US10378020B2 – August 13, 2019 – High level in vivo biosynthesis and isolation of water-soluble cannabinoids in plant systems

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Inventors :

Richard T. Sayre; Elton Carvalho Gonçalves; Tawanda Zidenga

Owner :

TRAIT BIOSCIENCES, INC.

Application Number :

US16110728

Document Number :

US10378020B2

Priority Date :

August 23, 2018

Filing Date :

August 23, 2018

Date of Grant/ Publication :

August 13, 2019

Class :

C12N15 / 82; C12N9 / 08

Abstract

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 a whole plant, or a cell suspension culture system.

Claim(s)

1. A method for the production of water-soluble cannabinoids in a Cannabis cell cytosol: genetically modifying a cultured Cannabis cell to transiently express: i. a nucleotide sequence encoding at least one cannabinoid synthase having its trichome targeting signal disrupted or removed; ii. a nucleotide sequence encoding a heterologous cytochrome P450 having hydroxylation or oxidation activity toward a cannabinoid to generate a hydroxylated cannabinoid or a cannabinoid carboxylic acid; iii. a nucleotide sequence encoding a heterologous P450 oxidoreductase that facilitates electron transfer from nicotinamide adenine dinucleotide phosphate (NADPH) to the cytochrome P450; and iv. a nucleotide sequence encoding a heterologous glycosyltransferase having glycosylation activity towards said cannabinoids, said hydroxylated cannabinoids, and said cannabinoid carboxylic acids.;
13. A method for producing water-soluble cannabinoids in the cytosol of a plant comprising the steps: genetically modifying at least one Cannabis plant through Agrobacterium -mediated transformation to transiently express: i. a nucleotide sequence encoding at least one cannabinoid synthase having its trichome targeting signal disrupted or removed; ii. a nucleotide sequence encoding a heterologous cytochrome P450 having hydroxylation or oxidation activity towards a cannabinoid to generate a hydroxylated cannabinoid, or a cannabinoid carboxylic acid; iii. a nucleotide sequence encoding a heterologous P450 oxidoreductase that facilitates electron transfer from NADPH to the cytochrome P450; and iv. a nucleotide sequence encoding a heterologous glycosyltransferase having glycosylation activity towards said cannabinoids, said hydroxylated cannabinoids, and said cannabinoid carboxylic acids.;
16. A method for producing water-soluble cannabinoids in a Cannabis plant cell comprising the steps: genetically modifying at least one Cannabis plant cell through Agrobacterium -mediated transformation to transiently express: i. a nucleotide sequence encoding at least one cannabinoid synthase having its trichome targeting signal disrupted or removed; ii. a nucleotide sequence encoding a heterologous cytochrome P450 having hydroxylation or oxidation activity towards a cannabinoid to generate a hydroxylated cannabinoid, or a cannabinoid carboxylic acid; iii. a nucleotide sequence encoding a heterologous P450 oxidoreductase that facilitates electron transfer from NADPH to the cytochrome P450; and iv. a nucleotide sequence encoding a heterologous glycosyltransferase having glycosylation activity towards said cannabinoids, said hydroxylated cannabinoids, and said cannabinoid carboxylic acids; v. a nucleotide sequence encoding an endogenous or heterologous myb transcription factor wherein said heterologous or heterologous myb transcription factor increases cannabinoid production by enhancing metabolite flux through the cannabinoid biosynthetic pathway; and vi. a nucleotide sequence encoding a heterologous catalase having reduction activity towards hydrogen peroxide generated by cannabinoid biosynthesis.;
17. A method for producing water-soluble cannabinoids in a Cannabis cell comprising the steps: genetically modifying at least one Cannabis cell to transiently express: i. a nucleotide sequence encoding at least one cannabinoid synthase having its trichome targeting signal disrupted or removed; ii. a nucleotide sequence encoding a heterologous cytochrome P450 having hydroxylation or oxidation activity towards a cannabinoid to generate a hydroxylated cannabinoid, or a cannabinoid carboxylic acid; iii. a nucleotide sequence encoding a heterologous P450 oxidoreductase that facilitates electron transfer from NADPH to the cytochrome P450; and iv. a nucleotide sequence encoding a heterologous glycosyltransferase having glycosylation activity towards said cannabinoids, said hydroxylated cannabinoids, and said cannabinoid carboxylic acids; v. a nucleotide sequence encoding an endogenous or heterologous myb transcription factor wherein said heterologous or heterologous myb transcription factor increases cannabinoid production by enhancing metabolite flux through the cannabinoid biosynthetic pathway; and vi. a nucleotide sequence encoding a heterologous catalase having reduction activity towards hydrogen peroxide generated by cannabinoid biosynthesis.

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