Application US20190316144

IN VIVO GENERATION OF WATER-SOLUBLE CANNABINOIDS IN PLANT CELL SUSPENSION CULTURES
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US20190316144

1. An in vivo method of producing water-soluble cannabinoids comprising the steps of: (6) (5)

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.

2. 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. (0)

3. 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. (0)

4. 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. (0)

5. The method of claim 1, wherein said nucleotide sequence encoding a heterologous cytochrome P450 is identified as SEQ ID NO. 1. (0)

6. The method of claim 1, wherein said nucleotide sequence encoding a heterologous P450 oxidoreductase is identified as SEQ ID NO. 3. (0)

7. 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. (1) (0)

9. An in vivo method for the generation of water-soluble cannabinoids comprising the steps of: (5) (3)

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.

10. 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. (0)

11. 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. (0)

12. 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. (1) (0)

14. 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. (1) (0)

16. 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. (2) (0)

20. A non-genetically modified in vivo method of producing water-soluble cannabinoids comprising the steps of: (1) (4)

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.

21. The method of claim 20, wherein said plant cell suspension cell culture from a non-cannabinoid producing plant comprises a tobacco cell suspension culture. (1) (0)

23. A method of producing water-soluble cannabinoids comprising the steps of: (4) (5)

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.

24. 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. (0)

25. 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 (1) (0)

27. 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. (1) (0)

29. 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. (1) (0)

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Application US20190316144

IN VIVO GENERATION OF WATER-SOLUBLE CANNABINOIDS IN PLANT CELL SUSPENSION CULTURES

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