, depending on the stereochemistry of the resulting product. A non-limiting example of this enzyme is cannabigerolic acid synthase (CBGAS).

[0472] The fusion protein may include a linker between the CTP and the enzymatic CBD biosynthetic pathway described herein.

[0473] In one embodiment, the fusion protein can comprise an N-terminal fusion or a C-terminal fusion. For example, the N-terminal fusion can comprise a chloroplast transport peptide and the C-terminal fusion can comprise a reporter protein known in the art, such as mNeonGreen or green fluorescent protein (GFP), red fluorescent protein (RFP), mCherry, luciferase, or β-galactosidase.

[0474] The amino acid sequence mutants for the polypeptides of the present invention can be prepared by introducing appropriate nucleotide changes into the nucleic acids defined herein or by synthesizing the desired polypeptide in vitro. Such mutants include, for example, deletions, insertions or substitutions of residues in the amino acid sequence. A combination of deletions, insertions and substitutions can be performed to achieve the final construct, provided that the final peptide product has the desired characteristics. Preferred amino acid sequence mutants have one, two, three, four or less than 10 amino acid changes relative to reference polypeptides such as the amino acids provided in SEQ ID NOs: 1 to 4.

[0475] Mutant (altered) polypeptides can be prepared using any technique known in the art, such as directed evolution, rational design strategies or mutagenesis (see below). Products derived from mutated / altered DNA can be easily screened using the techniques described herein to determine whether a product as defined herein is produced when expressed in a plant cell.

[0476] When designing amino acid sequence mutants, the location of the mutation site and the nature of the mutation will depend on the property to be modified. Mutation sites can be modified individually or sequentially, for example by (1) first selecting with conservative amino acids and then replacing with more radical choices based on the results obtained; (2) deleting the target residue or (3) inserting other residues adjacent to the targeted site.

[0477] Amino acid sequence deletions generally range from about 1 to 15 residues, more preferably about 1 to 10 residues and usually about 1 to 5 contiguous residues.

[0478] Substitution mutants have removed at least one amino acid residue in the polypeptide molecule, and different residues have been inserted in its position. When it is desired to maintain a certain activity, it is preferred that highly conservative amino acid positions in the related protein family are not substituted, or only conservative substitutions are performed. Examples of conservative substitutions are shown in Table 2 under the heading "Exemplary Substitutions".

[0479] In one embodiment, the mutant / variant polypeptide has one or two or three or four conservative amino acid changes when compared to a naturally occurring polypeptide. Detailed information on conservative amino acid changes is provided in Table 2. In a preferred embodiment, the changes are not in one or more motifs in a motif that is highly conserved between different polypeptides of the same class of enzymes. As will be appreciated by those skilled in the art, it is reasonably predicted that such minor changes will not alter the activity of the polypeptide when expressed in a recombinant cell.

[0480] The primary amino acid sequence of a polypeptide can be used to design variants / mutants thereof based on comparisons with closely related polypeptides. It will be appreciated by those skilled in the art that highly conserved residues in closely related proteins are less likely to be altered, particularly with non-conservative substitutions, and that activity is maintained compared to residues with a lower degree of conservation (see above).

[0481] Table 2: Exemplary substitutions.

[0482]

[0483]

[0484] Directed evolution

[0485] In directed evolution, random mutagenesis is applied to a protein, and a selection scheme is used to pick out variants with desired qualities, such as increased activity. Additional rounds of mutation and selection are then applied. A typical directed evolution strategy involves three steps:

[0486] 1) Diversification: Genes encoding proteins of interest are randomly mutated and / or recombined to generate large gene variant libraries. Variant gene libraries can be constructed by error-prone PCR (see, e.g., Leung, 1989; Cadwell and Joyce, 1992), from a pool of DNaseI-digested fragments prepared from a parental template (Stemmer, 1994a; Stemmer, 1994b; Crameri et al., 1998; Coco et al., 2001) from degenerate oligonucleotides (Ness et al., 2002, Coco, 2002) or from a mixture of both, or even from an undigested parental template (Zhao et al., 1998; Eggert et al., 2005; Jézéquek et al., 2008) and are usually assembled by PCR. Libraries can also be prepared from parental sequences recombined in vivo or in vitro by homologous or nonhomologous recombination (Ostermeier et al., 1999; Volkov et al., 1999; Sieber et al., 2001). Variant gene libraries can also be constructed by subcloning the gene of interest into a suitable vector, transforming the vector into a "mutant" strain such as E. coli XL-1red (Stratagene), and propagating the transformed bacteria for an appropriate number of generations. Variant gene libraries can also be constructed by DNA shuffling of the gene of interest (i.e., in vitro homologous recombination of selected mutant genes by random fragmentation and recombination) as described extensively by Harayama (1998).

[0487] 2) Selection: Screening or selection is used to test the presence of mutants (variants) with desired properties in the library. Screening can manually identify and isolate high-performance mutants, while selection can automatically eliminate all non-functional mutants. Screening can involve screening for the presence of known conserved amino acid motifs. Alternatively or in addition, screening can involve expressing the mutated polynucleotide in a host organism or part thereof and determining the level of activity.

[0488] 3) Amplification: Variants identified in a selection or screen are copied many times, allowing researchers to sequence their DNA to understand what mutation occurred.

[0489] These three steps are collectively referred to as a "round" of directed evolution. Most experiments will require more than one round. In these experiments, the "winners" from the previous round are diversified in the next round to create a new library. At the end of the experiment, all evolved protein or polynucleotide mutants are characterized using biochemical methods.

[0490] Reasonable design

[0491] Based on known information about protein structure and folding, proteins can be rationally designed. This can be achieved by designing from scratch (de novo design) or redesigning based on natural scaffolds (see, for example, Hellinga, 1997; Lu and Berry, 2007). Protein design generally involves identifying sequences that fold into a given or target structure, and can be done using computer models. Computational protein design algorithms search for sequences with low energy when folded into the target structure in sequence conformation space. Computational protein design algorithms use protein energetics models to evaluate how mutations affect the structure and function of proteins. These energy functions generally include a combination of molecular mechanics, statistics (i.e., knowledge-based) and other empirical terms. Suitable available software includes IPRO (Iterative Protein Redesign and Optimization), EGAD (Genetic Algorithm for Protein Design), Rosetta Design, Sharpen and Abalone.

[0492] Polynucleotides and genes

[0493] The present invention refers to various polynucleotides encoding polypeptides, which increase the production of cannabinoids, preferably CBGA. As used herein, "polynucleotide" or "nucleic acid" or "nucleic acid molecule" means a polymer of nucleotides, which can be DNA or RNA or a combination thereof, and includes genomic DNA, mRNA, cRNA and cDNA. Less preferred polynucleotides include tRNA, siRNA, shRNA and hpRNA. A given polynucleotide can be DNA or RNA of cellular, genomic or synthetic origin, prepared, for example, on an automatic synthesizer, and can be combined with carbohydrates, lipids, proteins or other substances, labeled with fluorescent or other groups, or connected to a solid phase carrier to perform a specific activity defined herein, or include one or more modified nucleotides that do not exist in nature, which is well known to those skilled in the art. The polymer can be single-stranded, substantially double-stranded or partially double-stranded. Base pairing as used herein refers to standard base pairing between nucleotides, including G:U base pairs. "Complementary" means that two polynucleotides can base pair (hybridize) along part of their length or along the full length of one or both. The term "polynucleotide" can be used interchangeably with the term "nucleic acid" in this article.

[0494] The polynucleotide or nucleic acid sequence of the present application can be a deoxyribonucleic acid (DNA) sequence or a ribonucleic acid (RNA) sequence, and can contain naturally occurring bases, including adenine, guanine, cytosine, thymidine and uracil. These sequences can also contain modified bases. Examples of such modified bases include aza and deaza adenine, guanine, cytosine, thymidine and uracil; and xanthine and hypoxanthine. Nucleic acids can be double-stranded or single-stranded, and represent sense strands or antisense strands. Further, the term "nucleic acid" includes complementary nucleic acid sequences.

[0495] As used herein, the term "nucleic acid molecule" or its derivatives are intended to include unmodified DNA or RNA or modified DNA or RNA. For example, nucleic acid molecules of the present disclosure consisting of single-stranded and double-stranded DNA, a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and RNA of a mixture of single-stranded and double-stranded regions, hybrid molecules including DNA and RNA may be useful, and the hybrid molecules may be single-stranded, or more typically double-stranded, or a mixture of single-stranded and double-stranded regions. In addition, it may be useful for nucleic acid molecules to consist of triple-stranded regions including RNA or DNA or both RNA and DNA. Nucleic acid molecules of the present disclosure may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. "Modified" bases include, for example, tritiated bases and uncommon bases such as inosine. DNA and RNA may be modified in various ways; therefore, "nucleic acid molecules" encompass chemical, enzymatic or metabolically modified forms. The term "polynucleotide" should have a corresponding meaning. In some embodiments, genetically engineered plants, parts thereof, algae or plastids include at least one polynucleotide described herein.

[0496] The term "isolated polynucleotide" means a polynucleotide that has been separated from polynucleotide sequences that are typically associated or linked to the polynucleotide in its native state if the polynucleotide occurs in nature. Preferably, if present in nature, the isolated polynucleotide is at least 90% free from other components with which it is naturally associated. Preferably, the polynucleotide does not occur in nature, for example by covalently joining two shorter polynucleotide sequences in a manner that does not occur in nature (chimeric polynucleotides).

[0497] A genomic form or clone of a gene containing a transcribed region may be interrupted by non-coding sequences known as "introns" or "intervening regions" or "intervening sequences", which may be homologous or heterologous to the "exons" of the gene. As used herein, an "intron" is a fragment of a gene that is transcribed as part of a primary RNA transcript but is not present in a mature mRNA molecule. Introns are removed or "spliced ​​out" from the nuclear transcript or primary transcript; therefore, introns are not present in the messenger RNA (mRNA). Introns may contain regulatory elements such as enhancers. As described herein, the barley CAD2 gene (both resistance and sensitivity alleles) contains two introns in its protein coding region. As used herein, an "exon" refers to a DNA region corresponding to an RNA sequence present in a mature mRNA or mature RNA molecule when the RNA molecule is not translated. mRNA acts during translation to specify the sequence or order of amino acids in a nascent polypeptide. The term "gene" includes synthetic or fusion molecules encoding all or part of the protein of the invention described herein and nucleotide sequences complementary to any of the above. The gene can be introduced into an appropriate vector for extrachromosomal maintenance in the cell or, preferably, for integration into the host genome.

[0498] As used herein, "chimeric gene" refers to any gene including a covalently joined sequence that is not joined in nature. Typically, chimeric genes include regulation and transcription or protein coding sequences that are not present together in nature. Therefore, chimeric genes can include regulatory sequences and coding sequences derived from different sources or regulatory sequences and coding sequences that are derived from the same source but arranged in a manner different from the manner present in nature. The term "endogenous" refers to a material that is usually present or produced in an unmodified plant that is in the same developmental stage as the studied plant in this article. "Endogenous gene" refers to a natural gene that is in its natural position in the genome of an organism. As used herein, "recombinant nucleic acid molecules", "recombinant polynucleotides" or their variants refer to nucleic acid molecules that have been constructed or modified by recombinant DNA / RNA technology. The term "exogenous polynucleotide" or "exogenous polynucleotide" or "heterologous polynucleotide" etc. refers to any nucleic acid that is introduced into the genome of a cell by experimental manipulation.

[0499] A foreign or exogenous gene can be a gene inserted into a non-native organism or cell, a native gene introduced into a new location within a natural host, or a chimeric gene. Alternatively, a foreign or exogenous gene can be the result of editing the genome of an organism or cell or progeny derived therefrom. A "transgene" is a gene introduced into the genome by a transformation procedure.

[0500] The terms "genetically engineered," "genetically modified," "gene modification," or variations thereof refer to any genetic manipulation performed by humans and include the introduction of genes into cells by transformation or transduction, gene editing, cisgenesis, mutating genes in cells, and altering or regulating the regulation of genes in cells or organisms to perform these manipulations on them or their progeny, etc.

[0501] In addition, the term "exogenous" in the context of a polynucleotide (nucleic acid) refers to a polynucleotide present in a cell that does not naturally include a polynucleotide. The cell may be a cell that includes a non-endogenous polynucleotide that causes a change in the amount of production of the encoded polypeptide, such as an exogenous polynucleotide that increases the expression of an endogenous polypeptide, or a cell that does not produce a polypeptide in its natural state. Alternatively, the cell may be a cell with a chromosomal position change. The increased yield of the polypeptide of the present invention is also referred to as "overexpression" herein. The exogenous polynucleotide of the present invention includes polynucleotides that have not been separated from other components of the transgenic (recombinant) cell or cell-free expression system in which it is present, as well as polynucleotides that are subsequently purified from at least some other components produced in such cells or cell-free systems. An exogenous polynucleotide (nucleic acid) may be a continuous segment of nucleotides present in nature, or may include two or more continuous segments of nucleotides from different sources (naturally occurring and / or synthetic), which are joined to form a single polynucleotide. Typically, such chimeric polynucleotides include at least an open reading frame encoding a polypeptide of the present invention, and the chimeric polynucleotide is operably connected to a promoter suitable for driving transcription of the open reading frame in the cell of interest.

[0502] The % identity of the polynucleotides is determined by GAP (Needleman and Wunsch, 1970) analysis (GCG program) with a gap creation penalty = 5 and a gap extension penalty = 0.3. The query sequence is at least 900 nucleotides in length, and the GAP analysis aligns the two sequences over a region of at least 900 nucleotides. Preferably, the query sequence is at least 975 nucleotides in length, and the GAP analysis aligns the two sequences over a region of at least 975 nucleotides. Even more preferably, the query sequence is at least 1,050 nucleotides in length, and the GAP analysis aligns the two sequences over a region of at least 1,050 nucleotides. Even more preferably, the GAP analysis aligns the two sequences over their entire length.

[0503] With regard to defined polynucleotides, it will be appreciated that higher % identity figures than those provided above will encompass preferred embodiments. Thus, where applicable, based on the minimum % identity figures, it is preferred that the polynucleotide comprises a polynucleotide sequence that is at least 50%, at least 60%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.8% and even more preferably at least 99.9% identical to the relevant designated SEQ ID NO.

[0504] When referring to polynucleotide identity herein, it is understood that the sequence identity given refers to the open reading frame sequence.

[0505] In further embodiments, the present invention relates to polynucleotides that are substantially identical or identical to the polynucleotides specifically described herein. As used herein, with respect to polynucleotides, the term "substantially identical" means the substitution of one or several (e.g., 2, 3, or 4) nucleotides while maintaining at least one activity of the native protein encoded by the polynucleotide. In addition, the term encompasses the addition or deletion of nucleotides that increases or decreases the size of the encoded native protein by one or several (e.g., 2, 3, or 4) amino acids while maintaining at least one activity of the native protein encoded by the polynucleotide.

[0506] The present invention also relates to the use of oligonucleotides, such as in the method for screening polynucleotides of the present invention or polynucleotides encoding polypeptides. As used herein, "oligonucleotides" are polynucleotides of up to 50 nucleotides in length. The minimum size of such oligonucleotides is the size required for forming a stable hybrid between the complementary sequence on the oligonucleotide and the nucleic acid molecule of the present invention. It can be RNA, DNA, or a combination or derivative of any one. Oligonucleotides are generally relatively short single-stranded molecules of 10 to 30 nucleotides, usually 15-25 nucleotides in length. When used as a guide, probe or primer for genome editing in an amplification reaction, the minimum size of such oligonucleotides is the size required for forming a stable hybrid between the complementary sequence on the oligonucleotide and the target nucleic acid molecule. Preferably, the length of the oligonucleotide is at least 15 nucleotides, more preferably at least 18 nucleotides, more preferably at least 19 nucleotides, more preferably at least 20 nucleotides, more preferably at least 22 nucleotides, even more preferably at least 25 nucleotides. The oligonucleotides used as probes of the present invention are generally conjugated with labels such as radioisotopes, enzymes, biotin, fluorescent molecules or chemiluminescent molecules.

[0507] As known to those skilled in the art, the sequence of the oligonucleotide primers described herein can be changed to a certain extent without affecting its usefulness to the method of the present invention. "Variants" (also referred to herein as "primers" or "probes" according to their uses) of the oligonucleotides disclosed herein that can be used for the method of the present invention include molecules of different sizes, and / or molecules that can hybridize with the genome of the genome close to the specific oligonucleotide molecules defined herein. For example, variants can include additional nucleotides (such as 1, 2, 3, 4 or more) or fewer nucleotides, as long as they still hybridize with the target area. In addition, several nucleotides can be replaced without affecting the ability of the oligonucleotide to hybridize with the target area. In addition, variants can be easily designed, and the variant hybridization is close to (for example, but not limited to within 50 nucleotides or within 100 nucleotides) the genomic region of the specific oligonucleotide hybridization defined herein.

[0508] The present invention comprises oligonucleotides, and the oligonucleotides can be used as guides for RNA-guided endonucleases, probes for identifying nucleic acid molecules, or primers for producing nucleic acid molecules. Probes and / or primers can be used to clone homologues of polynucleotides of the present invention from other species. In addition, hybridization techniques known in the art can also be used to screen genomes or cDNA libraries of such homologues.

[0509] The polynucleotides and oligonucleotides of the present invention include polynucleotides and oligonucleotides that hybridize under stringent conditions to one or more of the sequences disclosed herein or in the art that are capable of increasing cannabinoid production. As used herein, stringent conditions are as follows: (1) using low ionic strength and high temperature for washing, for example, 0.015 M NaCl / 0.0015 M sodium citrate / 0.1% NaDodSO4 at 50°C; (2) using a denaturing agent such as formamide during hybridization at 42°C, for example, 50% (vol / vol) formamide with 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate; or (3) using 50% formamide, 5x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5x Denhardt's solution (Denhardt's solution), sonicated salmon sperm DNA (50 g / ml), 0.1% SDS, and 10% dextran sulfate.

[0510] When compared to naturally occurring molecules, the polynucleotides of the present invention may have one or more mutations, which are deletions, insertions or substitutions of nucleotide residues. Mutants may be naturally occurring (that is, isolated from natural sources) or synthetic (e.g., by subjecting nucleic acids to site-directed mutagenesis). When compared to reference polynucleotides as defined herein, variants of the polynucleotides of the present invention include molecules of different sizes. For example, variants may include additional nucleotides (e.g., 1, 2, 3, 4 or more) or fewer nucleotides, as long as they encode functional proteins. In addition, several nucleotides may be substituted without affecting the integrity of the encoded protein. In addition, variants may include polynucleotides that encode the same polypeptide or amino acid sequence but the nucleotide sequence varies due to the redundancy of the gene code. The terms "polynucleotide variants" and "variants" also include naturally occurring allele variants.

[0511] Nucleic acid construct

[0512] The present invention further relates to nucleic acid constructs of the polynucleotides of the present invention, as well as vectors and host cells containing these polynucleotides, methods for their production and use, and uses thereof.

[0513] To enable expression of a polynucleotide in a plant, part thereof, algae or plastid thereof, a nucleic acid construct may include a coding region encoding a polynucleotide sequence as described herein, operably linked to one or more regulatory sequences, such as a promoter sequence, optionally a heterologous promoter sequence that functions in the plant, algae or plastid in which the nucleic acid construct is to be expressed.

[0514] A first polynucleotide is operably linked to a second polynucleotide when the first polynucleotide is in a functional relationship with the second polynucleotide. When produced recombinantly, operably linked polynucleotides are typically contiguous and, when necessary to join two protein coding regions, in the same reading frame (e.g., in a polycistronic ORF or joined by a peptide linker). However, polynucleotides do not need to be contiguous to be operably linked.

[0515] Regulatory sequences or control elements refer to nucleotide sequences that affect the timing and level / amount of transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters; translation leader sequences; introns; enhancers; stem-loop structures; repressor binding sequences; termination sequences; polyadenylation recognition sequences; etc. Specific regulatory sequences may be located upstream and / or downstream of the coding sequence to which they are operably linked.

[0516] As used herein, the term "cis-acting sequence", "cis-acting element" or "cis-regulatory region" or "regulatory region" or similar terms should be understood as any nucleotide sequence that, when properly positioned and linked relative to an expressible gene sequence, is capable of at least partially regulating the expression of a gene sequence. Those skilled in the art will appreciate that a cis-regulatory region may be capable of activating, silencing, enhancing, inhibiting or otherwise altering the expression level and / or cell type specificity and / or developmental specificity of a gene sequence at the transcriptional or post-transcriptional level. In a preferred embodiment of the invention, a cis-acting sequence is an activator sequence that enhances or stimulates the expression of an expressible gene sequence.

[0517] As used herein, the term "promoter" refers to a DNA region that may be located upstream of the transcription start point, and the region may involve the recognition and combination of RNA polymerase and other proteins to initiate transcription. The promoter may be operably connected to a coding sequence for expression in a cell, or the promoter may be operably connected to a polynucleotide encoding a signal sequence, which may be operably connected to a coding sequence for expression in a cell, preferably a plant or algae cell." plant promoter " may be a promoter that can initiate transcription in a plant cell. Similarly, an algae promoter may be a promoter that can initiate transcription in a plant cell. Examples of promoters that are subject to developmental control include promoters that preferentially initiate transcription in certain tissues such as leaves, roots, seeds, fibers, xylem vessels, tracheids, or sclerenchyma.

[0518] Promoters that initiate transcription only in certain tissues are referred to as "tissue specific". "Cell type specific" promoters primarily drive expression in certain cell types in one or more organs, such as trichomes or vascular cells in roots or leaves. Promoters that initiate transcription only in certain subcellular organelles (e.g., mitochondria or chloroplasts or other plastids) are referred to as "plastid-specific", "chloroplast-specific" or "mitochondrial-specific" promoters, respectively. It should be understood that some promoters can initiate transcription in more than one subcellular location.

[0519] In one embodiment, the nucleic acid construct contains a promoter or regulatory element operable in a plastid. Alternatively, the nucleic acid construct contains a promoter or regulatory element operable in the nucleus or cytosol.

[0520] An "inducible" promoter may be an environmentally controlled promoter. Examples of environmental conditions that may initiate transcription by an inducible promoter include anaerobic conditions and the presence of light. Tissue-specific, tissue-preferential, cell-type 25-specific and inducible promoters constitute the category of "non-constitutive" promoters. A "constitutive" promoter is a promoter that may be active under most environmental conditions or in most cell or tissue types.

[0521] Any inducible promoter can be used in some embodiments of the present invention (see Ward et al., 1993). With an inducible promoter, the transcription rate increases in response to an inducing agent. Exemplary inducible promoters include, but are not limited to, promoters from the ACEI system that respond to copper; the 1n2 gene from maize that responds to benzenesulfonamide herbicide safeners; the Tet repressor from Tn10; and inducible promoters from steroid hormone genes, whose transcriptional activity can be induced by glucocorticoids (Schena et al., 1991).

[0522] Exemplary constitutive promoters include, but are not limited to, promoters from plant viruses, such as the 35S promoter from cauliflower mosaic virus (CaMV); a promoter from the rice actin gene; an ubiquitin promoter; pEMU; MAS; a maize H3 histone promoter; and an ALS promoter, the Xbal / Ncol fragment 5' of the Brassica napus ALS3 structural gene (or a nucleotide sequence similar to the Xbal Ncol fragment) (WO 96 / 30530).

[0523] Additionally, in some embodiments of the present invention, any tissue-specific promoter may be used. Plants transformed with a nucleic acid construct comprising a coding sequence operably linked to a tissue-specific promoter may produce the product of the coding sequence exclusively or preferentially in a particular tissue. Exemplary tissue-specific or tissue-preferred promoters include, but are not limited to: root-preferred promoters, such as promoters from the phaseolin gene; leaf-specific and light-inducible promoters, such as promoters from cab or rubisco; anther-specific promoters, such as promoters from LAT52; pollen-specific promoters, such as promoters from ZmJ3; and microspore-preferred promoters, such as the apg promoter.

[0524] Promoters suitable for use in the nucleic acid constructs of the present invention include inducible, viral, synthetic or constitutive promoters, all of which are well known in the art.

[0525] In order to express in the specific tissues of plants such as leaves, seeds, roots or stems, it is preferred to have relatively high expression in these specific tissues for the promoter of the present invention. For this reason, it is possible to select from many promoters of genes with tissue or cell specificity or enhanced expression. The example of this column promoter comprises the chloroplast glutamine synthetase GS2 promoter from pea, the chloroplast fructose-1,6-bisphosphatase promoter from wheat, the nuclear photosynthetic ST-LS1 promoter from potato, the serine / threonine kinase promoter from Arabidopsis thaliana and the glucoamylase (CHS) promoter. Reportedly active in photosynthetically active tissues are the ribulose-1,5-bisphosphate carboxylase promoter from eastern larch (Larix laricina), the promoter of the Cab gene Cab6 from pine, the promoter of the Cab-1 gene from wheat, the promoter of the Cab-1 gene from spinach, the promoter of the Cab 1R gene from rice, the promoter of the pyruvate, orthophosphate dikinase (PPDK) from maize, the promoter from the tobacco Lhcb1*2 gene, the Arabidopsis thaliana Suc2 sucrose-H30 symporter promoter, and the promoters of thylakoid membrane protein genes from spinach (PsaD, PsaF, PsaE, PC, FNR, AtpC, AtpD, Cab, RbcS). Other promoters of chlorophyll α / β binding protein can also be used in the present invention, such as the promoters of the LhcB gene and the PsbP gene from white mustard (Sinapis alba).

[0526] Various plant gene promoters that are regulated in response to environmental, hormonal, chemical and / or developmental signals can also be used to express RNA binding protein genes in plant cells, including promoters regulated by: (1) heat; (2) light (e.g., pea RbcS-3A promoter, maize RbcS promoter); (3) hormones such as abscisic acid; (4) wounding (e.g., WunI); or (5) chemicals such as methyl jasmonate, salicylic acid, steroid hormones, alcohols, safeners, etc. (WO 97 / 06269); or (6) organ-specific promoters may also be used.

[0527] Other non-limiting examples describing such promoters include US Pat. Nos. 6,437,217 (maize RS81 promoter); 5,641,876 (rice actin promoter); 6,426,446 (maize RS324 promoter); 6,429,362 (maize PR-i promoter); 6,232,526 (maize A3 promoter); 6,177,611 (constitutive maize promoter); 5,322,938, 5,352,605, 5,359,142 and 5,530,196 (35S promoter); 6,433 ,252 (maize L3 oleosin promoter); 6,429,357 (rice actin 2 promoter and rice actin 2 intron); 6,294,714 (light-inducible 25 promoter); 6,140,078 (salt-inducible promoter); 6,252,138 (pathogen-inducible promoter); 6,175,060 (phosphate deficiency-inducible promoter); 6,388,170 (bidirectional promoter); 6,635,806 (γ-coixin promoter); and US09 / 757,089 (maize chloroplast aldolase promoter). Additional promoters include the nopaline synthase (NOS) promoter (Ebert et al., 1987) and the octopine synthase (OCS) promoter (both carried on a tumor-inducing plasmid of Agrobacterium tumefaciens); cauliflower mosaic virus group promoters, such as the cauliflower mosaic virus (CaMV) 19S promoter (Lawton et al., 1987); CaMV 35S promoter (Odell et al., 1985); Scrophularia mosaic virus 35S promoter (Walker et al., 1987); sucrose synthase promoter (Yang and Russell, 1990); R gene composite promoter (Chandler et al., 1989); chlorophyll a / b binding protein gene promoter; CaMV35S (US Pat. Nos. 5,322,938, 5,352,605, 5,359,142 and 5,530,196); FMV35S (US Pat. Nos. 6,051,753 and 5,378,619); PC1SV promoter (US Pat. No. 5,850,019); SCP1 promoter (US Pat. No. 6,677,503); and AGRtu.nos promoter (GenBank Accession No. V00087; Depicker et al., 1982; Bevan et al., 1983).

[0528] For expression in vegetative tissues, leaf-specific promoters such as the ribulose bisphosphate carboxylase (RBCS) promoter can be used. For example, the tomato RBCS1, RBCS2, and RBCS3A genes are expressed in leaves and light-grown seedlings (Meier et al., 1997). The ribulose bisphosphate carboxylase promoter described by Matsuoka et al. (1994) can be used, which is expressed at high levels almost exclusively in the mesophyll cells of leaves and sheaths. Another leaf-specific promoter is the light-harvesting chlorophyll a / b binding protein gene promoter (see Shiina et al., 1997). The Arabidopsis myb-related gene promoter (Atmyb5) described by Li et al. (1996) has leaf specificity. The Atmyb5 promoter is expressed in developing leaf trichomes, stipules, and epidermal cells on the margins of young rosettes and cauline leaves, as well as in immature seeds. Leaf promoters identified in maize by Busk et al. (1997) can also be used. Additional promoters for regulating endogenous pathways are described in et al., 1996, Noh and Amasino 1999, Borghi (2010), and Corrado and Karali (2009).

[0529] The additional regulatory sequences that can be optionally operably connected to the polynucleotide can include a 5'UTR between the promoter sequence and the coding sequence used as a translation leader sequence. The translation leader sequence is present in the fully processed mRNA, and it may affect the processing and / or RNA stability of the primary transcript. Examples of translation leader sequences include maize and petunia heat shock protein leaders (US5,362,865), plant virus coat protein leaders, plant rubisco leaders, etc. (see, for example, Turner and Foster, 1995). Non-limiting examples of 5'UTR include GmHsp (US5,659,122); PhDnaK (US5,362,865); AtAntl; TEV (Carrington and Freed, 1990); and AGRtunos (GenBank accession number V00087; and Bevan et al., 1983).

[0530] In some embodiments, the promoter region is derived from a chloroplast gene, such as the psbA gene from spinach or pea, the rbcL and atpB promoter regions from maize, and the rRNA promoter (from the plastid rrn operon). Examples of promoters are described in: Verma Daniell (2007); Rasala et al. (2011); Hanley-Bowdoin and Chua, (1987); Mullet et al. (1985); Hanley-Bowdoin (1986) PhD dissertation, The Rockefeller University; Krebbers et al. (1982); Zurawski et al. (1981); and Zurawski et al. (1982). Other promoters can be identified and the relative strength of the identified promoters assessed by placing the promoter 5' of interest on a promoterless marker gene and observing the effectiveness of transcription relative to that obtained from, for example, the promoter from the psbA gene, the strongest chloroplast promoter identified to date. The efficiency of coding region expression can additionally be increased by a variety of techniques. These include the use of multiple promoters inserted in tandem 5' to the DNA sequence of interest, such as a double psbA promoter, the addition of enhancer sequences, etc.

[0531] In a preferred embodiment, the nucleic acid construct is any one of the nucleic acid constructs listed in the examples, and can be optionally included in a vector. In another embodiment, the nucleic acid construct can include a long intergenic region (LIR), a promoter or a terminator described in the examples or known in the art.

[0532] In one aspect of the invention, methods are provided for integrating polynucleotides or nucleic acid constructs of the invention into nuclear genomic DNA or plastid (e.g., chloroplast) genomic DNA of plant or algae cells. In the former case, it is envisioned that the nucleic acid construct will contain polynucleotides encoding a fusion polypeptide comprising a plastid transport peptide. In the latter case, the nucleic acid construct will not necessarily contain polynucleotides encoding a fusion polypeptide comprising a plastid transport peptide.

[0533] When chloroplast genomic DNA integration is contemplated, one or more selective integration sites are required for integration of the construct into the chloroplast genome of the plant or algae. Examples of chloroplast integration regions include, but are not limited to, trnV-3'-rpsl2, trnI-trnA, and trnfM-trnG. Examples of specific chloroplast integration sites include, but are not limited to, trnH / pbA, trnG / trnfM, ycf3 / trnS, rbcL / accD, petA / psbJ, 5'rps12 / clpP, petD / rpoA, ndhB / rps7, 3'rpsl2 / trnV, trnV / rrnl6, rrnl6 / trnl, trnl / trnA, trnN / trnR, and rp32 / trnL.

[0534] Carrier

[0535] In another embodiment of the present invention, the polynucleotides described herein may be contained in a vector, preferably a vector suitable for expression in plants, algae or their plastids. In another embodiment, a host cell is transformed with a vector or nucleic acid molecule comprising a nucleic acid sequence as described herein. In another embodiment, the host cell is any microorganism described herein. As used herein, the term "vector" or "nucleic acid vector" means a nucleic acid molecule including regulatory elements and a site for introducing DNA such as a plasmid, which is used to introduce the DNA into a plant, algae, its cell or plastid. DNA can encode heterologous proteins, which can be expressed in plants, algae, its cells or plastids described herein, and used to increase the yield of cannabinoids described herein. Transgenic DNA can be integrated into the nuclear, mitochondrial or chloroplast genome by homologous or non-homologous recombination. DNA can also be replicated in an extrachromosomal vector without being integrated into the nuclear, mitochondrial or chloroplast genome. The vector can contain a single set of operably connected regulatory elements comprising a promoter, a 5' untranslated region (5'UTR), an insertion site for transgenic DNA, a 3' untranslated region (3'UTR) and a terminator sequence. Vectors useful in the methods of the present invention are well known in the art.

[0536] The carrier can be RNA or DNA, can be prokaryotic or eukaryotic, and is typically derived from a viral vector of a virus or plasmid. Plasmid vectors are typically included in other nucleotide sequences that provide easy selection, amplification and conversion of expression cassettes in prokaryotic cells, for example pUC derivative vectors, pSK derivative vectors, pGEM derivative vectors, pSP derivative vectors, pBS derivative vectors or binary vectors containing one or more T-DNA districts. Other nucleotide sequences include replication origins for providing autonomous replication of the carrier, preferably encoding selectable marker genes for antibiotic or herbicide resistance, providing multiple sites to insert the unique multiple cloning site of the nucleotide sequence encoded in the nucleic acid construct or the gene, and sequences that enhance prokaryotic and eukaryotic (especially plant) cell transformation.

[0537] In another embodiment, the vector is a commercially available vector. As used herein, the term "expression cassette" means a single operably linked set of regulatory elements comprising a promoter, a 5' untranslated region (5'UTR), an insertion site for transgenic DNA, a 3' untranslated region (3'UTR), and a terminator sequence. In one embodiment, the at least one polynucleotide is an episomal vector. In a preferred embodiment, the vector is any of the vectors listed in Table 3. In a more preferred embodiment, the vector comprises elements of a viral vector to enhance expression, for example, by utilizing the bean yellow dwarf virus (BeYDV) vector replication mechanism as described in Example 5 herein (Diamos and Mason 2019). In this regard, in a preferred embodiment, elements of the BeYDV comprising the replication mechanism can provide strong expression of the polynucleotides described herein. Specifically, in one embodiment, two replication proteins Rep and RepA produced on complementary sense DNA strands (C1 / C2 genes) can be utilized. Rep and RepA are produced from a single intron-containing transcript: RepA is the major protein product from the unspliced ​​transcript, while relatively rare intron excision alters the reading frame to produce Rep. In this example, the production of viral proteins is driven by a single bidirectional promoter in the long intergenic region (LIR), which also contains the viral origin of replication. The two different transcripts converge at a short intergenic region (SIR), which has a bidirectional transcription terminator signal and is suspected to be the origin of complementary chain synthesis (Liu et al., 1998).

[0538] In addition, polynucleotides, nucleic acid constructs or vectors can contain one or more introns before the cloning site or in the gene sequence to drive the strong expression of the gene of interest. The one or more introns can include introns of FBAC2-1TUFA-1, EIF6-1, RPS4-1, RbcS2-1, and RbcS2-2. Polynucleotides, nucleic acid constructs or vectors can contain more than one intron or more than one copy of the same intron. Polynucleotides, nucleic acid constructs or vectors can also contain suitable terminators, such as tEF-1a, t40SRPS8, tH4-1 B, ty-tubulin, tRBCMT, tFcpB, tFcpC, tFcpD, PAL, tFcpA, and tRbcS2. Selectable marker genes can also be linked to the vector, such as kanamycin resistance gene (also known as neomycin phosphotransferase gene II or nptII), zeocin resistance gene, hygromycin resistance gene, Basta resistance gene, hygromycin resistance gene, and the like.

[0539] According to the present invention, each expression vector may contain a promoter that drives transcription in a plant, algae or plant cell. As used herein, an "expression vector" is a DNA or RNA vector that can transform a host cell and achieve expression of one or more specific polynucleotides. Preferably, the expression vector is also capable of replicating within the host cell. The expression vector may be prokaryotic or eukaryotic, and is typically a virus or plasmid. The expression vector of the present invention is included in any vector that plays a role (i.e., direct gene expression) in algae and plant cells.

[0540] The polynucleotides, recombinant nucleic acid constructs or vectors of the present invention may also contain a selectable marker. The selectable marker may be used to monitor expression. Exemplary screenable markers include: a p-glucuronidase or uidA gene (GUS) encoding an enzyme with known chromogenic substrates (Jefferson et al., 1987); an R locus gene encoding a product that regulates the production of anthocyanin pigments (red) in plant tissues (Dellaporta et al., 1988); a β-lactamase gene (Sutcliffe et al., 1978); genes encoding 20 enzymes with known chromogenic substrates (e.g., PADAC, chromogenic cephalosporins); a luciferase gene (Ow et al., 1986); a xylE gene encoding a catechol dioxidase that converts chromogenic catechol (Zukowski et al., 1983); an amylase gene (Ikatu et al., 1990); a tyrosinase gene encoding an enzyme that oxidizes tyrosine to DOPA and dopaquinone, which are then condensed into melanin (Katz et al., 1983); and an α-galactosidase.

[0541] As used herein, the term "tag" refers to an amino acid sequence recognized by an antibody. The tag amino acid sequence is connected to the sequence of, for example, an enzyme, thereby allowing the enzyme to be detected or separated by binding between the tag and the tag-specific antibody. For example, common tags known in the art include 6His, MYC, FLAG, V5, HA, and HSV. These tags are useful when located at the N or C terminus.

[0542] As used herein, the term "reporter gene" refers to another molecule that allows detection of the reporter gene to which it is attached or associated, or a molecule that allows detection of an organism that includes the reporter gene. The reporter gene may comprise a fluorescent molecule comprising a fluorescent protein such as green fluorescent protein (GFP), yellow fluorescent protein (YFP), mNeonGreen, and red fluorescent protein (RFP). In some embodiments, the at least one nucleic acid molecule comprises one or more reporter gene sequences encoding a reporter gene having an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the nucleotide sequence as shown in SEQ ID NO: 16.

[0543] Nucleic acid sequences as described herein can be provided in a vector in different arrangements or combinations. Each individual sequence encoding a component of the cannabinoid biosynthetic pathway can be provided in a separate vector. Alternatively, multiple sequences can be provided together in the same vector. For example, polynucleotides encoding polyketide synthase and olivetate cyclase (OAC), acyl activating enzyme 1 (AAE1) and olivetol synthase (OLS) can be provided together in a first vector, and polynucleotides encoding isoprenyl transferase can be provided in a second vector. Alternatively, these sequences can be provided together in the same vector. When more than one polynucleotide is provided in the same vector, the sequences can be provided in separate expression cassettes, or provided together in the same expression cassette.

[0544] Increase RNA expression levels and stabilize expression

[0545] Post-transcriptional gene silencing (PTGS) is a nucleotide sequence-specific defense mechanism that can target both cellular and viral mRNAs for degradation. PTGS is present in plants that are stably or transiently transformed with recombinant polynucleotides and results in a reduction in the accumulation of RNA molecules similar to the introduced polynucleotide sequence. "Post-transcriptional" is understood to refer to a mechanism that operates at least partially but not necessarily completely after the initial RNA transcript is produced, for example during processing of the initial RNA transcript, or with RNA splicing or export to the cytoplasm, or within the cytoplasm through a complex associated with an Argonaute protein.

[0546] By limiting the expression of silencing suppressors in plants or parts thereof, the RNA molecule level can be increased, and / or the RNA molecule level can be stabilized in many generations. As used herein, "silencing suppressors" are any polynucleotides or polypeptides that can be expressed in plant cells that increase the level of expression products of genes introduced from different plant cells, specifically in repeated generations of initially genetically engineered plants. In one embodiment, the silencing suppressor is a viral silencing suppressor or a mutant thereof. A large number of viral silencing suppressors are known in the art and include, but are not limited to, P19, V2, P38, Pe-Po and RPV-P0. Examples of suitable viral silencing suppressors include those described in WO 2010 / 057246. Silencing suppressors can be stably expressed in plants or parts thereof of the present invention.

[0547] As used herein, the term "stable expression" or variants thereof refers to that the level of RNA molecules in progeny plants is substantially the same or higher in repeated generations, such as at least three generations, at least five generations, or at least ten generations, when compared to a corresponding plant lacking an exogenous polynucleotide encoding a silencing suppressor. However, the term does not exclude the possibility that there is some loss of RNA molecule levels in repeated generations when compared to the previous generation, such as a loss of not less than 10% per generation.

[0548] The inhibitory factor can be selected from any source, such as plants, viruses, mammals, etc. The inhibitory factor can be, for example, flock house virus B2, pothos latent virus P14, pothos latent virus AC2. African cassava mosaic virus AC4, Bhendi yellow vein mosaic disease C2, Bhendi yellow vein mosaic disease C4, Bhendi yellow vein mosaic disease βC1, tomato chlorosis virus p22, tomato chlorosis virus CP, tomato chlorosis virus CPm, tomato golden mosaic virus AL2, tomato leaf curl Java virus βC1, tomato yellow leaf curl virus V2, tomato yellow leaf curl virus-China C2, tomato yellow leaf curl virus Y10 isolate βC1, tomato yellow leaf curl Israel isolate V2, mungbean yellow mosaic virus-Vigna AC2, hibiscus chlorotic ringspot virus The following are the virus strains: turnip crinkle virus P38, turnip crinkle virus CP, cauliflower mosaic virus P6, beet yellows virus P21, citrus tristeza virus P20, citrus tristeza virus P23, citrus tristeza virus CP, cowpea mosaic virus SCP, sweet potato chlorotic stunt virus P22, cucumber mosaic virus 2b, tomato aspermy virus HC-Pro, beet curly top virus L2, soil borne wheat mosaic virus 19K, barley stripe mosaic virus Gammab, poa semilatent virusvirus) Gammab, peanut clump pecluvirus P15, rice dwarf virus Pns10, cucurbit aphid borne yellows virus P0, beet western yellows virus P0, potato virus X P25, cucumber vein yellowing virus P1b, plum pox virus HC-Pro, sugarcane mosaic virus HC-Pro, potato virus Y strain HC-Pro, tobacco etch virus P1 / HC-Pro, turnip mosaic virus P1 / HC-Pro, cocksfoot mottle virus P1, cocksfoot mottle virus-Norway isolate P1, rice yellow mottle virus virus P1, rice yellow mottle virus-Nigeria isolate P1, rice hojablanca virus NS3, rice stripe virus NS3, crucifer infecting tobacco mosaic virus 126K, crucifer infecting tobacco mosaic virus p122, tobacco mosaic virus p122, tobacco mosaic virus 126, tobacco mosaic virus 130K, tobacco rattle virus 16K, tomato bushy stunt virus P19, tomato spotted wilt virus NSs, apple chloroticleaf spot virus P50, grapevine virus Ap10, grapevine leafroll associated virus BYV p21The above list provides viruses and proteins (e.g., B2, P14, etc.) from which inhibitors can be obtained, or the names of the coding regions of inhibitors from each specific virus. As will be appreciated by those skilled in the art, other candidate silencing inhibitors can be obtained by examining the viral genome sequence for polypeptides encoded at the same position within the viral genome relative to the structure of the related viral genome including the known silencing inhibitor.

[0549] Silencing inhibitors can be classified based on their mode of action. When used in combination with gene silencing (exogenous polynucleotides encoding dsRNA), inhibitors such as V2 and the like that preferentially bind to double-stranded RNA molecules with protruding 5' ends relative to corresponding double-stranded RNA molecules with flat ends are particularly useful for enhancing transgenic expression. In the presence of exogenous polynucleotides encoding dsRNA, other inhibitors such as p19 and the like that preferentially bind to dsRNA molecules with a length of 21 base pairs relative to dsRNA molecules of different lengths can also allow transgenic expression, but generally to a lesser extent than, for example, V2. This allows the best combination of dsRNA, silencing inhibitors and overexpressed transgenics to be selected for a specific purpose. Such best combinations can be identified using the method of the present invention. Multiple copies of inhibitors can be used. Different inhibitors can be used together (e.g., in series).

[0550] In one embodiment, the silencing suppressor preferentially binds to a double-stranded RNA molecule with a protruding 5' end relative to a corresponding double-stranded RNA molecule with a blunt end. In this context, the corresponding double-stranded RNA molecule preferably has the same nucleotide sequence as the molecule with a 5' protruding end, but does not have a protruding 5' end. The binding assay is generally performed by any method known to those skilled in the art, such as in an in vitro assay.

[0551] In a specific example, the plant produces higher levels of an enzyme used to produce cannabinoids in plants such as tobacco or canola.

[0552] Codon optimization

[0553] It will be appreciated that it is possible to increase the expression of heterologous nucleic acids in a host organism or host cell by replacing a nucleotide sequence encoding a particular amino acid (i.e., a codon) with another codon that is better expressed in the host organism (i.e., codon optimization). One reason for this effect is the fact that different organisms exhibit preferences for different codons. In some embodiments, the heterologous polynucleotides disclosed herein are modified or optimized such that the nucleotide sequence reflects the codon preference of a specific host cell, preferably a plant or algae cell.

[0554] For example, in some embodiments, the polynucleotides can be modified or optimized for plant codon preferences, preferably vascular plants, more preferably Nicotiana, preferably Nicotiana benthamiana or Nicotiana tabacum. Alternatively, in some embodiments, the polynucleotides can be modified or optimized for algae codon preferences using methods known in the art for specific algae useful in the present invention, including Chlamydomonas sp., Dunaliella sp., and Chlorella sp. In another embodiment, the polynucleotides can be further modified or optimized for plastid expression in plants, parts thereof, or algae.

[0555] Statistical methods have been generated to analyze the codon usage bias in various plants or algae, and many computer algorithms have been developed to implement these statistical analyses in the design of codon-optimized gene sequences. Other modifications of codon usage that increase protein expression that are independent of codon bias have also been described.

[0556] It should be understood that the present disclosure encompasses degeneracy in codon usage, as understood by one of ordinary skill in the art and as presented in Table 2.

[0557] Table 2: Exemplary substitutions.

[0558]

[0559]

[0560] Recombinant cells

[0561] Another embodiment of the present invention comprises a recombinant cell comprising a host cell, preferably a plant or algae cell transformed with one or more exogenous polynucleotides, nucleic acid constructs or vectors of the present invention, or a progeny cell thereof. The transformation of a polynucleotide, nucleic acid construct or vector into a cell can be accomplished by any method for inserting a nucleic acid molecule into a cell. Transformation techniques include, but are not limited to, transfection, particle bombardment / biolistics, electroporation, microinjection, lipofection, adsorption, and protoplast fusion. In one embodiment, gene editing can be used to transform a target cell using, for example, a targeted nuclease such as TALEN, MADS7, Cpf1, or Cas9-CRISPR or an engineered nuclease derived therefrom.

[0562] Recombinant cells can remain unicellular, or can grow into tissues, organs or multicellular organisms. The transformed nucleic acid construct of the present invention can remain outside the chromosome, or can be integrated into one or more sites in the chromosome of the transformed (i.e., recombinant) cell in such a way that its expression capacity is retained. Preferred host cells are plants or algae cells, more preferably plant cells.

[0563] The host cell into which the polynucleotide is introduced may be an untransformed cell or a cell that has been transformed with at least one nucleic acid. Such nucleic acids may be related to lipid synthesis, cannabinoid synthesis, or may be unrelated. The host cell of the present invention may be endogenously (i.e., naturally) capable of producing a polypeptide as defined herein, in which case the recombinant cell derived therefrom has an enhanced ability to produce the polypeptide, or may only produce the polypeptide after transformation with at least one polynucleotide of the present invention. In one embodiment, the recombinant cell of the present invention has an enhanced ability to produce cannabinoids.

[0564] Nuclease genetic engineering

[0565] Endonucleases can be used to create single-strand or double-strand breaks in genomic DNA. Genomic DNA breaks in eukaryotic cells are repaired using either nonhomologous end joining (NHEJ) or homology-directed repair (HDR) pathways. NHEJ can result in incomplete repair, leading to unwanted mutations, and HDR can enable precise gene insertion by using exogenously provided repair DNA templates. CRISPR-associated (Cas) proteins have received a great deal of attention, and although transcription activator-like effector nucleases (TALENs) and zinc finger nucleases remain useful, the CRISPR-Cas system provides a simpler, more versatile, and less expensive tool for genome modification (Doudna and Charpentier, 2014).

[0566] CRISPR-Cas systems are classified into three main groups using various nucleases or combinations of nucleases. In class 1 CRISPR-Cas systems (types I, III, and IV), the effector module consists of a multiprotein complex, while class 2 systems (types II, V, and VI) use only one effector protein (Makarova et al., 2015). Cas contains genes that are coupled to or close to or located near the flanking CRISPR loci. Haft et al. (2005) provide a review of the Cas protein family.

[0567] The nuclease is guided by a synthetic small guide RNA (sgRNA or gRNA) that may or may not contain a tracRNA, thereby simplifying the CRISPR-Cas system to two genes: the endonuclease and the sgRNA (Jinek et al. 2012). The sgRNA is typically under the regulatory control of the U3 or U6 small nuclear RNA promoter. The sgRNA recognizes a specific gene and the portion of the gene that is used for targeting. The protospacer adjacent motif (PAM) is adjacent to the target site, thereby limiting the number of potential CRISPR-Cas targets in the genome, although amplification of the nuclease also increases the available number of PAMs. There are many web tools available for designing gRNAs, including CHOPCHOP (http: / / chopchop.cbu.uib.no), CRISPR Design https: / / omictools.com / crispr-design-tool, E-CRISP http: / / www.e-crisp.org / E-CRISP / , Geneious, or Benchling https: / / benchling.com / crispr.

[0568] The CRISPR-Cas system is the most commonly used system in eukaryotic work to date, using the Cas9 effector protein, typically using RNA-guided Streptococcus pyogenes Cas9 or optimized sequence variants in a variety of plant species (Luo et al., 2016). Luo et al. (2016) summarized many studies in which genes have been successfully targeted to various plant species, thereby producing insertion deletions and loss-of-function mutant phenotypes in endogenous gene open reading frames and / or promoters. Due to the cell wall on plant cells, the delivery of CRISPR-Cas mechanisms to cells and successful transgenic regeneration are usually delivered using Agrobacterium tumefaciens infection (Luo et al., 2016) or plasmid DNA particle bombardment or bioballistic delivery. Vectors suitable for cereal transformation include pCXUNcas9 (Sun et al., 2016) or pYLCRISPR / Cas9Pubi-H (Ma et al., 2015, accession number KR029109.1) available from Addgene.

[0569] Alternative CRISPR-Cas systems refer to effector enzymes containing a nuclease RuvC domain but without an HNH domain, the effector enzyme comprising a Cas12 enzyme, the Cas12 enzyme comprising Cas12a, Cas12b, Cas12f, Cpf1, C2c1, C2c3 and engineered derivatives. Cpf1 produces double-strand breaks in a staggered manner at the distal location of the PAM, and as a smaller endonuclease, can provide advantages for certain species (Begemann et al., 2017). Other CRISPR-Cas systems include RNA-guided RNases, the RNA-guided RNases comprising Cas13, Cas13a (C2c2), Cas13b, Cas13c.

[0570] Sequence insertion or integration

[0571] The CRISPR-Cas system can be combined with the provision of polynucleotides to direct homologous repair to insert sequences into the genome. Targeted genomic integration of plant transgenes enables the continuous addition of transgenes at the same locus. This "cis gene stacking" will greatly simplify subsequent breeding efforts, with all transgenes inherited as a single locus. When coupled with CRISPR / Cas9 cleavage at the target site, the transgene can be integrated into the locus through homology-directed repair promoted by flanking sequence homology. This approach can be used to quickly introduce new alleles without linkage drag, or to introduce allelic variants that do not occur naturally.

[0572] Nicking enzyme

[0573] The CRISPR-Cas II system uses a Cas9 nuclease with two enzymatic cleavage domains, RuvC and HNH domains. Mutations have been shown to change double-stranded cleavage to single-stranded cleavage and produce technical variants of Cas9 known as nickase or nuclease inactivation. The RuvC subdomain cuts non-complementary DNA strands, and the HNH subdomain cuts DNA strands complementary to the gRNA. Nickase or nuclease inactivated Cas9 retains DNA binding ability guided by gRNA. Mutations in subdomains are known in the art, such as Streptococcus pyogenes (S.pyogenes) Cas9 nucleases with D10A mutations or H840A mutations.

[0574] Genome base editing or modification

[0575] Base editors have been created by fusing deaminases to Cas9 domains (WO 2018 / 086623). By fusing deaminases, sequence targeting guided by gRNA can be used to convert targeted cytidine (C) to uracil (U) by deamination of cytidine in DNA. The cell's mismatch repair mechanism then replaces U with T. Suitable cytidine deaminases may include APOBEC1 deaminases, activation-induced cytidine deaminases (AID), APOBEC3G, and CDA1. Further, Cas9-deaminase fusions may be mutated Cas9s with nickase activity to produce single-strand breaks. It has been shown that nickase proteins may be more efficient in promoting homology-directed repair (Luo et al., 2016).

[0576] Vector-free genome editing or genome modification

[0577] Recently, a vector-free method using Cas9 / sgRNA ribonucleoprotein has been described as successfully reducing off-target events. The method requires in vitro expression of Cas9 ribonucleoprotein (RNP), which is transformed into cells or protoplasts and is integrated into the host genome independently of Cas9, thereby reducing the undesired side cutting associated with the random integration of the Cas9 gene. Only short side-joining sequences are required to form stable Cas9 and sgRNA stable ribonucleoproteins in vitro. Woo et al. (2015) produced preassembled Cas9 / sgRNA protein / RNA complexes and introduced them into protoplasts of Arabidopsis, rice, lettuce and tobacco, and observed up to 45% of targeted mutagenesis frequencies in regenerated plants. RNP and in vitro have been confirmed in several species including dicots (Woo et al., 2015) and monocots maize (Svitashev et al., 2016) and wheat (Liang et al., 2017). Genome editing in plants using CRISPR-Cas 9 in vitro transcripts or ribonucleoproteins is well described in Liang et al. (2018) and Liang et al. (2019).

[0578] Methods for gene insertion

[0579] Plant embryos can be bombarded with Cas9 genes and sgRNA genes targeting the integration site and DNA repair templates. The DNA repair template can be a synthetic DNA fragment or a 127-mer oligonucleotide, each of which encodes a cDNA or a gene of interest. The bombarded cells grow on tissue culture medium. DNA extracted from callus or T0 plant leaf tissue using the CTAB DNA extraction method can be analyzed by PCR to confirm gene integration. If pcr confirms the presence of the gene of interest, T1 plants are selected.

[0580] The method comprises introducing the polynucleotide of interest, which is referred to as donor DNA and endonuclease, into a plant cell. The endonuclease produces a break at the target site, thereby allowing the first homology region and the second homology region of the donor DNA and its corresponding homology genomic region to carry out homologous recombination. The genomic DNA of the cutting serves as a receptor for DNA sequence. The resulting DNA exchange between the donor and the genome causes the polynucleotide of interest of the donor DNA to be integrated into the chain break in the target site in the plant genome, thereby changing the original target site and producing a changed genomic sequence.

[0581] Donor DNA can be introduced by any means known in the art. For example, a plant having a target site is provided. The donor DNA can be provided to the plant by known transformation methods, including Agrobacterium-mediated transformation or bioballistic particle bombardment. RNA-guided Cas or Cpf1 endonucleases cut at the target site, and the donor DNA is inserted into the genome of the transformed plant.

[0582] Although homologous recombination occurs at a low frequency in somatic plant cells, the introduction of double-strand breaks (DSBs) at the target sites of selected endonucleases appears to increase / stimulate this process. Ongoing efforts to generate Cas, specifically Cas9, variants or alternatives such as Cpf1 or Cms1 may improve efficiency.

[0583] Genetically modified plants or algae

[0584] The present invention provides genetically engineered plants, algae or parts thereof or plastids thereof, wherein the plants, parts thereof, algae or plastids thereof are genetically engineered with one or more polynucleotides disclosed herein to increase the production of cannabinoids.

[0585] In one embodiment, the genetically engineered plant is a plant of a genus other than cannabis or a non-cannabis plant. In another embodiment, the genetically engineered plant part is an aerial plant part, preferably an aerial plant part of a vascular plant, more preferably a leaf or stem of a vascular plant. A non-limiting example is tobacco, preferably a leaf of Nicotiana benthamiana or Nicotiana tabacum. In the case of envisioning the use of plant parts in the present invention, suitable plant parts may include roots, stems, leaves, flowers, fruits and seeds, preferably leaves and seeds. When the term "aerial" plant part is used, this should be understood to mean the part of the plant above the ground, which includes stems, leaves, flowers, fruits and seeds.

[0586] In one embodiment, the genetically engineered plant is a lower plant (non-vascular) or a higher plant (vascular), preferably a vascular plant. Vascular plants should be understood to be these plants containing vascular tissues such as xylem (for transporting water) and phloem (for transporting minerals and nutrients), and are also referred to as vascular plants. Vascular plants are also constant water (capable of regulating water concentration), have true leaves, roots and stems, and include ferns, lycophytes, horsetails, seed plants, angiosperms and gymnosperms. In contrast, non-vascular plants or lower plants lack vascular tissue, are hydrophilic (lack of mechanisms to resist drying), lack true leaves, roots and stems, include mosses, liverworts and hornworts, and usually live in moist, swampy places. Those skilled in the art will appreciate that this distinction is well characterized in the art. It should be understood that plants known to belong to non-vascular and vascular classifications may be suitable for use according to the present invention.

[0587] In one embodiment, the plastid is not from algae. However, when algae are contemplated for use in the present invention, it should be understood that algae define a large and diverse group of photosynthetic eukaryotic organisms. Suitable algae for use in the present invention may include, but are not limited to, Euglenophyta (egg spores); Chrysophyta (golden brown algae and diatoms); Pyrrophyta (fire algae); Chlorophyta (green algae); Rhodophyta (red algae); Paeophyta (brown algae) and Xanthophyta (yellow-green algae). Specific non-limiting example algae include the following classes: Chlorophyceae, Eustigmatophyccae, Prymnesiophyceae, Bacillariophyceae, Diatoms capable of oil production include genera Amphipleura, Amphora, Chaetoceros, Cyclotella, Cymbella, Fragilaria, Hantzschia, Navicula, Nitzschia, Phaeodactylum, and Thalassiosira. The specific non-limiting examples of the Chlorophyta that can produce oil include Ankistrodesmus, Botryococcus, Chlorella, Chlorococcum, Dunaliella, Monoraphidium, Oocystis, Scenedesmus and Tetraselmis. On the one hand, Chlorophyta can be Chlorella or Dunalimis. The specific non-limiting examples of the Cyanobacteria that can produce oil include Oscillatoria and Synechococcus. The specific non-limiting examples of the Chrysophyta that can produce oil include Boekelovia. The specific non-limiting examples of the Haplophyta include Isochysis and Pleurochrysis.

[0588] Specific algae useful in the present invention include, for example, Chlamydomonas, such as Chlamydomonas reinhardtii, Dunaliella sp., such as Dunaliella salina, Dunaliella tertiolecta, D. acidophila, D. bardawil, D. bioculata, D. lateralis, D. maritima, D. minuta, D. parva, D. peircei, D. polymorpha, D. primolecta, D. pseudosalina, D. quartolecta, D. viridis, Haematococcus. sp.), Chlorella sp., such as Chlorella vulgaris, Chlorella sorokiniana or Chlorella protothecoides, Thraustochytrium sp., Schizochytrium sp., Volvox sp., Nannochloropsis sp., Botryococcus braunii, which may contain more than 60 wt % lipid, Phaeodactylum tricornutum, Thalassiosirapseudonana, Isochrysis sp., Pavlova sp., Chlorococcum sp., Ellipsoidion sp., Neochloris sp., Scenedesmus sp.

[0589] In another embodiment, the plant is a high biomass plant. When compared to a lower biomass plant, a high biomass plant should be understood as a plant that is capable of producing a higher amount of cannabinoids or industrial products described herein or known in the art. When compared to a lower biomass plant, a high biomass plant generally has a higher quality in a given area. It should be understood that a high biomass plant may be preferred for use in the present invention. Such high biomass plants may include, but are not limited to, Acrocomia aculeata (macauba palm), Arabidopsis thaliana, peanut (Arachis hypogaea or peanut), Astrocaryum murumuru or murumuru, Astrocaryum vulgare or tucuma, Indaia-rateiro (Attalea ageraensis), American oil palm (Attalea humilis or American oil palm), Attalea oleifera (andaia), Attalea phalerata or uricuri, Babassu palm (Attalea speciosa or babassu), oats (Avena sativa or oats), sugar beet (Beta vulgaris or sugar beet), Brassica sp. such as Brassica rapa (Brassica sp.), and sugar beet (Beta vulgaris or sugar beet). carinata), Brassica juncea, Brassica napobrassica, Brassica napus (canola), Camelina sativa (false flax), Cannabis sativa (hemp), Carthamus tinctorius (safflower), Caryocar brasiliense (pequi), Coconut (Cocos nucifera), Crambe abyssinica (Abyssinian kale), Cucumis melo (melon), Elaeis guineensis (African palm), Glycine max (soybean), Gossypium hirsutum (cotton), Helianthus sp.) such as sunflower (Helianthus annuus), barley (Hordeum vulgare), physic nut (Jatropha curcas), araranut-tree (Joannesia princeps), duckweed (Lemna sp.) (duckweed) such as Lemna aequinoctialis, Lemna disperma, Lemna ecuadoriensis, Lemna gibba (swelling duckweed), Lemna japonica, Lemna minor, Lemna minuta, Lemna obscura, Lemna paucicostata, Lemna perpusilla, Lemna tenera, Lemna trisulca, Lemna turionifera), Valdivian duckweed (Lemna valdiviana), Yungas rock duckweed (Lemna yungensis), Oiticica (Licaniarigida or oiticica), Flax (Linum usitatissimum or flax), Lupinus angustifolius or lupin, Mauritia flexuosa (buriti palm), Maximiliana maripa (inaja palm), Miscanthus sp. such as Miscanthus x giganteus and Miscanthus sinensis, Nicotiana sp. (tobacco) such as Nicotiana tabacum or Nicotiana benthamiana, Oenocarpus bacaba (bacaba-do-azeite), Oenocarpus bataua)(pataua), Oenocarpus distichus(bacaba-de-leque), Oryza sp.) (rice) such as Asian rice (Oryza sativa) and African cultivated rice (Oryza glaberrima), switchgrass (Panicum virgatum), Paraqueiba paraensis (mari), avocado (Persea amencana), Pongamia pinnata (Indian beech), Populus trichocarpa, Ricinus communis (castor), Saccharum sp. (sugarcane), sesame (Sesamumindicum), potato (Solanum tuberosum), sorghum (Sorghum sp.) such as sorghum (Sorghum bicolor or Sorghum vulgare), Theobromagrandiforum or cupuassu, Trifolium sp., Brazilian needle palm (Trithrinax brasiliensis), wheat (Triticum sp.) (wheat) such as common wheat (Triticum aestivum) and corn (Zea mays or corn).

[0590] In a preferred embodiment, according to the present invention, leaves of the high biomass plants listed above are used to produce cannabinoids. In another preferred embodiment, according to the present invention, chloroplasts of leaves of the high biomass plants listed above are used to produce cannabinoids.

[0591] As used herein, the terms "genetically engineered," "genetically modified," and derivatives thereof refer to plants, parts thereof, algae, or plastids thereof, whose genetic material has been altered using molecular biology techniques such as, but not limited to, molecular cloning, recombinant DNA methods, gene editing, transformation, and gene transfer. Genetically engineered plants, parts thereof, algae, or plastids thereof include living modified plants, parts thereof, algae, or plastids thereof, genetically engineered plants, parts thereof, algae, or plastids thereof, or transgenic plants, parts thereof, algae, or plastids thereof. Genetic alterations include additions, deletions, modifications, and / or mutations of genetic material. Such genetic engineering as described herein in the present disclosure increases the production of cannabinoids relative to corresponding wild-type plants, parts thereof, or algae that have not been genetically engineered to introduce polynucleotides of the cannabinoid pathway as described herein. The term wild type should be understood to mean the same plant or algae that has not been genetically engineered, and generally refers to the phenotype of the typical form of a given plant or algae occurring in nature.

[0592] The present disclosure provides a method for producing cannabinoids in a plant or algae, the method comprising culturing a plant or algae comprising a nucleic acid construct of the present invention under conditions sufficient to express the polypeptide in the plant or algae, wherein the polypeptide increases the production of cannabigerolic acid (CBGA) in the plant or algae when expressed in the presence of hexanoic acid (C6) and geranyl pyrophosphate (GPP) when compared to a wild-type plant or algae.

[0593] In one embodiment, the nuclear and / or plastid genome of a genetically engineered plant or algae comprises a polynucleotide. In some embodiments, the transgenic plant or algae is homozygous for the genetic modification. In some embodiments, the transgenic plant or algae is heterozygous for the genetic modification.

[0594] In one embodiment, the production of CBGA is increased by at least about 1.5 to about 2 times, about 2 to about 2.5 times, about 2.5 to about 3.0 times, about 3 to about 3.5 times, about 3.5 to about 4.0 times, about 4 to about 4.5 times, about 4.5 to about 5.0 times, about 5 to about 5.5 times, about 5.5 to about 6 times, about 6 to about 6.5 times, about 6.5 to about 7.0 times, about 7.0 to about 7.5 times, about 7.5 to about 8.0 times, about 8.0 to about 8.5 times, about 8.5 to about 9.0 times, about 9.0 to about 9.5 times, or more when compared to a wild-type plant, part thereof, algae or plastid thereof.

[0595] In another embodiment, the production of CBGA is increased by at least about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, about 90% to about 100%, about 100% to about 120%, about 120% to about 140%, about 150% to about 160%, about 170% to about 180%, about 190% to about 200%, about 210% to about 220%, about 230% to about 240%, about 250% to about 260%, about 270% to about 280%, about 290% to about 300%, about 310% to about 310%, about 320% to about 330%, about 340% to about 350%, about 360% to about 370%, about 380% to about 390%, about 400% to about 410%, about 420% to about 430%, about 440% to about 450%, about 460% to about 470%, about 480% to about 490%, about 500% to about 50 ... 140% to about 160%, about 160% to about 180%, about 180% to about 200%, about 200% to about 220%, about 220% to about 240%, about 240% to about 260%, about 260% to about 280%, about 280% to about 300%, about 300% to about 320%, about 320% to about 340%, about 340% to about 360%, about 360% to about 380%, about 400% to about 420%, or more.

[0596] Those skilled in the art will understand methods for calculating the yield of a given cannabinoid. For example, it is known that cannabis can produce up to 2.8t of dry flower per hectare at about 15% CBDA, producing about 420kg of the main cannabinoid per hectare. Assuming that tobacco biomass in the field is 10t of dry biomass / ha, about 4% based on dry weight provides a similar yield level. The technician will also understand that cannabis typically accumulates 2-5% CBGA.

[0597] In some embodiments, the genetically engineered plant is a transgenic version of a control, non-transgenic plant that normally produces cannabinoids. In another embodiment, the genetically engineered plant or algae is a transgenic version of a control, non-transgenic plant or algae that does not normally produce cannabinoids. In this case, one skilled in the art will understand the necessary components of the biosynthetic cannabinoid pathway that need to be expressed in the plant or algae to increase the production of cannabinoids, preferably CBGA, when compared to the wild-type plant or algae.

[0598] Methods for introducing exogenous polynucleotides into plant and algae cells are well known in the art. Such plant or algae cells are considered to be "transformed". Suitable methods may include viral infection (such as double-stranded DNA viruses), transfection, conjugation, protoplast fusion, electroporation, particle gun technology, calcium phosphate precipitation, direct microinjection, silicon carbide whisker technology, Agrobacterium-mediated transformation, CRISPR / Cas9-mediated genome editing, etc. The choice of method generally depends on the type of cell being transformed and the environment in which the transformation occurs (e.g., in vitro, in vitro or in vivo).

[0599] The CRISPR / Cas9 system can be used to generate transgenic (genetically engineered) plants of the present disclosure. The CRISPR / Cas9 system and methods are known in the art (see, e.g., Bortesi and Fischer, 2015; Fan et al., 2015).

[0600] Transformation methods based on soil bacteria Agrobacterium tumefaciens and its artificial variants / mutants may be the best characterized tools for introducing exogenous nucleic acids into vascular plants. The wild-type form of Agrobacterium contains Ti (tumor induction) plasmids, which guide the production of tumor-causing crown galls grown on host plants. The tumor-inducing T-DNA region of Ti plasmids is transferred to the plant genome and requires virulence genes and T-DNA borders encoded by Ti plasmids, which are a group of direct DNA repeats in the region to be transferred. The vector based on Agrobacterium is a modified form of Ti plasmids, in which the tumor-inducing function is replaced by the nucleotide sequence of interest introduced into the plant host. In this regard, transfer nucleic acids are usually flanked by two "border" sequences, although in some cases, a single border at one end can be used, and the second end of the nucleic acid transferred is randomly generated during the transfer process. The polynucleotide of interest is usually located between the left border-like sequence and the right border-like sequence.

[0601] Agrobacterium-mediated transformation generally employs a co-integrate vector or, for example, a binary vector system in which the components of the Ti plasmid are split between a helper vector that is permanently in the Agrobacterium host and carries the virulence genes, and a shuttle vector that contains the gene of interest bound by a T-DNA sequence. Various binary vectors are well known in the art and are commercially available, for example, from Clontech (Palo Alto, Calif.). For example, methods for co-cultivating Agrobacterium with cultured plant cells or wounded tissues such as leaf tissue, root explants, hypocotyls, stem tubers or rhizomes are also well known in the art (see, e.g., Glick and Thompson, (Eds.), Methods in Plant Molecular Biology and Biotechnology, Boca Raton, Fla.: CRC Press (1993). In general, it can be assumed that all bacteria that are able to enter the cytosol of a host cell and lyse within that cell compartment (e.g., S. flexneri or L. monocytogenes) should be able to transfer DNA.

[0602] Agrobacterium-mediated transformation can be used to generate a variety of transgenic vascular plants (Wang et al., 1995), including high biomass plants suitable for use in the present invention, including but not limited to tobacco and Brassica.

[0603] Microprojectile bombardment-mediated transformation can also be used to generate transgenic plants for testing. This method, first described by Klein et al. (1987), relies on microparticles such as gold or tungsten that are coated with the desired heterologous nucleic acid, such as by precipitation with calcium chloride, spermidine, or polyethylene glycol. Microprojectile bombardment particles are accelerated at high speed into angiosperm tissue using devices such as the BIOLISTIC PD-1000 (Biorad; Hercules Calif.).

[0604] Polynucleotide can be introduced into plant or algae in a way that heterologous nucleic acid can enter plant cell, for example, by in vivo or in vitro scheme. "In vivo" may mean that polynucleotide is applied to the living body of plant or algae, for example, infiltration. "In vitro" may mean that cell or explant is modified outside plant or algae, and then such cell or organ is regenerated into plant or algae. Many vectors or constructs suitable for stable transformation of plant or algae cells or for setting up transgenic plants or algae have been described, including Weissbach and Weissbach, (1989) Methods for Plant Molecular Biology Academic Press and Gelvin et al. (1990) Plant Molecular Biology Manual, Kluwer Academic Publishers described vector or construct. Specific examples may include examples of Ti plasmids derived from Agrobacterium tumefaciens, and examples disclosed by Herrera-Estrella et al. (1983), Bevan (1984) and Klee (1985). Alternatively, non-Ti vectors can be used to transfer DNA to plants and cells by using free DNA delivery techniques. By using these methods, transgenic plants such as wheat, rice (Christou, 1991) and corn (Gordon-Kamm, 1990) can be produced. Immature embryos can also be good target tissues for monocots, for direct DNA delivery techniques (Weeks et al., 1993; Vasil, 1993; Wan and Lemeaux, 1994) and for Agrobacterium-mediated DNA transfer (Ishida et al., 1996).

[0605] Disclosed methods for plastid transformation in higher plants include particle gun delivery of DNA containing selectable markers and targeting of DNA to the plastid genome by homologous recombination (US 5,451,513, US 5,545,818, US 5,877,402, US 5,932,479 and WO 99 / 05265). In addition, exemplary methods for introducing DNA into chloroplasts are bioballistic bombardment, polyethylene glycol transformation of protoplasts, and microinjection (Danieli et al., 1998; Staub et al., 2000; O'Neill et al., 1993; Knoblauch et al., 1999; US 5,545,817; US 5,576,198; WO 95 / 16783; Boynton et al., 1993, Svab et al., 1993, McBride et al., 1994).

[0606] Any carrier that is applicable to the polyethylene glycol transformation of bioballistic bombardment, protoplastid and the method for microinjection will be suitable as the targeting vector for chloroplast transformation.Any double-stranded DNA vector can be used as transformation vector, particularly when the introduction method does not utilize Agrobacterium.Alternately, the method for transforming plant of the present invention or algae can comprise any method of the description comprising the example herein.Can comprise any plant listed herein or known in the art through genetically engineered plant, preferably high biomass plant, more preferably tobacco, as Nicotiana benthamiana or common tobacco.

[0607] The present disclosure also provides transformed plant or algae cells, tissues, plants or algae, and products containing transformed plant or algae cells. The transformed cells, and tissues and parts thereof are characterized by the presence of polynucleotides integrated into the genome, and the plant or algae cells produce one or more polypeptides for producing cannabinoids. The recombinant plant cells of the present disclosure can be used as a recombinant cell population, or as a tissue, seed, whole plant, stem, fruit, leaf, root, flower, stem, tuber, grain, animal feed, plant field, etc.

[0608] The present disclosure also provides reproductive material of the subject transgenic plants or algae, wherein the reproductive material may include seeds, progeny plants and clonal material, wherein such material may produce plants or algae that produce cannabinoids according to methods known in the art or described herein.

[0609] In one embodiment, although the polynucleotide sequences described herein are preferably expressed in the plastids of a plant, most preferably in the chloroplasts of a plant, one skilled in the art will appreciate that other organelles containing oil droplets may be suitable for producing the cannabinoids described herein.

[0610] When the host cell used in the transformation process is omnipotent, plants or algae containing transgenic plastids (e.g., chloroplasts) can be produced. The procedures for regenerating transgenic plants from transformed cells or tissues are generally similar, with appropriate modifications within the capabilities of those skilled in the art. Regeneration of dicots such as sugar beet, Freytag et al. (1988); tobacco, Svab et al. (1990), or monocots such as wheat from anthers or embryos (see below) is generally successful.

[0611] If it is desired to transform plastids in other plant or algal material, such as anther culture-derived plants or embryo-derived callus, the plant material is placed in a convenient container, such as the culture dishes described above for isolated cells (see also US 6,680,426; Hanson et al. (2012); Golds et al. (1993)).

[0612] In case plastid is transformed, the cell of plant or algae can be reused for tissue culture, subsequently in desired local growth callus or regenerated plant or algae.Therefore, modified plant or algae cell can be repeatedly regenerated by using cell and tissue culture.In some cases, suitable breeding can be maintained from seed.In order to improve the ability of identifying transformed cells, it may be desirable to adopt selectable or screenable marker genes as previously described, and the conversion vector for producing transformant.When using selectable markers, by exposing cells to one or more selective agents, in the cell colony of potential conversion, identified transformed cells.When using screenable markers, cells can be screened for the marker gene traits expected.

[0613] Cells that survive after exposure to the selective agent, or cells that are scored as positive in the screening assay can be cultivated in a culture medium that supports plant or algae regeneration. In certain embodiments, any suitable plant or algae tissue culture medium (for example, MS and N6 culture medium) can be modified by comprising other materials such as growth regulators. Tissue can be maintained on the minimal medium with growth regulators, until there are enough tissues to be used for starting plant regeneration work, or after repeated rounds of artificial selection, until the morphology of the tissue is suitable for regeneration (for example, at least 2 weeks), then transferred to the culture medium that is conducive to bud formation. Regularly transfer culture, until enough buds are formed. Once buds are formed, the buds are transferred to the culture medium that is conducive to root formation. Once enough roots are formed, plants or algae can be transferred to soil for other growth and maturation.

[0614] To confirm the presence of the polynucleotide in the regenerated plant, various assays can be performed. Such assays include, for example: molecular biology assays such as Southern and Northern blotting, PCR and nucleic acid sequencing; plant part assays such as leaf or root assays.

[0615] According to the transformation method, transformant can comprise the cell as plastid and nuclear transformant, makes the marker relevant to the plastid transformation also may cause the cell selected as plastid and nuclear transformant.This problem can be solved by utilizing the preferred nucleic acid construct of the present invention described above, described preferred nucleic acid construct contains homologous recombination zone (also described as integration site), described homologous recombination zone can be integrated in the plastid genome but can not be integrated in the nuclear genome, and uses the promoter that only has activity in chloroplast.This makes selection method can select to produce polypeptide in plastid and do not produce the cell or plant of polypeptide outside plastid.

[0616] For example, the integration event can be analyzed by using, for example, specific oligonucleotide primers to the polynucleotides concerned. PCR genotyping is understood to include, but not limited to, polymerase chain reaction (PCR) amplification derived from the genomic DNA of the separated host plant callus, which is predicted to contain the polynucleotides concerned that are integrated into the genome, followed by standard cloning and sequence analysis of the PCR amplification product. The method of PCR genotyping has been well described (for example, Rios et al., 2002), and can be applied to the genomic DNA derived from any plant species (for example, corn (Z. mays) or soybean (G. max)) or tissue type, including cell culture.

[0617] Once a genetically engineered plant, algae, part thereof or plastid expressing one or more polynucleotides of the invention is produced, cannabinoids, preferably CBGA, may be extracted using methods known in the art or described herein.

[0618] In a non-limiting example, extraction from fresh or dried plants or algae parts thereof may involve a combination of steps including physical treatment of the material (e.g., ultrasound assisted, microwave assisted, sieving), solvent (e.g., ethanol, water, isopropyl) extraction, hydrocarbon or CO2 extraction (e.g., supercritical CO2 extraction), winterization and filtration, drying, distillation and / or chromatography, depending on the desired purity. Those skilled in the art will appreciate that the desired characteristics of the product will influence the method of extraction. For example, cannabinoids may be extracted in an acidic form by performing the processing steps at or below ambient temperature, or the cannabinoids may also be extracted in a neutral form by subjecting the material to higher temperatures to decarboxylation.

[0619] Specifically, the collected plant material is first vacuum dried and ground into a powder. The cannabinoids, preferably CBGA, are extracted using a supercritical gas (usually CO2) or an organic solvent (such as ethanol), thereby removing the lighter terpene fractions. The resulting crude cannabinoid extract is then winterized using cold ethanol to allow the elimination of waxes and lipids by precipitation. After filtration and removal of the alcohol by evaporation, the crude cannabinoid fraction ('resin') is concentrated by distillation to eliminate heavy terpenes and other residues. When it is desired to extract a specific cannabinoid molecule, the cannabinoid distillate is subjected to a further chromatography step, such as high performance liquid chromatography, centrifugal partition chromatography or supercritical fluid chromatography.

[0620] The extracted cannabinoids can then be converted to any of CBG, CBDA, CBD, CBCA, CBC, THCA, THC, or another cannabinoid known in the art using one or more known methods known in the art or described herein. Typically, such methods can include enzymatic conversion in vivo (e.g., by yeast feeding as disclosed in Zirpel et al. (2015)); by leaf infiltration, such as Geissler et al. (2018) and Gulck et al. (2020) or in vitro (Valliere et al., 2019, Valliere et al., 2020; Lange et al., 2016).

[0621] Composition

[0622] In one aspect of the present invention, a pharmaceutical composition obtained by the method of the present invention is provided. Preferably, the pharmaceutical composition is formulated with one or more pharmaceutically acceptable carriers known in the art.

[0623] The compositions described herein may be prepared for administration to a subject in need thereof orally, parenterally, by inhalation spray, adsorption, absorption, topically, rectally, nasally, buccally, vaginally, intracerebroventricularly, by implanted reservoir in a dosage formulation containing a conventional nontoxic pharmaceutically acceptable carrier, or by any other convenient dosage form known in the art, subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intracerebroventricular, intrasternal and intracranial injection or infusion technique.

[0624] Methods for preparing cannabinoids into a form suitable for administration to a subject (e.g., a pharmaceutical composition) are known in the art and include, for example, those described in Remington's Pharmaceutical Sciences (18th ed., Mack Publishing Co., Easton, Pa., 1990) and US Pharmacopeia: National Formulary (Mack Publishing Co., Easton, Pa., 1984).

[0625] The pharmaceutical composition of the present invention is particularly useful for topical administration. The composition for administration generally includes a solution of cannabinoids or their extracts dissolved in a pharmaceutically acceptable carrier, such as an aqueous carrier. Various aqueous carriers can be used, such as buffered saline, etc. The composition can contain pharmaceutically acceptable auxiliary substances required for close physiological conditions, such as pH regulators and buffers, toxicity regulators, etc., such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of the cannabinoids or their extracts of the present invention in these formulations can vary over a wide range and will be selected based on the volume of fluid, viscosity, body weight, etc., according to the specific mode of administration selected and the needs of the subject. Exemplary carriers include water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles such as mixed oils and ethyl oleate can also be used. Liposomes can also be used as carriers. The vehicle can contain a small amount of additives that enhance isotonicity and chemical stability, such as buffers and preservatives.

[0626] After formulation, the cannabinoids or extracts thereof of the present invention will be available for administration in a manner compatible with the dosage formulation and in therapeutically / prophylactically effective amounts. The formulations are readily administered in a variety of dosage forms, such as topical solutions of the type described above, but other pharmaceutically acceptable forms are also contemplated, such as tablets, pills, capsules or other oral administration solids, suppositories, vaginal suppositories, nasal solutions or sprays, aerosols, inhalants, liposomal forms, and the like. Pharmaceutical "slow release" capsules or compositions may also be used. Slow release formulations are generally designed to provide a constant level of drug over an extended period of time, and may be used to deliver the cannabinoids or extracts thereof of the present invention.

[0627] dose

[0628] The appropriate dose of the cannabinoid or its extract of the present invention will vary according to the specific cannabinoid or its extract and / or the subject being treated. A skilled physician has the ability to determine the appropriate dose, for example, by starting with a suboptimal dose and incrementally modifying the dose to determine the optimal or useful dose. Alternatively, in order to determine the appropriate dose for treatment / prevention, data from cell culture assays or animal studies are used, where the appropriate dose is within the range of circulating concentrations, including the ED50 of the active compound with little or no toxicity. The dose can vary within this range depending on the dosage form used and the route of administration utilized. The therapeutic / preventive effective dose can initially be estimated based on cell culture assays. In animal models, the dose can be formulated to achieve a circulating plasma concentration range containing the IC50 (i.e., the concentration and amount of the compound that achieves half the maximum inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine the useful dose in the human body. The level in plasma can be measured, for example, by high performance liquid chromatography.

[0629] In some examples, the methods of the present invention comprise administering a prophylactically or therapeutically effective amount of a cannabinoid, pharmaceutical composition, or extract thereof as described herein.

[0630] The term "therapeutically effective amount" refers to an amount that, when administered to a subject in need of treatment, improves the prognosis and / or status of the subject and / or reduces or inhibits one or more symptoms of the clinical conditions described herein to a level below that observed and accepted as a clinical diagnosis or clinical characteristic of the condition. The amount administered to the subject will depend on the specific characteristics of the condition to be treated, the type and stage of the condition to be treated, the mode of administration, and the characteristics of the subject, such as general health, other diseases, age, sex, genotype, and body weight. Those skilled in the art will be able to determine an appropriate dosage based on these and other factors. Therefore, the term should not be interpreted as limiting the present invention to a specific amount, for example, a weight or amount of protein, but rather the present invention encompasses any amount of cannabinoids or extracts thereof sufficient to achieve the described results in the subject.

[0631] The term "prophylactically effective amount" should be understood to refer to a sufficient amount of a protein to prevent or inhibit or delay the onset of detectable symptoms of one or more clinical conditions. Those skilled in the art will appreciate that such amounts will vary depending, for example, on the specific cannabinoid or extract thereof administered and / or the specific subject and / or the type or severity or level of the condition and / or the predisposition (genetic or otherwise) to the condition. Thus, the term should not be interpreted as limiting the invention to a specific amount, e.g., weight or amount of a cannabinoid or extract thereof, but rather the invention encompasses any amount of a cannabinoid or extract thereof sufficient to achieve the described result in a subject.

[0632] Medical Uses

[0633] It will be appreciated that the present invention may be used to treat any disease or condition responsive to treatment with the cannabinoids or extracts thereof described herein.

[0634] Diseases or conditions that may be treated and / or prevented using the cannabinoids of the present invention include, but are not limited to, chronic pain, neuropathic pain, cancer, nausea and / or vomiting associated with cancer chemotherapy, lack of appetite, multiple sclerosis, spasticity associated with multiple sclerosis or spinal cord injury epilepsy, Parkinson's disease, anorexia and / or weight loss, irritable bowel syndrome, Tourette syndrome, amyotrophic lateral sclerosis, Huntington's disease, dystonia, dementia, glaucoma, traumatic brain injury and / or intracranial hemorrhage, addiction, anxiety, depression, sleep disorders, post-traumatic stress disorder, microbial infections, including methicillin-resistant Staphylococcus aureus (MRSA) and biofilm-producing organisms, dermatitis, acne, schizophrenia and other psychotic disorders. Additionally, any other disease or condition known in the art to be susceptible to cannabinoid treatment may be conceived to be treated by the cannabinoids of the present invention.

[0635] A subject in need of treatment may experience a variety of symptoms, depending on the type of disease associated with inflammation. In one aspect, a subject in need of treatment may exhibit well-characterized symptoms associated with a disease or condition described herein.

[0636] The terms "treatment" or "treating" a subject encompass the administration of a cannabinoid of the present invention or an extract thereof to a subject, wherein the purpose is to delay, slow, stabilize, cure, heal, alleviate, mitigate, alter, remedy, lessen the worsening of, improve, advance or affect a condition or disease, or a symptom of a disease or condition.

[0637] The term "treat" refers to any indicator of successful treatment or improvement of an injury, pathology or condition, including any objective or subjective parameter, such as alleviation; remission; reduction in the rate of progression; reduction in the severity of the disease; stabilization, reduction of symptoms or damage. Specifically, treatment refers to the reduction of symptoms associated with a disease or condition.

[0638] The existence, improvement or treatment of a disease or condition can be determined by any clinical or biochemical correlation method known in the art. The correlation method can be the measurement of symptoms. For example, in the case of chronic pain, the improvement of symptoms may include joint pain relief, muscle pain relief, burning pain relief, fatigue relief, sleep apnea reduction, emotional problems reduction, including depression, anxiety and irritability. Alternatively, in the case of epilepsy, the improvement of symptoms may include confusion relief, trance relief, muscle stiffness relief, uncontrollable twitching movements of arms and legs reduction, loss of consciousness or perception reduction and emotional problems reduction, including anxiety and fear. Improvement or treatment can be determined directly by the subject or its sample or biopsy. Sample or biopsy may be diseased tissue.

[0639] Examples

[0640] Example 1 - Materials and Methods

[0641] Plant materials

[0642] Olivate (OA; 2,4-dihydroxy-6-pentyl-benzoic acid) for N. benthamiana permeation assay was purchased from Toronto Research Chemicals. Cannabigerolic acid (CBGA; 3-[(2E)-3,7-dimethyl-2,6-octadien-1-yl]-2,4-dihydroxy-6-pentyl-benzoic acid) was a 1 mg / mL certified reference material (CRM) in acetonitrile purchased from Cerilliant (TX, USA) and stored at -80 °C. Hexanoic acid (C6), geranyl pyrophosphate (GPP as triammonium salt), LCMS grade solvents (methanol, acetonitrile, isopropanol), formic acid, and ammonium acetate were purchased from Nu Chek Prep Inc (MN, USA) and ThermoFisher Scientific (MA, USA). Strata X-AW 33 μm polymeric weak anion column (100 mg / 6 mL; adsorbent mass / volume) was purchased from Phenomenex Australia Pty Ltd (Sydney, Australia). Antibiotics and their concentrations used in the Agrobacterium tumefaciens growth medium included Rifampicin (25 μg / ml), Kanamycin (50 μg / ml), Carbenicillin (50 μg / ml) and Gentamycin (15 μg / ml).

[0643] Gene constructs

[0644] All coding sequences were optimized using GeneArt (Thermo Fisher Scientific Inc, MA, USA) online algorithms for expression in Nicotiana benthamiana, which was bred to ensure compatibility with the Golden Gate (GG) cloning system (Weber et al., 2011). According to Engler et al. (2014), the optimized coding sequences were flanked by appropriate 4 nucleotide overhangs (SEQ ID 5-14). The 2x35S promoter and Nos terminator level 0 GG modules corresponded to pICH51288 and pICH41421, respectively (Engler et al., 2014). Modules were assembled into the pICH47732 backbone by GG cloning (Engler et al., 2014) to contain the optimized coding sequences of CsAAE1, CsOLS, CsOAC, or CsCBGAS, and the resulting expression vectors were named pCAN1-3 and pCAN7, respectively (see Table 3).

[0645] The N-terminus of the Rubisco small subunit (SSU) has previously been shown to redirect cytoplasmic proteins into the chloroplasts of plants (Lee et al., 2002). The level 0 GG module containing the coding sequence of the Rubisco small subunit chloroplast targeting peptide was based on pICH78133 as described by Engler et al. (2014) with minor modifications (SEQ ID NO: 15). The module was assembled into the pICH47732 backbone by GG cloning (Engler et al., 2014) to contain the coding sequence of CsAAE1, CsOLS, CsOAC or CsCBGAS downstream of the Rubisco SSU targeting peptide, and the resulting expression vectors were named pCAN4-6, respectively (Table 3).

[0646] The coding sequence of the mNeonGreen fluorescent protein (Shaner et al., 2013) was designed as a GG 0-level module (SEQ ID 16) with flanking overhangs that allow incorporation as a C-terminal fusion protein. The mNeonGreen protein was selected because it was previously shown to be a robust fluorescent reporter protein in plants and does not alter protein targeting (Stoddard and Rolland, 2019). The module was assembled into the pICH47732 backbone by GG cloning (Engler et al., 2014) to contain the coding sequence of one of CsAAE1, CsOLS, CsOAC, and CsCBGAS between the Rubisco SSU targeting peptide and the fluorescent protein, and the resulting expression vector was named pCAN11-13 (Table 3).

[0647] Three level 0 GG modules were designed to incorporate elements of bean yellow dwarf virus (BeYDV) to test whether recombinant protein expression levels can be improved (Baltes et al., 2014). Module EN38510 contains the BeYDV long intergenic region (LIR), flanked by GGAG and TACT GG sequences at the 5' and 3' ends, respectively (SEQ ID NO: 17). By using TACT and AATG as 5' and 3' GG linker sequences (SEQ ID NO: 18), the 2x35S promoter and tobacco mosaic virus (TMV) 5'UTR sequence were designed as GG modules compatible with EN38510 (EN38509). The alternative terminator module (EN38511) contains the 35S terminator sequence of pICH41414 (Engler et al., 2014), as well as the BeYDV short intergenic region (SIR), Rep / RepA coding sequence and LIR (SEQ ID NO: 19). According to Diamos and Mason (2019), the internal BsaI site in the Rep protein was removed and single nucleotide substitutions were introduced. The modules were assembled into the pICH47732 backbone by GG cloning (Engler et al., 2014) to provide a vector containing the coding sequences of CsAAE1, CsOLS, CsOAC, CsCBGAS, with module EN38510, module EN38509 and module EN38511. The resulting expression vector was named pCAN31-37 (see Table 3).

[0648] Gene synthesis was outsourced to GeneArt (Thermo Fisher Scientific, Massachusetts, USA) or Twist Bioscience (California, USA). The binary expression vector pICH47732 was used as the backbone for all GG assembly reactions (Engler et al., 2014) and is referred to herein as the 'standard vector'. A list of all expression vectors can be found in Table 3 below.

[0649] Table 3: List of expression constructs

[0650]

[0651]

[0652] Nicotiana benthamiana leaf penetration assay

[0653] Agrobacterium tumefaciens GV3101 culture containing independent expression vector is grown in 10mL LB substratum supplemented with antibiotic.After incubation 2 days at 28 ℃, acetosyringone is added in the culture with the ultimate concentration of 100 μM.Culture is incubated 3 hours at 28 ℃, and is collected by centrifuging 5 minutes with 4000rpm.Cell precipitation is resuspended in isopyknic infiltration buffer (5mM MgSO , 5mM MES, 5% ethanol, pH 5.7) gently, and described infiltration buffer contains 100 μM acetosyringone, 0.1% Tween 20 and suitable substrate (C6, GPP, OA), as described below.To mix through resuspended culture, to obtain the desired combination of different expression vectors, wherein the OD of every kind of culture in final infiltration solution is 1:1. 600 Set to 0.125. P19 was included in all infiltrations as a viral inhibitory protein as described by Wood et al. (2009).

[0654] Using 1mL syringe in conjunction with C6, GPP, OA and / or CBGA as substrate and / or positive control, the cell solution is infiltrated into the lower side of Nicotiana benthamiana leaf.Individual and / or co-infiltration substrate through infiltration comprises 1mM GPP, 2mM C6, 0.5mM OA or 0.2mM CBGA.Mark the leaf area through infiltration with pen, and plant is placed in cabinet (Conviron PGC20Flex; 23 ℃; 16 hour / 8 hour light / dark cycle).After initial infiltration for three days, re-infiltrate the leaf area through marking with infiltration buffer containing appropriate substrate.Collect the leaf spot (about 400mg fresh weight) through infiltration 4 days after gene infiltration.Sample is immediately frozen in liquid N2 quickly, and is stored at -80 ℃, until extraction.Negative control is made up of Agrobacterium tumefaciens expressing virus inhibitor protein P19 ('P19 '), infiltration buffer ('Inf neg ') not containing any Agrobacterium tumefaciens culture or buffer control ('blank ') during extraction.

[0655] Extraction of OA and CBGA

[0656] Cannabinoids are extracted from frozen infiltrated Nicotiana benthamiana leaf samples by adding 800 μ L 20% MEOH (prepared in ultrapure water) in 2 mL Eppendorf tubes. Samples were lysed using a Qiagen Tissuelyzer (3 minutes at 29.0 Hz) (Qiagen NV, Hilden, Germany) with two stainless steel ball bearings. After ultrasonic treatment for 10 minutes in an ice bath, the sample was immediately centrifuged for 5 minutes at 13,000 RPM. The extraction procedure was repeated twice and merged. 0.22 μ m nylon microcentrifuge filter was used to rotate 2 minutes at 12,000 RPM to filter the combined extracts. In the case of performing infiltration experiments with standard or BeYDV vectors, 10 or 6 replicates were combined. Next, the samples were transferred to 10 mL glass vials, dried under N2, and resuspended in an equal volume of 100 mM ammonium acetate buffer (pH 4.9 ± 0.5) buffer solution, followed by solid phase extraction purification.

[0657] By adding 6mL acetonitrile and 6mL 100mM ammonium acetate buffer (pH 4.9) Strata X-AW 33μm cartridges (Phenomenex, Sydney Australia) in Sydney, Australia) are pretreated and balanced. The combined sample is loaded onto the post, then washed with 6mL 100mM ammonium acetate (pH 4.9) and 6mL methanol. The sample is dried under vacuum for 5 minutes, then eluted with 6mL ammonium hydroxide: acetonitrile (5:95, v / v). Eluent is dried under 30 ℃ N2 flow, and is resuspended in acetonitrile, in the case of carrying out penetration experiment with standard and BeYDV derived carrier, respectively corresponding to 1 / 10 or 1 / 20 of initial loading volume. Take 50 μL aliquots and carry out instant LC-MS analysis, while remaining eluent is stored at -80 ℃.

[0658] LC-MS detection of OA and CBGA

[0659] Chromatographic separation of OA, CBGA and its glycosylated forms was achieved on an Agilent Zorbax Eclipse Plus C18 Rapid Separation HD column (2.1 x 150 mm; 1.8 μm particle size) (Agilent CA, USA) equipped with a thermostatic autosampler and column compartment, coupled to a guard column at a constant temperature of 40°C. Mobile phase A consisted of ultrapure water containing 0.1% formic acid, and mobile phase B consisted of acetonitrile containing 0.1% formic acid. The gradient separation started with an isocratic hold at 5% B for 2 minutes, followed by a linear gradient of 67% B at 5 minutes and 65% B at 9 minutes, reaching 100% B at 18 minutes and maintaining for 2 minutes. Reequilibration to the initial conditions was achieved by holding at 5% B for 2 minutes before the next injection. The flow rate was set to 0.3 mL / min using a UHPLC Vanquish binary pump (Thermo Scientific, MA, USA). Aliquots of 2 μL sample volume were injected, containing experimental negative and positive controls, procedural blanks, instrument blanks, and standards (OA, CBGA).

[0660] OA and CBGA and their glycosylated forms were detected on an Orbitrap Q-Exactive (Thermo Scientific, CA, USA) mass spectrometer equipped with a heated electrospray ionization (HESI) source, which was operated in positive ionization mode. The capillary and evaporator temperatures were 256°C and 412°C, respectively, the electrospray voltage was 3.5 kV, the sheath gas was 48 and the auxiliary gas was 11 (arbitrary units), and the S lens RF level was 50. The normalized collision energy was optimized at values ​​of 15 and 25. Data acquisition was set up at a resolving power of 70,000 FWHM at m / z 200 using the parallel reaction mode. The ionization was performed using the Pierce- TM LTQ ESI positive ion calibration solution (Thermo Scientific, San Jose, CA, USA) TM (Thermo Scientific TM , San Jose, CA, USA) for mass calibration. Detection was based on calculated [M+H] + Molecular ions where the precision is 2ppm and the retention time of the target compound exists in the inclusion list.

[0661] Mass calibration was performed prior to the run using Pierce™ LTQ ESI Positive Ion Calibration Solution (Thermo Scientific™, San Jose, CA, USA). Detection was based on the calculated [M+ H] + Molecular ions where the precision is 2ppm and the retention time of the target compound exists in the inclusion list.

[0662] Instrument control and data acquisition were achieved using Thermo Scientific Xcalibur (version 4.3.73.11, 2019) (Thermo Scientific, CA, USA), and data processing was performed using Freestyle software. Glycosylated and single forms of OA and CBGA were identified by comparing MS spectra and fragmentation patterns with those of reference standards and / or infiltrated leaves.

[0663] Serial dilutions of OA and CBGA with concentrations ranging from 0.0025 to 1 μg / mL were prepared based on the working standard solution. A calibration curve was constructed by using the peak area of ​​each OA (m / z 225.111) and CBGA (m / z 343.2268). The R obtained for the external standard linear calibration curve 2 ≥0.9998. Intra-day injection accuracy <4% CV (n=5). The instrument detection limit (LOD) of CBGA was 9 μg L- -1 , and the instrument detection limit of OA was 11 μg L- -1 , and is calculated based on the standard deviation of the responses and the slope.

[0664] Glycosylated forms of OA (OA-Glc) and CBGA (CBGA-Glc) were obtained by infiltration of leaves with 0.5 mM OA and 0.2 mM CBGA, respectively, followed by sample extraction as previously described. During extraction, the data obtained (area of ​​the chromatogram) were corrected for sample fresh weight and dilution / concentration factors.

[0665] Confocal microscopy

[0666] In order to verify that the designed enzyme is correctly retargeted to the chloroplast in Nicotiana benthamiana, the coding sequence of Rubisco SSU chloroplast targeting signal and mNeonGreen fluorescent protein is cloned into the N-terminal and C-terminal of CsAAE1, CsOLS and CsOAC in frame respectively. The mNeonGreen fusion construct containing hemagglutinin epitope (EN38113; SEQ ID 28) but not Rubisco SSU is used as a control to visualize cytoplasmic localization. As described above, in the absence of any substrate, Agrobacterium tumefaciens GV3101 culture is infiltrated into Nicotiana benthamiana. After three days, leaf disks with a diameter of about 0.5 cm are collected from the infiltrated leaves. The abaxial side of these discs was imaged with a Leica SP8 confocal laser scanning microscope (Leica Microsystems, Sydney, Australia) equipped with a 40x (NA=1.1) water immersion objective and a Leica application suite (LAS-X, Leica Microsystems, Sydney, Australia). Image z stacks were collected using a HyD detector and sequential scanning in separate tracks. In the first track, the tissue was excited at 506nm and the mNeonGreen signal was collected at 512-530nm with the transmitted light. The mNeonGreen detector was gated between 0.3ns and 6ns to remove cell wall autofluorescence. In the second track, the tissue was excited at 633nm and the chloroplast autofluorescence was collected at 650-690nm.

[0667] RNA expression

[0668] To quantify RNA expression levels in tobacco by digital droplet PCR, the method described in Vanhercke T et al., (2017) was followed.

[0669] Protein expression

[0670] Protein expression levels can be quantified by western analysis using HA-tag or mNeonGreen fusion proteins and antibodies against HA epitopes or mNeonGreen fluorescence. Alternatively, absolute quantification of protein levels expressed in leaves can be achieved by targeted LC-MS / MS methods (Colgrave et al., 2019).

[0671] Example 2- Biosynthetic pathways of OA and CBGA

[0672] The distinct intracellular localization patterns of cannabinoid biosynthetic enzymes suggest a model in which OA is generated from malonyl-CoA and C6 in the cytosol of cannabis trichome cells ( Figure 1OA is subsequently transported to chloroplasts by CsCBGAS for further conversion to CBGA. As a final step, CBGA is secreted into the apoplast storage cavity of the trichome head where it is converted into THCA, CBDA, and trace cannabinoids such as CDCA (Andre et al., 2016; Gülck and 2020; Arif et al., 2021).

[0673] Cannabinoid biosynthetic enzymes are thought to act on alternative substrates. The FA chain length promiscuity of cannabinoid biosynthetic enzymes was shown by Luo et al. (2019) in yeast feeding experiments.

[0674] Cannabiyl activating enzyme (CsAAE1; AFD33345.1; SEQ ID NO: 1) is a cytosolic CsAAE1 enzyme present in trichomes. It is a trichome-specific acyl-CoA synthase that converts C6 to CoA derivatives. CsAAE1 is a member of the acyl-activating enzyme (AAE) superfamily that activates carboxylic acids via an adenylate intermediate (Stout et al., 2012). This family of enzymes all have a highly conserved 12 amino acid residue AMP-binding motif (PROSITE PS00455) (Shockey and Browse, 2011). AAE1 belongs to the EC6.2.1.2 classification. CsAAE1 is known to accept FAs with different chain lengths (Stout et al., 2012).

[0675] Cannabis olive alcohol synthase (CsOLS; BAG14339.1; SEQ ID NO: 2) is one of two enzymes that work together to produce OA from C6-CoA and malonyl-CoA. Taura et al. (2009) identified the CsOLS gene as a polyketide synthase in cannabis trichomes that condenses C6-CoA and a variety of other short-chain fatty acids with malonyl-CoA. CsOLS can accept C4-C8CoA substrates (Taura et al., 2009). CsOLS is a cytoplasmic protein (Stout et al., 2012) and its crystal structure has recently been resolved (Kearsey et al., 2020). Non-natural variant OLS mutants have recently been described in WO2020 / 214951.

[0676] Cannabinol cyclase (CsOAC; AFN42527.1; SEQ ID NO: 3) is the second enzymatic step required for the production of OA. CsOAC is localized in the cytosol (Gagne et al., 2012). It refers to 3,5,7-trioxododecanoyl-CoA cyclase or 3,5,7-trioxoundecanoyl-CoA cyclase (EC 4.4.1.26), which is capable of converting 3,5,7-trioxododecanoyl-CoA to OA or 3,5,7-trioxoundecanoyl-CoA to divalinoic acid.

[0677] Cannabis CBGA synthase (CBGAS; DAC76710.1; SEQ ID NO: 4). In cannabis, OA is transported to chloroplasts by CsCBGAS for further conversion to CBGA. Recently, Luo et al. (2019) and Gulck et al. (2020) both identified CsPT4 as a functional CBGAS prenyltransferase in cannabis. Unlike CsOLS and CsOAC, CsCBGAS is a membrane-bound enzyme present in chloroplasts (Gulck et al., 2020). Valliere et al. (2019) identified an alternative prenyltransferase NphB from Streptomyces CL190 strain that is capable of producing CBGA. The group used structure-based protein design to improve the nonspecific CBGAS activity of soluble NphB. CBGAS refers to a cannabigerolic acid synthase (EC 2.5.1.102) that is capable of converting GPP and olivetolic acid (OA) or GPP and divaleric acid (DVA) into cannabigerolic acid (CBGA) or cannabigerolic acid (CBGVA).

[0678] Cannabis THCA and CBDA synthases (THCAS; CBDAS). The final step of the cannabinoid biosynthetic pathway consists of the conversion of CBGA to THC or CBD as the main cannabinoid end products by CsTHCAS or CsCBDAS, respectively. In cannabis, these two enzymes are located in the apoplast, which explains the accumulation of major cannabinoids such as THC and CBD at very high levels in extracellular storage globules (Sirikantaramas et al., 2004; Taura et al., 2007). CBDA synthase (EC1.21.3.8) is able to convert CBGA or CBGVA to cannabidiolic acid (CBDA) or cannabidiolic acid (CBDVA). CBDAS can accept CBGA (C5 tail) and CBGVA (C3 tail) in vitro (Valliere et al., 2019). THCA synthase (tetrahydrocannabinolic acid synthase EC1.21.3.7) is able to convert CBGA or CBGVA to THCA (tetrahydrocannabinolic acid) or THCVA (tetrahydrocannabinolic acid).

[0679] Example 3 - Functional CBGA biosynthetic pathway operating in the cytosol and chloroplasts

[0680] Penetration of OA or CBGA into N. benthamiana revealed rapid glycosylation of both molecules, confirming similar observations by Gulck et al. (2020) ( Figure 2 and 3 ).

[0681] The present inventors aimed to construct the CBGA biosynthetic pathway of cannabis in Nicotiana benthamiana leaves by transforming the enzymes in the biosynthetic pathway using the vectors described in Example 1. The enzymes for synthesizing OA were expressed in the cytosol, and the CBGA biosynthetic enzymes were targeted to the chloroplasts ( Figure 1 and 4 ). Since CsAAE1, CsOLS and CsOAC are localized in the cytosol in the trichomes of cannabis, the authors expressed codon-optimized versions of these three genes in Nicotiana benthamiana without any additional targeting peptides (pCAN1-3). In cannabis, CsCBGAS is localized in the chloroplast membrane due to the presence of a targeting peptide within the naturally occurring CBGAS sequence. To achieve chloroplast localization of recombinant CsCBGAS in Nicotiana benthamiana, the inventors expressed a codon-optimized version while retaining its own native plastid targeting sequence (pCAN7).

[0682] In the presence of C6 and GPP as substrates, when N. benthamiana was infiltrated with vectors pCAN1-3 and pCAN7, low levels of OA were detected that were absent in the corresponding P19 and blank infiltrations, used as negative controls ( Figure 6 ). Glycosylated forms of both OA and CBGA were also detected at low levels, indicating a functional recombinant CBGA biosynthetic pathway ( Figure 7 and 9 However, CBGA was not detected in its unmodified form ( Figure 8 ). This observation is consistent with the intrinsic rapid glycosylation of CBGA in Nicotiana benthamiana leaves.

[0683] Example 4 - Functional CBGA biosynthetic pathway targeted to chloroplasts

[0684] The functional pathway described in Example 3 relies on the biosynthesis of OA in the cytosol, followed by transport of this cannabinoid precursor to the chloroplasts for further conversion to CBGA by the membrane-bound CsCBGAS. The inventors hypothesized that, unlike cannabis trichomes, transport of OA to chloroplasts in Nicotiana benthamiana may create a metabolic bottleneck, thereby reducing the production of CBGA. In addition, if the CBGAS enzyme is unable to recognize the glycosylated form of OA as a substrate, the glycosylation of OA may lead to an additional metabolic 'dead end'. In order to improve the flux of OA to CBGA, the inventors designed an alternative CBGA biosynthetic pathway in which all enzymes are co-localized to the chloroplast. It is proposed that co-localization of all biosynthetic steps in the same organelle may result in better availability of the OA substrate for the CsCBGAS enzyme ( Figure 5 ). Therefore, the present inventors re-targeted each of CsAAE1 (pCAN5), CsOLS (pCAN6) and CsOAC (pCAN4) to the chloroplast by comprising a targeting peptide of Rubisco SSU as an N-terminal fusion.

[0685] Confocal microscopy confirmed the chloroplast targeting of CsAAE1, CsOLS, and CsOAC containing an N-terminal Rubisco SSU peptide ( Fig.10 ). The retargeted CsAAE1 proteins also showed some cytoplasmic localization. In the absence of the chloroplast targeting peptide, all three proteins were present in the cytosol as expected ( Fig.10 ).

[0686] In the presence of C6 and GPP as substrates, when N. benthamiana was infiltrated with vectors pCAN4-6 and pCAN7, both OA and CBGA as well as their glycosylated derivatives were produced at detectable levels ( Figure 6-9 ). The plastid-targeted pathway successfully produced detectable levels of CBGA, and glycosylated CBGA was significantly higher compared to the cytosol-chloroplast hybrid pathway of Example 3, indicating that flux was improved by re-targeting all enzymatic steps to the chloroplast. CBGA is the key cannabinoid end product of the pathway assembled in Nicotiana benthamiana, and the plastid pathway produced approximately 4 times more glycosylated OA than the cytosol-chloroplast model of Example 3. In the cytosol-chloroplast model of Example 3, CBGA was detectable at low levels in a glycosylated form, and its levels were significantly increased in the plastid-targeted pathway (estimated to be approximately 24-fold increase). In addition, unglycosylated CBGA was detected in the plastid-targeted pathway described in this example.

[0687] Based on confocal microscopy experiments, chloroplast targeting remains complex because although both OLS and OAC relocalize to chloroplasts, they appear to have different localization patterns within the chloroplasts despite the Rubisco chloroplast targeting peptide fused to each sequence. In addition, AAE1 fused to the same chloroplast targeting peptide still showed some localization in the cytosol. Nevertheless, the inventors have for the first time obtained a well-documented and functional CBGA biosynthetic cannabinoid pathway in a non-cannabis host plant.

[0688] Example 5 - Enhanced expression of a functional CBGA biosynthetic pathway operating in the cytoplasm and chloroplasts using a deconstructed viral expression vector.

[0689] Several methods are known to increase the expression of foreign proteins in plants, including the use of viral promoters suitable for a particular system (e.g., the CaMV 35S promoter, the maize-ubiquitin-1 promoter, or the Agrobacterium tumefaciens nopaline synthase (NOS) promoter) to increase transcription levels. It has been found that terminators such as the Ext terminator also improve protein expression. Plant viruses promoting protein expression are alternative mechanisms, particularly for transient expression. One such virus is the bean yellow dwarf virus (BeYDV), a Mastrevirus of the Geminiviridae family. BeYDV contains a single-stranded circular DNA genome and uses a rolling circle mechanism to replicate its genome, which has been shown to produce high yields of gene copies and thereby increase protein expression.

[0690] To test whether recombinant protein levels in N. benthamiana could be increased and cannabinoid production improved, a series of level 0 modules were designed that were compatible with GG cloning and contained elements of the Bean Yellow Dwarf Virus (BeYDV), as described in detail in Example 1. CsAAE1 (pCAN31), CsOLS (pCAN33), CsOAC (pCAN32), and CsCBGAS (pCAN34) were cloned in this new expression system and N. benthamiana leaves were infiltrated as described in Example 1 in the presence of C6 and GPP as substrates.

[0691] Compared to penetration using a standard expression vector (Example 3), the use of the BeYDV-adapted vector system resulted in a surprising and significant increase in the levels of OA, CBGA, and glycosylated forms of OA and CBGA ( Figure 6-9and 10). It is estimated that the OA production is increased by about 65 to 70 times due to the cytoplasmic-chloroplast expression model under the control of the viral vector expression system. In this experiment, the glycosylated form of OA in the cytoplasmic-chloroplast model increased by about 25 times under the control of the viral vector replication mechanism. CBGA was successfully detected, and the level of glycosylated form of CBGA was observed to be about 19 times the level from the results of Example 3. As expected, higher levels of glycosylated forms ( Figure 7 and 9 ), confirming the earlier results described in Example 3.

[0692] Example 6 - Enhanced expression of a functional CBGA biosynthetic pathway targeted to chloroplasts using a deconstructed viral expression vector.

[0693] Since retargeting of the pathway in chloroplasts (Example 4) and the use of BeYDV genetic elements to increase expression (Example 5) resulted in increased levels of OA and CBGA, a combination of these two strategies was undertaken to attempt to further increase CBGA yield and metabolic flux through the recombinant cannabinoid pathway in Nicotiana benthamiana. To this end, the Rubisco SSU chloroplast targeting sequence was cloned in frame with CsAAE1 (pCAN35), CsOLS (pCAN37) and CsOAC (pCAN36) in the new BeYDV-adapted expression system (see Table 3).

[0694] As described in Example 1, in the presence of C6 and GPP as substrates, when N. benthamiana was infiltrated with vectors pCAN35-37 and pCAN34, significantly increased levels of OA, CBGA and their glycosylated derivatives were observed ( Figure 6-9 and 10-11). Similar to earlier results obtained with a standard expression vector (Example 4), levels of all four cannabinoids were higher compared to the cytosol-chloroplast pathway (Example 5) using the same expression system. It is estimated that OA production was increased by approximately 65 to 70 times due to the viral vector expression system. In this experiment, the glycosylated form of OA in the chloroplast model was increased by approximately 40-45 times under the control of the viral vector replication machinery. CBGA (unglycosylated) was detected at levels approximately 10-13 times the levels of the results from Example 4, and the glycosylated form of CBGA was observed at levels approximately 2-3 times the levels of the results from Example 4.

[0695] Again, this demonstrates that the function of the CBGA biosynthetic pathway is improved when all enzymes are located in the same subcellular compartment, such as the chloroplast.

[0696] The inventors next sought to compare expression levels in one experiment using four independent biological replicates to allow comparisons between (1) cytoplasmic targeting versus chloroplast targeting, or (2) standard binary expression vectors versus deconstructed viral expression vectors. The same constructs used in the previous experiments were used here for the following four combinatorial gene penetrations: pCAN1+pCAN2+pCAN3+pCAN7 for cytoplasmic targeting using a binary vector ('Cyto'); pCAN4+pCAN5+pCAN6+pCAN7 for plastid targeting using a binary vector ('Plast'); pCAN31+pCAN32+pCAN33+pCAN37 for cytoplasmic targeting using a deconstructed viral vector ('Cyto VV'); and pCAN34+pCAN35+pCAN36+pCAN37 for plastid targeting using a deconstructed viral vector ('Plast VV').

[0697] As described in Example 3-6, Nicotiana benthamiana was infiltrated with an Agrobacterium strain carrying an expression vector. Each infiltration was combined with an Agrobacterium strain expressing the viral inhibitory protein p19, while an Agrobacterium strain expressing p19 and GFP was used as a negative control. As described in Example 3-6, substrates C6 and GPP were exogenously supplied.

[0698] Fig.11 The results presented demonstrate that accumulation of OA and CBGA and their glycosylated derivatives is higher when the recombinant proteins are targeted to chloroplasts compared to localization in the cytosol. Higher yields of OA and CBGA were also detected in both the cytosol and chloroplasts when the transgenes were expressed using deconstructed viral vectors compared to normal binary expression vectors.

[0699] Based on these results, the inventors proposed a method for the biosynthesis of CBGA in plant biomass, whereby it is possible to produce surprising synergistic effects using cytosol-chloroplast and / or chloroplast-targeted biosynthetic pathways that are enhanced by using viral vector replication machinery from geminiviruses such as BeYDV.

[0700] Example 7-C6 Engineering

[0701] Plastid targeting of the OA biosynthetic enzymes CsAAE1, CsOLS, and CsOAC has been shown to successfully produce OA, although at low levels in N. benthamiana leaves and mostly glycosylated with glucose (Example 4). Previous infiltration experiments herein relied on exogenously supplied C6:0 as a substrate for CsAAE1 to produce hexanoyl-CoA, which was subsequently converted to OA by the combined action of CsOLS and CsOAC. Plant leaves typically accumulate medium-chain length FAs, with the majority of carbon chain lengths being C16 or C18, while levels of C14 or shorter chain fatty acids are low. Very short chain length FAs (i.e., less than 10 carbon chain lengths), either in the acyl-CoA form or esterified to a glycerol backbone, are rare in leaves. Increased release of C6 in plastids may enhance OA synthesis by recombining the CsAAE1, CsOLS, and CsOAC biosynthetic enzymes.

[0702] Two approaches were designed to test the possibility of increasing C6 accumulation in N. benthamiana leaves, in combination with the expression of CsAAE1, CsOLS, and CsOAC, thereby enhancing OA synthesis. The first approach was designed to test the possibility of increasing the accumulation of cellular C6 content in N. benthamiana leaves, in combination with the expression of CsAAE1, CsOLS, and CsOAC, to enhance OA levels in the absence of an exogenous supply of C6 substrates. The Arabidopsis thaliana acyl-lipid thioesterase (AtALT4, At1g68280) has been shown to lead to the accumulation of short-chain fatty acids (SCFAs) containing 6 or 8 carbons in leaves and seeds (Kalinger et al., 2021). Transient expression of AtALT4 in N. benthamiana leaves also led to the accumulation of C6 fatty acids (Kalinger et al., 2021). AtALT4 contains a 47 amino acid transit peptide and is predicted to be localized in chloroplasts. Due to intron prediction, there are two reported sequence versions of AtALT4 in NCBI, NP_001319347 and F4HX80, named AtALT4.1 and AtALT4.2, respectively.

[0703] The second approach was to express Arabidopsis plastid lipase 1 (AtPLIP1, At3g61680, ACI49785) and tomato 13-lipoxygenase (TomLoxC, AAB65766). AtPLIP1 hydrolyzes C18:2 or C18:3 from the sn-1 position of a unique chloroplast-specific phosphatidylglycerol containing 16:1 Δ3transas its second acyl group (Wang, 2017). AtPLIP1 is a peripheral thylakoid membrane protein localized in chloroplasts. TomloxC is also predicted to be chloroplast-localized and encodes a 13-lipoxygenase that produces C5 and C6 fatty acid-derived volatiles by peroxidation of C18 polyunsaturated fatty acids in tomato leaves (Shen et al., 2014). Lipoxygenase activity is also thought to produce C6 from C18:2 in cannabis (Gülck and 2020).

[0704] For Nicotiana benthamiana, the coding sequences of AtALT4.1 (NP_001319347), AtALT4.2 (F4HX80), AtPLIP1 (ACI49785) and TomloxC (AAB65766, SEQ ID NO: 20-23, respectively) were codon optimized and synthesized. The codon optimized nucleotide sequences are listed as SEQ ID NO: 24-27. As described in Example 1, these coding sequences were cloned into the Golden Gate cloning vector pICH47732 under the 2x35S promoter. The resulting expression vector was named pCAN38-41 (Table 3). As described in Example 1, the same sequence was cloned into the Golden Gate cloning vector pICH47732 using modules EN38510, EN38509 and EN38511. The resulting expression vector was named pCAN42-45 (Table 3). These expression plasmids can be transformed into Agrobacterium tumefaciens GV3103, and the resulting strain combination is used for Nicotiana benthamiana leaf infiltration as described in Example 1. Total leaf FAs are analyzed by gas chromatography (GC) to detect the accumulation of short-chain FAs, especially C6. Expression of AtALT4.1, AtALT4.2, or AtPLIP1+TomloxC can also be combined with expression of CsAAE1, CsOLS, and CsOAC to produce OA in the absence of C6 feeding.

[0705] Results from experiments conducted in Nicotiana benthamiana showed that the codon-optimized sequence of AtALT4.1 (NP_001319347, SEQ ID NO: 24) was functional and resulted in the production of C6. AtALT4.2 showed activity in supporting C6 production. AtALT4.1 was used in the following experiments and is referred to as AtALT4.

[0706] AtALT4 (NP_001319347) was heterologously expressed in Nicotiana benthamiana leaves using a deconstructed viral expression vector in combination with chloroplast-targeted versions of the OA biosynthetic pathway (CsAAE1, CsOLS, and CsOAC) from a deconstructed viral expression vector. Fig.13, Plast VV) alone, the OA biosynthetic pathway combined with AtALT4 produced a greater amount of OA in the absence of C6 feeding ( Fig.13 , Plast VV+ALT).

[0707] Example 8 - Stable Tobacco Transformation

[0708] The codon optimized sequences of CsAAE1, CsOLS, CsOAC and CsCBGAS are cloned into plant binary expression vectors such as pORE04 (Coutu et al., 2007). In order to achieve the subcellular localization of CsAAE1, CsOLS and CsOAC in the cytosol or chloroplast, all three genes are cloned in the absence of a targeting peptide, or cloned in frame with a chloroplast targeting peptide such as Rubisco SSU as described in Examples 1 and 4. The promoter driving transgenic expression is constitutive (e.g., 35S, enTCUP2), tissue-specific (e.g., Rubisco SSU), developmentally regulated (e.g., SAG12), inducible or a combination thereof. The transformation and tissue culture regeneration of Nicotiana benthamiana mediated by Agrobacterium tumefaciens are carried out as described by Vanhercke et al. (2014). The confirmed transgenic events are transferred to a greenhouse and the increase in CBGA levels is analyzed after penetration with C6 and GPP substrates.

[0709] The same combination of transgene, promoter and subcellular targeting peptide as described above was cloned into a plant expression vector containing viral genetic elements to increase expression, similar to but not limited to Zhang and Masson (2006) or Dugdale et al., (2014). Confirmed transgenic events were transferred to a greenhouse and analyzed for increases in CBGA levels after infiltration with C6 and GPP substrates.

[0710] The same set of transgenes as described above were cloned into chloroplast transformation vectors and transplastomic Nicotiana benthamiana lines and tissue culture regeneration were generated (Svab and Maliga, 1993). Confirmed transgenic events were transferred to the greenhouse and analyzed for increases in CBGA levels after infiltration with C6 and GPP substrates.

[0711] The same group of transgenics were cloned into plant expression vectors with AtALT4, AtPLIP and TomLoxC or AtALT4 and AtPLIP and TomLoxC. Promoters, subcellular targeting and vector backbones were the same as described above. Transformation and tissue culture regeneration of Nicotiana benthamiana mediated by Agrobacterium tumefaciens were performed substantially as described by Vanhercke et al. (2014). The confirmed transgenic events were transferred to a greenhouse and analyzed for the increase of CBGA levels in the absence of exogenous C6 substrates in nutrient (e.g., leaf) tissues and seeds. CsAAE1, CsOLC, CsOAC and CsCBGAS were cloned into chloroplast transformation vectors with AtALT4, AtPLIP and TomLoxC or AtALT4 and AtPLIP and TomLoxC. Transplastomic Nicotiana benthamiana lines were produced substantially as described by Svab and Maliga (1993). The confirmed transgenic events were transferred to a greenhouse and analyzed for the increase of CBGA levels in the absence of exogenous C6 substrates in nutrient (e.g., leaf) tissues and seeds.

[0712] The present invention is the first demonstration of a functional CBGA cannabinoid biosynthetic pathway in a non-cannabis plant host. Specifically, for Nicotiana benthamiana, co-expression of codon optimization of the CsAAE, CsOLS, CsOAC and CsCBAS genes resulted in the synthesis of OA and glycosylated forms of OA and CBGA. It was further shown that retargeting CsAAE1, CsOLC and CsOAC to the chloroplasts of Nicotiana benthamiana cells further increased the yields of OA, CBGA and their respective glycosylated forms. When the recombinant cytoplasmic / chloroplast and chloroplast CBGA biosynthetic pathways were overexpressed using an improved vector incorporating specific BeYDV genetic elements, the levels of all four cannabinoids increased dramatically. Similarly, when all biosynthetic genes were co-located in the chloroplast, the levels of OA, CBGA and their glycosylated derivatives increased.

[0713] It will be appreciated by those skilled in the art that, without departing from the spirit or scope of the invention as broadly described, various changes and / or modifications may be made to the invention as shown in the specific embodiments. Therefore, the embodiments of the invention should be considered in all aspects as illustrative and not restrictive.

[0714] This application claims priority to Australian provisional patent application AU 2021904150 filed on December 20, 2021, the entire contents of which are incorporated herein by reference.

[0715] All publications discussed and / or cited herein are incorporated herein in their entirety.

[0716] Any discussion of documents, acts, materials, devices, articles of manufacture or the like that has been included in this specification is solely for the purpose of providing a context for the present invention. This should not be taken as an admission that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present invention as they existed before the priority date of each claim of this application.

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Claims

1. A genetically modified plastid of a plant or part thereof, comprising an acyl activating enzyme 1 (AAE1) polypeptide, an olivetol synthase (OLS) polypeptide and an olivetate cyclase (OAC) polypeptide, optionally further comprising a plastid-localized prenyltransferase polypeptide, preferably cannabigerolic acid synthase (CBGAS), wherein the polypeptide increases the production of cannabigerolic acid (CBGA) in the presence of hexanoic acid (C6) and geranyl pyrophosphate (GPP) when compared to plastids of a wild-type plant or part thereof.

2. A genetically modified plant or part thereof, comprising a polynucleotide encoding acyl activating enzyme 1 (AAE1), a polynucleotide encoding olivetol synthase (OLS) and a polynucleotide encoding olivetate cyclase (OAC), optionally further comprising a polynucleotide encoding a plastid-localized prenyltransferase polypeptide, preferably cannabigerolic acid synthase (CBGAS), wherein each polynucleotide is operably linked to a promoter capable of directing expression of said polynucleotide in said plant or part thereof, wherein the polynucleotide is codon-optimized for expression in the plant or part thereof, and The polypeptide encoded by the polynucleotide increases the production of cannabigerolic acid (CBGA) when expressed in the plant or part thereof in the presence of caproic acid (C6) and geranyl pyrophosphate (GPP) when compared to wild-type plants or parts thereof.

3. The genetically modified plant or part thereof according to claim 2, wherein the polynucleotide is comprised in a vector, preferably a viral vector, wherein the polynucleotide encodes a fusion polypeptide comprising a plastid transit peptide; and / or wherein the polypeptide encoded by the polynucleotide is expressed in the presence of a plastid-localized prenyltransferase; and The polypeptide encoded by the polynucleotide increases the production of cannabigerolic acid (CBGA) when expressed in the plant or part thereof in the presence of hexanoic acid (C6) and geranyl pyrophosphate (GPP) compared to wild-type plants or parts thereof.

4. The plastid according to claim 1 or the plant according to claim 2 or 3, or parts thereof, wherein the genetically modified part thereof is a vegetative plant part, preferably a vegetative plant part of a vascular plant.

5. The plastid of claim 1 or 4 or the plant or part thereof of any one of claims 2 to 4, wherein the genetically modified plant or part thereof is a high biomass plant, preferably selected from the group consisting of: Acrocomia aculeata (macauba palm), Arabidopsis thaliana, peanut (Arachis hypogaea), murumuru (Astrocaryummurumuru), tucuma (Astrocaryum vulgare), Indaia-rateiro (Attalea geraensis), American oil palm (Attalea humilis), andaia (Attalea oleifera), uricuri (Attalea phalerata), babassu (Attalea speciosa), oats (Avena sativa), beets (Betavulgaris) or sugar palm. beet, Brassica sp. such as Brassica carinata, Brassica juncea, Brassica napobrassica, Brassica napus (canola), Camelina sativa (false flax), Cannabis sativa (hemp), Carthamus tinctorius (safflower), Caryocar brasiliense (pequi), Coconut (Cocos nucifera), Crambe abyssinica (Abyssinian kale), Cucumis melo (melon), Elaeis guineensis (African palm), Glycine max (soybean), Gossypium hirsutum (cotton), Helianthus sp.) such as sunflower (Helianthus annuus), barley (Hordeum vulgare), physic nut (Jatropha curcas), arara nut-tree (Joannesia princeps), duckweed (Lemna sp.) (duckweed) such as Lemna aequinoctialis, Lemna disperma, Lemna ecuadoriensis, Lemna gibba (swelling duckweed), Lemna japonica, Lemna minor, Lemna minuta, Lemna obscura, Lemna paucicostata, Lemna perpusilla, Lemna tenera, Lemna trisulca, Lemna turionifera), Valdivian duckweed (Lemna valdiviana), Yungas rock duckweed (Lemna yungensis), Oti tree (Licania rigida or oiticica), Flax (Linumusitatissimum or flax), Lupinus angustifolius or lupin, Mauritia flexuosa (buriti palm), Maximiliana maripa (inaja palm), Miscanthus sp. such as Miscanthus x giganteus and Miscanthus sinensis, Nicotiana sp. (tobacco) such as Nicotiana tabacum or Nicotiana benthamiana, Oenocarpus bacaba (bacaba-do-azeite), Oenocarpus bataua)(pataua), Oenocarpus distichus(bacaba-de-leque), Oryza sp.) (rice) such as Asian rice (Oryza sativa) and African cultivated rice (Oryza glaberrima), switchgrass (Panicum virgatum), Paraqueiba paraensis (mari), avocado (Persea amencana), Pongamiapinnata (Indian beech), Populus trichocarpa, Ricinus communis (castor), Saccharum sp. (sugarcane), sesame (Sesamumindicum), potato (Solanum tuberosum), Sorghum sp. such as Sorghum bicolor (Sorghum vulgare), Theobroma grandiforum (cupuassu), Trifolium sp., Brazilian needle palm (Trithrinax brasiliensis) palm), Triticum sp. (wheat) such as common wheat (Triticum aestivum), and corn (Zea mays or corn).

6. The plastid or plant or part thereof according to claim 5, wherein the plant is tobacco, preferably Nicotiana benthamiana or Nicotiana tabacum.

7. The plastid according to any one of claims 1 or 4 to 6, wherein the polypeptide is encoded by one or more polynucleotides, wherein the one or more polynucleotides encode a fusion polypeptide comprising a plastid transit peptide; and Wherein the one or more polynucleotides are operably linked to a promoter capable of directing expression of the one or more polynucleotides in the plant or part thereof.

8. The plastid according to any one of claims 1 or 4 to 7 or the plant or part thereof according to any one of claims 2 to 6, wherein the increase in the production of CBGA in the plastid, plant or part thereof can be determined by chromatography.

9. The plant or part thereof according to any one of claims 2 to 6 or 8, wherein at least one polypeptide is active in the cytosol of the plant or part thereof.

10. The plant or part thereof according to any one of claims 2 to 6 or 8 to 9, wherein the polynucleotides are contained in the same or different nucleic acid constructs.

11. The plant or part thereof according to claim 10, wherein one or more polynucleotides, optionally each polynucleotide, encodes a fusion polypeptide comprising a plastid transport peptide, preferably a chloroplast transit peptide (CTP).

12. The plastid of any one of claims 1 or 4 to 8, wherein the plant or part thereof is a non-Cannabis plant.

13. The plant or part thereof according to any one of claims 2 to 6 or 8 to 11, wherein the plant or part thereof is a non-cannabis plant.

14. A plastid according to any one of claims 1 or 4 to 8 or 12 or a plant or part thereof according to any one of claims 2 to 6 or 8 to 13, wherein the polypeptide increases the production of CBGA in a vegetative part of the plant or part thereof.

15. The plastid or plant or part thereof of claim 14, wherein the production of CBGA is increased by at least about 1.5 to about 2 times, about 2 to about 2.5 times, about 2.5 to about 3.0 times, about 3 to about 3.5 times, about 3.5 to about 4.0 times, about 4 to about 4.5 times, about 4.5 to about 5.0 times, or about 5 to about 5.5 times or more when compared to a wild-type plastid, plant or part thereof.

16. The plastid or plant or part thereof of claim 14, wherein the production of CBGA is increased by at least about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, about 90% to about 100%, about 100% to about 120%, about 120% to about 130%, about 130% to about 150%, about 160% to about 170%, about 180% to about 190%, about 200% to about 210%, about 220% to about 230%, about 240% to about 250%, about 260% to about 270%, about 280% to about 290%, about 300% to about 310%, about 310% to about 320%, about 330% to about 340%, about 350% to about 360%, about 370% to about 380%, about 390% to about 400%, about 410% to about 420%, about 430% to about 440%, about 450% to about 460%, about 470% to about 480%, about 490% to about 500%, about 50 ... About 140%, about 140%-about 160%, about 160%-about 180%, about 180%-about 200%, about 200%-about 220%, about 220%-about 240%, about 240%-about 260%, about 260%-about 280%, about 280%-about 300%, about 300%-about 320%, about 320%-about 340%, about 340%-about 360%, about 360%-about 380%, about 400%-about 420%, or more.

17. A plastid according to any one of claims 1 or 4 to 8 or 12 or 14 to 16 or a plant or part thereof according to any one of claims 2 to 6 or 8 to 11 or 13 to 16, wherein the plastid is a chloroplast.

18. The plastid of claim 7 or the plant or part thereof of claim 11, wherein the plastid transport peptide is a chloroplast transit peptide (CTP).

19. The plastid of any one of claims 1 or 4 to 8 or 12 or 14 to 18, wherein at least two polypeptides, optionally each polypeptide, comprises a plastid transport peptide, preferably a chloroplast transit peptide (CTP).

20. The plastid of claim 19, wherein the CTP is a stromal targeting peptide comprising a Rubisco small subunit and a Rubisco large subunit, preferably the Rubisco small subunit.

21. The plastid of claim 20, wherein the Rubisco small subunit is located at the N-terminus of the polypeptide.

22. The plant or part thereof according to any one of claims 2 to 6 or 8 to 11 or 13 to 18, further comprising a polynucleotide encoding a prenyltransferase, preferably a cannabigerolic acid synthase (CBGAS), wherein the polynucleotide encodes a fusion polypeptide comprising a plastid transit peptide.

23. The plastid of any one of claims 1 or 4 to 8 or 12 or 14 to 21, further comprising a polypeptide encoded by a prenyltransferase, preferably cannabigerolic acid synthase (CBGAS).

24. The plant or part thereof of any one of claims 2 to 6 or 8 to 11 or 13 to 18 or 22, wherein the plant or part thereof further comprises at least one polynucleotide selected from the group consisting of a polynucleotide encoding an acyl-lipid thioesterase (ALT4), a polynucleotide encoding a plastidial lipase 1 (PLIP1), and a polynucleotide encoding a tomato 13-lipoxygenase (TomLoxC), optionally wherein the polynucleotide encodes a fusion polypeptide comprising a plastid transit peptide.

25. The plastid of any one of claims 1 or 4 to 8 or 12 or 14 to 21 or 23, wherein the plastid further comprises at least one polypeptide selected from the group consisting of: acyl-lipid thioesterase (ALT4), plastidial lipase 1 (PLIP1), and tomato 13-lipoxygenase (TomLoxC).

26. A nucleic acid construct encoding a polypeptide for expression in a plant or part thereof, comprising one or more or all of the polynucleotides encoding a polyketide synthase, a polyketide cyclase, an acyl activating enzyme, a prenyl transferase, a plastidial acyl-lipid thioesterase, a plastidial lipase, and a plastidial tomato 13-lipoxygenase; wherein the polynucleotide is operably linked to a promoter capable of directing expression of the one or more or all polynucleotides in the plant or part thereof, Optionally, wherein the one or more or all of the polynucleotides are operably linked to a polynucleotide encoding a plastid transit peptide; Optionally, wherein the nucleic acid construct encodes a fusion polypeptide comprising the plastid transit peptide, and When expressed, the polypeptide encoded by the polynucleotide increases the production of cannabigerolic acid (CBGA) compared to a wild-type plant or part thereof.

27. The nucleic acid according to claim 26, for expression in plastids of said plant or part thereof.

28. The nucleic acid construct according to claim 26 or 27, further comprising a polynucleotide encoding a silencing suppression polypeptide, preferably a p19 silencing suppression polypeptide.

29. according to each described nucleic acid construct in claim 26 to 28, wherein said nucleic acid construct is included in the carrier that is suitable for expressing in plant or its part.

30. The nucleic acid construct of claim 26, wherein: i) the polynucleotide encoding the polyketide synthase is olivetol synthase (OLS); ii) the polynucleotide encoding the acyl-activating enzyme is acyl-activating enzyme 1 (AAE1); iii) the polynucleotide encoding the polyketide cyclase is olivetate cyclase (OAC); Optionally wherein the nucleic acid construct comprises a polynucleotide encoding a silencing suppression polypeptide, preferably a p19 silencing suppression polypeptide, wherein one or more or all of the polynucleotides are operably linked to a promoter capable of directing expression of the polypeptide encoded by the polynucleotide in a plant or part thereof; Optionally, wherein the one or more or all of the polynucleotides are operably linked to a polynucleotide encoding a plastid transit peptide; and Optionally wherein the nucleic acid construct encodes a fusion polypeptide comprising a plastid transit peptide.

31. according to the nucleic acid construct described in any one of claims 26 to 30, wherein compared with wild-type plants or parts thereof, when expressed in plants or parts thereof, the output of CBGA increases by at least about 1.5-about 2 times, about 2-about 2.5 times, about 2.5-about 3.0 times, about 3-about 3.5 times, about 3.5-about 4.0 times, about 4-about 4.5 times, about 4.5-about 5.0 times or about 5-about 5.5 times or more times.

32. The nucleic acid construct of any one of claims 26 to 30, wherein when expressed in a plant or part thereof, the yield of CBGA is increased by at least about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to 70%, about 70% to about 80%, about 80% to about 90%, about 90% to about 100%, about 100% to about 120%, about 150% to about 200%, about 160% to about 210%, about 170% to about 220%, about 180% to about 230%, about 190% to about 300%, about 210% to about 300%, about 220% to about 300%, about 230% to about 300%, about 240% to about 300%, about 250% to about 300%, about 260% to about 300%, about 270% to about 300%, about 280% to about 300%, about 290% to about 300%, about 310% to about 310%, about 320% to about 310%, about 3 ... 120% to about 140%, about 140% to about 160%, about 160% to about 180%, about 180% to about 200%, about 200% to about 220%, about 220% to about 240%, about 240% to about 260%, about 260% to about 280%, about 280% to about 300%, about 300% to about 320%, about 320% to about 340%, about 340% to about 360%, about 360% to about 380%, about 400% to about 420%, or more.

33. A fusion polypeptide for expression in a plant or part thereof, the fusion polypeptide comprising: i) a polypeptide selected from the group consisting of a polyketide synthase, a polyketide cyclase, an acyl-activating enzyme, a prenyl transferase, a plastidial acyl-lipid thioesterase, a plastidial lipase or a plastidial tomato 13-lipoxygenase; and ii) plastid transport sequences, wherein the polypeptide of (i) increases the production of cannabigerolic acid (CBGA) in the presence of hexanoic acid (C6) and geranyl pyrophosphate (GPP) when compared to wild-type plants or parts thereof.

34. The fusion polypeptide of claim 33, wherein the plastid trafficking sequence is a chloroplast transit peptide (CTP).

35. A plasmid comprising the fusion polypeptide according to claim 33 or 34.

36. A plant, part thereof or cell thereof, comprising the fusion polypeptide according to claim 33 or 34.

37. The nucleic acid construct of any one of claims 26 to 32 or the fusion polypeptide of claim 33 or 34, wherein the polyketide synthase is olivetol synthase (OLS), the polyketide cyclase is olivetate cyclase (OAC), the acyl activating enzyme is acyl activating enzyme 1 (AAE1), the plastidial acyl-lipid thioesterase is ALT4, the plastidial lipase is PLIP1, the plastidial tomato 13-lipoxygenase is TomLoxC, and the prenyltransferase is cannabigerolic acid synthase (CBGAS), preferably wherein the plastid transport peptide is a chloroplast transit peptide (CTP).

38. The plastid of any one of claims 1 or 4 to 8 or 12 or 14 to 21 or 23 or 25, the plant or part thereof of any one of claims 2 to 6 or 8 to 11 or 13 to 18 or 22 or 24, or the nucleic acid construct or fusion polypeptide of claim 37, wherein one or more or all of: i) the polynucleotide encoding AAE1 comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8% or identical to the sequence shown in any one of SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7; ii) the polynucleotide encoding OLS comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8% or identical to the sequence shown in any one of SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10; iii) the polynucleotide encoding OAC comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8% or identical to the sequence shown in any one of SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13; iv) the polynucleotide encoding the plastid transit peptide comprises a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8% or identical to the sequence shown in SEQ ID NO:

15.

39. The plant or part thereof of claim 22, the plastid of claim 23, the nucleic acid construct of any one of claims 26 to 32 or 37, or the fusion protein of any one of claims 33 to 35, wherein the polynucleotide encoding the prenyl transferase comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8% or identical to the sequence shown in SEQ ID NO:

14.

40. The plant or part thereof according to claim 24, the plastid according to claim 25, or the nucleic acid construct or fusion protein according to claim 37, wherein: i) the polynucleotide encoding ALT4 comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8% or identical to the sequence shown in SEQ ID NO: 24 or 25; ii) the polynucleotide encoding PLIP1 comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8% or identical to the sequence shown in SEQ ID NO: 26; and iii) the polynucleotide encoding TomLoxC comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8% or identical to the sequence shown in SEQ ID NO:

27.

41. A method for producing cannabigerolic acid (CBGA) in a plant or a plant part thereof, the method comprising culturing a plant or a part thereof comprising a nucleic acid construct according to any one of claims 26 to 32 or 37 to 40 under conditions sufficient to express the polypeptide in the plant or plant part, wherein the polypeptide increases the production of cannabigerolic acid (CBGA) in the plant or plant part when expressed in the presence of hexanoic acid (C6) and geranyl pyrophosphate (GPP) when compared to a wild-type plant or plant part.

42. The method of claim 41, further comprising providing exogenous C6, malonyl-CoA and / or GPP to the plant or plant part, optionally in the form of a plant feed, an amino acid supplement or a C6 and / or malonyl-CoA and / or GPP substrate.

43. The method of claim 41 or 42, wherein the polypeptide is expressed in the plastid of the plant or plant part.

44. The method according to any one of claims 41 to 43, further comprising introducing the nucleic acid construct according to any one of claims 26 to 32 or 37 to 40 into the plant or plant part.

45. The method of any one of claims 41 to 44, wherein CBGA is produced in a vegetative part of the plant.

46. ​​The method of claim 45, wherein the vegetative parts comprise leaves of the plant.

47. The method of claim 45 or 46, wherein the increase in the production of CBGA in the vegetative part of the plant can be determined by chromatography.

48. Cannabigerolic acid (CBGA) or cannabigerol (CBG) produced by the method of any one of claims 41 to 47.

49. A cannabigerol (CBG) produced by the method of any one of claims 41 to 47.

50. The method of any one of claims 41 to 47, further comprising producing cannabinoids, the method comprising: i) recovering the CBGA produced by the method according to any one of claims 41 to 47; as well as ii) optionally feeding said CBGA to a genetically modified microorganism, preferably a yeast or a bacterium; and iii) optionally subjecting said CBGA to a chemical, physical or biochemical transformation using said CBGA as a starting material, thereby producing said cannabinoid.

51. The method of claim 50, comprising producing at least one of cannabigerol (CBG), tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA) and cannabichromene acid (CBCA), preferably cannabidiolic acid (CBDA), optionally comprising producing tetrahydrocannabinol (THC), cannabidiol (CBD) and cannabichromene (CBC), preferably cannabidiol (CBG) or cannabidiol (CBD).

52. The method of claim 50 or 51 further comprising a CBGA decarboxylation step, preferably by incubating the CBGA with heat or light.

53. A cannabidiolic acid (CBDA) or cannabidiol (CBD) produced by the method according to any one of claims 50 to 52.

54. Cannabichromene acid (CBCA) or cannabichromene (CBC) produced by the method according to any one of claims 50 to 53.

55. Tetrahydrocannabinolic acid (THCA) or tetrahydrocannabinol (THC) produced by the method of any one of claims 50 to 52.

56. A method of producing a genetically modified plant, the method comprising: i) regenerating a plant from a plant transformed with a nucleic acid construct according to any one of claims 26 to 32 or 37 to 40; ii) optionally collecting seeds from said plant; and / or iii) optionally producing one or more progeny plants from the genetically modified plant, thereby producing the genetically modified plant.

57. A method of producing a genetically modified seed, the method comprising: i) regenerating a plant from a plant cell transformed with a nucleic acid construct according to any one of claims 26 to 32 or 37 to 40; as well as ii) collecting seeds from said plants, thereby producing genetically modified seeds.

58. The method of claim 56 or 57, further comprising introducing the nucleic acid construct of any one of claims 26 to 32 or 37 to 40 into the plant or a cell thereof.

59. The method of claim 58, wherein the encoded polypeptide is expressed in the vegetative parts of the plant, preferably the leaves.

60. The method according to any one of claims 56 to 59, wherein the plant is a vascular plant, preferably a vascular plant of the genus Nicotiana, more preferably Nicotiana benthamiana or Nicotiana tabacum.

61. A method of producing a genetically modified plant, the method comprising: i) crossing two parental plants, wherein at least one plant comprises a genetic modification introduced by a nucleic acid construct according to any one of claims 26 to 32 or 37 to 40; ii) screening one or more progeny plants from the cross in i) for the presence or absence of the genetic modification; as well as iii) selecting progeny plants comprising said genetic modification, thereby producing said plants.

62. The method of claim 61, wherein the method further comprises: iv) crossing a first genetically modified parent plant with a second parent plant, wherein the first genetically modified parent plant is a plant comprising a genetic modification introduced by a nucleic acid construct according to any one of claims 26 to 32 or 37 to 40; and v) backcrossing the progeny plant of said cross of step (iv) with a plant of the same genotype as said second parent plant to produce a plant having most of the genotype of said second parent and including said genetic modification.

63. The method of claim 61 or 62, wherein said vegetative part, preferably said leaves of said plant comprises said genetic modification.

64. A genetically modified and / or recombinant plant cell for producing cannabigerolic acid (CBGA), the genetically modified and / or recombinant plant cell comprising a nucleic acid construct according to any one of claims 26 to 32 or 37 to 40.

65. A genetically modified plant for producing cannabigerolic acid (CBGA), the genetically modified plant comprising the nucleic acid construct according to any one of claims 26 to 32 or 37 to 40.

66. A plant part of the genetically modified plant according to claim 65.

67. The plant of claim 66, wherein said plant is a seed comprising said genetic modification.

68. A method of producing a plant part from a genetically modified plant, the method comprising: i) growing the plant according to any one of claims 61 to 63; as well as ii) collecting said plant parts.

69. Use of a nucleic acid construct according to any one of claims 26 to 32 or 37 to 40 for producing a recombinant plant cell or a genetically modified plant.

70. A crop or plant population comprising: (i) a plant according to any one of claims 2 to 6 or 8 to 11 or 13 to 18 or 22 or 24, a plastid according to any one of claims 1 or 4 to 8 or 12 or 14 to 21 or 23 or 25, a nucleic acid construct according to any one of claims 26 to 32 or 37 to 40, or a fusion polypeptide according to any one of claims 33, 34 or 37 to 40; or (ii) a plant according to any one of claims 2 to 6 or 8 to 11 or 13 to 18 or 22 or 24, a plastid according to any one of claims 1 or 4 to 8 or 12 or 14 to 21 or 23 or 25, a nucleic acid construct according to any one of claims 26 to 32 or 37 to 40, or a fusion polypeptide according to any one of claims 33, 34 or 37 to 40, and wild-type plants.

71. The crop or plant population of claim 70, wherein the crop or field comprises vegetative plant parts, preferably vascular plants, more preferably vegetative plant parts of the Nicotiana genus, most preferably Nicotiana benthamiana or Nicotiana tabacum plants.

72. The crop or plant population of claim 71 further comprising non-vascular plants.

73. A crop or plant population according to any one of claims 70 to 72, wherein the plants comprise a higher yield of CBGA when compared to wild-type plants, optionally ascertainable by chromatography.

74. An industrial plant biomass obtained from leaves of a genetically modified plant according to any one of claims 2 to 6 or 8 to 11 or 13 to 18 or 22 to 24, wherein the leaves of the plant biomass comprise a higher yield of CBGA when compared to leaves of a wild-type plant, optionally capable of being determined by chromatography.

75. A composition comprising a plastid according to any one of claims 1 or 4 to 8 or 12 or 14 to 21 or 23 or 25, a nucleic acid construct according to any one of claims 26 to 32 or 37 to 40 or a plant cell according to claim 64 and one or more acceptable carriers.

76. A method for identifying a polynucleotide encoding a polypeptide for producing cannabigerolic acid (CBGA) in a plant, part thereof, or plastid or cell thereof, the method comprising: i) obtaining a nucleic acid construct according to any one of claims 26 to 32 or 37 to 40; ii) introducing the nucleic acid construct into a plant, part thereof, or plastid or cell thereof; iii) determining whether the level of CBGA is increased relative to a corresponding wild type plant, part thereof, or plastid or cell thereof lacking the nucleic acid construct; and iv) Optionally, selecting a polynucleotide that, when expressed, produces a polypeptide for producing CBGA.

77. The method of claim 76, wherein the CBGA is produced in plastids of the vegetative part of the plant, optionally detectable by chromatography.

78. A method for producing an industrial product, the method comprising the steps of: (i) obtaining a plant or part thereof according to any one of claims 2 to 6 or 8 to 11 or 13 to 18 or 20 or 22 or 24 or a plastid according to any one of claims 1 or 4 to 8 or 12 or 14 to 21 or 23 or 25; (ii) treating the plant or its parts or plastids; (iii) transforming at least some of the cannabinoids in the plant, part or plastid thereof of step (i), or the treated plant, part or plastid thereof obtained by step (ii), by applying thermal, chemical or enzymatic means, or any combination thereof, to produce cannabinoids in situ; and (iv) recovering said cannabinoids, thereby producing said industrial product.

79. The method of claim 78, wherein the industrial product is cannabigerolic acid (CBGA) or cannabigerol (CBG).

80. A method for producing extracted cannabigerolic acid (CBGA), the method comprising the steps of: (i) obtaining a plant or part thereof according to any one of claims 2 to 6 or 8 to 11 or 13 to 18 or 20 or 22 or 24 or a plastid according to any one of claims 1 or 4 to 8 or 12 or 14 to 21 or 23 or 25; (ii) extracting CBGA from the plant, part or plastid thereof; as well as (iii) recovering the extracted CBGA, thereby producing extracted CBGA.

81. The method of claim 80, wherein the extracted CBGA comprises olivetic acid (OA).

82. A method according to claim 80 or 81, wherein the extraction process of step (ii) comprises one or more or all of the following: (a) grinding of the dried material; (b) solvent (e.g., ethanol, hydrocarbon) or supercritical CO2 extraction; (c) winterization and filtration; (d) drying; (e) distillation; and / or (f) chromatography.

83. A method for producing an extract enriched in cannabigerolic acid (CBGA), the method comprising the steps of: (i) obtaining a plant or part thereof according to any one of claims 2 to 6 or 8 to 11 or 13 to 18 or 20 or 22 or 24 or a plastid according to any one of claims 1 or 4 to 8 or 12 or 14 to 21 or 23 or 25; (ii) treating the plant, part or plastid thereof of step (i); (iii) centrifuging the treated plant, part or plastid from step (ii) to obtain an extract; (iv) filtering the extract obtained by step (iii); (v) drying the filtered extract of step (iv) and resuspending it in a buffer solution; (vi) purifying the extract of step (v) using a solid phase extraction column; and and (vii) recovering a CBGA-enriched extract, thereby producing said CBGA-enriched extract.

84. A method for producing a pharmaceutical composition, the method comprising: (i) obtaining the extract of any one of claims 80 to 83 or the cannabinoid of any one of claims 48 to 49 or 53 to 55; as well as (ii) formulating the extract or cannabinoid with one or more pharmaceutically acceptable carriers, thereby producing a pharmaceutical composition.

85. A pharmaceutical composition obtained from claim 84.

86. A method of treating and / or preventing a condition or disease responsive to cannabinoid treatment in a subject, the method comprising administering to the subject a therapeutically effective amount of a cannabinoid according to any one of claims 48 to 49, 53 to 55, an extract according to any one of claims 80 to 83 or a pharmaceutical composition according to claim 85, thereby treating and / or preventing a condition or disease responsive to cannabinoid treatment.

87. Use of a therapeutically effective amount of a cannabinoid as claimed in any one of claims 48 to 49, 53 to 55, an extract as claimed in any one of claims 80 to 83 or a pharmaceutical composition as claimed in claim 85 for the preparation of a medicament for the treatment of a condition or disease responsive to cannabinoid treatment.

88. The method of claim 86 or the use of claim 87, wherein the condition or disease is selected from the group consisting of chronic pain, neuropathic pain, cancer, nausea and / or vomiting associated with cancer chemotherapy, loss of appetite, multiple sclerosis, spasticity associated with multiple sclerosis or spinal cord injury epilepsy, Parkinson's disease, anorexia and / or weight loss, irritable bowel syndrome, Tourette syndrome, amyotrophic lateral sclerosis, Huntington's disease, dystonia, dementia, glaucoma, traumatic brain injury and / or intracranial hemorrhage, addiction, anxiety, depression, sleep disorders, post-traumatic stress disorder, microbial infections including methicillin-resistant Staphylococcus aureus (MRSA) and biofilm-producing organisms, dermatitis, acne, schizophrenia and other psychiatric disorders.

89. Use of a plant or part thereof according to any one of claims 2 to 6 or 8 to 11 or 13 to 18 or 22 or 24, or a plastid according to any one of claims 1 or 4 to 8 or 12 or 14 to 21 or 23 or 25, for the preparation of an industrial product, preferably a cannabinoid, more preferably cannabigerolic acid (CBGA) and / or cannabidiol (CBG).

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