High level in vivo biosynthesis and isolation of water-soluble cannabinoids in stably transformed plant systems
The inventive technology relates to systems and methods for enhanced in vivo production, accumulation and modification of cannabinoids. In one embodiment, the invention may include systems and methods for enhanced in vivo biosynthesis of chemically-modified water-soluble cannabinoids in stably transformed whole plant, or a cell suspension culture system.
1. A method for the production of water-soluble cannabinoids in the cytosol of a Cannabis cell comprising expressing in the Cannabis sativa cell a nucleotide sequence, operably linked to a promoter, encoding:
i. a cannabinoid synthase endogenous to said Cannabis sativa cell, having its trichome targeting signal disrupted or removed, wherein the cannabinoid synthase is a tetrahydrocannabinolic acid synthase (THCA synthase) polypeptide having the amino acid sequence of SEQ ID NO: 46, or an amino acid sequence having 98% sequence identity with SEQ ID NO: 46, and a cannabidiolic acid synthase (CBDA synthase) polypeptide having the amino acid sequence of SEQ ID NO: 23, or an amino acid sequence having 98% sequence identity with SEQ ID NO: 23;
ii. a heterologous glycosyltransferase, selected from the group consisting of: SEQ ID NO: 7, 27, 29, 31, 33, 35, 37, and 39;
iii. a myb transcription factor having SEQ ID NO: 11, or wherein the nucleotide sequence encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ 12 and 43-45;
iv. a heterologous catalase enzyme;
v. a heterologous cytochrome P450 according to SEQ ID NO: 1; and
vi. a heterologous P450 oxidoreductase according to SEQ ID NO: 3.
2. The method of claim 1 , wherein said catalase is selected from the group consisting of: SEQ ID NO: 13 and 15.
3. A method for producing water-soluble cannabinoids comprising expressing in a Cannabis sativa plant cell:
i. a nucleotide sequence, operably linked to a promoter, encoding a cannabinoid synthase from Cannabis sativa , having its trichome targeting signal disrupted or removed, wherein the cannabinoid synthase is a THCA synthase polypeptide having the amino acid sequence of SEQ ID NO: 46, or an amino acid sequence having 98% sequence identity with SEQ ID NO: 46, and a CBDA synthase polypeptide having the amino acid sequence of SEQ ID NO: 23, or an amino acid sequence having 98% sequence identity with SEQ ID NO: 23;
ii. a nucleotide sequence, operably linked to a promoter, encoding a heterologous glycosyltransferase selected from the group consisting of: SEQ ID NO: 7, 27, 29, 31, 33, 35, 37 and 39;
iii. a nucleotide sequence, operably linked to a promoter, encoding a myb transcription factor having SEQ ID NO: 11, or wherein the nucleotide sequence encodes a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:12 and 43-45; and
iv. a nucleotide sequence, operably linked to a promoter, encoding a heterologous catalase enzyme; and
wherein the cannabinoid synthase synthesizes a cannabinoid in the cytosol of said Cannabis sativa cell selected from the group consisting of: cannabidiol (CBD), cannabidiolic acid (CBDA), tetrahydrocannabinolic acid (THCA) and tetrahydrocannabinol (THC), and wherein the cannabinoid is further glycosylated in the cytosol of said Cannabis sativa cell.
4. A method for the production of water-soluble cannabinoids comprising expressing in a Cannabis cell:
i. a nucleotide sequence encoding a CBDA synthase polypeptide from Cannabis sativa having the amino acid sequence of SEQ ID NO: 23, or an amino acid sequence having 98% sequence identity with SEQ ID NO: 23, having its trichome targeting signal disrupted or removed;
ii. a nucleotide sequence, operably linked to a promoter, encoding a heterologous glycosyltransferase selected from the group consisting of: SEQ ID NO: 29 and 31;
iii. a nucleotide sequence, operably linked to a promoter, encoding a myb transcription factor having SEQ ID NO: 11, or wherein the nucleotide sequence encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 12 and 43-45; and
iv. a nucleotide sequence, operably linked to a promoter, encoding a heterologous catalase enzyme;
wherein said CBDA synthase synthesizes CBDA in the cytosol of said Cannabis cell, and wherein said CBDA is further glycosylated in the cytosol of said Cannabis cell forming a CBDA glycoside or a CBD glycoside.
5. The method of claim 4 , further comprising the step of expressing a nucleotide sequence according to SEQ ID NO: 1, operably linked to a promoter, encoding a heterologous cytochrome P450.
6. The method of claim 4 , further comprising the step of expressing a nucleotide sequence according to SEQ ID NO: 3, operably linked to a promoter, encoding a heterologous P450 oxidoreductase.
7. The method of claim 4 , wherein said catalase enzyme is selected from the group consisting of: the nucleotide sequence of SEQ ID NO: 13 and 15.