Genetically modified yeast for the production of cannabigerol acid, cannabichromenic acid, and related cannabinoids.

Abstract

The present invention relates generally to production methods, enzymes and recombinant yeast strains for the biosynthesis of clinically important cannabinoid compounds. TIFF2023511109000035.tif102170

Description

Technical Field

[0001] Cross - reference to related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 963,448, filed on January 20, 2020, which is hereby incorporated by reference in its entirety for all purposes.

[0002] Field of the Invention The present invention generally relates to production methods, enzymes, and recombinant yeast strains for the biosynthesis of cannabigerolic acid, cannabichromenic acid, and related cannabinoids.

Background Art

[0003] Background of the Invention The Cannabis sativa species has been widely cultivated and utilized worldwide for several applications. Currently, cannabinoids are mainly isolated by cultivating large areas of hemp or cannabis plants in agricultural activities around the world. Despite being clinically important, the level of production methods, including synthetic chemical processes, is low.

[0004] Synthetic biology enables the biosynthesis of individual cannabinoids using isolated genetic pathways in engineered microorganisms, enabling the commercial production and mass production of natural cannabinoids and their analogs as high - purity compounds with sufficient biological and pharmacological activities.

Summary of the Invention

[0005] ]> Summary of aspects of the invention This summary highlights certain aspects of the present invention and does not include a description of all aspects of the present invention.

[0006] In one aspect, this disclosure provides methods and materials for producing cannabinoid compounds of interest, such as cannabigerol acid (CBGA) and cannabichromene acid (CBCA), and their decarboxylated derivatives, cannabigerol (CBG) and cannabichromene (CBC). In one aspect, a method for obtaining enantiomerically pure CBC is provided herein. In a further aspect, methods for producing related compounds such as cannabidiolic acid (CBDA), tetrahydrocannabinolic acid (THCA), and their decarboxylated derivatives, tetrahydrocannabinol (THC) and cannabidiol (CBD), are provided herein. This disclosure further provides methods and materials for producing cannabigerovalic acid (CBGVA), cannabiclomevalic acid (CBCVA), tetrahydrocannabivalic acid (THCVA), and cannabidiolvalic acid (CBDVA), and their corresponding decarboxylated derivatives.

[0007] In another aspect, a method for obtaining high-yield CBG from the chemical decarboxylation of CBGA in a reaction involving an organic solvent, such as an alcohol or an alcohol-water mixture, in the presence of a metal catalyst is provided herein. In yet another aspect, a method for obtaining high-yield CBG and additional cannabinoids using a decarboxylase enzyme is provided herein.

[0008] In some embodiments, oleic acid is supplied to a yeast culture preparation that has been genetically modified to express a prenyltransferase, such as the polypeptide of amino acid SEQ ID NO:1; or a polypeptide comprising the amino acid sequence of SEQ ID NO:2, and CBCA synthase, and further modified to overexpress a member of the mevalonate pathway family. Such cells produce enantiomerically pure CBCA, for example, with a purity of greater than 90%, greater than 95%, or greater than 99%. The CBCA can then be decarboxylated enzymatically or chemically to yield CBC. In some embodiments, the chemical decarboxylation is carried out as described in the previous paragraph. In further embodiments, enzymatically pure CBDA or THCA can be prepared using a yeast culture preparation modified to express CBDA synthase or THCA synthase rather than CBCA synthase.

[0009] Oleic acid from any source can be used as a feedstock. In some embodiments, oleic acid is produced from a genetically engineered host cell modified to produce oleic acid, such as a genetically engineered yeast cell. Such cells are described in WO 2018 / 209143. In some embodiments, the yeast culture preparation is modified to produce oleic acid or divalent acid, for example, modified to express an acyl-CoA synthetase, such as an acyl-CoA synthetase that converts hexanoic acid or butanoic acid to hexanoyl-CoA or butanoyl-CoA, oleic acid synthase, and oleic acid cyclase. [Invention 1001] The first exogenous polynucleotide encoding prenyltransferase, and A second exogenous polynucleotide encoding CBCA synthase, CBDA synthase, or THCA synthase. Modified recombinant yeast host cells, including those containing this cell type. [Invention 1002] A modified recombinant yeast host cell according to the present invention 1001, wherein the amino acid sequence of prenyltransferase is SEQ ID NO:1. [Invention 1003] Modified recombinant yeast host cells according to the present invention 1001, wherein the amino acid sequence of prenyltransferase contains SEQ ID NO:2. [Invention 1004] A modified recombinant yeast host cell according to Invention 1001 or 1002, wherein the second exogenous polynucleotide encodes a CBCA synthase, and the CBCA synthase contains an amino acid sequence having at least 95% identity with any one of SEQ ID NO:3-9. [Invention 1005] A modified recombinant yeast host cell according to Invention 1001, wherein the amino acid sequence of prenyltransferase is SEQ ID NO:1 or the amino acid sequence of prenyltransferase comprises SEQ ID NO:2, and the second exogenous polynucleotide encodes CBDA synthase or THCA synthase. [Invention 1006] A modified recombinant yeast host cell according to any of invention 1001 to 1005, which has been genetically modified to overexpress mevalonate pathway enzymes. [Invention 1007] A modified recombinant yeast host cell according to the present invention 1006, wherein one or more mevalonate pathway enzymes are endogenous enzymes. [Invention 1008] A modified recombinant yeast host cell according to Invention 1006, wherein one or more mevalonate pathways are exogenous enzymes not naturally expressed in yeast host cells. [Invention 1009] Modified recombinant yeast host cells according to any of invention 1001-1008, which are modified to overexpress erg10, erg13, thmgr, erg12, erg8, mvd1, idi1, and erg20 F96WN127W, as well as the mvaE and mvaS genes derived from Enterococcus faecalis. [Invention 1010] Methods for producing cannabinoids, including the following steps: Expressing a polynucleotide encoding prenyltransferase, It expresses a polynucleotide encoding CBCA synthase, CBDA synthase, or THCA synthase. A step of providing genetically modified recombinant yeast host cells with olivetolic acid or divalic acid, or a fluorinated or chlorinated analog thereof, for the production of the corresponding acid cannabinoid CBCA, CBDA, or THCA, or a fluorinated or chlorinated analog thereof, wherein the acid cannabinoid is produced by prenylation of olivetolic acid or divalic acid, or an analog thereof; A step of enzymatically or chemically decarboxylating the acidic cannabinoid in order to produce the corresponding compound CBC, CBD, or THC, or an analogue thereof. [Invention 1011] The method of the present invention 1010, wherein recombinant yeast host cells express a polynucleotide encoding CBCA synthase. [Invention 1012] The method of the present invention 1011, wherein the produced CBCA has a CBCA enantiomer purity of over 96%. [Invention 1013] The method of the present invention 1010, 1011, or 1012, wherein the decarboxylation step is performed on an extract of yeast host cells prepared using an extraction reagent containing an organic solvent or an organic solvent / water mixture. [Invention 1014] Any method according to item 1010 to 1013 of the present invention, wherein the decarboxylation step includes incubating the extract at a temperature of 20°C to 100°C in the presence of a metal catalyst. [Invention 1015] The method of the present invention 1014, wherein the metal catalyst is zinc, molybdenum, nickel, copper, platinum, palladium, or iron. [Invention 1016] The method of the present invention 1014 or 1015, wherein the metal catalyst is provided as a metal-loaded zeolite catalyst. [Invention 1017] The method of the present invention 1010 or 1011, wherein the decarboxylation step is carried out with a decarboxylase enzyme. [Invention 1018] The method of Invention 1017, wherein the decarboxylase is Aspergillus nidulans orsB decarboxylase, Aspergillus clavatus-derived PatG enzyme, or Enterobacter cloacae-derived 3,4-dihydroxybenzoic acid decarboxylase. [Invention 1019] Roseburia hominis expresses a polynucleotide encoding an acyl-CoA synthase selected from the group consisting of butanoyl-CoA transferase, revS, CsAAE3, and CsAAE1; It expresses a polynucleotide encoding olivetolate synthase; and It expresses a polynucleotide that encodes olivetolate cyclase. A method according to any of items 1010 to 1018 of the present invention, wherein olivetolic acid or divalic acid, or an analog thereof, is produced by a host cell that has been genetically modified in such a manner. [Invention 1020] The method of the present invention 1019, wherein the acyl-CoA synthase is Rosebria hominis butanoyl-CoA transferase. [Invention 1021] The method of the present invention 1019, wherein the acyl-CoA synthase comprises an amino acid sequence having at least 95% identity with SEQ ID NO:20; the olivetolate synthase comprises an amino acid sequence having at least 95% amino acid sequence identity with SEQ ID NO:13; and the olivetolate cyclase comprises an amino acid sequence having at least 95% identity with SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:16. [Invention 1022] A method for obtaining a yield of 60% or more of CBG from CBGA-producing genetically modified yeast cells, including the following steps: The steps include: preparing a yeast cell extract using an extraction reagent containing an organic solvent or an organic solvent / water mixture; and The step of incubating the extract at a temperature of 20°C to 100°C in the presence of a metal catalyst. [Invention 1023] The method of the present invention 1022, wherein the organic solvent is an alcohol-water mixture. [Invention 1024] The method of the present invention 1022 or 1023, wherein the metal catalyst is zinc, molybdenum, nickel, copper, platinum, palladium, or iron; or a salt thereof. [Invention 1025] A method according to any one of the present invention 1022 to 1024, wherein the metal catalyst is provided as a metal-loaded zeolite catalyst. [Invention 1026] A modified recombinant yeast host cell containing an exogenous polynucleotide encoding prenyltransferase, The amino acid sequence of the prenyltransferase is SEQ ID NO:1. The aforementioned modified recombinant yeast host cells. [Invention 1027] A modified recombinant yeast host cell containing an exogenous polynucleotide encoding a prenyltransferase polypeptide, The prenyltransferase polypeptide contains the amino acid sequence of SEQ ID NO:2. The aforementioned modified recombinant yeast host cells. [Invention 1028] A modified recombinant yeast host cell according to Invention 1026 or 1027, further comprising a second exogenous polynucleotide encoding CBCA synthase, CBDA synthase, or THCA synthase. [Brief explanation of the drawing]

[0010] [Figure 1] Describe the biosynthetic scheme for producing CBGA and CBCA. [Figure 2] This document outlines analytical and preparative HPLC procedures for identifying biologically active CBC enantiomers. [Figure 3] This diagram illustrates the fermentation results of CBGA production for conversion to CBCA using CBCA synthase. [Modes for carrying out the invention]

[0011] Detailed description of the invention Technical terms Unless otherwise defined, all technical terms, notations, and other scientific terms used herein are intended to have the meanings that a person skilled in the art would ordinarily understand. In some cases, terms that have a commonly understood meaning are defined herein for clarity and / or ease of reference, and the inclusion of such terms herein should not necessarily be construed as representing a substantial difference from the meanings commonly understood in the art.

[0012] As used herein, the terms “cannabinoid,” “cannabinoid compound,” and “cannabinoid product” are used interchangeably to refer to molecules comprising a polyketide moiety, e.g., olivetolic acid or another 2-alkyl-4,6-dihydroxybenzoic acid, e.g., divalic acid, and a terpene-derived moiety, e.g., a geranyl group. The geranyl group is derived from the diphosphate ester of geraniol, known as geranyl pyrophosphate, and can react with olivetolic acid-type compounds to form the acidic cannabinoid cannabigerol acid (CBGA) and CBGA analogs. CBGA can be converted enzymatically (e.g., by decarboxylation by in vivo or in vitro enzymatic treatment) or chemically (e.g., by decarboxylation using a metal catalyst and heating) to further bioactive cannabinoids. Similarly, 19-carbon precursors CBGVA and CBGVA analogs can be generated by this disclosure and converted to further bioactive cannabinoids by the methods described herein. Halogenated, for example, fluorinated or chlorinated, deuterated, or tritiated analogs can be produced using the methods of the present invention with substrates and reagents described in PCT application number PCT / US2019 / 059237, which is incorporated herein by reference. TIFF0007875122000001.tif38128

[0013] The term cannabinoid includes acidic cannabinoids and neutral cannabinoids. The term "acidic cannabinoid" refers to cannabinoids that have a carboxylic acid moiety. The carboxylic acid moiety can be in protonated form (i.e., as -COOH) or in deprotonated form (i.e., as the carboxylic acid ion -COO). - They can exist as such. Examples of acidic cannabinoids include, but are not limited to, cannabigerolic acid, cannabidiolic acid, cannabichromenic acid, and Δ9-tetrahydrocannabinolic acid. The term "neutral cannabinoid" does not include the carboxylic acid moiety (i.e., partial -COOH or -COO). - This refers to cannabinoids (excluding those containing hydroxylase). Examples of neutral cannabinoids include, but are not limited to, cannabigerol, cannabidiol, cannabichromene, and Δ9-tetrahydrocannabinol.

[0014] The term "2-alkyl-4,6-dihydroxybenzoic acid" refers to the following structure This refers to a compound containing TIFF0007875122000002.tif18128, where R is C1~C 20The alkyl group is, in some embodiments, halogenated, hydroxylated, deuterated, and / or tritiated. Examples of 2-alkyl-4,6-dihydroxybenzoic acid include, but are not limited to, olivetolic acid (i.e., 2-pentyl-4,6-dihydroxybenzoic acid; CAS registry number 491-72-5) and divalic acid (i.e., 2-propyl-4,6-dihydroxybenzoic acid; CAS registry number 4707-50-0). Olivetolic acid analogs include other 2-alkyl-4,6-dihydroxybenzoic acid derivatives, as well as substituted resorcinols, including, but not limited to, 5-halomethylresorcinol, 5-haloethylresorcinol, 5-halopropylresorcinol, 5-halohexylresorcinol, 5-haloheptylresorcinol, 5-halooctylresorcinol, and 5-halononylresorcinol. Other analogues include deuterated or tritiated forms (from)

[0015] The term "prenyl moiety" refers to a substituent containing at least one methylbutenyl group (e.g., 2-methylbuta-2-en-1-yl group). In many cases, terpene natural products and other compounds are obtained by biochemically synthesizing the prenyl moiety from isopentenyl pyrophosphate and / or isopentenyl diphosphate. Examples of prenyl moieties include, but are not limited to, prenyl, geranyl, myrcenyl, osimenyl, farnesyl, and geranylgeranyl.

[0016] The term "geraniol" refers to (2E)-3,7-dimethyl-2,6-octadien-1-ol (CAS registry number 106-24-1). The term "geranylation" refers to the covalent bonding of a 3,7-dimethyl-2,6-octadien-1-yl group to a molecule such as 2-alkyl-4,6-hydroxybenzoic acid. Geranylation can be carried out chemically or enzymatically, as described herein.

[0017] The term "2-alkyl-4,6-dihydroxybenzoic acid" refers to the following structure Refers to a compound having TIFF0007875122000003.tif18128, wherein R is C1-C 20 an alkyl group. Examples of 2-alkyl-4,6-dihydroxybenzoic acid include, but are not limited to, oleuroic acid (i.e., 2-pentyl-4,6-dihydroxybenzoic acid; CAS registration number 491-72-5) and divaric acid (i.e., 2-propyl-4,6-dihydroxybenzoic acid; CAS registration number 4707-50-0). Examples of oleuroic acid analogs include other 2-alkyl-4,6-dihydroxybenzoic acid derivatives, as well as substituted resorcinols such as 5-methylresorcinol, 5-ethylresorcinol, 5-propylresorcinol, 5-hexylresorcinol, 5-heptylresorcinol, 5-octylresorcinol, and 5-nonylresorcinol.

[0018] The term "alkyl" refers to a straight-chain or branched saturated aliphatic group, either by itself or as part of another substituent. Alkyl can have any number of carbons, e.g., C 1~2 、C 1~3 、C 1~4 、C 1~5 、C 1~6 、C 1~7 、C 1~8 、C 1~9 、C 1~10 、C 2~3 、C 2~4 、C 2~5 、C 2~6 、C 3~4 、C 3~5 、C 3~6 、C 4~5 、C 4~6 、and C 5~6 can be included. For example, C 1~6 alkyl includes, but is not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, etc. Alkyl can also refer to an alkyl group having up to 20 carbons, such as, but not limited to, heptyl, octyl, nonyl, decyl, etc.

[0019] The term "alkenyl" refers to an alkyl group as defined herein, comprising one or more carbon-carbon double bonds, either by itself or as part of another substituent. Examples of alkenyl groups include, but are not limited to, vinyl (i.e., ethenyl), clotyl (i.e., buta-2-en-1-yl), and penta-1,3-dien-1-yl. The alkenyl moiety may be further substituted with, for example, aryl substituents (e.g., phenyl or hydroxyphenyl in the case of 4-hydroxystyryl).

[0020] The terms "halogen" and "halo" refer to a fluorine atom, chlorine atom, bromine atom, or iodine atom, either by itself or as part of another substituent.

[0021] The term "haloalkyl" refers to an alkyl group in which some or all of its hydrogen atoms are replaced by halogen atoms, either by themselves or as part of another substituent. Like alkyl groups, a haloalkyl group can have any suitable number of carbon atoms, for example, C 1~6 They may have such properties. For example, haloalkyls include trifluoromethyl and fluoromethyl. In some cases, the term "perfluoro" is used to define a compound or group in which all hydrogens are replaced by fluorine. For example, perfluoromethyl refers to 1,1,1-trifluoromethyl.

[0022] The term "hydroxyalkyl" refers to an alkyl group in which some or all of its hydrogen atoms are replaced by a hydroxyl group (i.e., an -OH group), either by itself or as part of another substituent. Like alkyl and haloalkyl groups, a hydroxyalkyl group can have any suitable number of carbon atoms, e.g., C 1~6 It may have.

[0023] The term "deuterated" refers to the replacement of one or more hydrogen atoms with one or more deuterium atoms (i.e., 2 This refers to a substituent (e.g., an alkyl group) that has a hydrogen atom.

[0024] The term "tritiated" refers to the replacement of one or more hydrogen atoms with one or more tritium atoms (i.e., 3 This refers to a substituent (e.g., an alkyl group) that has a hydrogen atom.

[0025] An "organic solvent" refers to a carbon-containing substance that is liquid at ambient temperature and pressure and substantially free of water. Examples of organic solvents include, but are not limited to, alcohols, toluene, methylene chloride, ethyl acetate, acetonitrile, tetrahydrofuran, benzene, chloroform, diethyl ether, dimethylformamide, dimethyl sulfoxide, and petroleum ether.

[0026] The term "acid" refers to a substance that can form a conjugate base of an acid by donating a proton (i.e., a hydrogen cation). Examples of acids include, but are not limited to, mineral acids (e.g., hydrochloric acid, sulfuric acid), carboxylic acids (e.g., acetic acid, formic acid), and sulfonic acids (e.g., methanesulfonic acid, p-toluenesulfonic acid).

[0027] Throughout this specification and the claims, the term “comprise,” or variations such as “comprises” and “comprising,” shall be understood to imply the inclusion of the integer or group of integers described, but not the exclusion of any other integer or group of integers.

[0028] The term “identical” or “identity” percentage for two or more polypeptide sequences refers to two or more sequences or subsequences that, when compared and aligned with respect to the maximum match across a given region, have a predetermined percentage of identical amino acid residues across that region (e.g., at least 70%, at least 75%, at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity). Alignment for determining amino acid sequence identity percentage can be performed in a variety of ways, including, among others, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Geneious, or Megalign (DNASTAR) software. Examples of suitable algorithms for determining sequence identity and similarity percentages include the BLAST 2.0 algorithm described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410 (1990). EMBOSS-Water can also be used to improve alignment accuracy, as can be found at the European Molecular Biology Laboratory - European Bioinformatics Institute (EMBL-EBI). This program uses a Smith-Waterman-based algorithm for global pairwise sequence alignment. An alignment option allows for increasing the gap opening penalty to prevent the introduction of false gaps. For the purposes of this application, EMBOSS-Water is a preferred algorithm for sequence alignment to determine identity percentage.

[0029] As used herein, “conservative” substitution means the substitution of an amino acid in which the charge, hydrophobicity, and / or size of the side chain group are preserved. An exemplary set of amino acids that are interchangeable with each other includes (i) positively charged amino acids such as Lys, Arg, and His; (ii) negatively charged amino acids such as Glu and Asp; (iii) aromatic amino acids Phe, Tyr, and Trp; (iv) nitrogen ring amino acids His and Trp; (v) aliphatic amino acids Gly, Ala, Val, Leu, and Ile; (vi) slightly polar amino acids Met and Cys; (vii) small side chain amino acids Ser, Thr, Asp, Asn, Gly, Ala, Glu, Gln, and Pro; (viii) small hydroxyl amino acids Ser and Thr; and sulfur-containing amino acids Cys and Met. References to amino acid charge in this paragraph refer to the charge at pH 7.0.

[0030] Abbreviations are used in certain cases. For example, the term "CBGA" refers to cannabigerolic acid. Similarly, "OA" refers to olivetolic acid; "CBG" refers to cannabigerol; "CBDA" refers to cannabidiolic acid; "CBD" refers to cannabidiol; and "THC" refers to Δ 9 -Tetrahydrocannabinol (Δ 9 -THC) refers to "Δ 8 -THC" is Δ 8 - Refers to tetrahydrocannabinol; "THCA" means Δ 9 -Tetrahydrocannabinolic acid (Δ 9 -THCA) refers to; "Δ 8 -THCA is Δ 8 - Refers to tetrahydrocannabinolic acid; "CBCA" refers to cannabichromeneic acid; "CBC" refers to cannabichromene; "CBGV" refers to cannabigerovaline; "CBGVA" refers to cannabigerovalic acid; "CBCV" refers to cannabiclomevalin; "CBCVA" refers to cannabiclomevalic acid; "THCV" refers to Δ 9 -Tetrahydrocannabivarin (Δ 9-THCV) refers to Δ 9 -Tetrahydrocannabivaric acid (Δ 9 -THCV) refers to; "GOT" refers to geranyl pyrophosphate olivetolate geranyltransferase; "YAC" refers to yeast artificial chromosome; "IRES" or "intrasequence ribosome entry site" refers to a special sequence that directly promotes ribosome binding and mRNA translation independently of the cap structure; and "HPLC" refers to high-performance liquid chromatography.

[0031] In this specification and the appended claims, the singular forms “a,” “and,” and “the” refer to multiple subjects unless otherwise clearly indicated by the context.

[0032] As used herein, the terms “about” and “approximately” refer to a closed range around a particular number when used to modify that number. For example, if the value is “X”, then “about X” or “approximately X” would refer to values ​​between 0.9X and 1.1X, such as 0.95X and 1.05X, or 0.98X and 1.02X, or 0.99X and 1.01X. Any reference to "about X" or "approximately X" must specifically indicate at least the values ​​X, 0.9X, 0.91X, 0.92X, 0.93X, 0.94X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, 1.05X, 1.06X, 1.07X, 1.08X, 1.09X, and 1.1X, as well as any values ​​within this range.

[0033] For example, the techniques and methods described or referenced herein using conventional methodologies, such as those described in Green et al., Molecular Cloning: A Laboratory Manual 4th edition (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; and Ausubel, et al., Current Protocols in Molecular Biology, through December 2019, John Wiley & Sons, Inc., are generally well understood and commonly used by those skilled in the art. Unless otherwise stated, methods including the use of commercially available kits and reagents are commonly performed according to the manufacturer's specified protocols and / or parameters. Therefore, before describing the methods, expression systems, and uses of this invention, it should be understood that the present invention is not limited to, and is therefore naturally subject to variation, the specific methodologies, protocols, host cell lines, species or genera, constructs, and reagents described.

[0034] overview In one aspect, a method for producing enantiomerically pure forms of CBC, or CBD or THC, is described herein. In some embodiments, the enantiomerically pure forms have at least about 96%, 97%, 98%, 99%, or more of the active CBC enantiomer. In a preferred embodiment, the method involves coupling geranyl pyrophosphate to olivetolic acid in a yeast cell culture containing yeast genetically modified to express a member of the mevalonate pathway to produce CBGA. In some embodiments, CBGA is produced at levels greater than about 1 gram / L. In some embodiments, the yeast cells are further manipulated to convert CBGA to CBCA. CBGA or CBCA (or CBDA or THCA) can also be enzymatically or chemically converted to the corresponding neutral cannabinoid, e.g., CBG or CBC (or CBD or THC). In some embodiments, CBCA is decarboxylated to provide an enantiomerically pure preparation of active CBC. An exemplary pathway for producing CBCA is shown in Figure 1. In some embodiments, the same modifications are made to produce valine-type cannabinoids, using either exogenously supplied divalic acid or divalic acid biosynthesized in the same lineage instead of olivetolic acid. In some embodiments, halogenated or otherwise modified olivetolic acid or divalic acid may be used.

[0035] In a further context, a method for decarboxylating CBGA is described herein, which includes the step of incubating CBGA in a reaction carried out at a temperature, for example, about 20°C to about 100°C, or about 30°C to about 60 or 80°C, in the presence of a metal catalyst and an aqueous solution or organic solvent, such as an alcohol or an alcohol-aqueous solution. In some embodiments, CBGA is decarboxylated with high efficiency, such that at least 70%, or at least 80%, or at least 90% of the CBGA is converted to CBG.

[0036] Offering olivetolic acid or divalic acid In some embodiments, olivetolic acid is enzymatically coupled to geranyl pyrophosate by a prenyltransferase, e.g., GOT (such as a prenyltransferase polypeptide containing the amino acid sequence SEQ ID NO:1 or SEQ ID NO:2) to produce CBGA as an analog compound. In some embodiments, the prenyltransferase includes a region. In some embodiments, olivetolic acid is supplied to recombinant yeast host cells at concentrations ranging, for example, from about 1 to about 6 mM. In other embodiments, yeast host cells are modified to biosynthesize olivetolic acid by manipulating the cells to express cyclase enzymes, e.g., acyl-CoA synthase, e.g., hexanoyl-CoA synthase; type III PKS, such as olivetolic acid synthase or engineered variants thereof; and olivetolic acid cyclase or engineered variants thereof. Such enzymes are described in WO 2018 / 209143, which is incorporated by reference.

[0037] In some embodiments, divalic acid is enzymatically coupled to geranyl pyrophosate by prenyltransferase, e.g., GOT (such as a prenyltransferase polypeptide containing the amino acid sequence of amino acid sequence SEQ ID NO:1 or SEQ ID NO:2) to produce CBGVA. In some embodiments, the prenyltransferase contains region 80–398 of the mature GOT3 sequence. In some embodiments, divalic acid is supplied to recombinant yeast host cells at concentrations ranging, for example, from about 1 to about 6 mM. In other embodiments, yeast host cells are modified to biosynthesize divalic acid by manipulating the cells to express cyclase enzymes such as acyl-coA synthetase, e.g., butanoyl-coA synthase; type III PKS, such as olivetolate synthase or engineered variants thereof; and olivetolate cyclase or engineered variants thereof. Such enzymes are described in WO 2018 / 209143, which is incorporated by reference.

[0038] Acyl-CoA synthase for expression in recombinant host cells As used herein, the term "acyl-activating enzyme" refers to either "CoA transferase" or "CoA ligase." The term "acyl-CoA synthase" is used synonymously with "acyl-activating enzyme." Such enzymes can perform the conversion.

[0039] In some embodiments, host cells are genetically modified to express revS polypeptides derived from the genus Streptomyces (see, e.g., Miyazawa et al, J. Biol. Chem. 290:26994-27001, 2015) or their variants, such as exogenous polynucleotides encoding native homologs, homologous species, or non-native variants with acyl-CoA synthetase activity. In some embodiments, the polynucleotide encodes a polypeptide having at least 70% identity, or at least 75% identity, or at least about 80% or more identity (e.g., about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) with the sequence described in SEQ ID NO:10. In some embodiments, the polynucleotide encodes a RevS polypeptide having about 75%, 80%, 85%, 90%, 95%, or more identity with the sequence described in SEQ ID NO:10. In some embodiments, the non-natural variant includes one or more modifications, such as substitutions, including conservative substitutions, in regions outside the AMP-binding motif or catalytic site, for example.

[0040] In some embodiments, host cells are genetically modified to express an exogenous polynucleotide encoding a cannabis sativa-derived acyl-activating enzyme (CsAAE3) or a variant thereof, e.g., a native homolog, homologous species, or non-native variant having acyl-CoA synthetase activity. In some embodiments, the CsAAE3 polypeptide encoded by the polynucleotide comprises an amino acid sequence having at least 70% identity, or at least 75% identity, or at least about 80% or more identity (e.g., about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) with the sequence described in SEQ ID NO:11. In some embodiments, the acyl-CoA synthetase polynucleotide encodes CsAAE3, or its homologue or a non-natural product, comprising an amino acid sequence having approximately 75%, 80%, 85%, 90%, 95%, or more identity with the sequence described in SEQ ID NO:11. In some embodiments, the non-natural variant includes one or more modifications, such as substitutions (e.g., conservative substitutions), compared to SEQ ID NO:11, for example, in the region outside the AMP-binding motif or catalytic site.

[0041] In some embodiments, host cells are genetically modified to express an exogenous polynucleotide encoding a cannabis sativa-derived acyl-activating enzyme (CsAAE1) or a variant thereof, e.g., a native homolog, homologous species, or non-native variant having acyl-CoA synthetase activity. In some embodiments, the CsAAE1 polypeptide encoded by the polynucleotide comprises an amino acid sequence having at least 70% identity, or at least 75% identity, or at least about 80% or more identity (e.g., about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) with the sequence described in SEQ ID NO:12. In some embodiments, the acyl-CoA synthetase polynucleotide encodes CsAAE1 or its homologues, comprising an amino acid sequence having approximately 75%, 80%, 85%, 90%, 95%, or more identity with the sequence described in SEQ ID NO:12. In some embodiments, the CsAAE1 polynucleotide encodes a polypeptide in which the transmembrane domain is deleted. In some embodiments, the non-natural variant includes one or more modifications, such as substitutions, such as conservative substitutions, compared to SEQ ID NO:12, for example, in the region outside the AMP-binding motif or catalytic site.

[0042] In some embodiments, for example, for the production of olivetolic acid or divalic acid, host cells are genetically modified to express butyryl-CoA transferase derived from Roseburia hominis (see, e.g., Charrier, et al, Microbiology 152:179-185, 2006) or a variant thereof, such as an exogenous polynucleotide encoding a native homolog, homologous species, or non-native variant having butyryl-CoA transferase activity. In some embodiments, the polynucleotide encodes a polypeptide having at least 70% identity, or at least 75% identity, or at least about 80% or more identity (e.g., about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) with the sequence described in SEQ ID NO:20. In some embodiments, the polynucleotide encodes a RevS polypeptide having about 75%, 80%, 85%, 90%, 95%, or more identity with the sequence described in SEQ ID NO:20. In some embodiments, the non-natural variant includes one or more modifications, such as substitutions, including conservative substitutions, in regions outside the AMP-binding motif or catalytic site, for example.

[0043] Additional examples of organisms expressing CoA-transferases for use in this method can be found in the Comprehensive Enzyme Information System (BRENDA) under enzyme numbers EC 2.8.3.8 (CoA acetate-transferase), EC 2.8.3.1 (CoA propionic acid-transferase), and EC 2.8.3.9 (CoA butyrate-acetoacetate-transferase). These organisms include, but are not limited to, the following: species and strains of the genus Anaerostipes (e.g., A. caccae; A. caccae DSM 14662), species and strains of the genus Anaerobutyricum (e.g., A. hallii; A. hallii M72 / 1), species and strains of the genus Anaerotignum (e.g., A. propionicum), species and strains of the genus Aspergillus (e.g., A. nidulans), and species and strains of the genus Butyrivibrio (e.g., B. fibrisolvens); B. fibrisorbens 16 / 4), Clostridium species and strains (e.g., C. kluyveri), Coprococcus species and strains (e.g., Coprococcus species L2-50), Cupriavidus species (e.g., C. necator H16), Escherichia species and strains (e.g., Escherichia coli K-12); Eubacterium species and strains (e.g., E. rectale, E. rectale DSM 17629), Faecalibacterium species and strains (e.g., F. prausnitzii; F. prausnitzii A2-165); F. Prausnitzii L2-6; F.This includes species and strains of the genus *Plausnitzi* (M21 / 2), *Megasphaera* (e.g., *M. elsdenii*), *Propionibacterium* (e.g., *P. freudenreichii*), and *Roseburia* (e.g., *R. hominis*, *R. intestinalis*; *R. intestinalis* L1-82; *R. inulinivorans*; *R. inulinivorans* A2-194; and *Roseburia* species A2-181).

[0044] A non-specific example of a particular CoA-transferase is listed below. TIFF0007875122000004.tif165166

[0045] In some embodiments, the CoA transferase comprises an Escherichia coli acetyl-CoA:acetoacetyl-CoA transferase polypeptide sequence, e.g., SEQ ID NO:21 and SEQ ID NO:22. In some embodiments, the CoA transferase comprises a C. nekatol H16 propionic acid CoA-transferase polypeptide sequence, e.g., as described in SEQ ID NO:23. In some embodiments, the CoA transferase comprises an amino acid sequence having at least 60% or more identity with the sequence described in SEQ ID NO: 21, 22, or 23 (e.g., at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity). In some embodiments, the CoA transferase has at least 70%, 75%, 80%, 85%, 90%, 95%, or more identity with the sequence described in SEQ ID NO: 21, 22, or 23. In some embodiments, the CoA transferase contains the amino acid sequence of SEQ ID NO: 21, 22, or 23.

[0046] In some embodiments, acyl-CoA ligases are used to convert aliphatic carboxylic acids to acyl-CoA thioesters. In some embodiments, the CoA ligase is selected from the group consisting of Mycobacterium avium mig medium-chain acyl-CoA ligase, A. thaliana AT4g05160 coumarate acyl-CoA ligase, S. cerevisiae FAA2 medium-chain acyl-CoA ligase, and Escherichia coli FADK acyl-CoA ligase.

[0047] In some embodiments, the CoA ligase comprises, for example, the M. avium mig medium-chain acyl-CoA ligase polypeptide sequence described in SEQ ID NO:24. In some embodiments, the CoA ligase comprises, for example, the A. saliana AT4g05160 coumarate acyl-CoA ligase polypeptide sequence described in SEQ ID NO:25. In some embodiments, the CoA ligase comprises, for example, the S. cerevisiae FAA2 medium-chain acyl-CoA ligase polypeptide sequence described in SEQ ID NO:26. In some embodiments, the CoA ligase comprises, for example, the Escherichia coli FADK acyl-CoA ligase polypeptide sequence described in SEQ ID NO:27. In some embodiments, the CoA ligase contains an amino acid sequence having at least 60% or more identity with the sequence described in any one of SEQ ID NO: 24, 25, 26, or 27 (e.g., at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity). In some embodiments, the CoA ligase has at least 70%, 75%, 80%, 85%, 90%, 95%, or more identity with the sequence described in SEQ ID NO: 24, 25, 26, or 27. In some embodiments, the CoA ligase contains the amino acid sequence of SEQ ID NO: 24, 25, 26, or 7. In some embodiments,

[0048] In some embodiments, aliphatic carboxylic acids are C 1~5 It is a carboxylic acid.

[0049] In some embodiments, aliphatic carboxylic acids are C 6~20 It is a carboxylic acid.

[0050] In some embodiments, aliphatic carboxylic acids include carbon-carbon double bonds, hydroxyl groups, halogens, deuterium, tritium, or combinations thereof.

[0051] Olivetolate synthase for expression in recombinant host cells In some embodiments, host cells are further genetically modified to express olivetolate synthase, or a variant thereof, such as an exogenous polynucleotide encoding a native homolog or homologous molecular species or a non-native variant with polyketide synthase activity. Olivetolate synthase (Taura et al. FEBS Letters 583:2061-2066, 2009), also known as 3,5,7-trioxododecanoyl-CoA synthase or UniProtKB-B1Q2B6, is a type III PKS that catalyzes the condensation of three molecules of acyl-CoA and malonyl-CoA to form the 3,5,7-trioxoalkanoyl-CoA tetraketide shown below: In formula TIFF0007875122000005.tif11128, "CoA" is coenzyme A and "R" is an alkyl group. For example, when hexanoic acid is used as a starting feedstock for cannabinoid production, the hexanoyl-CoA formed by the above acyl-CoA synthetase, e.g., revS or CsAAE3, is condensed with three molecules of malonyl-CoA to form 3,5,7-trioxododecanoyl-CoA (i.e., "R" is an n-pentyl group).

[0052] In some embodiments, the olivetolate synthase polynucleotide encodes a polypeptide comprising an amino acid sequence having at least 70% identity, or at least 75% identity, or at least about 80% or more identity (e.g., about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) with the sequence described in SEQ ID NO:13. In some embodiments, the olivetolate synthase polynucleotide encodes a type III PKS comprising an amino acid sequence having about 75%, 80%, 85%, 90%, 95%, or more identity with the sequence described in SEQ ID NO:13.

[0053] 2-alkyl-4,6-dihydroxybenzoate cyclase expressed in recombinant host cells The host cells according to the present invention may be further modified to express an exogenous polynucleotide encoding 2-alkyl-4,6-dihydroxybenzoate cyclase (e.g., olivetolate cyclase). In some embodiments, 2-alkyl-4,6-dihydroxybenzoate cyclase is a dimeric α+β barrel (DABB) protein domain similar to DABB-type polyketide cyclases from the genus Streptomyces. Olivetolate cyclase has been described, for example, by Gagne et al. (Proc. Nat. Acad. Sci. USA 109 (31): 12811-12816; 2012).

[0054] In some embodiments, a polynucleotide encoding 2-alkyl-4,6-dihydroxybenzoic acid cyclase encodes a polypeptide having at least 70% identity, or at least 75% identity, or at least about 80% or more identity (e.g., about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) with the sequence described in SEQ ID NO: 14, 15, or 16.

[0055] In some embodiments, instead of utilizing acyl-CoA synthase to enzymatically produce thioesters from carboxylic acids, chemically synthesized thioesters are used as starting materials.

[0056] During the culture and production of prenylated compounds, exogenous prenyl species such as geraniol can also be supplied to host cells. Alternatively, host cells can be cultured in a medium containing high levels of prenyl precursors, such as prenol, isoprenol, or geraniol. In methods involving multiple precursor supply (MPF), five-carbon prenols and isoprenols can be enzymatically converted to monophosphate levels (i.e., dimethylallyl monophosphate and isopentenyl monophosphate), then to diphosphate levels (i.e., dimethylallyl pyrophosphate and isopentenyl pyrophosphate), and then combined to form ten-carbon geranyl pyrophosphate.

[0057] In some embodiments, the starting carboxylic acid is hexanoic acid or butanoic acid, yielding precursors for the final production of cannabigerolic acid or cannabigerovaric acid type molecules, as well as their decarboxylated and other chemically converted derivatives. In some embodiments, the starting carboxylic acid is an analog compound that is fluorinated or chlorinated at various positions, or deuterated or tritiated at various positions.

[0058] Production of CBGA, CBCA, CBDA, and THCA In a typical embodiment, recombinant yeast host cells provided herein can be genetically modified to express a prenyltransferase that catalyzes the coupling of geranyl pyrophosphate to olivetolic acid or divalic acid (or an analog thereof) to form cannabinoid compounds, such as CBGA or CBGVA (or CBGA or CBGVA analog compounds). Examples of prenyltransferases include geranyl pyrophosphate:olivetolic acid geranyltransferase (GOT; EC 2.5.1.102) described by Fellermeier and Zenk (FEBS Letters 427:283-285; 1998), and asaprenyltransferases described in WO 2018 / 200888 and WO 2019 / 071000. Streptomyces prenyltransferase containing NphB, as described by Kumano et al. (Bioorg Med Chem. 16(17): 8117-8126; 2008), may be used in accordance with the present invention. In some embodiments, the prenyltransferase is fnq26, i.e., Streptomyces cinnamonensis-derived flaviolin linalyltransferase.

[0059] In some embodiments, yeast host cells are modified to express GOT to catalyze the coupling of geranyl pyrophosphate to olivetolic acid (or olivetolic acid analogs, e.g., fluorinated or chlorinated analogs). In some embodiments, the amino acid sequence of GOT is SEQ ID NO:1. In some embodiments, the GOT polypeptide comprises the amino acid sequence of SEQ ID NO:2.

[0060] In some embodiments, divalic acid (or analogues of divalic acid, e.g., fluorinated or chlorinated analogues) is used as an intermediate compound to which geranyl pyrophosphate is coupled to produce CBGVA (or a corresponding analogue).

[0061] In some embodiments, the yeast host strain is further modified to convert CBGA, CBGVA, or analogues of CBGA or CBGVA to a second acidic cannabinoid. In some such embodiments, the expression system is on the same vector, on a different vector, or integrated into the host cell genome. In other embodiments, the expression system for conversion activity encodes one of the C. sativa enzymes CBCA synthase, THCA synthase, or CBDA synthase. In some embodiments, the synthase is a hop-derived homolog, e.g., a hop-derived CBDA synthase homolog. In some embodiments, the expression system encodes a hop CBDA homolog having at least 70% identity, or at least 75% identity, or at least about 80% or more identity (e.g., about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) with the sequence described in SEQ ID NO: 17, 18, or 19.

[0062] CBCAS may be expressed as a fusion protein lacking its own signal peptide, or it may be expressed with its own signal peptide or a heterologous signal peptide or its amino-terminal hydrophobic domain.

[0063] In other embodiments, an HDEL or KDEL endoplasmic reticulum-retaining sequence is fused to an expressed GOT, CBCAS, or GOT / CBCAS mutant enzyme. In some embodiments, the GOT and CBCAS constructs may be modified to introduce targeted or random mutations in the expressed enzyme so that the expressed enzyme has desirable properties for cannabinoid acid production.

[0064] In some embodiments, the CBCAS signal peptide is an endogenous signal peptide used by the cannabis plant, or it may be replaced by a yeast or heterologous targeting sequence, such as a yeast α factor pre-sequence or pre-pro sequence, a yeast proteinase A pre-sequence or pre-pro sequence, or a sequence derived from the cannabis GOT (also referred to herein as "CsPT4") enzyme, such as a hydrophobic region starting around amino acid number 80 of the mature GOT3 enzyme. Other preferred signal peptides include the S. cerevisiae pdi1 signal sequence or the Aspergillus japonica-derived berberine bridge-associated easE signal sequence. The CBCAS gene construct may be modified by altering the sequence to remove N-linked glycosylation sites in the protein. All permutations and combinations of glycosylation site modifications may be investigated for increased or optimal activity. In other embodiments, fusion proteins such as hSOD may be incorporated into the expressed construct. CBCA, THCA, or CBDA synthase gene constructs can be similarly modified.

[0065] Exemplary CBCAS polypeptide sequences are provided in SEQ ID NO:3-9. In some embodiments, a polynucleotide encoding CBCAS encodes a polypeptide having at least 70% identity, or at least 75% identity, or at least about 80% or more identity (e.g., about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) with the region of any one of the CBCAS polypeptides in SEQ ID NO:3-9, excluding the signal sequence or ER retention sequence. In some embodiments, the polypeptide has about 75%, 80%, 85%, 90%, 95%, or more identity with the sequence described in any one of SEQ ID NO:3-9.

[0066] In some embodiments, acidic cannabinoids, such as CBGA or CBCA, can be decarboxylated to form neutral cannabinoid compounds, such as CBG or CBC, using a decarboxylase, such as Aspergillus nidulans orsB decarboxylase, or its homologue or modified variant. Alternatively, acidic cannabinoids can be decarboxylated by maintaining them at a high temperature (e.g., about 40°C, 50°C, or 100°C) using a metal catalyst. Thus, in a further aspect, provided herein are chemical and biochemical methods for the decarboxylation of cannabinoid acids such as THCA, CBDA, CBGA, CBCA, THCVA, CBDVA, CBGVA, and CBCVA, as well as further cannabinoid acid analogs. In some embodiments, CBGA is converted to CBG using an organic solvent and a metal catalyst.

[0067] In some embodiments, decarboxylation is carried out using a metal catalyst and heat, and may utilize whole centrifuged yeast cells expressing and retaining cannabinoid acids, or may use cannabinoid acids extracted from yeast cells using an extraction reagent comprising an organic solvent, water / aqueous buffer, or a mixture thereof as a substrate. In preferred embodiments, such organic solvents include alcohols such as ethanol, propanol, isopropanol, or butanol. The metal catalyst includes any metal catalyst suitable for decarboxylation of acidic cannabinoids. In some embodiments, the metal catalyst includes zinc, magnesium, molybdenum, nickel, copper, platinum, palladium, or iron. The catalyst may be a hydroxide (-OH), amine (-NR3, where each R group is independently H, an optionally substituted alkyl, or an optionally substituted aryl), thiol (-SR, where R is an optionally substituted alkyl or an optionally substituted aryl), or halide (e.g., F - Cl - , Br - , and I -The catalyst may further include, but is not limited to, one or more metal ligands, including, organic acids (e.g., acetoacetic acid, hydroxamic acid, etc.), chelating agents (e.g., aminopolycarboxylates, e.g., ethylenediaminetetraacetic acid (EDTA) and nitrilotriacetic acid (NTA), and heterocyclic compounds (e.g., phenanthroline, bipyridine, etc.). In some embodiments, the metal catalyst is a metal salt. In some embodiments, the metal salt is a zinc salt. In some embodiments, the metal salt is a palladium salt or a platinum salt. The metal catalyst may exist in solution in solid form, for example, as zinc dust or zinc oxide, or in the form of a metal-doped zeolite catalyst, such as HZSM-5, which is supported with a metal such as zinc, magnesium, molybdenum, nickel, copper, platinum, palladium, or iron. Similarly, the zeolite catalyst may be used alone, in the presence of the aforementioned metal salt solution, or in non-aqueous organic solvents, including anhydrous organic solvents. The decarboxylation reaction is typically carried out at a temperature in the range of approximately 25°C to about 100°C and a pH in the range of about 3 to about 12, for a few minutes to several hours, for example, 2 to 6 hours, or longer, for example, 12, 18, 24, 36, or 48 hours. Various decarboxylation methods are also described, for example, in U.S. Patent Application Publication No. 20180016216 and Wang et al., Cannabis and Cannabinoid Res. Volume 1.1, 2016).

[0068] Any suitable organic solvent can be used in the method of the present invention. Suitable solvents include, but are not limited to, alcohols such as methanol, ethanol, propanol, isopropanol, and butanol. In some embodiments, the organic solvent is hexane or heptane. In some embodiments, the solvent is toluene, methylene chloride, ethyl acetate, acetonitrile, tetrahydrofuran, benzene, ethylbenzene, xylene (i.e., m-xylene, o-xylene, p-xylene, or any combination thereof), chloroform, diethyl ether, dimethylformamide, dimethyl sulfoxide, petroleum ether, and mixtures thereof. Aqueous organic solvent mixtures (i.e., mixtures of water and water-miscible organic solvents such as tetrahydrofuran or dimethylformamide) can also be used. Generally, the ratio of solvent to 2-alkyl-4,6-dihydroxybenzoic acid is in the range of about 1:1 to about 1000:1 by weight. The ratio of solvent to 2-alkyl-4,6-dihydroxybenzoic acid can be, for example, about 100:1 by weight, or about 10:1 by weight, or about 5:1 by weight. In certain embodiments, 2-alkyl-4,6-dihydroxybenzoic acid is present in a yeast mixture (e.g., dried yeast cells, or a wet yeast cell pellet taken from a culture). In some such embodiments, the reaction mixture includes host cells (e.g., dried yeast cells). The ratio of solvent to yeast mixture (e.g., dried yeast cells) can range from about 1:1 to about 1000:1 by weight. The ratio of solvent to yeast mixture can be, for example, about 100:1 by weight, or about 10:1 by weight, or about 5:1 by weight, or about 2:1 by weight.

[0069] Enzymatic methods for the decarboxylation of cannabinoid acids include the use of recombinant aromatic decarboxylase enzymes, as described by Payer et al., Advanced Synth. & Catal. 361:2402-2420, 2019. In some embodiments, the PatG enzyme from Aspergillus clavatus, the orsB orceric acid decarboxylase from Aspergillus nidurans, or the 3,4-dihydroxybenzoic acid decarboxylase from Enterobacter cloacae may be used. In some embodiments, the decarboxylase is a wild-type enzyme. In other embodiments, such enzymes are variants engineered through amino acid mutations to have greater decarboxylase activity or other optimal parameters, such as modified thermal or pH optimal conditions. In some embodiments, the decarboxylase contacts the target cannabinoid acid as a lysate from the engineered microorganism or whole-cell in the presence of zymolyase. In some embodiments, the decarboxylase enzyme is expressed by genetically modified host cells, such as a strain of S. cerevisiae yeast.

[0070] In yet another embodiment, the conversion of a first intermediate cannabinoid to a second cannabinoid via the action of a wild-type or mutant cannabinoid or cannabinoid acid synthase, either within the same manipulated host cell or through co-culture with two or more recombinant host cell strains, such as yeast.

[0071] As described above, in some embodiments, host cells, such as a yeast strain, transformed or genome-integrated with a polynucleotide segment, plasmid, or vector containing each of the above genes are transformed with another expression system for the conversion of CBGA or a CBGA analog to a second acidic cannabinoid. In some such embodiments, the expression system is on the same vector, on separate vectors, or integrated into the host cell genome. In other embodiments, the expression system for conversion activity encodes one of the C. sativa enzymes: CBCA synthase, THCA synthase, or CBDA synthase.

[0072] For large-scale downstream processing, the producing yeast cells may be centrifuged to separate and purify the cannabinoid acids localized in the culture medium, for example, by ion-exchange chromatography, hydrophobic or chelate chromatography, or selective extraction procedures to obtain highly purified cannabinoid acids. Similarly, yeast-associated cannabinoid acids may be purified using such techniques, or decarboxylated prior to the use of such techniques.

[0073] The cannabinoid compounds of interest and cannabinoid compound intermediates are produced using the expression systems described herein. Such compounds include, but are not limited to, CBGA, CBG, CBCA, CBC, THCA, THC, CBDA, and CBD, as well as analog compounds, including, for example, halogenated, deuterated, or tritiated compounds. In some embodiments, each step of the metabolic pathway producing the cannabinoid compound of interest takes place in the modified recombinant cells described herein. In other embodiments, at least one step of the metabolic pathway takes place in the modified recombinant cells described herein, and at least one step of the metabolic pathway takes place extracellularly, for example, in a host cell extract or in co-cultured modified recombinant cells. The compounds produced at each step of the metabolic pathway may also be called “intermediates,” “intermediate compounds,” or “compound intermediates.”

[0074] host cell In some embodiments, the methods of the present invention are carried out using yeast or filamentous fungal host cells such as Aspergillus host cells. Suitable yeast genera for use as host cells include, but are not limited to, those of the genera Saccharomyces, Schizosaccharomyces, Candida, Hansenula, Pichia, Kluyveromyces, Yarrowia, and Phaffia. Suitable yeast species include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida albicans, Hansenula polymorpha, Pichia pastoris, Papilio canadensis, Kluyveromyces marxianus, Kluyveromyces lactis, Phaffia rhodozyma, and Yarrowia liporitica.Examples of filamentous fungal genera that can be used as host cells include Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysoporium, Coprinus, Coriolus, and Corina. Corynascus, Chaertomium, Cryptococcus, Filobasidium, Fusarium, Gibberella, Humicola, Magnaporthe, Mucor, Myceliophthora, Mucor Genus Mucor, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Scytaldium, Sue Examples of cells from the genera Schizophyllum, Sporotrichum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, and Trichoderma include, but are not limited to, these. Exemplary filamentous fungal species include Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, and Aspergillus japonicus.japonicus), Aspergillus nidurans, Aspergillus niger, Aspergillus oryzae, Chrysosporium lucknowense, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Bjerkandera adusta, Ceriporiopsis aneirina Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis librosarivulosa), Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Coprinus cinereus, Coriolus hirsutus, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Neurospora intermedia, Penicillium purpurogenum, Penicillium canesens canescens), Penicillium solitum, Penicillium funiculosum, Phanerochaete chrysosporium, Phlebia radiate, Pleurotus eryngii, Talaromyces flavus, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum Examples include *Trichoderma longibrachiatum*, *Trichoderma reesei*, and *Trichoderma viride*.

[0075] In some embodiments, the host cells are selected from the group consisting of Saccharomyces cerevisiae, Cliberomyces lactis, Cliberomyces marxianus, Pichia pastris, Yarowia liporitica, Hansenula polymorpha, and the Aspergillus genus.

[0076] In the above embodiment, the gene may be encoded by a chemosynthetic gene encoding a wild-type or mutant enzyme derived from a species of hemp, Arabidopsis thaliana, Pseudomonas, or Dictyostelinum, through yeast codon optimization.

[0077] In one aspect, the present invention provides vectors and modified recombinant host cells for the expression of polypeptides having the enzymatic activity described herein. In some embodiments, the vector of the present invention comprises a select gene, a yeast 2-micron sequence, and a polynucleotide encoding a polypeptide, the polynucleotide functionally linked to an alcohol dehydrogenase 2 promoter. In some of these embodiments, the polynucleotide comprises one or more yeast-preferred codons substituted with native codons. In some embodiments, the vector does not grow and / or amplify in bacterial host cells. In some embodiments, the select gene is the URA3, HIS3, TRP1, or LEU2 gene.

[0078] As detailed above, in some embodiments, the polypeptide comprises geranyl pyrophosphate geranyltransferase (EC 2.5.1.102, GOT) or a functionally active portion thereof. In some of the latter embodiments, GOT may be cleaved at the amino terminus and / or carboxyl terminus. In some embodiments, GOT may be expressed directly using appropriately positioned methionine start codons and appropriately positioned stop codons. In further embodiments, GOT may be expressed as a fusion protein with a partner that confers enhanced expression of active GOT. Suitable fusion partners include human superoxide dismutase (hSOD) or any other superoxide dismutase, yeast maltose-binding protein (MBP), human or yeast ubiquitin (Ub), human catalase (hCAT), S. cerevisiae catalase T (CTT1), S. cerevisiae peroxisome catalase A (CTA1), transferrin, human serum albumin (HSA), or any other SOD, catalase, or fusion partner. Further fusion partners include human galectin, E. coli MBP, yeast prepro-α factor, and GB1.

[0079] In some aspects, yeast cells are cir 0 In some of these embodiments, the yeast cells are protease-deficient and / or strain BJ2168 (ATCC 208287). In some embodiments, the yeast strain is a modified industrial ethanol-producing strain and / or strain "Super Alcohol-Activated Dried Yeast" (Angel Yeast Co., Ltd. Yichang, Hubei 443003, PRChina). Such strains are cr 0 Modified by curing, it has selectable marker mutations in the genome (e.g., URA3, HIS3, TRP1, and LEU2) that are well known to those skilled in the art.

[0080] In some embodiments, the final concentration of the desired cannabinoid-producing enzyme in the yeast culture is at least about 0.1 g / liter, 1 g / liter, 2 g / liter, or 4 g / liter. In some embodiments, the desired polypeptide is a fusion polypeptide. In some embodiments of the method of the present invention, the nucleotide sequence encoding the desired polypeptide is further functionally linked to an alcohol dehydrogenase 2 terminator. In some embodiments, dissolved oxygen is continuously present in the culture medium at a concentration of about 50% or more. In some embodiments, the polynucleotide further comprises a polynucleotide encoding the yeast URA3 polypeptide. In some embodiments, yeast cells are BJ2168[cir 0 This refers to Saccharomyces species, such as TRP revertant mutants. In some embodiments, glucose provides about 100% of the oxidizable substrate for respiration. In one embodiment of the method of the present invention, a polynucleotide further encodes a signal sequence or prepro-secretion sequence functionally linked to the yeast alcohol dehydrogenase-2 promoter.

[0081] The present invention relates to compositions and methods for polypeptide production in plasmid-transformed yeast, wherein the plasmid used herein is an episomal expression plasmid and comprises a yeast promoter and terminator, a gene for the polypeptide to be produced, which may optionally contain one or more yeast-preferred codons substituting naturally occurring native codons, and an origin of replication such as a 2-micron DNA sequence of an endogenous yeast plasmid. Preferably, the yeast promoter is a regulated promoter, e.g., the ADH2 promoter; generally, the terminator is compatible with, but not required to be compatible with, the promoter, e.g., the ADH2 terminator. In other embodiments, but not limited to, the terminator may be used, such as a S. cerevisiae CYC, GPD, PYK, PGK, or ADH1 gene terminator. In some embodiments, the plasmid may contain a yeast pre- or pre-pro-signaling (leader) sequence if it is desired that the polypeptide be secreted extracellularly or target a yeast vacuole or membrane; however, many polypeptides, such as GOT or a GOT fusion protein with a leader sequence, are not used because it is desired that the polypeptide be maintained intracellularly so that it can be used to catalyze the conversion of soluble substrates. In some embodiments, the plasmid may not be able to grow and / or amplify in a bacterial host cell, for example, because it substantially does not contain the bacterial sequence necessary for growth and / or amplification in bacterial cells. The plasmid is introduced into a suitable yeast strain; in some embodiments, this is circle zero (CIR 0), a protease-deficient yeast strain; in some embodiments, the original strain contains an endogenous yeast plasmid, which is then cured before transformation. Transformed yeast can be selected by growth on a medium suitable for the plasmid selection marker, e.g., leucine-deficient or uracil-deficient medium. In productive fermentation, if a controlled promoter, e.g., the ADH2 promoter, is used, the promoter can be modified to suppress recombinant polypeptide synthesis. In embodiments where the ADH2 promoter is used, protein production under the control of the promoter is suppressed at high glucose levels, providing continuous expression under glucose-restricted growth conditions. The feed-batch fermentation process described herein allows for the regulation of the amount of glucose supplied to the yeast culture, and thus allows for the control of protein expression.

[0082] The plasmid of the present invention includes an origin of replication. In one embodiment, a 2-micron DNA sequence necessary for autonomous replication in yeast is utilized, making it possible to maintain the plasmid as an extrachromosomal element that can be stably maintained in the host yeast. In several embodiments, a full-length yeast 2-micron sequence may be used, although functional fragments are also intended. The full-length yeast 2-micron sequence can be cloned, for example, from a S. cerevisiae genomic DNA preparation containing 2-micron DNA; see, for example, Barr et al. (eds), Chapters 9 and 10.

[0083] In a further embodiment, a strain producing CBGA is transformed with a plasmid expressing cannabichromenate synthase (CBCAS) as described above. In embodiments producing CBCA, THCA, or CBDA, the strain is transformed with a plasmid expressing the corresponding synthase. The synthase may also be expressed as a fusion protein or with native or heterologous signal peptides, as desired, and may be modified to induce targeted or random mutations in the expressed enzyme so that the enzyme has desirable properties for cannabinoid acid production.

[0084] Promoters used to drive the transcription of genes in S. cerevisiae and other yeasts are well known in the art and include DNA elements controlled by glucose concentration in the growth medium, such as the alcohol dehydrogenase 2 (ADH2) promoter. When conditional expression is required, other regulatory or inductive promoters are used, such as promoters that drive the expression of the GAL1, MET25, and CUP1 genes. GAL1 and CUP1 are induced by galactose and copper, respectively, while MET25 is induced in the absence of methionine.

[0085] In some embodiments, one or more exogenous polynucleotides are functionally linked to a glucose-regulating promoter. In some embodiments, the expression of one or more exogenous polynucleotides is driven by an alcohol dehydrogenase 2 promoter.

[0086] Other promoters constitutively drive transcription. These promoters include, but are not limited to, regulatory elements of highly expressed yeast glycolytic enzymes such as glyceraldehyde-3-phosphate dehydrogenase (GPD), phosphoglycerate kinase (PGK), pyruvate kinase (PYK), triose phosphate isomerase (TPI), and alcohol dehydrogenase 1 (ADH1). Another potent constitutive promoter that can be used is the promoter derived from the S. cerevisiae transcription elongation factor EF-1α gene (TEF1) (Partow et al., Yeast. 2010, (11):955-64).

[0087] In other embodiments, host cells can increase cannabinoid production by increasing the precursor pool, etc. Heterogeneous or chemosynthetic genes of enzymes such as malonyl-CoA synthase, acetyl-CoA carboxylase, acetyl-CoA synthase-1 and -2, gene products in the mevalonate pathway, e.g., acetoacetyl-CoA thiolase, HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, mevalonate pyrophosphate decarboxylase, isopentenyl diphosphate isomerase, and mutant farnesyl pyrophosphate synthase (ERG20; Zhao et al., 2016) from Saccharomyces or other eukaryotic or prokaryotic species can be introduced onto high-level expression plasmid vectors or through genomic integration using methods well known to those skilled in the art. Therefore, in some embodiments, the enzyme may be naturally present in the cell, but the cell is engineered to produce higher levels than wild-type cells, while in other embodiments, the enzyme is not naturally present in the host cell but is heterologous to the host cell, e.g., of another species. Such methods may include the use of CRISPR-Cas-9 technology, yeast artificial chromosomes (YACs), or retrotransposons. Alternatively, if natural to the host organism, these genes can also be upregulated by gene element integration methods known to those skilled in the art.

[0088] Furthermore, similar manipulations can be used in other contexts to reduce the production of natural products that utilize carbon sources, such as ethanol, which leads to a decrease in the utilization of those carbon sources for cannabinoid production. These genes may be completely “knocked out” by deletion from the genome, or their activity may be reduced through decreased promoter strength, mutations, etc. Examples of these genes include the genes for the enzymes ADH1 and / or ADH6. Other genes that can be “knocked out” include genes involved in the ergosterol pathway, such as ERG9, wild-type ERG20, and two of the most promising aromatic decarboxylase genes in yeast, PAD1 and FDC1.

[0089] In certain aspects of the present invention, a yeast strain overexpressing an integrated or modified gene of the mevalonate pathway is used to produce cannabinoid acids by biosynthesizing aromatic polyketide precursors, olivetolic acid, divalic acid, and geranyl diphosphate for attachment to analogs. In one exemplary embodiment, strain Y385 coupled with plasmid pBM308L was used for CBGA production. Unless otherwise specified, strain Y385 has the following integrations using sequences based on S. cerevisiae: HO locus locations: pTEF1-IDI1; pADH2-tHMGR; pADH2-ERG13; pTEF2-ERG20 (F96W, N127W); ROX1 locus locations: pTEF2-ERG8; pADH2-ERG10; pADH2-tHMGR; pTDH3-MVD1; YFL041W locus locations: pMLS1-ERG20 (F96W, N127W); pICL1-ERG13; pADH2 (S.para)-tHMGR; pFBA1-MatB (S.co); REI1 locus locations: pMLS1-ERG12; pFBA1-MVD1; pADH2-mvaE (E.fa); pICL1-mvaS (E.fa); pTEF1-ERG8; PRB1 locus location: pURA3-URA3; pTEF1-ADR1; pFBA1-PDC (Z.mo).

[0090] In some embodiments, host cells, such as yeast cells producing CBGA or its halogenated, deuterated, or tritiated analogs, are engineered to overexpress enzymes of the mevalonate pathway. Such enzymes include, for example, Erg10, Erg13, HMGR, Erg12, Erg8, Mvd1, Idi1, and Erg20. See, for example, U.S. Patent No. 6,689,593, incorporated by reference.

[0091] Further embodiments include genes for accessory enzymes aimed at promoting the production of the final product cannabinoid. One such enzyme, catalase, is involved in the oxidative cyclization of CBGA and its analogues, such as cannabidiolic acid synthase (Taura et al., 2007), Δ 9 -It can neutralize hydrogen peroxide produced by tetrahydrocannabinolate synthase (Sirikantaramas et al., 2004) and cannabichromenate synthase (Morimoto et al., 1998).

[0092] In a further embodiment, the engineered host cell contains endogenous or heterologous genes that are upregulated or downregulated to optimize, for example, the precursor pool for cannabinoid biosynthesis. Furthermore, additional heterologous gene products can be expressed to exhibit “accessory” functions within the cell, e.g., CBDA, Δ 9 Overexpressed catalase can be expressed to neutralize the hydrogen peroxide formed during the oxidative cyclization step in which important acidic cannabinoids such as THCA and CBCA are obtained. "Accessory" genes and their expression products may be obtained by integration into the yeast genome through techniques well known in the art, and may be expressed from plasmids (also known as yeast expression vectors), yeast artificial chromosomes (YACs), or yeast transposons.

[0093] In some embodiments, as further described below, host cells, such as yeast strains, transformed or genome-integrated with plasmids or vectors containing each of the above genes are transformed with another expression system for the conversion of CBGA or CBGA analogs to a second acidic cannabinoid. In some such embodiments, the expression system is on the same vector, on separate vectors, or integrated into the host cell genome.

[0094] The engineered cells of the present invention that produce cannabinoids can be prepared by transforming host cells through genomic integration or by using an episomal plasmid (also called an expression vector, or simply a vector) with at least one nucleotide sequence-coding enzyme involved in the engineered metabolic pathway. As used herein, the terms “nucleotide sequence,” “nucleic acid sequence,” and “gene construct” are interchangeable and mean a polymer of single-stranded or double-stranded RNA or DNA optionally containing synthetic, non-natural, or modified nucleotide bases. A nucleotide sequence may include one or more segments of cDNA, genomic DNA, synthetic DNA, or RNA. In some embodiments, the nucleotide sequence is codon-optimized to reflect the normal codon usage of the host cell without modifying the polypeptide encoded by the nucleotide sequence. In certain embodiments, the terms “codon-optimized” or “codon-optimized” mean modifying the codon content of a nucleic acid sequence without modifying the sequence of the polypeptide encoded by the nucleic acid to enhance expression in a particular host cell. In certain embodiments, this term is intended to encompass the modification of codon content in a nucleic acid sequence as a means of regulating the expression level of a polypeptide (e.g., increasing or decreasing the expression level). Thus, nucleic acid sequences encoding enzymes involved in the engineered metabolic pathway are described. In some embodiments, metabolically engineered cells may express one or more polypeptides exhibiting the enzymatic activity necessary to carry out the steps described below. In some embodiments, nucleotide sequences are synthesized and codon-optimized for expression in yeast according to the method described in U.S. Patent No. 7,561,972.

[0095] For example, a particular cell may contain one, two, three, four, five, or more than five nucleic acid sequences, each nucleic acid sequence encoding a polypeptide necessary to produce a cannabinoid compound or a cannabinoid compound intermediate as described herein. Alternatively, a single nucleic acid molecule may encode one or more polypeptides. For example, a single nucleic acid molecule may contain nucleic acid sequences encoding two, three, four, or even five different polypeptides. Nucleic acid sequences useful for the present invention as described herein can be obtained from a variety of sources, such as cDNA sequence amplification, DNA libraries, novel synthesis, or excision of genomic segments. Sequences obtained from these sources can then be modified using standard molecular biological techniques and / or recombinant DNA techniques to produce nucleic acid sequences with the desired modifications. Exemplary methods for modifying nucleic acid sequences include, for example, site-directed mutagenesis, PCR mutagenesis, deletion, insertion, substitution, exchange, homologous recombination, site-directed recombination, or various combinations thereof using restriction enzymes, optionally combined with ligation. In other embodiments, the nucleic acid sequence may be a synthetic nucleic acid sequence. Synthetic polynucleotide sequences can be produced using various methods described in U.S. Patent No. 7,323,320 and U.S. Patent Application Publications 2006 / 0160138 and 2007 / 0269870. Methods for transforming yeast cells are well known in the art.

[0096] Fermentation conditions In general, cannabinoid production by the methods provided herein involves culturing host cells (e.g., yeast or filamentous fungi) engineered to include the expression system described above. In some embodiments, the carbon source for yeast growth may be, for example, sugars such as glucose, sucrose, xylose, or other sustainable raw sugars, such as raw sugars derived from cellulose sources. In other embodiments, the carbon source used may be methanol, glycerol, ethanol, or acetate. In some embodiments, the raw material composition is experimentally refined to optimize the yeast growth level and the final cannabinoid production level, which are measured using analytical techniques such as HPLC. In such embodiments, the method involves the use of glucose / ethanol or glucose / acetate mixtures where the molar ratio of glucose to two carbon sources (ethanol or acetate) is in the range of 50 / 50, 60 / 40, 80 / 20, or 90 / 10. The raw material supply may be optimized to induce a glucose-regulating promoter and to maximize the production of acetyl-CoA and malonyl-CoA precursors in the producing strain.

[0097] Fermentation methods can be adapted to specific yeast strains by differences in carbon utilization pathways or expression regulation modes. For example, fermentation of Saccharomyces yeasts may require a single glucose supply, a complex nitrogen source (e.g., casein hydrolysate), and multiple vitamin supplements. This is in contrast to the methylotropic yeast Pichia pastris, which requires only simple ammonium (nitrogen) salts, but may require glycerol, methanol, and trace mineral raw materials for optimal growth and expression. See, for example, Elliott et al. J. Protein Chem. (1990) 9:95 104, U.S. Patent No. 5,324,639, and Fieschko et al. Biotechnol. Bioeng. (1987) 29:1113 1121. Culture media may contain components such as yeast extract and peptone. Microorganisms can be cultured in common fermentation modes, including but not limited to batch, fed-batch, and continuous-flow.

[0098] In some embodiments, the rate of glucose addition to the fermenter is adjusted so that the glucose addition rate is approximately equal to the rate of glucose consumption by the yeast. Under such conditions, the amount of glucose or ethanol does not accumulate significantly. The glucose addition rate in this case may depend on factors including, but not limited to, the specific yeast strain, fermentation temperature, and the physical dimensions of the fermentation apparatus.

[0099] Using a multiple precursor feeding (MPF) method (see WO 2018 / 209143 incorporated by reference), in a batch setting, the precursor olivetolic acid (or another 2-alkyl-4,6-dihydroxybenzoic acid, e.g., an olivetolic acid analog such as divalic acid), prenol, isoprenol, or geraniol may be present at concentrations of 0.1–50 g / L (e.g., 1–10 g / L). In a fed-batch setting, the precursors can be slowly fed into the fermentation apparatus over 2–20 hours so that 1–100 g / L (e.g., 1–10 g / L, or 10–100 g / L) of each essential precursor is ultimately added.

[0100] Similarly, carboxylic acid starting materials, such as hexanoic acid, butanoic acid, and pentanoic acid, may be present at concentrations of 0.1 to 50 g / L (e.g., 1 to 10 g / L). In a fed-add batch configuration, the carboxylic acid can be slowly supplied to the fermentation apparatus over 2 to 20 hours so that 1 to 100 g / L (e.g., 1 to 10 g / L, or 10 to 100 g / L) of carboxylic acid is ultimately added.

[0101] Culture conditions such as expression time, temperature, and pH can be adjusted to obtain high yields of target cannabinoid intermediates (e.g., olivetolic acid or divalic acid) and / or target cannabinoid products (e.g., CBGA, CBGVA). Generally, host cells are cultured at a temperature in the range of approximately 20°C to approximately 40°C for a period ranging from several hours to more than one day (e.g., 24, 30, 36, or 48 hours), depending on the specific host cells used, in the presence of starting materials such as olivetolic acid, hexanoic acid, prenol, or isoprenol. For example, S. cerevisiae can be cultured at 25–32°C for 24–40 hours (e.g., 30 hours). The pH of the culture medium can be maintained at a specific level by adding acids, bases, and / or buffers. In some embodiments, culturing yeast at a pH of 6 or higher can reduce the production of undesirable by-products such as olivetolic acid. In some embodiments, the pH of the yeast culture is in the range of approximately 6–8. In some embodiments, the pH of the yeast culture is approximately 6.5. In some embodiments, the pH of the yeast culture is approximately 7. In some embodiments, the pH of the yeast culture is approximately 8.

[0102] In some embodiments, recombinant yeast cells are genetically modified to produce the cannabinoid or cannabinoid acid product or intermediate of interest at levels of at least about 0.1 g / L, at least about 0.5 g / L, at least about 0.75 g / L, at least about 1 g / L, at least about 1.5 g / L, at least about 2 g / L, at least about 2.5 g / L, at least about 3 g / L, at least about 3.5 g / L, at least about 4 g / L, at least about 4.5 g / L, at least about 5 g / L, at least about 5.5 g / L, at least about 6 g / L, at least about 7 g / L, at least about 8 g / L, at least about 9 g / L, or at least 10 g / L when cultured in vivo in a suitable precursor-containing medium as described above. In some embodiments, recombinant yeast cells are genetically modified to produce a cannabinoid product or intermediate of interest at levels of at least about 20 g / L, at least about 30 g / L, at least about 50 g / L, or at least about 80 g / L when cultured in vivo in a suitable medium.

[0103] Cannabinoid production can be carried out in any container that allows for cell growth and / or incubation. For example, the reaction mixture may be a bioreactor, cell culture flask or cell culture plate, multi-well plate (e.g., 96, 384, 1056-well microtiter plate, etc.), culture flask, fermenter, or other container for cell growth or incubation. The biologically produced product of interest can be isolated from the luminescent medium or cell extract using methods known in the art. For example, solids or cell fragments can be removed by centrifugation or filtration. The product of interest can be isolated by, for example, distillation, liquid-liquid extraction, membrane evaporation, adsorption, or other methods.

[0104] The present invention, as described exemplary herein, can be appropriately implemented in the absence of any one or more elements or limitations not specifically disclosed herein. Therefore, for example, in each case herein, any of the terms “contains,” “essentially consisting of,” and “consisting of” can be replaced with any of the other two terms. Thus, for example, some embodiments may encompass host cells “containing” some components, other embodiments may encompass host cells “essentially consisting of” the same components, and yet another embodiment may encompass host cells “consisting of” the same components. The terms and expressions used are descriptive terms, not restrictive terms, and in the use of these terms and expressions, there is no intention to exclude any equivalent or part of the features presented and described, but rather it is recognized that various modifications are possible within the scope of the claimed invention. Therefore, while the present invention is specifically disclosed by preferred embodiments and optional features, those skilled in the art should understand that modifications and variations of the concepts disclosed herein are possible, and that these modifications and variations are considered to be within the scope of the invention as defined by the appended claims.

[0105] The written description above is considered sufficient to enable those skilled in the art to carry out the present invention. The following examples are provided for illustrative purposes only and are not intended in any way to limit the scope of the present invention. In fact, various modifications of the present invention other than those shown and described herein will be apparent to those skilled in the art from the above description and are within the scope of the appended claims.

[0106] Where references are made herein to patent specifications, other external documents, or other sources, these are generally intended to provide context for describing the features of the present invention. Unless otherwise specifically stated, references to such external documents should not be construed, in any scope, as an acknowledgment that these documents or sources constitute prior art or form part of the common general knowledge in the art. All patents, patent applications, and references cited herein are incorporated herein by reference in their entirety. [Examples]

[0107] Example 1. Construction of a plasmid expressing a truncated GOT3 gene and production of CBGA. This example describes the construction and use of plasmid pBM308L. This plasmid is an episome expression plasmid constructed using the ADH2 promoter and terminator sequences adjacent to the human superoxide dismutase (hSOD) gene, which are in-frame fused to the GOT3 minigene encoding amino acid numbers 80-398 of the CsPT4 sequence, the LEU2 gene, and a 2-micron sequence de novo cloned from yeast genomic DNA by PCR. The CsPT4 gene of C. sativa was chemically synthesized by GenScript Corporation using yeast-preferred codons.

[0108] The 2-micron sequence within this plasmid contains the origin for autonomous replication in yeast. The LEU2 gene facilitates the selection of transformed cells and plasmid retention during growth in leucine-deficient medium. GOT3 expression is regulated by the glucose-regulated ADH2 promoter. Cell growth under glucose-restricted conditions induces continuous expression of the hSOD / GOT3 fusion protein, while expression is inhibited when cells grow in excess glucose. The ADH2 terminator is required to terminate transcription of the rAAT gene in yeast.

[0109] Plasmid pBM308L was transformed into strain Y385. Strain Y385 is a derivative of "Super Alcohol-Activated Dried Yeast" (Angel Yeast Co., Ltd. Yichang, Hubei 443003, PRChina). In addition to the introduced selection markers (URA3 and LEU2), strain Y385 also contains the entire mevalonate pathway for geranyl pyrophosphate biosynthesis, as well as the ADR1 transcription factor and pyruvate decarboxylase enzyme (PDC) genes.

[0110] The culture was grown overnight in 3 mL of Leu-Minimal Medium. Next, 500 μl of the overnight culture was inoculated into 5 mL of YPD (2% D, 10 mM riboflavin and 50 μM pantothenic acid). After another 24 hours, when the optical density reached 15, 2.69 mg of olivetolic acid (OA) (80 mM OA (75% EtOH) 150 μl) was added to the culture medium. Further OA was added at 44 and 81 hours to obtain a total of 6.2 mg of OA (80 mM 350 μl).

[0111] CBGA, CBFA, and CBIA production were recorded over time (Figure 3). At 90 hours post-inoculation, the flask was left at 4°C and sampled. CBGA was measured at 820 mg / L. Final OD 600 The concentration was 21.2. A total flask extraction was performed, and CBGA was measured at 843 mg / L. Furthermore, lower levels of the minority products CBFA and CBIA were detected. CBFA and CBIA are longer and shorter side-chain homologs of CBGA, in which the geranyl group is replaced by a farnesyl group or an isopentenyl group, respectively (Pollastro et al., J. Natural Products 74:2019-2022, 2011).

[0112] Example 2. Extraction and purification of CBGA derived from recombinant S. cerevisiae cells. The cell cultures prepared as described in Example 1 were centrifuged and extracted with 50% isopropanol / water. The yeast medium supernatant was collected and subjected to ion-exchange chromatography, followed by further downstream processing steps to obtain CBGA with a purity of over 98%. The isopropanol / water extract was converted to CBG as described in Example 6.

[0113] Example 3. Production of enantiomerically pure CBCA in S. cerevisiae The yeast cell strain described in Example 1 was co-transformed with plasmid pBM703U, which contains a synthetic DNA sequence encoding the full-length CBCA synthase gene (encoding the CBCAS sequence at SEQ ID NO:9) under the control of the ADH2 promoter, with yeast-preferred codons. The cells were grown as described in Example 1, and the total culture extract (50% isopropanol / medium) was analyzed by HPLC. Under normal reversed-phase HPLC conditions, CBCA was found to be synthesized in good yield. When CBCA was decarboxylated and analyzed using a chiral (amylose-based) HPLC column, surprisingly, the only enantiomer biosynthesized was found to be the active CBC molecule. Previous studies had shown that CBC isolated from cannabis plants consists of both enantiomers of CBC. In contrast, Figure 2 shows that when the CBCAS enzyme is expressed in yeast, under the conditions used for decarboxylation from CBCA to CBC in this experiment, known epimerization is permitted, resulting in CBCA with enantiomer purity exceeding 96%, and most likely close to 100%.

[0114] Example 4. Decarboxylation of CBGA in CBGA-containing yeast cells in the presence of a metal salt. Decarboxylation of CBGA from centrifuged yeast as described in Example 1 was achieved on a small scale by centrifugation of 1 mL of whole yeast cell culture followed by resuspension in 10 mM, 50 mM, or 100 mM metal salt solutions, with or without the addition of zeolite (10 mg). The suspension with 50 mM zinc sulfate and 5 mM sodium hydroxide was heated overnight at 70°C, and the yield of CBG, as measured by HPLC, was found to be over 90%.

[0115] Example 5. Decarboxylation of CBCA in CBCA-containing yeast cells in the presence of a metal salt. Enantiomerically pure CBC is prepared similarly using the cells described in Example 3, as in Example 4. The reaction proceeds in the same manner as CBGA decarboxylation, except that CBCA decarboxylation is faster and yields higher results.

[0116] Example 6. Increased CBGA yield by supplying ethanol as a surplus carbon source. In an experiment similar to Example 1, ethanol was added as a surplus carbon source (as a 1.5 mM bolus of olivetolic acid in 50% ethanol). After overnight growth, six such additions were made at 12-hour intervals. The medium also contained Tween 20 (1%). In this experiment, extraction and HPLC analysis revealed an increase in the yield of 1.40 g / L CBGA compared to the yield in Example 1.

[0117] Example 7. Fusion of cleaved GOT3 with SOD increases yield. Using small-scale, non-glucose-supplied shaking flask experiments, hSOD-GOT3 fusion constructs containing cleavage-type GOT3 (80-398) fusion constructs were directly compared to the same cleavage-type enzyme lacking an hSOD fusion partner. The results showed a significant increase in CBGA levels when using the hSOD fusion construct (346.5 mg / L vs. 73.2 mg / L).

[0118] Example 8. Further GOT3 cutting The GOT3 enzyme is known to contain several putative transmembrane domains. In this experiment, we evaluated additional GOT3 constructs with alternative cleavage compared to GOT3 (80-398). In this experimental set, plasmid 308L (Example 1) was replaced with plasmids pBM309L, pBM316L, and pBM318L containing the GOT3 gene, as follows: pBM309L with a longer N-terminal deletion encoding GOT3 amino acid numbers 113-398; pBM316L encoding GOT3 amino acid numbers 80-269; and pBM318L encoding GOT3 amino acid numbers 80-339. HPLC analysis revealed CBGA expression levels that were barely detectable or undetectable.

[0119] Example 9. Decarboxylation of cannabinoid acids CBGA and CBCA after extraction from yeast cells. Decarboxylation in isopropanol / water was shown to proceed similarly when cannabinoid acids were isolated from yeast cells by vortexing in a 50% isopropanol / water mixture, centrifuging, and treating the clear solution with a metal or metal salt as described above.

[0120] Example 10. Production of CBGVA Yeast cells expressing the C. sativa olivetolate PKS system (C. sativa tetraketide synthase (TKS) and engineered C. sativa cyclase) and transformed with a DNA construct for butanoyl-CoA production using Rosebria hominis butanoyl-CoA transferase, or with a construct encoding CsAAE3 or revS, were grown overnight in 3 mL of Leu-,Ura-minimal medium. 300 μl of the overnight culture was then inoculated into 3 mL culture tubes of YPD (2% D, 10 mM riboflavin and 50 μM pantothenic acid). Cells were grown overnight at 30°C and 250 rpm. A 2 mM butanoic acid bolus from 1 M ethanol solution was supplied in the morning and evening, and the cultures were grown overnight. The following morning and evening, 2 mM butanoic acid was supplied from a 0.3 M butanoic acid stock diluted in ethanol, and the culture was grown overnight. The supply of 2 mM butanoic acid (0.3 M stock in ethanol) was repeated until day 3. The culture was extracted as described above, and the production of divalic acid (dVA) and divalinol (dVL) was measured by HPLC at 72 hours. The yield of dVA was 628 mg / L, and the yield of dVL was 93 mg / L. When this experiment was repeated in a 2 L (2 liter) glucose-supplied fermenter, the yield of dVA increased to approximately 1.4 g / L. This yield was higher than the yield obtained using revS or CsAAE3 constructs.

[0121] Example 11. Production of CBGVA using GOT3 cleavage (80-398) Y371 strains transformed with plasmid 308L expressing GOT3 (amino acid numbers 80-398) were grown overnight in 3 mL of Leu-Minimal Medium. 500 μl of the overnight culture was then inoculated into 5 mL flasks of YPD (2% D, 10 mM riboflavin, 50 μM pantothenic acid, 1% Tween 20). Cells were grown overnight at 30°C and 250 rpm. 0.5 mM crude dVA extract dissolved in EtOH was added in the morning and evening (total 1 mM dVA). Cells were grown for a further 48-72 hours, and CBGVA production was measured by HPLC. CBGVA was obtained at a concentration of 148 mg / L.

[0122] Example 12. Production of THCA, CBCA, CBDA, THCVA, CBCVA, and CBDVA: Overnight cultures of yeast strains expressing appropriate cannabinoid acid synthases were grown in 3 mL of appropriate dropout minimum medium. 500 μl of the overnight culture was then inoculated into 5 mL flasks of YPD (2% D, 10 mM riboflavin, 50 μM pantothenic acid, 1% Tween 20). Cells were grown for 72 hours at 15°C at 250 rpm. Cells were then transferred to 30°C and supplied with 2 mM OA or dVA dissolved in 50% EtOH. Cells were grown for a further 48–72 hours at 30°C, the culture was extracted, and cannabinoid acid production was measured by HPLC. The yields of cannabinoid acids from these small-scale shaking flask experiments were 301 mg / L (THCA), 309 mg / L (CBCA), 163 mg / L (CBDA), 84 mg / L (THCVA), 56 mg / L (CBCVA), and 16 mg / L (CBDVA).

[0123] Example 13. Production of olivetolic acid analogs and olivetolic acid analogs in recombinant yeast Yeast cells expressing the C. sativa olivetolate PKS system were transformed with DNA constructs of various CoA transferases or CoA ligases and cultured as described in Example 10, supplied with a range of fatty acid substrates in addition to butanoic acid. As shown in the table below, R. hominis-butyryl-CoA:acetate-CoA-transferase was found to be particularly useful in the production of various olivetolate analogs and olivetolate analogs.

[0124] (Table 1) Production of olivetolic acid, olivetolic acid and their analogues, such as divalic acid and divalinol, using selected acyl-CoA transferases and ligases. TIFF0007875122000006.tif124167 1 C. sativa CsAAE3 2 Streptomyces SN-593 revS medium-chain fatty acid acyl-CoA ligase 3M. avium mig medium-chain acyl-CoA ligauze 4 S. cerevisiae FAA2 medium-chain acyl-CoA ligauze 5 A. Sariana AT4g05160 Acyl-CoA Coumarate Ligase 6 E. coli FADK acyl-CoA ligauze 7 R. hominis butyryl-CoA:CoA acetate transferase 8 C. Necatol propionic acid-CoA transferase a OA analog = yield of olivetolic acid analog (mg / L) b OL analog = Olivetol analog yield (mg / L)

[0125] The examples and embodiments described herein are for illustrative purposes only, and it will be understood that various modifications or changes will be suggested to those skilled in the art and will be included in the spirit and scope of this application and the appended claims. All publications, patents, accession numbers, and patent applications referenced herein are incorporated herein in their entirety by reference for all purposes.

[0126] Example sequence SEQ ID NO:1 GOT cleavage sequence; amino acid numbers 80-398 of the CsPT4 GOT protein; the M at position 1 of SEQ ID NO:1 is the initiation methionine encoded by the polynucleotide construct expressing the polypeptide, and position 80 of amino acid numbers 80-398 of the mature GOT sequence corresponds to the residue "S" at position 2 of SEQ ID NO:1. TIFF0007875122000007.tif41160 SEQ ID NO:2 shows the hSOD-GOT3 amino acid sequence fused to region 80-398 of the GOT protein sequence. The hSOD sequence is underlined. TIFF0007875122000008.tif62160 SEQ ID NO:3 Prepro α-CBCAS protein sequence; the prepro sequence is underlined. The start of the mature polypeptide sequence is shown in bold. TIFF0007875122000009.tif79160 SEQ ID NO:4 Prepro α-CBCAS-HDEL (protein sequence). The prepro sequence and HDEL sequence are underlined. TIFF0007875122000010.tif77160 SEQ ID NO:5 Pdi1-CBCAS protein sequence. The Saccharomyces cerevisiae Pdi1 signal sequence is underlined. TIFF0007875122000011.tif70160 SEQ ID NO:6 EasE-CBCAS protein sequence. The berberine bridge-related EasE signaling sequence from Aspergillus japonica is underlined. TIFF0007875122000012.tif70160 SEQ ID NO:7 Prepro α-CBCAS protein sequence (amino acid numbers 87-545 of SEQ ID NO:9). The prepro sequence is underlined. TIFF0007875122000013.tif70160 SEQ ID NO:8 CBCAS; amino acid numbers 87-545 (of SEQ ID NO:9) containing a methionine start codon. TIFF0007875122000014.tif62160 SEQ ID NO:9 CBCAS Synthase Full-Length Amino Acid Sequence TIFF0007875122000015.tif70160 SEQ ID NO:10 Exemplary RevS polypeptide sequence GenBank BAK64635.1 TIFF0007875122000016.tif77160 SEQ ID NO:11 Exemplary Cannabis sativa CsAAE3 polypeptide sequence; GenBank AFD33347.1 TIFF0007875122000017.tif70160 SEQ ID NO:12 Exemplary Cannabis sativa CSAAE1 polypeptide sequence; GenBank AFD33345.1 The transmembrane domain is underlined. TIFF0007875122000018.tif92160 SEQ ID NO:13 Exemplary olivetolate synthase polypeptide; UniProtKB / Swiss-Prot: B1Q2B6.1 TIFF0007875122000019.tif47159 SEQ ID NO:14 Exemplary olivetolate cyclase polypeptide sequence; UniProtKB / Swiss-Prot: I6WU39.1 TIFF0007875122000020.tif11158 SEQ ID NO:15 is an olivetolate cyclase polypeptide sequence that lacks N-terminal methionine and C-terminal lysine compared to SEQ ID NO:14. TIFF0007875122000021.tif11159 SEQ ID NO:16, compared to SEQ ID NO:5, is a cleavage-type cyclase with 95 aa, mediated by the N-terminal methionine and the C-terminal 5-amino acid sequence YTPRK. TIFF0007875122000022.tif11159 SEQ ID NO:17 Translation of protein sequence from hop CBDAS homologous nucleotide sequence HL.Tea.v1.0.G019551 TIFF0007875122000023.tif63160 SEQ ID NO:18 Nucleotide sequence HL.Tea.v1.0.G019636.1 in hops. Protein sequence translation from CBDAS homolog. TIFF0007875122000024.tif70160 SEQ ID NO:19 Nucleotide sequence HL.Tea.v1.0.G037793.1 in hops. Protein sequence translation from CBDAS homolog. TIFF0007875122000025.tif70159 SEQ ID NO:20 Rosebria hominis UniProtKB-G@SYC0 protein sequence TIFF0007875122000026.tif55160 SEQ ID NO:21 Escherichia coli Acetyl-CoA:Acetoacetyl-CoA Transferase AtoA TIFF0007875122000027.tif26159 SEQ ID NO:22 Escherichia coli Acetyl-CoA:Acetoacetyl-CoA transferase AtoA TIFF0007875122000028.tif25160 SEQ ID NO:23 C. Nekatol H16 Propionic Acid CoA-Transferase TIFF0007875122000029.tif70160 SEQ ID NO:24 M. avium mig medium-chain acyl-CoA ligauze TIFF0007875122000030.tif70160 SEQ ID NO:25 A. Sariana AT4g05160 Acyl-CoA Coumarate Ligase TIFF0007875122000031.tif70160 SEQ ID NO:26 S. cerevisiae FAA2 medium-chain acyl-CoA ligauze TIFF0007875122000032.tif92160 SEQ ID NO:27 Escherichia coli FADK acyl-CoA ligase TIFF0007875122000033.tif70160

Claims

1. The first exogenous polynucleotide encoding prenyltransferase, and A second exogenous polynucleotide encoding CBCA synthase, CBDA synthase, or THCA synthase. A modified recombinant yeast host cell, which includes, The prenyltransferase comprises geranyl-pyrophosphate-olivetolate geranyltransferase (GOT), and the GOT is expressed as a fusion protein with a partner that confers enhanced expression of active GOT, and The partner that confers enhanced expression of the active GOT is selected from the group consisting of superoxide dismutase, yeast maltose-binding protein (MBP), human or yeast ubiquitin (Ub), catalase, Saccharomyces cerevisiae catalase T (CTT1), Saccharomyces cerevisiae peroxisome catalase A (CTA1), transferrin, human serum albumin (HSA), human galectin, Escherichia coli maltose-binding protein (MBP), yeast prepro-α factor, and GB1. The aforementioned modified recombinant yeast host cells.

2. The modified recombinant yeast host cell according to claim 1, wherein the superoxide dismutase is human superoxide dismutase (hSOD).

3. The modified recombinant yeast host cell according to claim 1, wherein the catalase is human catalase (hCAT).

4. (a) The second exogenous polynucleotide encodes a CBCA synthase, and the CBCA synthase contains an amino acid sequence that has at least 95% identity with one of SEQ ID NO: 3 to 9. (b) The amino acid sequence of the prenyltransferase contains SEQ ID NO:2, or (c) The amino acid sequence of the prenyltransferase contains SEQ ID NO:2; and the second exogenous polynucleotide encodes CBDA synthase or THCA synthase. Modified recombinant yeast host cells according to claim 1.

5. A modified recombinant yeast host cell according to any one of claims 1 to 4, which is genetically modified to overexpress mevalonate pathway enzymes.

6. The modified recombinant yeast host cell according to claim 5, wherein one or more mevalonate pathway enzymes are endogenous enzymes.

7. The modified recombinant yeast host cell according to claim 5, wherein one or more mevalonate pathway enzymes are exogenous enzymes not naturally expressed in yeast host cells.

8. Modified recombinant yeast host cells according to any one of claims 1 to 7, which are modified to overexpress erg10, erg13, thmgr, erg12, erg8, mvd1, idi1, and erg20 F96WN127W, as well as the mvaE and mvaS genes derived from Enterococcus faecalis.

9. Methods for producing cannabinoids, including the following steps: A step of providing modified recombinant yeast host cells according to claim 1 with olivetolic acid or divalic acid, or a fluorinated or chlorinated analog thereof, for the production of the corresponding acid cannabinoid CBCA, CBDA, or THCA, or a fluorinated or chlorinated analog thereof, wherein the acid cannabinoid is produced by prenylation of olivetolic acid or divalic acid, or an analog thereof; A step of enzymatically or chemically decarboxylating the acidic cannabinoid in order to produce the corresponding compound CBC, CBD, or THC, or an analogue thereof.

10. The method according to claim 9, wherein the superoxide dismutase is human superoxide dismutase (hSOD).

11. The method according to claim 9, wherein the catalase is human catalase (hCAT).

12. The method according to any one of claims 9 to 11, wherein recombinant yeast host cells express a polynucleotide encoding CBCA synthase.

13. The method according to claim 12, wherein the produced CBCA has an enantiomer purity of more than 96%.

14. The method according to any one of claims 9 to 13, wherein the decarboxylation step is performed on an extract of yeast host cells prepared using an extraction reagent containing an organic solvent or an organic solvent / water mixture.

15. The method according to any one of claims 9 to 14, wherein the decarboxylation step comprises incubating the extract at a temperature of 20°C to 100°C in the presence of a metal catalyst.

16. The method according to claim 15, wherein the metal catalyst is zinc, molybdenum, nickel, copper, platinum, palladium, or iron.

17. The method according to claim 15 or 16, wherein the metal catalyst is provided as a metal-loaded zeolite catalyst.

18. The method according to any one of claims 9 to 13, wherein the decarboxylation step is carried out with a decarboxylase enzyme.

19. The method according to claim 18, wherein the decarboxylase is Aspergillus nidulans orsB decarboxylase, Aspergillus clavatus-derived PatG enzyme, or Enterobacter cloacae-derived 3,4-dihydroxybenzoic acid decarboxylase.

20. Roseburia hominis expresses a polynucleotide encoding an acyl-CoA synthase selected from the group consisting of butanoyl-CoA transferase, revS, CsAAE3, and CsAAE1; It expresses a polynucleotide encoding olivetolate synthase; and It expresses a polynucleotide that encodes olivetolate cyclase. The method according to any one of claims 9 to 19, wherein olivetolic acid or divalic acid, or an analog thereof, is produced by a host cell that has been genetically modified in such a manner.

21. The method according to claim 20, wherein the acyl-CoA synthase is rosebria hominis butanoyl-CoA transferase.

22. The method according to claim 20, wherein the acyl-CoA synthase comprises an amino acid sequence having at least 95% identity with SEQ ID NO:20; the olivetolate synthase comprises an amino acid sequence having at least 95% amino acid sequence identity with SEQ ID NO:13; and the olivetolate cyclase comprises an amino acid sequence having at least 95% identity with SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:

16.

23. A method for obtaining a yield of 60% or more of CBG from CBGA from modified recombinant yeast host cells according to claim 1 that produce CBGA, comprising the following steps: The steps include: preparing a yeast cell extract using an extraction reagent containing an organic solvent / water mixture, and A step of incubating the extract at a temperature of 20°C to 100°C in the presence of a metal catalyst.

24. The method according to claim 23, wherein the organic solvent is an alcohol-water mixture.

25. The method according to claim 23 or 24, wherein the metal catalyst is zinc, molybdenum, nickel, copper, platinum, palladium, or iron; or a salt thereof.

26. The method according to any one of claims 23 to 25, wherein the metal catalyst is provided as a metal-loaded zeolite catalyst.

27. A modified recombinant yeast host cell containing an exogenous polynucleotide encoding prenyltransferase, The prenyltransferase comprises geranyl-pyrophosphate-olivetolate geranyltransferase (GOT), and the GOT is expressed as a fusion protein with a partner that confers enhanced expression of active GOT, and The partner that confers enhanced expression of the active GOT is selected from the group consisting of superoxide dismutase, yeast maltose-binding protein (MBP), human or yeast ubiquitin (Ub), catalase, Saccharomyces cerevisiae catalase T (CTT1), Saccharomyces cerevisiae peroxisome catalase A (CTA1), transferrin, human serum albumin (HSA), human galectin, Escherichia coli maltose-binding protein (MBP), yeast prepro-α factor, and GB1. The aforementioned modified recombinant yeast host cells.

28. The modified recombinant yeast host cell according to claim 27, wherein the amino acid sequence of the prenyltransferase includes SEQ ID NO:

2.

29. A modified recombinant yeast host cell according to claim 27 or 28, further comprising a second exogenous polynucleotide encoding CBCA synthase, CBDA synthase, or THCA synthase.

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