Upregulation of UDP-glucose production and methods of use in a yeast-based cannabinoid glycosylation system

Abstract

The inventive technology described herein includes systems, methods, and compositions for the upregulation of Uridine diphosphate glucose (UDPG) recycling in a yeast cell, by expressing a heterologous nucleotide sequence, operably linked to a promoter, encoding a phosphoglucomutase enzyme which catalyzes the conversion from glucose-6-phosphate to glucose- 1 -phosphate, and a UDP-glucose pyrophosphorylase enzyme which catalyzes the formation of UDPG from glucose 1 -phosphate and UTP. The increased levels of UDPG substrate produced by the yeast cell may be used as a substrate for a co-expressed UDP -glycosyltransferases enzyme capable of generating cannabinoid glycoside compounds.

Description

[0001] UPREGULATION OF UDP-GLUCOSE PRODUCTION AND METHODS OF USE IN A YEAST-BASED CANNABINOID GLYCOSYLATION SYSTEM CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of and priority to U.S. Provisional Application No.

[0003] 63 / 762,262, filed February 24, 2025. The entire specification and figures of the above-referenced application are hereby incorporated, in their entirety by reference.

[0004] SEQUENCE LISTING

[0005] The instant application contains contents of the electronic sequence listing (90425.00355-Sequence-Listing.xml; Size: 21,301 bytes; and Date of Creation: February 24, 2026) is herein incorporated by reference in its entirety.

[0006] TECHNICAL FIELD

[0007] The present invention relates generally to the fields of molecular biology and genetic metabolic engineering, and in particular novel systems, methods, and compositions for the upregulation Uridine diphosphate glucose (UDPG) recycling of yeast cells configured to produce cannabinoid glycosides.

[0008] BACKGROUND

[0009] Phytocannabinoids are a class of specialized compounds synthesized by Cannabis sativa (generally refer to herein as Cannabis, or hemp). They are formed by condensation of terpene and phenol precursors. They include these more abundant forms: A9-tetrahydrocannabinol (THC), cannabidiol (CBD), cannabichromene (CBC), and cannabigerol (CBG). Another cannabinoid, cannabinol (CBN), is formed from THC as a degradation product and can be detected in some plant strains. Typically, THC, CBD, CBC, and CBG occur together in different ratios in the various plant strains. These 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 as a result of decarboxylation. In general, the carboxylic acids have the function of a biosynthetic precursor. For example, the tetrahydrocannabinols A9- and A8-THC arise in vivo from the THC carboxylic acids by decarboxylation and likewise, CBD from the associated cannabidiolic acid. Notably, cannabinoid may be derived from natural sources, such as Cannabis plants, or through synthetic production methods.Importantly, 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. As described in, US8410064 by Pandya et al., cannabinoids may be subject to cytochrome P450 oxidation and subsequent UDP-glucuronosyltransferase dependent glucuronidation in the body after consumption. The resulting glucuronide of the oxidized cannabinoids is the main metabolite found in urine, and thus, this solubilization process plays a critical role in the metabolic clearance of cannabinoids. In another embodiment outlined in PCT / US 18 / 24409 and PCT / US18 / 41710 (both of which are incorporated herein in their entirety by reference, including examples 1-19, and all specific materials and methods, as well as sequence listings), by Sayre etal., cannabinoids may be glycosylated to form water-soluble glycoside compounds, also referred to herein as cannabinoid glycosides. As shown in Figure 3, such water-soluble cannabinoid glycosides may include one or more sugar moi eties, and preferably 1-3 sugar moi eties, and even more preferably 2 or more sugar moieties. Naturally, such ranges are exemplary only, and should not be construes as limiting embodiments as a cannabinoid glycosides may include a plurality of sugar moieties.

[0010] As again described by Sayre et al., PCT / US2018 / 041710 (incorporated herein by reference), cannabinoid compounds, such as CBD or THC, may be incubated in a yeast cell culture expressing a heterologous UDP-glycosyltransferases, , also referred to herein as (UGT) enzyme having glycosylation activity toward the cannabinoid compounds. As can be seen by this example, the presence of UDPG is required for this glycosylation process, such that increasing its availability may allow for the increased production of cannabinoid glycosides. As can be seen, there exists a need for a novel systems and methods to upregulate UDPG production in yeast cells to provide increased levels of UDPG substrate for a co-expressed UGT’s configured to generate cannabinoid glycoside compounds.

[0011] SUMMARY OF THE INVENTION

[0012] One aspect of the invention includes systems, methods, and compositions for the upregulation of UDPG recycling in a yeast cell, which in one preferred embodiment may increase levels of UDPG substrate for a co-expressed UGT’s configured to generate cannabinoid glycoside compounds.

[0013] Another aspect of the invention includes systems, methods, and compositions for the upregulation of UDPG recycling in a yeast cell, by expressing a heterologous nucleotide sequence,operably linked to a promoter, encoding a phosphoglucomutase enzyme which catalyzes the conversion from glucose-6-phosphate to glucose- 1 -phosphate, and a UDP-glucose pyrophosphorylase enzyme which catalyzes the formation of UDPG from glucose 1 -phosphate and UTP. Notably, the UGP1 enzyme’s reaction is in equilibrium, such that by pulling glucose 1-phosphate into UDPG with UGP, the present inventors can drive the reaction in the glucose-6-phosphate > glucose 1 -phosphate direction.

[0014] Another aspect of the invention includes systems, methods, and compositions for the upregulation of UDPG recycling in a yeast cell, and preferably a Pichia pastoris yeast cell, expressing a heterologous UGT having activity towards one or more cannabinoids, by expressing a heterologous nucleotide sequence, operably linked to a promoter, encoding a phosphoglucomutase (PGM2) (SEQ ID NO. 1) from Saccharomyces cerevisiae, which catalyzes the conversion from glucose-6-phosphate to glucose- 1 -phosphate, and a UDP -glucose pyrophosphorylase (UGP1) protein (SEQ ID NO. 5) from S. cerevisiae, which catalyzes the formation of UDPG from glucose 1 -phosphate and UTP.

[0015] Another aspect of the invention includes systems, methods, and compositions for the production of cannabinoid glycosides, the method comprising the steps of generating a yeast cell, and preferably a yeast cell culture, expressing a heterologous nucleotide sequence, operably linked to a promoter, encoding a phosphoglucomutase enzyme, and / or UDP -glucose pyrophosphorylase, and a UGT having activity towards one or more cannabinoids; introducing one or more cannabinoids to the yeast cell, and glycosylating the cannabinoid forming a cannabinoid glycoside. The cannabinoid glycoside may further be isolated.

[0016] Another aspect of the invention includes systems, methods, and compositions for the production of cannabinoid glycosides, the method comprising the steps of generating a yeast cell, and preferably a yeast cell culture, expressing a heterologous nucleotide sequence, operably linked to a promoter, encoding a phosphoglucomutase (PGM2) (SEQ ID NO. 1), a UDP-glucose pyrophosphorylase (UGP1) protein (SEQ ID NO. 5), and a UGT, for example one or more cannabinoid incorporated herein by reference, having activity towards one or more cannabinoids; introducing one or more cannabinoids to the yeast cell, and glycosylating the cannabinoid forming a cannabinoid glycoside.

[0017] The present disclosure relates to engineered yeast cells and methods that increase intracellular recycling of glucose to UDP-glucose (UDPG) to support downstream glycosylationreactions, including the enzymatic glycosylation of cannabinoid compounds. In one aspect, a yeast cell is provided that expresses a heterologous nucleotide sequence operably linked to a promoter encoding one or more enzymes selected from: Phosphoglucomutase (PGM), or a fragment or variant thereof; and UDP-glucose pyrophosphorylase (UGP), or a fragment or variant thereof; wherein expression of PGM and / or UGP upregulates glucose-to-UDPG recycling in the yeast cell.

[0018] In certain embodiments, the yeast cell is a Pichia pastoris cell. In some embodiments, the PGM enzyme comprises PGM2 from Saccharomyces cerevisiae, including PGM2 according to SEQ ID NO. 2 or a sequence >80% identical to any of SEQ ID NOs. 1-4. In some embodiments, the UGP enzyme comprises UGP1 from S. cerevisiae, including UGP1 according to SEQ ID NOs.

[0019] 4-5 or a sequence >80% identical thereto. In various embodiments, the heterologous nucleotide sequence is codon-optimized for expression in yeast.

[0020] In another aspect, a yeast cell is provided that co-expresses (i) PGM as above (e.g., catalyzing conversion of glucose-6-phosphate to glucose- 1 -phosphate), (ii) UGP as above (e.g., catalyzing formation of UDPG from glucose- 1 -phosphate and UTP), and (iii) a UDP-glucosyltransferase (UGT) having glycosylation activity toward one or more cannabinoid compounds. In some embodiments, the UGT is selected from sequences having glycosylation activity toward a cannabinoid as disclosed in U.S. Application No. 17 / 189,063 (e.g., SEQ ID NOs.

[0021] 1-9181, 9208, 9210, 9212, 9214, 9216, 9218, 9220, 9236, 9238, 9240, 9242, 9244, each incorporated herein by reference). In further embodiments, the PGM and UGP are PGM according to SEQ ID NO. 1 and UGP according to SEQ ID NO. 4.

[0022] Methods

[0023] In a further aspect, a method of upregulating glucose-to-UDPG recycling is provided, comprising providing a yeast cell that expresses a heterologous nucleotide sequence operably linked to a promoter encoding PGM and / or UGP as described herein, such that PGM and / or UGP expression increases UDPG recycling in the yeast cell. In certain embodiments, the yeast cell is Pichia pastoris; in some embodiments, the PGM is PGM2 from S. cerevisiae (e.g., SEQ ID NOs.

[0024] 1-2 or sequences >80% identical thereto), and / or the UGP is UGP1 from S. cerevisiae (e.g., SEQ ID NOs. 4-5 or sequences >80% identical thereto). In some embodiments, the heterologous nucleotide sequence is codon-optimized for yeast expression.

[0025] In yet another aspect, a method of glycosylating cannabinoid compounds is provided for a cannabinoid glycosylation system, comprising: (i) providing a yeast cell culture that expressesPGM and / or UGP as above to enhance UDPG supply and further expresses a UGT having glycosylation activity toward one or more cannabinoids; (ii) incubating one or more cannabinoid compounds having at least one glycosylation site with the yeast cell culture (e.g., by introducing a quantity of the cannabinoid to a fermenter containing the culture); and (iii) glycosylating the cannabinoid compounds.

[0026] In various embodiments, the one or more cannabinoid compounds include cannabinoids having at least one glycosylation site, such as cannabidiol (CBD), cannabidiolic acid (CBDA), A9-tetrahydrocannabinol (THC), tetrahydrocannabinolic acid (THCA), and other cannabinoids including, for example, A8-THC, 11 -hydroxy- A9-THC, cannabichromene (CBC), cannabigerol (CBG), cannabigerolic acid (CBGA), cannabinol (CBN), cannabicyclol (CBL), cannabivarin (CBV), tetrahydrocannabivarin (THCV), cannabigerivarin (CBGV), cannabichromevarin (CBCV), cannabidivarin (CBDV), cannabielsoin (CBE), cannabifuran (CBF), and cannabinodiol (CBDN).

[0027] Across the aspects above, codon optimization for yeast, selection of Pichia pastoris as host, and the use of S. cerevisiae PGM2 and UGP1 are exemplified embodiments that can be employed singly or in combination to improve UDPG availability and cannabinoid glycosylation efficiency.

[0028] Additional aspects of the invention may become evident based on the specification and figures presented below.

[0029] BRIEF DESCRIPTION OF THE FIGURES

[0030] Figure 1. Exemplary chemical structure of UDPG molecule.

[0031] Figure 2. Diagram outlining UDPG metabolism in yeast.

[0032] Figure 3. Exemplary chemical formula of glycosylated molecule of CBD having a: (A) single sugar moiety (CBD-2’ -glucoside or CBDIXGly), and (B) a molecule of CBD having two sugar moi eties (CBD-2’, 6’ -diglucoside or CBD2XGly).

[0033] Figure 4. (A) Schematic diagram of yeast-based cannabinoid glycosylation system in one embodiment thereof; (B) time course outlining batch-fed yeast-based cannabinoid glycosylation system in one embodiment thereof.

[0034] Figure 5. Comparative metabolomic analysis between S. cerevisiae and P. pastoris showing steady-state levels of various secondary metabolites, including higher steady-state concentration of UDPG in S. cerevisiae compared to P. pastoris. (Camicer, M, et al. (2012). Development of quantitative metabolomics for Pichia pastoris. Metabolomics 8, 284-298.)Figure 6. Schematic diagram of metabolically engineered yeast cell configured to heterologously express a PGM2 and UGP1 genes from S. cerevisiae

[0035] Figure 7. Express! on / Integration vector configured to express PGM2 and UGP1 genes from ,S'. cerevisiae in P. pastoris and up-regulate UDPG recycling in one embodiment thereof.

[0036] Figure 8. Exemplary cannabinoid compounds having one or more identified glycosylation sites.

[0037] DETAILED DESCRIPTION OF THE INVENTION

[0038] As shown in Figure 2, as part of process of UDPG recycling metabolism in yeast, glucose (Glu) is transported into the cell by a hexose transporter (HXT), or produced by gluconeogenesis (GNG), and phosphorylated to glucose-6-phosphate by a hexose kinase (HXK). It is then isomerized to glucose- 1 -phosphate by phosphoglucomutase (PGM). It is then activated to the nucleotide sugar with UTP by UDP-glucose pyrophosphorylase (UGP). After the glucose is covalently attached to an aglycone by a UDP-glycosyltransferases (UGT) enzyme, the free uridine diphosphate (UDP) is recycled to uridine triphosphate (UTP) by the action of nucleotide diphosphate kinase (NDK). In one embodiment of the invention, a yeast cell may be engineered to upregulate UDPG recycling, which in one preferred embodiment may increase levels of UDPG substrate for co-expressed UGTs configured to generate cannabinoid glycosides. In one preferred embodiment, a yeast cell, 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 species, mixes of species, hybrids of different species or clones may be used to generate a suspension culture. In certain cases, such fermenters may include large industrial-scale fermenters allowing for a large quantity of yeast cells to be grown. In this embodiment, 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, which may establish a cell culture having a consistent rate of UDPG production. 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.

[0039] As noted, in certain embodiment, the production of cannabinoid glycosides may be generated from cannabinoid compounds derived from of Cannabis, such as Cannabis sativa or hemp, as well as other plants which may be utilized with the inventive technology. In certain otherembodiment, the production of cannabinoid glycosides may be generated from cannabinoid compounds generated synthetically. In alternative embodiments, cannabinoid glycosides may be generated from cannabinoid compounds synthesized in vitro, or for example in a bioreactor system. In alternative embodiments, cannabinoid glycosides may be generated from cannabinoid compounds produced in a yeast cell, for example in a yeast cell that has been genetically modified to include one or more cannabinoid biosynthesis pathways. In certain preferred embodiments, Cannabis plant material may be harvested and undergo cannabinoid extraction through one or more of the methods generally known in the art. These extracted cannabinoids may be introduced into a genetically modified yeast suspension cell culture to be further modified, in some embodiment to express one or more heterologous UGTs having glycosylation activity directed towards one or more cannabinoids such as CBD or THC and the like. This water-soluble cannabinoid glycoside may preferably be a THC-glycoside or a CBD glycoside, or even a THCA-glycoside or a CBDA-glycoside, among others.

[0040] The water-soluble cannabinoid glycosides may be extracted from the cell cultures supematant / media, or from the cells. In this preferred embodiment, a transformed yeast cells may be lysed such that accumulated cannabinoid glycosides are released to the surrounding lysate. Additional steps may include treating this lysate. In other embodiment, 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 may be further isolated and purified. In one preferred embodiment, the culture’s supematant / media, or the cell’s lysate maybe processed utilizing affinity chromatography, or other purification methods. In this preferred embodiment, an affinity column having a ligand configured to bind with one or more of the cannabinoid glycosides, for example, through association with the glycoside functional group, among others, 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. In some embodiments, non-binding and non-specific binding proteins that may have been present in the lysate may be removed. Finally, 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.In yet another separate embodiment, the water-soluble cannabinoid glycosides may be passively and / or actively excreted from a cell, and preferably a yeast cell.

[0041] In one exemplary model, an ATP -binding cassette transporter (ABC transporters) or other similar molecular structure may recognize the glucuronic acid functional group (conjugate) on the transiently modified cannabinoid and actively transport it into the surrounding media. (Examples and sequences for ABC transporters are generally described in Sayre et al. PCT / US 18 / 24409 and PCT / US18 / 41710, such examples and sequences being incorporated herein by reference). In this embodiment, a yeast cell culture may be allowed to grow until an output parameter is reached. In one example, an output parameter may include allowing the yeast cell culture to grow until a desired cell / optical density is reached, or a desired level of cannabinoid glycosides is reached. In this embodiment, the culture media containing the cannabinoid glycosides may be harvested for later cannabinoid extraction. In some embodiments, this harvested media may be treated in a manner similar to the lysate generally described above. Additionally, the transiently modified cannabinoids present in the raw and / or treated media may be isolated and purified, for example, through affinity chromatography in a manner similar to that described above.

[0042] In another embodiment, the invention may include a method of upregulating UDPG recycling in a yeast cell, which in one preferred embodiment may increase levels of UDPG substrate for a co-expressed UGT’s configured to generate cannabinoid glycoside compounds.

[0043] In another embodiment, the invention may include a yeast cell expressing a heterologous nucleotide sequence, operably linked to a promoter, encoding a phosphoglucomutase enzyme that catalyzes the conversion from glucose-6-phosphate to glucose- 1 -phosphate.

[0044] In another embodiment, the invention may include a method of upregulating the conversion of glucose-6-phosphate to glucose- 1 -phosphate in a yeast cell by expressing a heterologous nucleotide sequence, operably linked to a promoter, encoding a phosphoglucomutase enzyme that catalyzes the conversion from glucose-6-phosphate to glucose- 1 -phosphate.

[0045] In another embodiment, the invention may include a yeast cell, and preferably aP. pastoris yeast cell, expressing a heterologous nucleotide sequence, operably linked to a promoter, encoding phosphoglucomutase (PGM2) (SEQ ID NO. 1) from S. cerevisiae, which catalyzes the conversion from glucose-6-phosphate to glucose- 1 -phosphate.

[0046] In another embodiment, the invention may include a method of upregulating the conversion of glucose-6-phosphate to glucose- 1 -phosphate in a yeast cell, and preferably a P. pastoris yeastcell, expressing a heterologous nucleotide sequence, operably linked to a promoter, encoding phosphoglucomutase (PGM2) (SEQ ID NO. 1) from S. cerevisiae, which catalyzes the conversion from glucose-6-phosphate to glucose- 1 -phosphate.

[0047] In another embodiment, the invention may include a yeast cell expressing a heterologous nucleotide sequence, operably linked to a promoter, encoding a pyrophosphorylase enzyme that catalyzes the formation of UDPG from glucose 1 -phosphate and UTP.

[0048] In another embodiment, the invention may include a method of upregulating the formation of UDPG from glucose 1 -phosphate and UTP by expressing in a yeast cell a heterologous nucleotide sequence, operably linked to a promoter, encoding a pyrophosphorylase enzyme that catalyzes the formation of UDPG from glucose 1 -phosphate and UTP.

[0049] In another embodiment, the invention may include a yeast cell, and preferably a / < pastoris yeast cell, expressing a heterologous nucleotide sequence, operably linked to a promoter, encoding UDP-glucose pyrophosphorylase (UGP1) protein (SEQ ID NO. 5) from S. cerevisiae, which catalyzes the formation of UDPG from glucose 1 -phosphate and UTP.

[0050] In another embodiment, the invention may include a method of upregulating the formation of UDPG from glucose 1 -phosphate and UTP by expressing in a yeast cell a heterologous nucleotide sequence, operably linked to a promoter, UDP-glucose pyrophosphorylase (UGP1) protein (SEQ ID NO. 5) from S. cerevisiae, which catalyzes the formation of UDPG from glucose 1 -phosphate and UTP.

[0051] In another embodiment, the invention may include a yeast cell expressing a heterologous nucleotide sequence, operably linked to a promoter, encoding a phosphoglucomutase enzyme that catalyzes the conversion from glucose-6-phosphate to glucose- 1 -phosphate, and pyrophosphorylase enzyme that catalyzes the formation of UDPG from glucose 1 -phosphate and UTP.

[0052] In another embodiment, the invention may include a yeast cell, and preferably a / < pastoris yeast cell, expressing a heterologous nucleotide sequence, operably linked to a promoter, encoding:

[0053] - phosphoglucomutase (PGM2) (SEQ ID NO. 1) from . cerevisiae, which catalyzes the conversion from glucose-6-phosphate to glucose- 1 -phosphate;

[0054] and- UDP-glucose pyrophosphorylase (UGP1) protein (SEQ ID NO. 5) from S. cerevisiae, which catalyzes the formation of UDPG from glucose 1-phosphate and UTP.

[0055] In another embodiment, the invention may include a method of upregulating UDPG recycling in a yeast cell, expressing a heterologous nucleotide sequence, operably linked to a promoter, encoding a phosphoglucomutase enzyme that catalyzes the conversion from glucose-6-phosphate to glucose- 1-phosphate, and pyrophosphorylase enzyme that catalyzes the formation of UDPG from glucose 1-phosphate and UTP.

[0056] In another embodiment, the invention may include a method of upregulating UDPG recycling in a yeast cell, expressing a heterologous nucleotide sequence, operably linked to a promoter, encoding:

[0057] - phosphoglucomutase (PGM2) (SEQ ID NO. 1) from 5. cerevisiae which catalyzes the conversion from glucose-6-phosphate to glucose- 1-phosphate; and

[0058] - UDP-glucose pyrophosphorylase (UGP1) protein (SEQ ID NO. 5) from S.

[0059] cerevisiae, which catalyzes the formation of UDPG from glucose 1-phosphate and UTP.

[0060] In another example, the invention may include an expression vector expressing PGM2 (SEQ ID NO. 1) and UGP1 (SEQ ID NO. 4) genes from S. cerevisiae, that may be integrated into a UGT-expressing yeast cell, and preferably a P. Pastoris yeast cell, expressing a heterologous nucleotide sequence, operably linked to a promoter, encoding a UGT having glycosylation activity towards one or more cannabinoid compounds.

[0061] In another embodiment, the increased levels of UDPG in the yeast cell may be used as a substrate by a co-expressed UGT having glycosylation activity towards a cannabinoid compound, to form cannabinoid glycoside compounds. One embodiment of the present invention incorporates by reference the sequences for novel UDP-glucose glucosyl transferases (UDP-UGTs or UGTs) enzymes having glycosylation activity towards one or more cannabinoid compounds identified in 17 / 189,063. Specifically, in one preferred embodiment, the present invention incorporates by reference amino acid sequences identified as SEQ ID NOs. 1-9181, Table 1-2, and UGTs having 90% sequence identity with SEQ ID NOs. 1-9181, that have glycosylation activity towards one or more cannabinoid compounds, and preferably THC and CBD identified in 17 / 189,063. Forexample, in this embodiment a yeast cell, such as a P. pastoris yeast cell, may be 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. SEQ ID NOs. 1-9181, and a nucleotide sequence encoding an amino acid sequence having 90% sequence identity with SEQ ID NOs. 1-9181, operably linked to a promoter identified in 17 / 189,063. In another embodiment of the present invention incorporates by reference the sequences for novel UDP-glucosyltransferases (UDP-UGTs or UGTs) enzymes having glycosylation activity towards one or more cannabinoid compounds identified in PCT / US2018 / 041710, Specifically, in one preferred embodiment, the present invention incorporates by reference SEQ ID NOs. 7, 27, 29, 31, 33, 35, 37, 39, 55, 57, 59, 61, and 63. For example, in this embodiment a yeast cell, such as a P. pastoris yeast cell, may be 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. 7, 27, 29, 31, 33, 35, 37, 39, 55, 57, 59, 61, and 63, and a nucleotide sequence encoding an amino acid sequence having 90% sequence identity with SEQ ID NOs. SEQ ID NOs. 7, 27, 29, 31, 33, 35, 37, 39, 55, 57, 59, 61, and 63, operably linked to a promoter identified in PCT / US2018 / 041710.

[0062] In another embodiment, the invention may include a yeast cell expressing a nucleotide sequence, operably linked to a promoter, encoding an endogenous phosphoglucomutase enzyme that catalyzes the conversion from glucose-6-phosphate to glucose- 1 -phosphate, and an endogenous pyrophosphorylase enzyme that catalyzes the formation of UDPG from glucose 1-phosphate and UTP. In a preferred embodiment, the endogenous enzyme may be endogenous from Pichia pastoris, S. cerevisiae, or K. marxianus.

[0063] In another example, the invention may include an expression vector expressing PGM2 (SEQ ID NO. 1) and UGP1 (SEQ ID NO. 4) genes from S. cerevisiae, that may be integrated into a UGT-expressing yeast cell, and preferably a S. cerevisiae yeast cell, also expressing a nucleotide sequence, operably linked to a promoter, encoding a heterologous UGT having glycosylation activity towards one or more cannabinoid compounds.

[0064] DEFINITIONS

[0065] As used herein, “upregulate” means a positive regulatory effect on gene expression. As further used herein, “upregulate” means increased conversion of glucose to UDPG in a cell, andalso means an increase production of intermediates and UDPG in a cell, and preferably a yeast cell.

[0066] As used herein, a “cannabinoid” is a chemical compound (such as cannabinol, THC or cannabidiol) that may be produced synthetically, or may be found in the plant species Cannabis among others like: Echinacea, Acmella Oleracea, Helichrysum Umbraculigerunr, Radula Marginata (Liverwort) and Theobroma Cacao, and metabolites and synthetic analogues thereof that may or may not have psychoactive properties. A phytocannabinoids includes cannabinoid derived from plants. 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). Hence 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.

[0067] 5,227,537, incorporated by reference); (3S,4R)-7-hydroxy-A6-tetrahydrocannabinol homologs and derivatives described in U.S. Pat. No. 4,876,276, incorporated by reference; (+)-4-[4-DMH-2,6-diacetoxy-phenyl]-2-carboxy-6,6-dimethylbicyclo[3.1.1]hept-2-en, and other 4-phenylpinene derivatives disclosed in U.S. Pat. No. 5,434,295, which is incorporated by reference; and cannabidiol (-)(CBD) analogs such as (-)CBD-monomethylether, (-)CBD dimethyl ether; (-)CBD diacetate; (-)3'-acetyl-CBD monoacetate; and ±AF11, all of which are disclosed in Consroe et al., J. Clin. Phannacol. 21:428S-436S, 1981, which is also incorporated by reference. Many other cannabinoids are similarly disclosed in Agurell et al., Pharmacol. Rev. 38:31-43, 1986, which is also incorporated by reference.

[0068] Examples of 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-tetrahydrocannabivarin, delta-9- tetrahydrocannabiorcolic acid, delta-9-tetrahydrocannabiorcol,delta-7-cis-iso- tetrahydrocannabivarin, delta-8-tetrahydrocannabiniolic acid, delta-8- tetrahydrocannabinol, cannabicyclolic acid, cannabicylovarin, cannabielsoic acid A, cannabielsoic acid B, cannabinolic acid, cannabinol methylether, cannabinol-C4, cannabinol-C2, cannabiorcol, 10-ethoxy-9-hydroxy-delta-6a-tetrahydrocannabinol, 8,9-dihydroxy-delta-6a-tetrahydrocannabinol, cannabitriol varin, ethoxy- cannabitriolvarin, dehydrocannabifuran, cannabifuran, cannabichromanon, cannabicitran, 10-oxo-delta-6a-tetrahydrocannabinol, delta-9-cis- tetrahydrocannabinol, 3, 4, 5, 6-tetrahydro-7-hydroxy-alpha-alpha-2-trimethyl-9-n- propyl-2, 6-methano-2H-l -benzox ocin-5-methanol-cannabiripsol,trihydroxy-delta-9-tetrahydrocannabinol, and cannabinol. Examples of cannabinoids within the context of this disclosure include tetrahydrocannabinol and cannabidiol. The term “cannabinoid” may also include different modified forms of a cannabinoid such as a hydroxylated cannabinoid or cannabinoid carboxylic acid. For example, if 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.

[0069] The term “endocannabinoid” refers to compounds including arachidonoyl ethanolamide (anandamide, AEA), 2-arachidonoyl ethanolamide (2-AG), 1 -arachidonoyl ethanolamide (1 -AG), and docosahexaenoyl ethanolamide (DHEA, synaptamide), oleoyl ethanolamide (OEA), eicsapentaenoyl ethanolamide, prostaglandin ethanolamide, docosahexaenoyl ethanolamide, linolenoyl ethanolamide, 5(Z),8(Z),1 1 (Z)- eicosatrienoic acid ethanolamide (mead acid ethanol ami de), heptadecanoul ethanolamide, stearoyl ethanolamide, docosaenoyl ethanolamide, nervonoyl ethanolamide, tricosanoyl ethanolamide, lignoceroyl ethanolamide, myristoyl ethanolamide, pentadecanoyl ethanolamide, palmitoleoyl ethanolamide, docosahexaenoic acid (DHA). Particularly preferred endocannabinoids are AEA, 2-AG, 1 -AG, and DHEA.

[0070] Hydroxylation is a chemical process that introduces a hydroxyl group (-OH) into an organic compound. Acetylation is a chemical reaction that adds an acetyl chemical group. Glycosylation is the coupling of a glycosyl donor, to a glycosyl acceptor forming a glycoside. “Glycosylation site” means an position on a molecule that is recognized by a UGT enzyme as a position for attachment of a sugar residue.

[0071] A protein has “homology” or is “homologous” 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. Alternatively, a protein has homology to a second protein if the two proteins have “similar” aminoacid sequences. (Thus, 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. For example, 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%. The property of substantial homology is closely related to specific hybridization. For example, 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. In another embodiment, 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.

[0072] As used herein, “fragment” or “functional fragment” refers to a portion of a peptide or nucleotide sequence that still retains the activity of the wild-type whole.

[0073] The term “homolog”, used with respect to an original enzyme or gene of a first family or species, refers to distinct enzymes or genes of a second family or species which are determined by functional, structural or genomic analyses to be an enzyme or gene of the second family or species which corresponds to the original enzyme or gene of the first family or species. Most often, homologs will have functional, structural or genomic similarities. Techniques are known by which homologs of an enzyme or gene can readily be cloned using genetic probes and PCR. Identity of cloned sequences as homolog can be confirmed using functional assays and / or by genomic mapping of the genes.

[0074] The term “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,” or “control elements,” refer to nucleotide sequences that influence the timing and level / amount of transcription, RNA processing or stability, or translation of theassociated 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.

[0075] As used herein, the term “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. Tissuespecific, 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.

[0076] In a preferred embodiment, the invention may use a strong promoters from P. Pastoris. For example, expression of UGP1 and / or PGM2 may be operably linked to a MDH3 gene promoter (SEQ ID NO. 7) and / or TEF2 gene promoter (SEQ ID NO. 8).

[0077] As used herein, the term “transformation” or “genetically modified” refers to the transfer of one or more nucleic acid molecule(s) into a cell. A yeast cell is “transformed” or “genetically modified” by a nucleic acid molecule transduced into the cell when the nucleic acid molecule becomes stably replicated by the cell. As used herein, the term “transformation” or “genetically modified” encompasses all techniques by which a nucleic acid molecule can be introduced into, such as a yeast cell, including both transient and stable transformation. Transformation methods and techniques are readily known to those of ordinary skill in the art and further are readily reproduced through commercially available kits for the same.

[0078] The term “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. There are various types of vectors including virus, plasmid, bacteriophages, cosmids, and bacteria. An “expression vector” is nucleic acid capable ofreplicating 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. Thus, 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.” In contrast, as described in the examples herein, 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).

[0079] As is known in the art, 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. For example, 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.

[0080] Unless otherwise indicated, 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. Specifically, 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.Amino acid Nucleic acid codons

[0081]

[0082] Moreover, because the proteins are described herein, one can chemically synthesize a polynucleotide which encodes these polypeptides / chimeric proteins. 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. Chrom. 255:137-149 (1983).

[0083] The term “expression,” as used herein, or “expression of a coding sequence” (for example, a gene or a transgene) refer 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. Regulation of gene expression occurs, for example, through controls acting on transcription, translation, RNA transport and processing, degradation of intermediary molecules such as mRNA, or through activation, inactivation, compartmentalization, or degradation of specific protein molecules after they have been made, or by combinations thereof. Gene expression can be measured at the 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).

[0084] The term “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). The term “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. The term “ribonucleic acid” (RNA) 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). The term “deoxyribonucleic acid” (DNA) is inclusive of cDNA, genomic DNA, and DNA-RNA hybrids. The terms “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.

[0085] The term “gene” or “sequence” 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). The term “structural gene” as used herein is intended to mean a DNA sequence that is transcribed into mRNA which is then translatedinto a sequence of amino acids characteristic of a specific polypeptide. It should be noted that any reference to a SEQ ID, or sequence specifically encompasses that sequence, as well as all corresponding sequences that correspond to that first sequence. For example, for any amino acid sequence identified, the specific specifically includes all compatible nucleotide (DNA and RNA) sequences that give rise to that amino acid sequence or protein, and vice versa.

[0086] 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, intemucleotide modifications (e.g., uncharged linkages: for example, methyl phosphonates, phosphotri esters, phosphoramidates, carbamates, etc.; charged linkages: for example, phosphorothioates, phosphorodithioates, etc.; pendent moieties: for example, peptides; intercal ators: for example, acridine, psoralen, etc.; chelators; alkylators; and modified linkages: for example, alpha anomeric nucleic acids, etc.). The term “nucleic acid molecule” also includes any topological conformation, including single-stranded, double-stranded, partially duplexed, triplexed, hair-pinned, circular, and padlocked conformations.

[0087] The term “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.

[0088] 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. As used herein, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise.

[0089] As used herein, “heterologous” or “exogenous” in reference to a nucleic acid is a nucleic acid that originates from a foreign species, oris 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.By “host cell” is meant a cell which contains an introduced nucleic acid construct and supports the replication and / or expression of the construct.

[0090] REFERENCES

[0091] 1. Pandey, RP, Parajuli, P, Koirala, N, Lee, JH, Park, YI, and Sohng, JK (2014). Glucosylation of isoflavonoids in engineered Escherichia coli. Mol Cells 37, 172-177. PMID: 24599002

[0092] 2. Yamaguchi, T, and Asano, Y (2018). Prunasin production using engineered Escherichia coli expressing UGT85A47 from Japanese apricot and UDP-glucose biosynthetic enzyme genes. Biosci Biotechnol Biochem 82, 2021-2029. PMID: 30027801

[0093] 3. Shrestha, A, Pandey, RP, Dhakal, D, Parajuli, P, and Sohng, JK (2018). Biosynthesis of flavone C-glucosides in engineered Escherichia coli. Appl Microbiol Biotechnol 102, 1251-1267. PMID: 29308528

[0094] 4. Wang, JH, Wang, D, Li, WX, Huang, Y, Dai, ZB, and Zhang, XL (2019).

[0095] [Optimization of UDP-glucose supply module and production of ginsenoside Fl in Saccharomyces cerevisiae]. Zhongguo Zhong Yao Za Zhi 44, 4596-4604. PMID: 31872653

[0096] 5. De Bruyn, F, Maertens, J, Beauprez, J, Soetaert, W, and De Mey, M (2015). Biotechnological advances in UDP-sugar based glycosylation of small molecules. Biotechnology Advances 33, 288-302. PMID:

[0097] 6. Wang, P, etal. (2019). Synthesizing ginsenoside Rh2 in Saccharomyces cerevisiae cell factory at high-efficiency. Cell Discov 5, 5. PMID: 30652026

[0098] 7. Terasaka, K, Mizutani, Y, Nagatsu, A, and Mizukami, H (2012). In situ UDP-glucose regeneration unravels diverse functions of plant secondary product glycosyltransferases. FEBS Letters 586, 4344-4350. PMID:

[0099] 8. Zhuang, Y, et al- (2017). Biosynthesis of plant-derived ginsenoside Rh2 in yeast via repurposing a key promiscuous microbial enzyme. Metabolic Engineering 42, 25-32.

Claims

CLAIMSWhat is claimed is:

1. A yeast cell expressing a heterologous nucleotide sequence, operably linked to a promoter encoding:- a phosphoglucomutase (PGM) enzyme, or a fragment thereof, and / or- a UDP -glucose pyrophosphorylase (UGP) enzyme, or a fragment thereof; and- wherein said PGM and / or UGP enzymes upregulate glucose to UDP-Glucose (UDPG) recycling in said yeast cell.

2. The yeast cell of claim 1, wherein said yeast cell comprises a Pichia pastoris yeast cell.

3. The yeast cell of any of claims 1-2, wherein said PGM enzyme comprises a PGM2 enzyme from Saccharomyces cerevisiae.

4. The yeast cell of any of claims 1-3, wherein said PGM enzyme comprises a PGM2 enzyme according to SEQ ID NO. 2, or a sequence having 80% or more sequence identity with SEQ ID NO. 1-4.

5. The yeast cell of any of claims 1-2, wherein said UGP enzyme comprises a UGP1 enzyme from Saccharomyces cerevisiae.

6. The yeast cell of any of claims 1-2 and 5, wherein said UGP enzyme comprises a UGP1 enzyme according to SEQ ID NO. 4-5, or a sequence having 80% or more sequence identity with SEQ ID NO. 4-5.

7. The yeast cell of any of claims 1 and 4-6, wherein the nucleotide sequence is codon optimized for expression in a yeast cell.

8. A yeast cell expressing a heterologous nucleotide sequence, operably linked to a promoter encoding:- a phosphoglucomutase (PGM) enzyme, or a fragment thereof, which catalyzes the conversion from glucose-6-phosphate to glucose- 1 -phosphate, and / or- a UDP-glucose pyrophosphorylase (UGP) enzyme, or a fragment thereof, which catalyzes the formation of UDPG from glucose 1 -phosphate and UTP; and- a UDP-glucosyltransferases (UGT) having glycosylation activity towards one or more cannabinoid compounds.

9. The yeast cell of claim 8, wherein said yeast cell comprises a Pichia pastoris yeast cell.

10. The yeast cell of any of claims 8-9, wherein said PGM enzyme comprises a PGM2 enzyme from Saccharomyces cerevisiae.

11. The yeast cell of any of claims 9-10, wherein said PGM enzyme comprises a PGM2 enzyme according to SEQ ID NO. 1-2, or a sequence having 80% or more sequence identity with SEQ ID NO. 1-1.

12. The yeast cell of any of claims 8-9, wherein said UGP enzyme comprises a UGP1 enzyme from Saccharomyces cerevisiae.

13. The yeast cell of any of claims 8-9 and 12, wherein said UGP enzyme comprises a UGP1 enzyme according to SEQ ID NO. 4-5, or a sequence having 80% or more sequence identity with SEQ ID NO. 4-5.

14. The yeast cell of any of claims 8 and 11-13, wherein the nucleotide sequence is codon optimized for expression in a yeast cell.

15. The yeast cell of any of claim 8-14, wherein said one or more cannabinoid compounds comprises a cannabinoid compound having at least one glycosylation site.

16. The yeast cell of any of claim 8-14, wherein said one or more cannabinoid compounds comprises a cannabinoid compound selected from the group consisting of: cannabidiol (CBD),cannabidiolic acid (CBDA), delta-9-tetrahydrocannabinol (THC), and tetrahydrocannabinolic acid (THCA).

17. The yeast cell of any of claim 8-14, wherein said one or more cannabinoid compounds comprises a cannabinoid compound selected from the group consisting of: delta-A9— tetrahydrocannabinol (THC), delta- 8— tetrahydrocannabinol (Delta-8-THC), 11 -Hydroxy- A9-tetrahydrocannabinol (11-OH-THC), tetrahydrocannabinolic acid (THCA), cannabidiol (CBD), cannabichromene (CBC), cannabigerol (CBG), cannabigerolic acid (CBGA), cannabinol (CBN), cannabidiolic acid (CBDA), cannabidiolic acid (CBDA), cannabicyclol (CBL), cannabivarin (CBV), tetrahydrocannabivarin (THCV), cannabigerivarin (CBGV), cannabichromevarin (CBCV), cannabidivarin (CBDV), cannabicyclol (CBL), cannabielsoin (CBE), cannabifuran (CBF); and cannabinodiol (CBDN).

18. A yeast cell expressing a nucleotide sequence, operably linked to a promoter, encoding:- a phosphoglucomutase (PGM) enzyme according to SEQ ID NO. 1;- a UDP -glucose pyrophosphorylase (UGP) enzyme according to SEQ ID NO. 4;- wherein said PGM and / or UGP enzymes upregulate glucose to UDP-Glucose (UDPG) recycling in said yeast cell; and- a UDP-glucosyltransferases (UGT) having glycosylation activity towards one or more cannabinoid compounds.

19. A method upregulating glucose to UDP-Glucose (UDPG) recycling comprising the steps of:- providing a yeast cell expressing a heterologous nucleotide sequence, operably linked to a promoter encoding:- a phosphoglucomutase (PGM) enzyme, or a fragment thereof, and / or - a UDP -glucose pyrophosphorylase (UGP) enzyme, or a fragment thereof; - wherein said PGM and / or UGP enzymes upregulate glucose to UDP-Glucose (UDPG) recycling in said yeast cell.

20. The method of claim 19, wherein said yeast cell comprises aPichia pastoris yeast cell.

21. The method of any of claims 19-29, wherein said PGM enzyme comprises a PGM2 enzyme from Saccharomyces cerevisiae.

22. The method of any of claims 19-21, wherein said PGM enzyme comprises a PGM2 enzyme according to SEQ ID NO. 1-2, or a sequence having 80% or more sequence identity with SEQ ID NO. 1-2.

23. The method of any of claims 19-20, wherein said UGP enzyme comprises a UGP1 enzyme from Saccharomyces cerevisiae.

24. The method cell of any of claims 19-20 and 23, wherein said UGP enzyme comprises a UGP1 enzyme according to SEQ ID NO. 4-5, or a sequence having 80% or more sequence identity with SEQ ID NO. 4-5.

25. The method of any of claims 19 and 22-24, wherein the nucleotide sequence is codon optimized for expression in a yeast cell.

26. A method upregulating glucose to UDP-Glucose (UDPG) recycling in a cannabinoid glycosylation system comprising the steps of:- providing a yeast cell culture expressing a heterologous nucleotide sequence, operably linked to a promoter encoding:- a phosphoglucomutase (PGM) enzyme, or a fragment thereof, which catalyzes the conversion from glucose-6-phosphate to glucose- 1 -phosphate, and / or - a UDP -glucose pyrophosphorylase (UGP) enzyme, or a fragment thereof, which catalyzes the formation of UDPG from glucose 1 -phosphate and UTP; and - a UDP-glucosyltransferases (UGT) having glycosylation activity towards one or more cannabinoid compounds.- incubating one or more cannabinoid compounds having at least one glycosylation site in said yeast cell culture; and- wherein said cannabinoid compounds is converted to a cannabinoid glycoside via the action of the UGT.

27. The method of claim 26, wherein said yeast cell comprises aPichia pastoris yeast cell.

28. The method of any of claims 26-27, wherein said PGM enzyme comprises a PGM2 enzyme from Saccharomyces cerevisiae.

29. The method of any of claims 27-28, wherein said PGM enzyme comprises a PGM2 enzyme according to SEQ ID NO. 1-2, or a sequence having 80% or more sequence identity with SEQ ID NO. 1-2.

30. The yeast of any of claims 26-27, wherein said UGP enzyme comprises a UGP1 enzyme from Saccharomyces cerevisiae.

31. The method of any of claims 26-27 and 30, wherein said UGP enzyme comprises a UGP1 enzyme according to SEQ ID NO. 4-5, or a sequence having 80% or more sequence identity with SEQ ID NO. 4-5.

32. The method of any of claims 26 and 29-31, wherein the nucleotide sequence is codon optimized for expression in a yeast cell.

33. The method of any of claim 26-32, wherein said one or more cannabinoid compounds comprises a cannabinoid compound having at least one glycosylation site.

34. The method of any of claim 26-32, wherein said one or more cannabinoid compounds comprises a cannabinoid compound selected from the group consisting of: cannabidiol (CBD), cannabidiolic acid (CBD A), delta-9-tetrahydrocannabinol (THC), and tetrahydrocannabinolic acid (THCA).

35. The method of any of claim 26-32, wherein said one or more cannabinoid compounds comprises a cannabinoid compound selected from the group consisting of: delta-A9- tetrahydrocannabinol (THC), delta- A8— tetrahydrocannabinol (Delta-8-THC), 11 -Hydroxy- A9-tetrahydrocannabinol (11-OH-THC), tetrahydrocannabinolic acid (THCA), cannabidiol (CBD), cannabichromene (CBC), cannabigerol (CBG), cannabigerolic acid (CBGA), cannabinol (CBN), cannabidiolic acid (CBDA), cannabidiolic acid (CBDA), cannabicyclol (CBL), cannabivarin (CBV), tetrahydrocannabivarin (THCV), cannabigerivarin (CBGV), cannabichromevarin (CBCV), cannabidivarin (CBDV), cannabicyclol (CBL), cannabielsoin (CBE), cannabifuran (CBF); and cannabinodiol (CBDN).

36. The method of claim 26, wherein said step of incubating comprising introducing a quantity of a cannabinoid to a fermenter containing said yeast cell culture.

37. The method of claim 36, and further comprising the step of isolating cannabinoid compounds yeast cell culture.

38. TheUGT of any of claims 8, 18 and 26, wherein saidUGT is selected from the group consisting of: SEQ ID NO. having glycosylation activity towards a cannabinoid, wherein said UGT is selected from the group consisting of: SEQ ID NOs. 1-9181, and SEQ ID NO. 9208, SEQ ID NO.9210, SEQ ID NO. 9212, SEQ ID NO. 9214, SEQ ID NO. 9216, SEQ ID NO. 9218, SEQ ID NO.9220, SEQ ID NO. 9236, SEQ ID NO. 9238, SEQ ID NO. 9240, SEQ ID NO. 9242, and SEQ ID NO. 9244, as specifically incorporated by reference from US Application No. 17 / 189,063.

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