WO2024259172A1 - Synthèse améliorée de composés glycosidiques cannabinoïdes - Google Patents

Synthèse améliorée de composés glycosidiques cannabinoïdes Download PDF

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Publication number
WO2024259172A1
WO2024259172A1 PCT/US2024/033912 US2024033912W WO2024259172A1 WO 2024259172 A1 WO2024259172 A1 WO 2024259172A1 US 2024033912 W US2024033912 W US 2024033912W WO 2024259172 A1 WO2024259172 A1 WO 2024259172A1
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cannabinoid
cell culture
ugt
glycoside
udp
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Syed Ammar HUSSAIN
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Trait Biosciences Inc
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Trait Biosciences Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)

Definitions

  • the present invention relates generally to the identification novel UDP-glucosyltransferase enzymes having specific activity towards cannabinoid compounds.
  • the present invention further relates generally to the use of novel UGT enzymes having specific activity towards cannabinoid compounds to generate water-soluble cannabinoid glycoside compounds.
  • BACKGROUND Cannabinoids are a class of specialized compounds synthesized by Cannabis. They are formed by condensation of terpene and phenol precursors. They include these more abundant forms: ⁇ 9 -tetrahydrocannabinol (THC), cannabidiol (CBD), cannabichromene (CBC), and cannabigerol (CBG).
  • cannabinol is formed from THC as a degradation product and can be detected in some plant strains.
  • THC, CBD, CBC, and CBG occur together in different ratios in the various plant strains.
  • cannabinoids are generally lipophilic, nitrogen-free, mostly phenolic compounds and are derived biogenetically from a monoterpene and phenol, the acid cannabinoids from a monoterpene and phenol carboxylic acid and have a C21 base.
  • Cannabinoids also find their corresponding carboxylic acids in plant products. In general, the carboxylic acids have the function of a biosynthetic precursor.
  • the tetrahydrocannabinols ⁇ 9 – and ⁇ 8 -THC arise in vivo from the THC carboxylic acids by decarboxylation and likewise, CBD from the associated cannabidiolic acid.
  • cannabinoids are hydrophobic small molecules and, as a result, are highly insoluble. Due to this insolubility, cannabinoids such as THC and CBD may need to be efficiently solubilized to facilitate transport, storage, and adsorption through certain tissues and organs.
  • cannabinoids in the human body goes through the classic two-phases detoxification process of oxidation followed by glucuronidation – which is a form of glycosylation involving the addition of a sugar from UDP-Glucuronic Acid to a cannabinoid.
  • glucuronidation – is a form of glycosylation involving the addition of a sugar from UDP-Glucuronic Acid to a cannabinoid.
  • the chemical structures of UDP-glucuronic acid and UDP-glucose are similar.
  • cannabinoids may be subject to cytochrome P450 oxidation and subsequent UDP-glucuronosyltransferase dependent glucuronidation in the body after consumption.
  • cannabinoids may be glycosylated to form water-soluble glycoside compounds.
  • water- soluble cannabinoid glycoside may include one or more sugar moieties, and preferably 1-3 sugar moieties, also referred to a glycosylation sites.
  • the stability of nanoemulsions is inherently unstable, and may be disturbed by slight fluctuations in temperature and pH and is further subject to the “oswald ripening effect” or ORE.
  • ORE describes the process whereby molecules on the surface of particles are more energetically unstable than those within. Therefore, the unstable surface molecules often go into solution shrinking the particle over time and increasing the number of free molecules in solution. When the solution is supersaturated with the molecules of the shrinking particles, those free molecules will redeposit on the larger particles. Thus, small particles decrease in size until they disappear, and large particles grow even larger. This shrinking and growing of particles will result in a larger mean diameter of a particle size distribution (PSD). Over time, this causes emulsion instability and eventually phase separation.
  • PSD particle size distribution
  • nanoemulsions may not be safe for human consumption.
  • nanoemulsions were first developed as a method to deliver small quantities of pharmaceutical compounds having poor solubility.
  • the ability to “hide” a compound, such as a cannabinoid, in a nanoemulsion may allow the cannabinoid to be delivered to parts of the body where it was previously prevented from entering, as well as accumulating in tissues and organs where cannabinoids and nanoparticles would not typically be found.
  • such nanoemulsions, as well as other water-compatible strategies do not address one of the major- shortcomings of cannabinoid-infused commercial consumables, namely the strong unpleasant smell and taste.
  • UDP-UGTs or UGTs novel UDP- glucosyltransferase
  • the present invention includes the identification of novel UGTs according to the nucleotide sequences identified as SEQ ID NOs.1, and UGTs having at least 90% sequence identity with SEQ ID NOs. 1, that have glycosylation activity towards one or more cannabinoid compounds, and preferably CBD.
  • the UGT of the invention cany generate a cannabinoid, and preferably CBD having between 1-4 UDP sugar moieties, referred to generally as CBD-1G, CBD-2G, CBD-3G, and CBD- 4G.
  • the present invention includes the identification of novel UGTs according to the amino acid sequences identified as SEQ ID NOs.
  • the UGT of the invention cany generate a cannabinoid, and preferably CBD having between 1-4 UDP sugar moieties, referred to generally as CBD-1G, CBD-2G, CBD-3G, and CBD-4G.
  • the present invention includes the identification of novel UGTs according to the amino acid sequences identified as SEQ ID NOs.4, 6, 8 and 10, and UGTs having at least 90% sequence identity with SEQ ID NOs.4, 6, 8 and 10, that have glycosylation activity towards one or more cannabinoid compounds, and preferably CBN.
  • the UGT of the invention cany generate a cannabinoid, and preferably CBD having at least one UDP sugar moiety, referred to generally as CBN-1G.
  • the present invention includes the identification of novel UGTs according to the nucleotide sequences identified as SEQ ID NOs.3, 5, 7, and 9, and UGTs having at least 90% sequence identity with SEQ ID NOs.3, 5, 7, and 9, that have glycosylation activity towards one or more cannabinoid compounds, and preferably CBN.
  • the UGT of the invention cany generate a cannabinoid, and preferably CBD having at least one UDP sugar moieties, referred to generally as CBN-1G.
  • One aspect of the present invention further relates generally to the use of novel UGT enzymes having specific activity towards one or more cannabinoid compounds to generate water- soluble cannabinoid glycoside compounds in in vitro, ex vivo, and in vivo systems.
  • the present invention use of novel UGT enzymes according to the amino acid sequences identified as SEQ ID NO.1, and UGTs having at least 90% sequence identity with SEQ ID NO. 1, that have glycosylation activity towards one or more cannabinoid compounds, and preferably CBD, in in vitro, ex vivo, and in vivo systems.
  • an in vivo system may include a whole organism system, such as a plant, or cell culture, such as a plant cell culture, an algal cell culture, a fungi cell culture, or a microorganism cell culture, such as a bacterial or yeast cell culture.
  • Another aspect of the current inventive technology includes the isolated amino acid sequences encoding one or more UGTs that have glycosylation activity towards one or more cannabinoid compounds according to SEQ ID NOs. 1, and amino acid sequence having at least 90% sequence identity with SEQ ID NO.1.
  • Another aspect of the current inventive technology includes a nucleotide sequence encoding one or more UGTs that have glycosylation activity towards one or more cannabinoid compounds according to SEQ ID NOs.
  • Another aspect of the current inventive technology includes an expression vector having a nucleotide sequence encoding one or more UGTs that have glycosylation activity towards one or more cannabinoid compounds according to SEQ ID NOs.1, and a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity with SEQ ID NOs.2, operably linked to a promoter.
  • Another aspect of the current inventive technology includes one or more organisms, such as a plant, plant cell, bacteria, algae, fungi, or yeast cell, transformed by an expression vector having a nucleotide sequence encoding one or more UGTs that have glycosylation activity towards one or more cannabinoid compounds according to SEQ ID NOs. 1, and a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity with SEQ ID NOs. 2, operably linked to a promoter.
  • One aspect of the current inventive technology includes improved systems and methods for the bioconversion of cannabinoid compounds into water-soluble cannabinoid glycosides, or water-soluble cannabinoid glycosides in a bacterial, yeast, or plant cell culture system.
  • a preferred plant cell culture system may include a Cannabis suspension cell culture, or a tobacco plant cell culture. Additional aspects of the invention may become evident based on the specification and figures presented below.
  • BRIEF DESCRIPTION OF THE FIGURES Figure 1: shows exemplary chemical structures for cannabidiol (CBD) glycosides having between 1-4 UDP-glucose moieties, specifically CBD-1G, CBD-2G, CBD-3G, and CBD-4G generated by GT3 (SEQ ID NO.1, 2)
  • Figure 2 shows HPLC analysis of CBD glycosides having between 1-4 UDP-glucose moieties, specifically CBD-1G, CBD-2G, CBD-3G, and CBD-4G generated by GT3 (SEQ ID NO. 1, 2) from a CBD substrate.
  • CBD cannabidiol
  • Figure 3 shows bioconversion of CBD into a CBD glycoside having a single UDP sugar moiety (CBD-1G) by UGTs 1-12.
  • Figure 4 shows bioconversion of CBD into a CBD glycoside having two UDP sugar moiety (CBD-2G) by UGTs 1-12, where GT3 demonstrated highest production of CBD-2G.
  • Figure 5 shows bioconversion of CBD into a CBD glycoside having three UDP sugar moiety (CBD-3G) by UGTs 1-12, where GT3 demonstrated highest production of CBD-3G.
  • Figure 6 shows bioconversion of CBD into a CBD glycoside having four UDP sugar moiety (CBD-4G) by UGTs 1-12, where GT3 demonstrated highest production of CBD-4G.
  • Figure 7A-E shows HPLC analysis of CBN glycosides generated having 1 UDP- glucose moiety (CBN1) from a CBN substrate and a UDP-glucose catalyzed by GT4 (SEQ ID NO. 3, 4); (B) shows HPLC analysis of CBN glycosides generated having 1 UDP-glucose moiety (CBN1) from a CBN substrate and a UDP-glucose catalyzed by GT5 (SEQ ID NO. 5, 6); (C) shows HPLC analysis of CBN glycosides generated having 1 UDP-glucose moiety (CBN1) from a CBN substrate and a UDP-glucose catalyzed by GT6 (SEQ ID NO.
  • One embodiment of the present invention relates generally to the identification novel UDP- glucosyltransferases (UDP-UGTs or UGTs) enzymes having glycosylation activity towards one or more cannabinoid compounds.
  • the present invention includes the identification of novel UGTs identified as GT3, that catalyzes the bioconversion of a CBD substrate and UDP-glucose to a CBD glycoside having between 1-4 UDP sugar moieties, referred to generally as CBD-1G, CBD-2G, CBD-3G, and CBD-4G.
  • UDP-UGTs or UGTs novel UDP-glucosyltransferases
  • the present invention includes the identification of novel UGTs according to SEQ ID NOs. 1, and UGTs having at least 90% sequence identity with SEQ ID NOs.1, that catalyzes the bioconversion of a CBD substrate and UDP-glucose to a CBD glycoside having between 1-4 UDP sugar moieties, referred to generally as CBD-1G, CBD-2G, CBD-3G, and CBD-4G.
  • Another embodiment of the present invention relates generally to the identification novel UDP-glucosyltransferases (UDP-UGTs or UGTs) enzymes having glycosylation activity towards one or more cannabinoid compounds.
  • the present invention includes the identification of novel UGTs according to SEQ ID NOs.2, or a fragment thereof, and UGTs having at least 90% sequence identity with SEQ ID NOs.2, that catalyzes the bioconversion of a CBD substrate and UDP-glucose to a CBD glycoside having between 1-4 UDP sugar moieties, referred to generally as CBD-1G, CBD-2G, CBD-3G, and CBD-4G.
  • the present invention includes the identification of novel UGTs identified as GT4, GT5, GT6, and GT8, that catalyzes the bioconversion of a CBN substrate and UDP-glucose to a CBD glycoside having at least one UDP sugar moiety, referred to generally as CBN-1G.
  • Another embodiment of the present invention relates generally to the identification novel UDP-glucosyltransferases (UDP-UGTs or UGTs) enzymes having glycosylation activity towards one or more cannabinoid compounds.
  • the present invention includes the identification of novel UGTs according to SEQ ID NOs.4, 6, 8, and 10, and UGTs having at least 90% sequence identity with SEQ ID NOs.
  • CBN-1G that catalyzes the bioconversion of a CBN substrate and UDP-glucose to a CBD glycoside having at least one UDP sugar moiety, referred to generally as CBN-1G.
  • Another embodiment of the present invention relates generally to the identification novel UDP-glucosyltransferases (UDP-UGTs or UGTs) enzymes having glycosylation activity towards one or more cannabinoid compounds.
  • the present invention includes the identification of novel UGTs according to SEQ ID NOs.4, 6, 8 and 10, or a fragment thereof, and UGTs having at least 90% sequence identity with SEQ ID NOs.
  • the present invention relates generally to the use of novel UGT enzymes having specific activity towards one or more cannabinoid compounds to generate water- soluble cannabinoid glycoside compounds in in vitro, ex vivo, and in vivo systems.
  • the present invention includes a novel UGT enzyme according to the amino acid sequences identified as SEQ ID NOs. 2, or a fragment thereof, or UGTs having at least 90% sequence identity with SEQ ID NOs.
  • an in vivo system may include a whole organism system, such as a plant, or cell culture, such as a plant cell culture, an algal cell culture, a fungi cell culture, or a microorganism cell culture, such as a bacterial or yeast cell culture.
  • a whole organism system such as a plant, or cell culture, such as a plant cell culture, an algal cell culture, a fungi cell culture, or a microorganism cell culture, such as a bacterial or yeast cell culture.
  • One embodiment of the present invention further relates generally to novel methods of generating water-soluble cannabinoid glycoside compounds.
  • the invention novel methods of generating water-soluble CBD-glycoside comprising the step of introducing a CBD substrate to a UGT enzyme according to the amino acid sequences identified as SEQ ID NOs. 2, or a fragment thereof, or UGTs having at least 90% sequence identity with SEQ ID NOs. 2, in in vitro, ex vivo, and in vivo systems.
  • the invention novel methods of generating water-soluble CBN-glycoside comprising the step of introducing a CBN substrate to a UGT enzyme according to the amino acid sequences identified as SEQ ID NOs.
  • an in vivo system may include a whole organism system, such as a plant, or cell culture, such as a plant cell culture, an algal cell culture, a fungi cell culture, or a microorganism cell culture, such as a bacterial or yeast cell culture.
  • an ex vivo system may include a bioreactor system.
  • an in vitro system may include chemical conversion of cannabinoids into water-soluble cannabinoid glycoside compounds.
  • a plant, a plant cell, an algal cell, a fungi, a bacteria, or a yeast cell may be genetically modified to express a nucleotide sequence encoding one or more UGTs that have glycosylation activity towards one or more cannabinoid compounds, and preferably a UGT selected from the group of nucleotide sequences selected from the amino acid sequence according to SEQ ID NOs.2, 4, 6, 8 and 10, or a fragment thereof, and/or an amino acid sequence having at least 90% sequence identity with SEQ ID NOs.2, 4, 6, 8 and 10.
  • a plant, a plant cell, an algal cell, a fungi, a bacteria, or a yeast cell may be genetically modified to express a nucleotide sequence encoding one or more UGTs that have glycosylation activity towards one or more cannabinoid compounds, and preferably a UGT selected from the group of nucleotide sequences selected from SEQ ID NOs.1, 3, 5, 7, and 9, and/or a nucleotide sequence having at least 90% sequence identity with SEQ ID NOs.1, 3, 5, 7, and 9.
  • Another embodiment of the current inventive technology may include the generation of genetically modified organisms configured to produce water-soluble CBD glycoside compounds having between 1-4 UDP-sugar moieties.
  • a plant, a plant cell, an algal cell, a fungi, a bacteria, or a yeast cell may be genetically modified to express a nucleotide sequence according to SEQ ID NO.1, and/or a nucleotide sequence having at least 90% sequence identity with SEQ ID NOs.1.
  • Another embodiment of the current inventive technology may include the generation of genetically modified organisms configured to produce water-soluble CBD glycoside compounds having between 1-4 UDP-sugar moieties.
  • a plant, a plant cell, an algal cell, a fungi, a bacteria, or a yeast cell may be genetically modified to express a nucleotide sequence encoding a peptide having an amino acid sequence according to SEQ ID NO.2, and/or amino acid sequence having at least 90% sequence identity with SEQ ID NO.2.
  • Another embodiment of the current inventive technology may include the generation of genetically modified organisms configured to produce water-soluble CBN glycoside compounds having at least one UDP-sugar moiety.
  • a plant, a plant cell, an algal cell, a fungi, a bacteria, or a yeast cell may be genetically modified to express a nucleotide sequence selected from SEQ ID NO.
  • Another embodiment of the current inventive technology may include the generation of genetically modified organisms configured to produce water-soluble CBD glycoside compounds having least one UDP-sugar moiety.
  • a plant, a plant cell, an algal cell, a fungi, a bacteria, or a yeast cell may be genetically modified to express a nucleotide sequence encoding a peptide having an amino acid sequence according to SEQ ID NO.4, 6, 8 and 10, and/or amino acid sequence having at least 90% sequence identity with SEQ ID NOs.4, 6, 8 and 10.
  • Another embodiment of the current inventive technology includes the isolated peptide having an amino acid sequence encoding a UGT having glycosylation activity towards one or more cannabinoid compounds, and preferably CBD according to SEQ ID NO.2, or a fragment thereof, and/or an amino acid sequence having at least 90% sequence identity with SEQ ID NO.1.
  • Another embodiment of the current inventive technology includes the isolated nucleotide sequence encoding a UGT having glycosylation activity towards one or more cannabinoid compounds, and preferably CBD according to SEQ ID NO.1, and/or a nucleotide sequence having at least 90% sequence identity with SEQ ID NO.1.
  • Another embodiment of the current inventive technology includes an expression vector having a nucleotide sequence, operably linked to a promoter, encoding a UGT having glycosylation activity towards one or more cannabinoid compounds, and preferably CBD according to SEQ ID NO.2, or a fragment thereof, and/or an amino acid sequence having at least 90% sequence identity with SEQ ID NO.2.
  • Another embodiment of the current inventive technology includes an expression vector having a nucleotide sequence, operably linked to a promoter, encoding a UGT having glycosylation activity towards one or more cannabinoid compounds, and preferably CBD according to SEQ ID NO.1, and/or a nucleotide sequence having at least 90% sequence identity with SEQ ID NO.1.
  • Another embodiment of the current inventive technology includes one or more organisms, such as a plant, plant cell, bacteria, algae, fungi, or yeast cell, transformed by an expression vector having a nucleotide sequence encoding a UGT having glycosylation activity towards one or more cannabinoid compounds, and preferably CBD according to SEQ ID NO.2, or a fragment thereof, and/or an amino acid sequence having at least 90% sequence identity with SEQ ID NO.2.
  • organisms such as a plant, plant cell, bacteria, algae, fungi, or yeast cell
  • an expression vector having a nucleotide sequence encoding a UGT having glycosylation activity towards one or more cannabinoid compounds, and preferably CBD according to SEQ ID NO.2, or a fragment thereof, and/or an amino acid sequence having at least 90% sequence identity with SEQ ID NO.2.
  • Another embodiment of the current inventive technology includes one or more organisms, such as a plant, plant cell, bacteria, algae, fungi, or yeast cell, transformed by an expression vector having a nucleotide sequence encoding a UGT having glycosylation activity towards one or more cannabinoid compounds, and preferably CBD according to SEQ ID NO. 1, and/or a nucleotide sequence having at least 90% sequence identity with SEQ ID NO.1.
  • Another embodiment of the current inventive technology includes the isolated peptide having an amino acid sequence encoding a UGT having glycosylation activity towards one or more cannabinoid compounds, and preferably CBN according to SEQ ID NO.
  • Another embodiment of the current inventive technology includes the isolated nucleotide sequence encoding a UGT having glycosylation activity towards one or more cannabinoid compounds, and preferably CBN according to SEQ ID NO. 3, 5, 7, and 9, and/or a nucleotide sequence having at least 90% sequence identity with SEQ ID NO.3, 5, 7, and 9.
  • Another embodiment of the current inventive technology includes an expression vector having a nucleotide sequence, operably linked to a promoter, encoding a UGT having glycosylation activity towards one or more cannabinoid compounds, and preferably CBN according to SEQ ID NO.
  • Another embodiment of the current inventive technology includes an expression vector having a nucleotide sequence, operably linked to a promoter, encoding a UGT having glycosylation activity towards one or more cannabinoid compounds, and preferably CBN according to SEQ ID NO.3, 5, 7, and 9, and/or a nucleotide sequence having at least 90% sequence identity with SEQ ID NO.3, 5, 7, and 9.
  • Another embodiment of the current inventive technology includes one or more organisms, such as a plant, plant cell, bacteria, algae, fungi, or yeast cell, transformed by an expression vector having a nucleotide sequence encoding a UGT having glycosylation activity towards one or more cannabinoid compounds, and preferably CBN according to SEQ ID NO. 4, 6, 8, and 10, or a fragment thereof, and/or an amino acid sequence having at least 90% sequence identity with SEQ ID NO.4, 6, 8, and 10.
  • organisms such as a plant, plant cell, bacteria, algae, fungi, or yeast cell
  • an expression vector having a nucleotide sequence encoding a UGT having glycosylation activity towards one or more cannabinoid compounds, and preferably CBN according to SEQ ID NO. 4, 6, 8, and 10, or a fragment thereof, and/or an amino acid sequence having at least 90% sequence identity with SEQ ID NO.4, 6, 8, and 10.
  • Another embodiment of the current inventive technology includes one or more organisms, such as a plant, plant cell, bacteria, algae, fungi, or yeast cell, transformed by an expression vector having a nucleotide sequence encoding a UGT having glycosylation activity towards one or more cannabinoid compounds, and preferably CBN according to SEQ ID NO. 3, 5, 7, and 9, and/or a nucleotide sequence having at least 90% sequence identity with SEQ ID NO.3, 5, 7, and 9.
  • the invention novel systems, methods and compositions for the production of water-soluble cannabinoid glycosides in yeast or bacteria cells.
  • a plant such as yeast or bacterial cell
  • a culture of yeast cells such as Saccharomyces cerevisiae, Kluyveromyces marxianus, or Pichia pastoris or other suitable yeast species, may be established in a fermenter or other similar apparatus. It should be noted that the use of the above identified example in this embodiment is exemplary only, as various yeast strains, mixes of strains, hybrids of different strains or clones may be used to generate a suspension culture.
  • such fermenters may include large industrial-scale fermenters allowing for a large quantity of yeast and bacterial cells to be grown.
  • it may be possible to culture a large quantity of cells from a single-strain of, for example, S. cerevisiae, P. pastoris, or K. marxianus, or a bacterium such as E. Coli, or Bacillus subtilis, which may establish a cell culture having a consistent rate of cannabinoid modification.
  • Such cultured growth may be continuously sustained with the continual addition of nutrient and other growth factors being added to the culture.
  • Such features may be automated or accomplished manually.
  • the water-soluble cannabinoids such as CBD-1G, CBD-2G, CBD-3G, and CBD-4G
  • a transformed yeast or bacteria cells may be lysed such that accumulated cannabinoid glycosides are released to the surrounding lysate. Additional steps may include treating this lysate. Examples of such treatment may include filtering, centrifugation or screening to remove extraneous cellular material as well as chemical treatments to improve later cannabinoid glycoside yields.
  • the cannabinoid glycosides, such as CBD-1G, CBD-2G, CBD-3G, and CBD-4G may be further isolated and purified.
  • the culture’s supernatant/media, or the cell’s lysate may be processed utilizing affinity chromatography or other purification methods.
  • an affinity column having a ligand configured to bind with one or more of the cannabinoid glycosides such that they may be immobilized or coupled to a solid support.
  • the material may then be passed over the column such that the cannabinoid glycosides, having specific binding affinity to the ligand become bound and immobilized.
  • non- binding and non-specific binding proteins that may have been present in the lysate may be removed.
  • the cannabinoid glycosides may be eluted or displaced from the affinity column by, for example, a corresponding sugar or other compound that may displace or disrupt the cannabinoid-ligand bond.
  • the eluted cannabinoid glycosides may be collected and further purified or processed.
  • Another embodiment of the current invention may include systems, methods and compositions for the generation of water soluble cannabinoid glycoside compounds in whole plants and plant cell cultures.
  • this N-terminal trichome targeting sequence or domain may generally include the first 28 amino acid residues of a generalized synthase and may be coupled with a UGT, and preferably a UGT selected from the group consisting of SEQ ID NO.2, 4, 6, 8 and 10, or a fragment thereof.
  • N-terminal trichome targeting sequence for THCA synthase and CBDA synthase are identified by Sayre et al., PCT/US18/41710, such sequences being specifically incorporated here by reference.
  • This extracellular targeting sequence may be recognized by the plant cell and cause the transport of the UGT from the cytoplasm to the plant’s trichrome, and in particular the storage compartment of the plant trichrome where extracellular cannabinoid glycosylation may occur.
  • one or more UGT and preferably a UGT selected from the group consisting of SEQ ID NO.2, 4, 6, 8 and 10, or a fragment thereof., may either be engineered to express all or part of the N-terminal extracellular targeting sequence as present in an exemplary synthase enzyme.
  • a trichome structure such as in Cannabis, will have limited substrate for a UGT to use to effectuate glycosylation.
  • the invention may include systems, methods and compositions to increase substrates for UGTs in a plant trichome structure.
  • an exogenous or endogenous UDP-glucose/UDP- galactose transporter may be expressed in a trichome producing plant, such as Cannabis plant.
  • a trichome producing plant such as Cannabis plant.
  • the UDP-glucose/UDP-galactose transporter may be modified to include a plasma-membrane targeting sequence and/or domain, exemplary sequences being identified by Sayre et al., PCT/US18/41710, such sequences being specifically incorporated here by reference.
  • the UDP-glucose/UDP- galactose transporter may allow the artificial fusion protein to be anchored to the plasma membrane.
  • sugar substrates from the cytosol may pass through the plasma membrane bound UDP-glucose/UDP-galactose transporter into the trichome.
  • substrates for UGTs may be localized to the trichome and allowed to accumulate further allowing enhanced glycosylation of cannabinoids in the trichome.
  • such plants or cell cultures may be genetically modified to direct cannabinoid synthesis to the cytosol, as opposed to a trichome structure.
  • cannabinoid biosynthesis may be redirected from the plant’s trichome to be localized in the plant cell’s cytosol.
  • a cytosolic cannabinoid production system may be established as described in PCT/US18/24409 and PCT/US18/41710, both by Sayre et al. (these applications are both incorporated by reference with respect to their disclosure related to cytosolic cannabinoid production and/or modification in whole, and plant cell systems).
  • a cytosolic cannabinoid production system may include the in vivo creation of one or more recombinant proteins that may allow cannabinoid biosynthesis to be localized to the cytosol where one or more heterologous UGT proteins may also be expressed and present in the cytosol.
  • This inventive feature allows not only higher levels of cannabinoid production and accumulation, but efficient production of cannabinoids in suspension cell cultures.
  • this inventive feature allows cannabinoid glycoside production and accumulation without a trichome structure in whole plants, allowing cells that would not traditionally produce cannabinoids, such as cells in Cannabis leaves and stalks, to become cannabinoid-producing cells
  • one or more cannabinoid synthases may be modified to remove all or part of an N-terminal extracellular trichome targeting.
  • Exemplary N- terminal trichome targeting sequence for THCA synthase and CBDA synthase are identified by Sayre et al., PCT/US18/41710. Co-expression with this cytosolic-targeted synthase with a heterologous UGT may allow the localization of cannabinoid synthesis to the cytosol.
  • cannabinoid biosynthesis may be coupled with cannabinoid glycosylation in a cell cytosol.
  • a UGT for example SEQ ID NOs. 2, 4, 6, 8 and 10, or a fragment thereof
  • cytosolic targeted UGT enzymes may be co-expressed with heterologous catalase and cannabinoid transporters or other genes that may reduce cannabinoid biosynthesis toxicity and/or facilitate transport through or out of the cell.
  • a catalase enzyme may be co-expressed with a UGT of the invention.
  • a heterologous catalase is selected from the group of catalase sequences identified in PCT/US18/24409 and PCT/US18/41710, both by Sayre et al., such catalase sequences being incorporated herein by reference.
  • Such cytosolic targeted enzymes may also be co-expressed with one or more myb transcriptions factors that may enhance metabolite flux through the cannabinoid biosynthetic pathway which may increase cannabinoid production.
  • a myb transcription factor may be endogenous to Cannabis, or an ortholog thereof.
  • myb transcription factor may include those identified by Sayre et al., PCT/US18/41710, such specific sequences being incorporated herein by reference.
  • one or more endogenous cannabinoid synthase genes may be disrupted and/or knocked out and replaced with cytosolic-targeted cannabinoid synthase proteins as described herein.
  • the disrupted endogenous cannabinoid synthase gene(s) may be the same or different than the expressed cytosolic-targeted cannabinoid synthase protein.
  • one or more endogenous cannabinoid synthase genes may be disrupted and/or knocked out in a Cannabis plant or suspension cell culture wherein one or more cannabinoid synthase genes has been disrupted and/or knocked out is selected from the group consisting of: a CBG synthase gene; a THCA synthase, a CBDA synthase, and a CBCA synthase.
  • the Cannabis plant or suspension cell culture may express a polynucleotide encoding one or more cannabinoid synthases having its trichome targeting sequence disrupted and/or removed which may be selected from the group consisting of: a CBG synthase gene having its trichome targeting sequence disrupted and/or removed; a THCA synthase having its trichome targeting sequence disrupted and/or removed; a CBDA synthase having its trichome targeting sequence disrupted and/or removed; and a CBCA synthase having its trichome targeting sequence disrupted and/or removed.
  • a CBG synthase gene having its trichome targeting sequence disrupted and/or removed
  • a THCA synthase having its trichome targeting sequence disrupted and/or removed
  • CBDA synthase having its trichome targeting sequence disrupted and/or removed
  • CBCA synthase having its trichome targeting sequence disrupted and/or removed.
  • a “cannabinoid” is a chemical compound (such as cannabinol, THC or cannabidiol) that is found in the plant species Cannabis among others like: Echinacea; Acmella Oleracea; Helichrysum Umbraculigerum; Radula Marginata (Liverwort) and Theobroma Cacao, and metabolites and synthetic analogues thereof that may or may not have psychoactive properties.
  • Cannabinoids therefore include (without limitation) compounds (such as THC) that have high affinity for the cannabinoid receptor (for example Ki ⁇ 250 nM), and compounds that do not have significant affinity for the cannabinoid receptor (such as cannabidiol, CBD).
  • Cannabinoids also include compounds that have a characteristic dibenzopyran ring structure (of the type seen in THC) and cannabinoids which do not possess a pyran ring (such as cannabidiol).
  • a partial list of cannabinoids includes THC, CBD, dimethyl heptylpentyl cannabidiol (DMHP-CBD), 6,12- dihydro-6-hydroxy-cannabidiol (described in U.S. Pat. No.5,227,537, incorporated by reference); (3S,4R)-7-hydroxy- ⁇ 6-tetrahydrocannabinol homologs and derivatives described in U.S. Pat. No.
  • cannabinoids are tetrahydrocannabinol, cannabidiol, cannabigerol, cannabichromene, cannabicyclol, cannabivarin, cannabielsoin, cannabicitran, cannabigerolic acid, cannabigerolic acid monomethylether, cannabigerol monomethylether, cannabigerovarinic acid, cannabigerovarin, cannabichromenic acid, cannabichromevarinic acid, cannabichromevarin, cannabidolic acid, cannabidiol monomethylether, cannabidiol-C4, cannabidivarinic acid, cannabidiorcol, delta-9-tetrahydrocannabinolic acid A, delta-9- tetrahydrocannabinolic acid B, delta-9-tetrahydrocannabinolic acid-C4, delta-9- tetrahydrocannabivarinic acid,delta-9-
  • cannabinoids within the context of this disclosure include tetrahydrocannabinol and cannabidiol.
  • cannabinoid may also include different modified forms of a cannabinoid such as a hydroxylated cannabinoid or cannabinoid carboxylic acid.
  • a UGT were to be capable of glycosylating a cannabinoid, it would include the term cannabinoid as defined elsewhere, as well as the aforementioned modified forms. It may further include multiple glycosylation moieties.
  • a protein has “homology” or “homology” to a second protein if the amino acid sequence encoded by a gene has a similar amino acid sequence to that of the second gene.
  • a protein has homology to a second protein if the two proteins have “similar” amino acid sequences.
  • the term “homologous proteins” is defined to mean that the two proteins have similar amino acid sequences). More specifically, in certain embodiments, the term “homologous” with regard to a contiguous nucleic acid sequence, refers to contiguous nucleotide sequences that hybridize under appropriate conditions to the reference nucleic acid sequence.
  • homologous sequences may have from about 75%-100, or more generally 80% to 100% sequence identity, such as about 81%; about 82%; about 83%; about 84%; about 85%; about 86%; about 87%; about 88%; about 89%; about 90%; about 91%; about 92%; about 93%; about 94% about 95%; about 96%; about 97%; about 98%; about 98.5%; about 99%; about 99.5%; and about 100%.
  • sequence identity such as about 81%; about 82%; about 83%; about 84%; about 85%; about 86%; about 87%; about 88%; about 89%; about 90%; about 91%; about 92%; about 93%; about 94% about 95%; about 96%; about 97%; about 98%; about 98.5%; about 99%; about 99.5%; and about 100%.
  • sequence identity such as about 81%; about 82%; about 83%; about 84%; about 85%; about 86%; about 87%; about 88%; about 89%; about 90%
  • a nucleic acid molecule is specifically hybridizable when there is a sufficient degree of complementarity to avoid non-specific binding of the nucleic acid to non-target sequences under conditions where specific binding is desired, for example, under stringent hybridization conditions, and would fall within the range of a homolog.
  • expression optimization for example for a mammalian lipocalin or odorant binding protein, to be expressed in yeast may be considered homologous and having a variable sequence identity due to the variable codon positions. Additional embodiments may also include homology to include redundant nucleotide codons.
  • operably linked when used in reference to a regulatory sequence and a coding sequence, means that the regulatory sequence affects the expression of the linked coding sequence.
  • regulatory sequences refer to nucleotide sequences that influence 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. Particular regulatory sequences may be located upstream and/or downstream of a coding sequence operably linked thereto. Also, particular regulatory sequences operably linked to a coding sequence may be located on the associated complementary strand of a double-stranded nucleic acid molecule.
  • promoter refers to a region of DNA that may be upstream from the start of transcription, and that may be involved in recognition and binding of RNA polymerase and other proteins to initiate transcription.
  • a promoter may be operably linked to a coding sequence for expression in a cell, or a promoter may be operably linked to a nucleotide sequence encoding a signal sequence which may be operably linked to a coding sequence for expression in a cell.
  • An “inducible” promoter may be a promoter which may be under environmental control. Tissue-specific, tissue-preferred, cell type specific, and inducible promoters constitute the class of “non-constitutive” promoters.
  • a “constitutive” promoter is a promoter which may be active under most environmental conditions or in most cell or tissue types.
  • transformation or “genetically modified” refers to the transfer of one or more nucleic acid molecule(s) into a cell.
  • a plant is “transformed” or “genetically modified” by a nucleic acid molecule transduced into the plant when the nucleic acid molecule becomes stably replicated by the plant.
  • the term “transformation” or “genetically modified” encompasses all techniques by which a nucleic acid molecule can be introduced into, such as a plant.
  • vector refers to some means by which DNA, RNA, a protein, or polypeptide can be introduced into a host.
  • the polynucleotides, protein, and polypeptide which are to be introduced into a host can be therapeutic or prophylactic in nature; can encode or be an antigen; or can be regulatory in nature, etc.
  • vectors including virus, plasmid, bacteriophages, cosmids, and bacteria.
  • An “expression vector” is nucleic acid capable of replicating in a selected host cell or organism.
  • An expression vector can replicate as an autonomous structure, or alternatively can integrate, in whole or in part, into the host cell chromosomes or the nucleic acids of an organelle, or it is used as a shuttle for delivering foreign DNA to cells, and thus replicate along with the host cell genome.
  • an expression vector are polynucleotides capable of replicating in a selected host cell, organelle, or organism, e.g., a plasmid, virus, artificial chromosome, nucleic acid fragment, and for which certain genes on the expression vector (including genes of interest) are transcribed and translated into a polypeptide or protein within the cell, organelle or organism; or any suitable construct known in the art, which comprises an “expression cassette.”
  • a “cassette” is a polynucleotide containing a section of an expression vector of this invention. The use of a cassette assists in the assembly of the expression vectors.
  • An expression vector is a replicon, such as plasmid, phage, virus, chimeric virus, or cosmid, and which contains the desired polynucleotide sequence operably linked to the expression control sequence(s).
  • a replicon such as plasmid, phage, virus, chimeric virus, or cosmid
  • different organisms preferentially utilize different codons for generating polypeptides.
  • Such “codon usage” preferences may be used in the design of nucleic acid molecules encoding the proteins and chimeras of the invention in order to optimize expression in a particular host cell system.
  • all nucleotides of the present invention may be optimized for expression in a select organisms, such as a Cannabis plant, yeast, algae, fungi, and bacteria.
  • a polynucleotide sequence is operably linked to an expression control sequence(s) (e.g., a promoter and, optionally, an enhancer) when the expression control sequence controls and regulates the transcription and/or translation of that polynucleotide sequence.
  • an expression control sequence e.g., a promoter and, optionally, an enhancer
  • a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), the complementary (or complement) sequence, and the reverse complement sequence, as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (see e.g., Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). Because of the degeneracy of nucleic acid codons, one can use various different polynucleotides to encode identical polypeptides. The Table below, contains information about which nucleic acid codons encode which amino acids.
  • Oligonucleotides and polynucleotides that are not commercially available can be chemically synthesized e.g., according to the solid phase phosphoramidite triester method first described by Beaucage and Caruthers, Tetrahedron Letts.22:1859-1862 (1981), or using an automated synthesizer, as described in Van Devanter et al., Nucleic Acids Res. 12:6159- 6168 (1984). Other methods for synthesizing oligonucleotides and polynucleotides are known in the art. Purification of oligonucleotides is by either native acrylamide gel electrophoresis or by anion-exchange HPLC as described in Pearson & Reanier, J.
  • plant or “plant system” includes whole plants, plant organs, progeny of whole plants or plant organs, embryos, somatic embryos, embryo-like structures, protocorms, protocorm- like bodies (PLBs), and culture and/or suspensions of plant cells.
  • Plant organs comprise, e.g., shoot vegetative organs/structures (e.g., leaves, stems and tubers), roots, flowers and floral organs/structures (e.g., bracts, sepals, petals, stamens, carpels, anthers and ovules), seed (including embryo, endosperm, and seed coat) and fruit (the mature ovary), plant tissue (e.g., vascular tissue, ground tissue, and the like) and cells (e.g., guard cells, egg cells, trichomes and the like).
  • the invention may also include Cannabaceae and other Cannabis strains, such as C. sativa generally.
  • expression refers to the process by which the coded information of a nucleic acid transcriptional unit (including, e.g., genomic DNA or cDNA) is converted into an operational, non- operational, or structural part of a cell, often including the synthesis of a protein.
  • Gene expression can be influenced by external signals; for example, exposure of a cell, tissue, or organism to an agent that increases or decreases gene expression. Expression of a gene can also be regulated anywhere in the pathway from DNA to RNA to protein.
  • RNA level or the protein level by any method known in the art, including, without limitation, Northern blot, RT-PCR, Western blot, or in vitro, in situ, or in vivo protein activity assay(s).
  • nucleic acid or “nucleic acid molecules” include single- and double-stranded forms of DNA; single-stranded forms of RNA; and double-stranded forms of RNA (dsRNA).
  • nucleotide sequence or “nucleic acid sequence” refers to both the sense and antisense strands of a nucleic acid as either individual single strands or in the duplex.
  • ribonucleic acid is inclusive of iRNA (inhibitory RNA), dsRNA (double stranded RNA), siRNA (small interfering RNA), mRNA (messenger RNA), miRNA (micro-RNA), hpRNA (hairpin RNA), tRNA (transfer RNA), whether charged or discharged with a corresponding acetylated amino acid), and cRNA (complementary RNA).
  • DNA deoxyribonucleic acid
  • nucleic acid segment and “nucleotide sequence segment,” or more generally “segment,” will be understood by those in the art as a functional term that includes both genomic sequences, ribosomal RNA sequences, transfer RNA sequences, messenger RNA sequences, operon sequences, and smaller engineered nucleotide sequences that encoded or may be adapted to encode, peptides, polypeptides, or proteins.
  • gene refers to a coding region operably joined to appropriate regulatory sequences capable of regulating the expression of the gene product (e.g., a polypeptide or a functional RNA) in some manner.
  • a gene includes untranslated regulatory regions of DNA (e.g., promoters, enhancers, repressors, etc.) preceding (up-stream) and following (down-stream) the coding region (open reading frame, ORF) as well as, where applicable, intervening sequences (i.e., introns) between individual coding regions (i.e., exons).
  • structural gene as used herein is intended to mean a DNA sequence that is transcribed into mRNA which is then translated into a sequence of amino acids characteristic of a specific polypeptide.
  • fragment refers to a portion of a peptide or nucleotide sequence that still retains the activity of the whole.
  • disclosure of a DNA sequence also include the corresponding RNA and amino acid sequence including all redundant codons and conservative amino acid substitutions
  • disclosure of a RNA sequence also include the corresponding DNA and amino acid sequence including all redundant codons and conservative amino acid substitutions
  • disclosure of amino acid sequence also include the corresponding RNA and DNA sequence including all redundant codons and conservative amino acid substitutions and vice versa.
  • a nucleic acid molecule may include either or both naturally occurring and modified nucleotides linked together by naturally occurring and/or non-naturally occurring nucleotide linkages.
  • Nucleic acid molecules may be modified chemically or biochemically, or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art.
  • Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications (e.g., uncharged linkages: for example, methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.; charged linkages: for example, phosphorothioates, phosphorodithioates, etc.; pendent moieties: for example, peptides; intercalators: for example, acridine, psoralen, etc.; chelators; alkylators; and modified linkages: for example, alpha anomeric nucleic acids, etc.).
  • internucleotide modifications e.g., uncharged linkages: for example, methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.; charged linkages: for example, phosphorothioates, phosphorodithioates, etc.; pendent moieties
  • nucleic acid molecule also includes any topological conformation, including single-stranded, double-stranded, partially duplexed, triplexed, hair-pinned, circular, and padlocked conformations.
  • sequence identity or “identity,” as used herein in the context of two nucleic acid or polypeptide sequences, refers to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window.
  • heterologous or “exogenous” in reference to a nucleic acid is a nucleic acid that originates from a foreign species, or is synthetically designed, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention.
  • a heterologous protein may originate from a foreign species or, if from the same species, is substantially modified from its original form by deliberate human intervention.
  • kits and reagents are generally carried out in accordance with manufacturer defined protocols and/or parameters unless otherwise noted.
  • use herein of the singular is to be read to include the plural and vice versa.
  • the term “a” or “an” used in relation to an entity is to be read to refer to one or more of that entity.
  • the terms “a” (or “an”), “one or more,” and “at least one” are used interchangeably herein.
  • the terms “approximately” and “about” refer to a quantity, level, value, or amount that varies by as much as 30%, or in another embodiment by as much as 20%, and in a third embodiment by as much as 10% to a reference quantity, level, value or amount.
  • the term “comprise,” or variations thereof such as “comprises” or “comprising,” are to be read to indicate the inclusion of any recited integer (e.g., a feature, element, characteristic, property, method/process step or limitation) or group of integers (e.g., features, element, characteristics, properties, method/process steps or limitations) but not the exclusion of any other integer or group of integers.
  • the term “comprising” is inclusive or open-ended and does not exclude additional, unrecited integers or method/process steps.

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Abstract

La présente invention concerne de manière générale l'identification des nouvelles enzymes UDP-glucosyltransférases ayant une activité spécifique vis-à-vis de composés cannabinoïdes. La présente invention concerne en outre de manière générale l'utilisation de nouvelles enzymes UGT ayant une activité spécifique vis-à-vis de composés cannabinoïdes pour générer des composés glycosidiques cannabinoïdes solubles dans l'eau.
PCT/US2024/033912 2023-06-14 2024-06-13 Synthèse améliorée de composés glycosidiques cannabinoïdes Ceased WO2024259172A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002010210A2 (fr) * 2001-08-28 2002-02-07 Bayer Cropscience Ag Polypeptides permettant d'identifier de nouveaux composes herbicides actifs
US7847156B2 (en) * 2003-10-20 2010-12-07 Cropdesign N.V. Plants having improved growth characteristics and methods for making the same
WO2018204483A2 (fr) * 2017-05-03 2018-11-08 Senomyx, Inc. Procédés de fabrication d'édulcorants à haute intensité
WO2021174092A1 (fr) * 2020-02-28 2021-09-02 Trait Biosciences, Inc. Nouveaux systèmes, procédés et compositions pour la glycosylation de composés cannabinoïdes

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002010210A2 (fr) * 2001-08-28 2002-02-07 Bayer Cropscience Ag Polypeptides permettant d'identifier de nouveaux composes herbicides actifs
US7847156B2 (en) * 2003-10-20 2010-12-07 Cropdesign N.V. Plants having improved growth characteristics and methods for making the same
WO2018204483A2 (fr) * 2017-05-03 2018-11-08 Senomyx, Inc. Procédés de fabrication d'édulcorants à haute intensité
WO2021174092A1 (fr) * 2020-02-28 2021-09-02 Trait Biosciences, Inc. Nouveaux systèmes, procédés et compositions pour la glycosylation de composés cannabinoïdes

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