# US 2019/0316144 A1 · In vivo generation of water-soluble cannabinoids in plant cell suspension cultures > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2019/0316144 A1 - Document ID: US20190316144A1 - Status: Published application - Kind: A1 - Application number: 16/426,059 - Filed: 2019-05-30 - Published: 2019-10-17 - Earliest claimed: 2017-07-11 - Assignees: Trait Biosciences, Inc. - Applicants: Trait Biosciences Inc. - Inventors: Richard T. Sayre; Elton Carvalho Gonçalves; Tawanda Zidenga; Stephanie Willette - Web page: https://patentgrep.com/patent/US20190316144A1 ## CPC - C12N 15/8257: {for the production of primary gene products, e.g. pharmaceutical products, interferon} - A01H 4/00: Plant reproduction by tissue culture techniques {; Tissue culture techniques therefor} - A61K 31/658: {o-phenolic cannabinoids, e.g. cannabidiol, cannabigerolic acid, cannabichromene or tetrahydrocannabinol} - C12N 9/0042: {NADPH-cytochrome P450 reductase (1.6.2.4)} - C12N 9/1051: {Hexosyltransferases (2.4.1)} - C12N 15/8201: {Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation} - C12N 15/8243: {involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine} - C12Y 106/02004: NADPH-hemoprotein reductase (1.6.2.4), i.e. NADP-cytochrome P450-reductase - C12Y 204/01: Hexosyltransferases (2.4.1) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - A01H: NEW PLANTS OR {NON-TRANSGENIC} PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) - C12Y: ENZYMES ## Abstract The present invention includes systems, methods and compositions for the in vivo bioconversion of water-soluble cannabinoids in plant cell suspension cultures. ## Claims ### Claim 1 An in vivo method of producing water-soluble cannabinoids comprising the steps of: genetically modifying a tobacco cell to express: a nucleotide sequence encoding a heterologous cytochrome P450 operably linked to a promoter having hydroxylation or oxidation activity toward one or more cannabinoids to generate a hydroxylated cannabinoid or a cannabinoid carboxylic acid; a nucleotide sequence encoding a heterologous P450 oxidoreductase operably linked to a promoter that facilitates electron transfer from nicotinamide adenine dinucleotide phosphate (NADPH) to the cytochrome P450; wherein said tobacco cell expresses at least one endogenous glycosyltransferase protein having glycosylation activity towards one or more cannabinoids. establishing a suspension cell culture of said genetically modified tobacco cells; introducing one or more cannabinoids to said suspension cell culture of said genetically modified tobacco cells; chemically converting said one or more cannabinoids into one or more water soluble glycosylated cannabinoids; and isolating said water soluble glycosylated cannabinoids. ### Claim 2 (depends on 1) The method of claim 1, wherein said endogenous glycosyltransferase protein comprises an endogenous glycosyltransferase protein selected from the group consisting of: NtGT3; NtGT4; and NtGT5, and a homolog or ortholog of NtGT3, NtGT4, and NtGT5 having glycosylation activity towards one or more cannabinoids. ### Claim 3 (depends on 1) The method of claim 1, wherein said endogenous glycosyltransferase comprises an endogenous glycosyltransferase protein selected from the group of amino acid sequences consisting of: SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 36. ### Claim 4 (depends on 1) The method of claim 1, further comprising the step of genetically modifying said tobacco cell to overexpress a nucleotide sequence encoding one or more endogenous glycosyltransferases having glycosylation activity towards one or more cannabinoids, wherein said nucleotide sequence is operably linked to a promoter. ### Claim 5 (depends on 1) The method of claim 1, wherein said nucleotide sequence encoding a heterologous cytochrome P450 is identified as SEQ ID NO. 1. ### Claim 6 (depends on 1) The method of claim 1, wherein said nucleotide sequence encoding a heterologous P450 oxidoreductase is identified as SEQ ID NO. 3. ### Claim 7 (depends on 1) The method of claim 1, further comprising the step of genetically modifying said tobacco cell to express a nucleotide sequence, operably linked to a promoter, encoding a heterologous ABC transporter that facilitates the transport of said glycosylated cannabinoids out of said genetically modified tobacco cell. ### Claim 8 (depends on 7) The method of claim 7, wherein said nucleotide sequence encoding a heterologous ABC transporter is selected from the group consisting of: the nucleotide sequence of SEQ ID NO. 9, and the nucleotide sequence of SEQ ID NO. 67. ### Claim 9 An in vivo method for the generation of water-soluble cannabinoids comprising the steps of: establishing a suspension cell culture of tobacco cells that expresses at least one endogenous glycosyltransferase protein having glycosylation activity towards one or more cannabinoids; introducing one or more cannabinoids to said suspension cell culture of tobacco cells; and chemically converting said one or more cannabinoids into one or more water soluble glycosylated cannabinoids through the action of said at least one endogenous glycosyltransferase. ### Claim 10 (depends on 9) The method of claim 9, wherein said endogenous glycosyltransferase protein comprises an endogenous glycosyltransferase protein selected from the group consisting of: NtGT3; NtGT4; and NtGT5, and a homolog or ortholog of NtGT3, NtGT4, and NtGT5 having glycosylation activity towards one or more cannabinoids. ### Claim 11 (depends on 9) The method of claim 9, wherein said endogenous glycosyltransferase protein comprises an endogenous glycosyltransferase protein selected from the group of amino acid sequences consisting of: SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 36. ### Claim 12 (depends on 9) The method of claim 9, further comprising the step of genetically modifying said tobacco cells in said suspension culture to overexpress one or more endogenous glycosyltransferase proteins. ### Claim 13 (depends on 12) The method of claim 12, wherein said endogenous glycosyltransferase protein comprises an endogenous glycosyltransferase protein selected from the group consisting of: NtGT3; NtGT4; and NtGT5, and a homolog or ortholog of NtGT3, NtGT4, and NtGT5 having glycosylation activity towards one or more cannabinoids. ### Claim 14 (depends on 9) The method of claim 9, further comprising the step of genetically modifying said tobacco cells in said suspension culture to express a heterologous ABC transporter protein to facilitate the transport of said glycosylated cannabinoids out of said tobacco cell. ### Claim 15 (depends on 14) The method of claim 14, wherein said multi-drug ABC transporter protein is selected from the group consisting of: SEQ ID NO. 10, and SEQ ID NO. 68. ### Claim 16 (depends on 9) The method of claim 9, and further comprising the step of genetically modifying said tobacco cells in said suspension culture to express a heterologous cytochrome P450 protein wherein said heterologous cytochrome P450 hydroxylates said one or more cannabinoids to form a hydroxylated cannabinoid and/or oxidizes a said hydroxylated cannabinoid to form a cannabinoid carboxylic acid. ### Claim 17 (depends on 16) The method of claim 16, wherein said heterologous cytochrome P450 protein is identified as SEQ ID NO. 2. ### Claim 18 (depends on 16) The method of claim 16, further comprising the step of genetically modifying said tobacco cells in said suspension culture to express a heterologous P450 oxidoreductase protein wherein said heterologous P450 oxidoreductase protein facilitates electron transfer from a nicotinamide adenine dinucleotide phosphate (NADPH) to said cytochrome P450. ### Claim 19 (depends on 18) The method of claim 18, wherein said heterologous P450 oxidoreductase protein is identified as SEQ ID NO. 4. ### Claim 20 A non-genetically modified in vivo method of producing water-soluble cannabinoids comprising the steps of: establishing a plant cell suspension cell culture from a non-cannabinoid producing plant that expresses at least one endogenous glycosyltransferase having glycosylation activity towards one or more cannabinoids; introducing one or more cannabinoids to said plant cell suspension culture; chemically converting said one or more cannabinoids into one or more water soluble glycosylated cannabinoids through the action of said at least one endogenous glycosyltransferase; isolating said glycosylated cannabinoids from said plant cell suspension culture. ### Claim 21 (depends on 20) The method of claim 20, wherein said plant cell suspension cell culture from a non-cannabinoid producing plant comprises a tobacco cell suspension culture. ### Claim 22 (depends on 21) The method of claim 21, wherein said endogenous glycosyltransferase comprises an endogenous glycosyltransferase selected from the group consisting of: NtGT3; NtGT4; and NtGT5, and a homolog or ortholog of NtGT3, NtGT4, and NtGT5 having glycosylation activity towards one or more cannabinoids. ### Claim 23 A method of producing water-soluble cannabinoids comprising the steps of: genetically modifying a plant cell to express a nucleotide sequence operably linked to a promoter encoding a heterologous glycosyltransferase from a tobacco plant having glycosylation activity towards one or more cannabinoids; establishing a suspension cell culture of said genetically modified plant cells; introducing one or more cannabinoids to said suspension cell culture of said genetically modified plant cells; chemically converting said one or more cannabinoids into one or more water soluble glycosylated cannabinoids; and isolating said glycosylated cannabinoids from said plant cell suspension culture. ### Claim 24 (depends on 23) The method of claim 23, wherein said heterologous glycosyltransferase from a tobacco plant comprises a heterologous glycosyltransferase from a tobacco plant selected from the group consisting of: NtGT3; NtGT4; and NtGT5, and a homolog or ortholog of NtGT3, NtGT4, and NtGT5 having glycosylation activity towards one or more cannabinoids. ### Claim 25 (depends on 23) The method of claim 23, further comprising the step of genetically modifying said plant cells to express a nucleotide sequence encoding a heterologous ABC transporter that facilitates the transport of said glycosylated cannabinoids out of said genetically modifying plant cell, wherein said nucleotide sequence is operably linked to a promoter ### Claim 26 (depends on 25) The method of claim 25, wherein said nucleotide sequence encoding a heterologous ABC transporter is selected from the group consisting of: the nucleotide sequence of SEQ ID NO. 9, and the nucleotide sequence of SEQ ID NO. 67. ### Claim 27 (depends on 23) The method of claim 23, further comprising the step of genetically modifying said plant cells to express a heterologous cytochrome P450 protein wherein said heterologous cytochrome P450 hydroxylates said one or more cannabinoids to form a hydroxylated cannabinoid and/or oxidizes a said hydroxylated cannabinoid to form a cannabinoid carboxylic acid. ### Claim 28 (depends on 27) The method of claim 27, wherein said heterologous cytochrome P450 protein is identified as SEQ ID NO. 2. ### Claim 29 (depends on 23) The method of claim 23, further comprising the step of genetically modifying said plant cells to express a heterologous P450 oxidoreductase protein wherein said heterologous P450 oxidoreductase protein facilitates electron transfer from a nicotinamide adenine dinucleotide phosphate (NADPH) to said cytochrome P450. ### Claim 30 (depends on 29) The method of claim 29 wherein said heterologous P450 oxidoreductase protein is identified as SEQ ID NO. 4.