Biosynthesis of substituted compounds and cannabinoids

Abstract

The present invention provides a system and method for prenylation and a recombinant pathway for the production of compounds, cannabinoids, cannabinoid precursors, and other prenylated chemicals in a cell-free system. The present invention further includes enzymes and recombinant microorganisms that catalyze the reaction. The compounds, cannabinoids, cannabinoid precursors, and other prenylated chemicals are operable to be substituted with at least one deuterium, at least one tritium, at least one halogen, at least one hydroxyl group, and / or at least one additional isotope.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application is related to and claims priority to the following U.S. patent applications: This application claims priority to and the benefit of U.S. patent application Ser. No. 18 / 227,719, filed July 28, 2023, which claims priority to and the benefit of U.S. provisional application Ser. No. 63 / 370,070, filed August 1, 2022, each of which is incorporated herein by reference in its entirety. This application also claims priority to and the benefit of U.S. provisional application Ser. No. 63 / 370,070.

[0002] (Reference to sequence listing) An official copy of the sequence listing will be submitted simultaneously with the specification via EFS-Web as an XML file with the file name "4482003.xml," a creation date of July 28, 2023, and a file size of 133 kilobytes. The sequence listing submitted via EFS-Web is a part of the specification and is incorporated herein by reference in its entirety. [Background technology]

[0003] 1. Field of the Invention

[0004] The present invention relates to cannabinoids, and more particularly to substituted compounds and cannabinoids, and their biosynthesis.

[0005] 2. Description of the Prior Art

[0006] Prenylation of natural compounds increases structural diversity, alters biological activity, and enhances therapeutic efficacy. Prenylated compounds often have low natural abundance or are difficult to isolate. Some prenylated natural products include a wide range of bioactive molecules with proven medicinal properties. Examples include prenylflavonoids, prenylstilbenoids, and cannabinoids.

[0007] Cannabinoids are a broad class of plant-derived, bioactive natural products that modulate cannabinoid receptors (CB1 and CB2) in the human endocannabinoid system. Cannabinoids are promising pharmacological agents, with over 100 clinical trials underway to test their therapeutic potential as antiemetics, anticonvulsants, analgesics, and antidepressants. Additionally, three cannabinoid therapies have been approved by the FDA for the treatment of chemotherapy-induced nausea, MS spasticity, and seizures associated with severe epilepsy.

[0008] Despite their therapeutic potential, the production of pharmaceutical-grade (>99%) cannabinoids remains a significant technical challenge. Cannabis plants, such as marijuana and hemp, produce a variety of low-abundance cannabinoids, along with high levels of tetrahydrocannabinolic acid (THCA) and cannabidiolic acid (CBDA). However, even highly expressed cannabinoids like CBDA and THCA are difficult to isolate due to the high structural similarity of contaminating cannabinoids and the variability in cannabinoid composition within each crop. These challenges are exacerbated when attempting to isolate rare cannabinoids. Furthermore, current cannabis cultivation methods pose serious environmental problems. As a result, there has been significant interest in developing alternative methods for the production of cannabinoids and cannabinoid analogs.

[0009] Additionally, it is commonly known in the art to synthesize deuterated cannabinoids.

[0010] Prior art documents include the following:

[0011] U.S. Patent No. 5,036,014, filed January 31, 1989, and issued July 30, 1991, by inventors Elsohly et al., entitled "Deuterated cannabinoids as standards for the analysis of tetrahydrocannabinol and its metabolites in biological fluids," relates to new internal standards for use in gas chromatography / mass spectrometry test methods comprising deuterated cannabinoids developed for the analysis of tetrahydrocannabinol and its metabolites in biological fluids.

[0012] U.S. Patent No. 5,633,357, filed March 4, 1994, and issued May 27, 1997, for "Synthesis of Carboxylic Acid Glucuronides," by inventors Tius et al., describes a method for producing carboxylic acid glucuronic acids by reacting a carboxylic acid precursor with a blocked sugar epoxide precursor. Also disclosed are deuterated 11-nor-Δ8- or Δ9-THC carboxylic acid glucuronide, 5'-deuterated 11-nor-Δ8- or Δ9-THC carboxylic acid, or 5'-deuterated Δ8- or Δ9-THC glucuronide, each having a deuterated hydrocarbon chain. These compositions are useful as GC-MS standards, in methods for preparing antibodies reactive with THC glucuronides, and in GC-MS diagnostic methods for THC metabolites.

[0013] U.S. Patent No. 10,837,031, filed May 10, 2018, and issued November 22, 2018, by inventors Barr et al., for "Recombinant production systems for prenylated polyketides of the cannabinoid family," relates to production methods, enzymes, and recombinant yeast strains for the biosynthesis of clinically important prenylated polyketides of the cannabinoid family. Using readily available starting materials, heterologous enzymes are used to induce cannabinoid biosynthesis in yeast.

[0014] WIPO International Publication No. 2021034403, "Cannabinoid acid ester compositions and uses thereof," by inventors Swisa et al., filed June 19, 2020, and published February 25, 2021, relates to pharmaceutical compositions containing cannabinoid acid ester compounds, alone or in combination with one or more additional cannabinoid compounds. The document discloses that the cannabinoid acid ester compounds are tetrahydrocannabinolic acid (THCA) esters. The document also discloses that the cannabinoid acid ester compounds are cannabigerolic acid (CBGA) esters. The document also discloses that the cannabinoid acid ester compounds are cannabinolic acid (CBNA) esters. Various therapeutic applications for the cannabinoid acid ester compounds and pharmaceutical compositions are also provided, including combination therapies using the cannabinoid acid ester compounds and one or more additional therapeutic agents.

[0015] WIPO International Publication No. 2020186010, "Cannabinoid acid ester compositions and uses thereof," by Robinson et al., filed March 12, 2020, and published September 17, 2020, relates to pharmaceutical compositions containing cannabinoid acid ester compounds, alone or in combination with one or more additional cannabinoid compounds. The publication discloses that the cannabinoid acid ester compounds are cannabidiolic acid esters. Various therapeutic applications for the cannabinoid acid ester compounds and pharmaceutical compositions are also provided, including combination therapies using the cannabinoid acid ester compounds with one or more additional therapeutic agents.

[0016] U.S. Patent Application Publication No. 20210403408, "Cannabinoid analogs and methods for their preparation," by inventors Barr et al., filed April 29, 2021, and published June 30, 2021, relates to cannabinoid analogs, including halogenated, hydroxylated, deuterated, and tritiated cannabinoid analogs. Cannabinoid analogs can be prepared by partial or complete expression in engineered host cells, such as recombinant yeast cells, optionally in combination with chemical synthesis steps.

[0017] U.S. Patent Application Publication No. 20210230113, filed January 22, 2021, and published July 29, 2021, by inventor Filer, entitled "Cannabinoid Derivatives," relates to 8,9-dihydrocannabinoid derivatives, deuterated cannabinoid derivatives, and tritiated cannabinoid derivatives. The disclosure also provides compositions, methods of preparation, and manufacturing processes for the derivatives.

[0018] WIPO WO 2021000053, "Cannabinoid Derivatives," filed July 3, 2020, and published January 7, 2021, by inventors Omeara et al., relates to cannabinoid derivatives, pharmaceutical compositions comprising the derivatives, and methods of using the derivatives in the treatment or prevention of diseases associated with cannabinoid receptors. The claimed cannabinoid derivative is a compound represented by the following formula:

[0019] [ka]

[0020] WIPO International Publication No. 2021046640, "Cannabinoid derivatives and precursors, and asymmetric synthesis for the same," by inventors Abdur-Rashid et al., filed September 9, 2020, and published March 18, 2021, relates to novel cannabinoid derivatives and precursors and catalytic asymmetric processes for their preparation. The disclosure also relates to pharmaceutical compositions of the novel cannabinoid derivatives, as well as medicinal and analytical uses. For example, the disclosure relates to the preparation of novel precursors and the use of such precursor compounds for the preparation of isotopically labeled cannabinoid products using chiral and achiral catalysts and catalytic processes. Deuterium-, carbon-13-, and carbon-14-containing compounds can be prepared and purified prior to transformation into the desired individual deuterated cannabinoid products.

[0021] WIPO International Publication No. 2021113669, "Cannabinoids and uses thereof," by inventors Deng et al., filed December 4, 2020, and published June 10, 2021, relates to cannabinoid compounds, pharmaceutical compositions comprising one or more cannabinoid compounds, and uses of pharmaceutical compositions comprising one or more cannabinoid compounds for the treatment of a disease or condition (e.g., a fibrotic or inflammatory disease) in a subject in need thereof.

[0022] WIPO International Publication No. 2022082313, "Compositions and methods for treating neuronal disorders with cannabinoids," by inventors Hsu et al., filed October 21, 2021, and published April 28, 2022, relates to methods and compositions comprising cannabinoid compounds for providing neuroprotection and / or stimulating neuritogenesis. The cannabinoid compounds are compounds shown below, where R1 is COOH or H, R2 is C3H7 or C5H11, R3 is H or Me, and R4 and R5 are Me or (CH2)2CH=C(CH3)2, such as CBGA, derivatives thereof, prodrugs thereof, or combinations thereof, which can be used to treat neurodegenerative disorders or promote neurite outgrowth and / or restore neuritogenesis in patients in need thereof.

[0023] [ka]

[0024] WIPO International Publication No. 2021150636, "Genetically modified yeast for the production of cannabigerolic acid, cannabichromenic acid and related cannabinoids," by inventors Barr et al., filed January 20, 2021, and published July 29, 2021, relates to production methods, enzymes, and recombinant yeast strains for the biosynthesis of clinically important cannabinoid compounds.

[0025] U.S. Patent Publication No. 20100298579, "Process for preparing synthetic cannabinoids," by inventors Steup et al., filed April 29, 2010, and published November 25, 2010, relates to organic synthesis, particularly to a method for preparing cannabinoids. The method described is applicable to all stereoisomers and congeners of cannabinoids. To this end, the publication provides methods for preparing the compounds in two or three chemical synthesis steps.

[0026] U.S. Patent Publication No. 20100152283, filed December 17, 2009, and published June 17, 2010, by inventors Gant et al., entitled "Tetrahydrocannabinol modulators of cannabinoid receptors," relates to tetrahydrocannabinol modulators of cannabinoid receptors, pharmaceutical compositions thereof, and methods of use thereof.

[0027] WIPO International Publication No. 2020102430, "Use of type I and type II polyketide synthases for the production of cannabinoids and cannabinoid analogs," by inventors Barr et al., filed November 13, 2019, and published May 22, 2020, relates to production methods, enzymes, and recombinant yeast strains for the biosynthesis of clinically important prenylated polyketides of the cannabinoid family. Using readily available starting materials, heterologous enzymes are used to induce cannabinoid biosynthesis in yeast.

[0028] U.S. Patent Publication No. 20210040512, "Recombinant production systems for prenylated polyketides of the cannabinoid family," by inventors Barr et al., filed October 21, 2020, and published February 11, 2021, relates to production methods, enzymes, and recombinant yeast strains for the biosynthesis of clinically important prenylated polyketides of the cannabinoid family. Using readily available starting materials, cannabinoid biosynthesis in yeast is induced using heterologous enzymes.

[0029] WIPO International Publication No. 2021133989, "Preparation of cannabichromene and related cannabinoids," by inventor Marlowe, filed December 23, 2020, and published July 1, 2021, relates to a method for preparing cannabichromene and related cannabinoid compounds. The method includes forming a reaction mixture containing 3,7-dimethylocta-2,6-dienal, a diamine, and olivetol or a related starting material, and maintaining the reaction mixture under conditions sufficient to form a desired product. The disclosed methods may also include a one-pot conversion of a cannabichromene-type product to a cannabinol-type product.

[0030] WIPO International Publication No. 2021102567, "Cannabigerol derivatives and use thereof as cannabinoid receptor modulators," by inventors Ahmar et al., filed November 25, 2020, and published June 3, 2021, relates to the synthesis of a series of pentylbenzene-1,3-diol compounds represented by the following formula: These compounds bind to cannabinoid 1 and 2 receptors (CB1 and CB2) and are therefore presumed to be useful for modulating the activity of these receptors. Therefore, these synthetic cannabinoids are expected to be used in the treatment of various CB1- and CB2-mediated disorders.

[0031] [ka]

[0032] WIPO International Publication No. 2021050786, "Cannabinoid compositions with improved organoleptic and therapeutic properties, method of production, and use thereof," by inventors Alarcon et al., filed September 10, 2020, and published March 18, 2021, relates to compositions comprising cannabinoids, terpenoids, and other flavonoids. This specification also provides methods for producing compositions comprising cannabinoids, terpenoids, and other flavonoids on an industrial scale. The compositions provided herein possess desirable organoleptic properties and therapeutic effects when ingested or topically applied. The compositions of this disclosure are useful in inhaled, ingested, or topical products for the relief and treatment of various acute or chronic diseases.

[0033] WIPO WO 2021222288, filed April 27, 2021, and published November 4, 2021, by inventors Feng et al., entitled "Compositions and methods for enhancing recombinant biosynthesis of cannabinoids," relates to recombinant host cells comprising a pathway capable of producing cannabinoids and a heterologous nucleic acid encoding a protein not included in the pathway that enhances the host cell's ability to produce cannabinoids. The disclosure also provides methods for producing cannabinoids using the host cells.

[0034] WIPO WO 2021195517, "Compositions and methods for recombinant biosynthesis of cannabinoids," by inventor Schuetz, filed March 26, 2021, and published September 30, 2021, relates to recombinant host cells comprising a pathway capable of producing cannabinoids and nucleic acids derived from Cannabis trichome mRNA that do not encode enzymes in the pathway but enhance the host cell's ability to produce cannabinoids. The disclosure also provides methods for producing cannabinoids using the host cells.

[0035] U.S. Patent Publication No. 20110311474, filed December 1, 2010, and published April 23, 2013, entitled "Novel tricyclic compounds," by inventors Wishart et al., relates to the compounds shown below, their pharmaceutically acceptable salts, prodrugs, biologically active metabolites, and stereoisomers and isomers thereof, where variables are defined in this disclosure. The compounds of this disclosure are useful for the treatment of immunological and oncological diseases.

[0036] [ka]

[0037] WIPO International Publication No. 2021183448, entitled "Optimized olivetolic acid cyclase polypeptides," filed March 8, 2021, and published September 16, 2021, by inventors Horwitz et al., relates to modified variants of olivetolic acid cyclase polypeptides, the modified variants having the amino acid sequence of SEQ ID NO:1 with at least one amino acid substitution, a nucleic acid comprising a nucleotide sequence encoding the modified variant, methods for producing modified host cells comprising the nucleic acid, modified host cells expressing the modified variants, methods for producing olivetolic acid, olivetolic acid derivatives, cannabinoids or cannabinoid derivatives, and methods for screening for modified variants of olivetolic acid cyclase polypeptides.

[0038] U.S. Patent Publication No. 20200254041, filed October 5, 2018, and published June 2, 2020, by inventors Leone-Bay et al., entitled "Rapid onset and extended action plant-based and synthetic cannabinoid formulations," relates to plant-derived pharmaceutical compounds or dietary supplements and synthetic cannabinoid formulations that have rapid onset and extended action. The rapid onset is provided by N-acylated fatty acid amino acids and / or penetration enhancers. The extended action is provided by one or more sustained-release systems.

[0039] U.S. Patent Publication No. 20210251947, filed February 9, 2021, and published August 19, 2021, by inventor Elkarim, entitled "Stable formulations of dronabinol," describes cannabinoid formulations that are stable at room temperature for at least about one to two years, methods for making the same, and methods of treatment using the formulations. The publication discloses that the compositions are oxidatively stable formulations of dronabinol. Summary of the Invention

[0040] The present invention relates to substituted compounds and cannabinoids, and their biosynthesis.

[0041] It is an object of the present invention to provide substituted compounds and cannabinoids with more favorable pharmacokinetics, as well as systems and methods for their biosynthesis.

[0042] In one embodiment, the present invention provides a cannabinoid composition comprising a cannabinoid derived from a deuterated fatty acid, the compound comprising a deuterated carbon chain, the deuterated carbon chain comprising at least one deuterated carbon.

[0043] In another embodiment, the present invention provides a method for producing a phospholipid-containing enzyme comprising the steps of phosphorylating prenol and / or isoprenol using hydroxyethylthiazole kinase (ThiM) to produce isopentenyl phosphate, isomerizing the isopentenyl phosphate to produce isopentenyl diphosphate, which can be phosphorylated in the presence of inositol polyphosphate kinase to produce dimethylallyl diphosphate (DMAPP), and synthesizing geranyl pyrophosphate from the isopentenyl diphosphate and / or DMAPP in the presence of farnesyl pyrophosphate synthase. The present invention provides a method for synthesizing a cannabinoid compound or a derivative thereof, the method comprising the steps of: activating a deuterated fatty acid to produce a deuterated CoA thioester; activating an acid to produce a second CoA thioester; synthesizing the deuterated CoA thioester and the second CoA thioester; cyclizing the synthesized products to produce deuterated olivetolic acid; prenylating the deuterated olivetolic acid in the presence of geranyl pyrophosphate to produce deuterated cannabigerolic acid; and cyclizing the deuterated cannabigerolic acid to produce a deuterated cannabinoid.

[0044] In yet another embodiment, the present invention provides a method for producing a cannabinoid or derivative thereof by cloning at least one polynucleotide sequence encoding at least one enzyme into at least one microorganism to produce a modified microorganism, wherein the modified microorganism has increased expression of at least one enzyme compared to an unmodified parent microorganism; lysing at least one cell of the modified microorganism to obtain at least one enzyme, wherein the at least one enzyme is used in a cannabinoid biosynthetic pathway; and producing a cannabinoid or derivative thereof using the cannabinoid biosynthetic pathway. The method further provides a method for producing a cannabinoid or derivative thereof by activating a deuterated fatty acid in the presence of an acyl-activating enzyme (AAE)3 to produce a deuterated CoA thiol. The present invention provides a cell-free method for synthesizing a cannabinoid or a derivative thereof, the method comprising the steps of: producing an ester; activating the acid in the presence of a corresponding CoA synthetase to produce a second CoA thioester; synthesizing the deuterated CoA thioester and the second CoA thioester in the presence of olivetol synthase (OLS); cyclizing the synthesis product in the presence of olivetolate cyclase (OAC) to produce deuterated olivetolic acid; prenylating the deuterated olivetolic acid in the presence of geranyl pyrophosphate to produce deuterated cannabigerolic acid; and cyclizing the deuterated cannabigerolic acid to produce a deuterated cannabinoid.

[0045] These and other aspects of the present invention will become apparent to those skilled in the art from the following description of the preferred embodiments taken in conjunction with the drawings supporting the claimed invention. [Brief explanation of the drawings]

[0046] [Figure 1] This shows the metabolic pathway of cannabidiol (CBD). [Figure 2] Figure 2A shows an exemplary biosynthetic pathway of the present disclosure for the production of a prenylated natural product. Figure 2B shows an exemplary biosynthetic pathway of the present disclosure for the production of a prenylated natural product, continuing from Figure 2A. Figure 2C shows a more detailed diagram of the pathway shown in Figures 2A-2B. [Figure 3]Figure 3A shows pyruvate dehydrogenase (PDH) activity measured in the presence of various aromatic polyketides and 2% ethanol. Figure 3B shows a comparison of the final titers achieved with the complete pathway utilizing PDH and the PDH bypass system. Figure 3C shows the amount of 5-prenyl-1,6-DHN and CBGA produced over time in the PDH bypass system using wild-type (WT) NphB. Figure 3D shows the results of pathways using NphB, AtaPT, or NovQ prenyltransferases with various aromatic substrates. [Figure 4] Figure 4A shows a model of olivetrate in the active site of WT NphB. Figure 4B shows the results of an activity assay to determine the approximate activity of NphB mutants using olivetrate as a substrate. Figure 4C shows gas chromatography-mass spectrometry (GC-MS) chromatograms of the entire pathway reaction products using the M23 mutant and WT NphB compared to the CBGA standard. [Figure 5] Figure 5A shows the cell-free enzymatic production of cannabinoid precursors from glucose over time. Figure 5B shows the nonanfuro cannabigerolic acid (CBGA) capture system. Figure 5C shows the production of cannabinoids over time using cannabidiolic acid synthase (CBDAS). [Figure 6] Figure 6A shows a schematic of the MatB transferase pathway. Figure 6B shows a schematic of the MdcA transferase pathway. Figure 6C shows additional details of exemplary steps in the polyketide module of the pathway shown in Figures 6A-6B. [Figure 7] A schematic representation of the pathway from (iso)prenol to geranyl pyrophosphate (GPP) is shown. [Figure 8] Figure 1 shows the various canonical (eukaryotic) and non-canonical (archaeal I and II) mevalonate pathways that function to generate IPP / DMAPP from acetyl-CoA (or mevalonate). [Figure 9] As a control, Fourier transform mass spectrometry (FTMS-pESI) spectral data by probe electrospray ionization of acetonitrile with 0.1% formic acid added is shown. [Figure 10] FTMS-pESI spectral data of cannabigerolic acid (CBGA) in positive mode. [Figure 11] FTMS-pESI spectral data of CBGA in negative mode. [Figure 12] FTMS-pESI spectral data of CBGA in control and positive modes are shown. [Figure 13] FTMS-pESI spectral data of CBGA in control and positive modes are shown. [Figure 14] FTMS-pESI spectral data of deuterated CBGA (dCBGA) in positive mode. [Figure 15] FTMS-pESI spectral data of deuterated CBGA in negative mode. [Figure 16] Additional FTMS-pESI spectral data of deuterated CBGA in positive mode is shown. [Figure 17] Additional FTMS-pESI spectral data of deuterated CBGA in negative mode is shown. [Figure 18] nLC-MS / MS data of CBGA is shown. [Figure 19] nLC-MS / MS data of dCBGA is shown. [Figure 20] Shown is nLC-MS / MS data after 1 hour of the first sample of CBGA. [Figure 21] nLC-MS / MS data of the second sample of CBGA after 1 hour is shown. [Figure 22] nLC-MS / MS data of the third sample of CBGA after 1 hour is shown. [Figure 23] nLC-MS / MS data after 1 hour of the first sample of dCBGA is shown. [Figure 24] nLC-MS / MS data after 1 hour of the second sample of dCBGA is shown. [Figure 25]nLC-MS / MS data after 1 hour of the third sample of dCBGA is shown. [Figure 26] A graph comparing CBGA and dCBGA is shown. [Figure 27] Figure 27A shows one embodiment of a cell-free biosynthetic pathway for deuterated cannabinoids. Figure 27B shows one embodiment of a cell-free biosynthetic pathway for deuterated cannabinoids, including a pathway for producing GPP. [Figure 28] Figure 28A shows one embodiment of a deuterated methyl compound, and Figure 28B shows examples of methylation of various small molecules using d3-SAM and a methyltransferase. [Figure 29] An example of deuterated methylated psilocybin using specific methyltransferases is shown. [Figure 30] An example of a methylated chrysoeriol that has been deuterated using a specific methyltransferase is shown. [Figure 31] An example of the synthesis of a deuterated glucoside is shown below. [Figure 32] HPLC (high performance liquid chromatography) traces of cell-free production of multiple cannabinoids are shown. [Figure 33] 1 shows a bar graph indicating the progress made during route optimization. [Figure 34] Figure 34A is a bar graph showing OLS variants and their corresponding CBGA production levels. Figure 34B is a bar graph showing OLS variants and their corresponding CBGA production levels at enzyme loadings. [Figure 35] 1 shows the time course of a CBGA synthesis reaction according to one embodiment of the present invention. [Figure 36] 1 shows an HPLC trace of CBGA and THCA activity according to one embodiment of the present invention. [Figure 37] 1 shows an HPLC trace of THC in toluene according to one embodiment of the present invention. [Figure 38] A comparison of the enzyme activity of S-adenosyl-L-methionine-d3 and S-adenosyl-L-methionine is shown. DETAILED DESCRIPTION OF THE INVENTION

[0047] The present invention relates generally to substituted compounds and cannabinoids, and their biosynthesis.

[0048] In one embodiment, the present invention provides a cannabinoid composition comprising a cannabinoid derived from a deuterated fatty acid, the compound comprising a deuterated carbon chain, the deuterated carbon chain comprising at least one deuterated carbon.

[0049] In another embodiment, the present invention provides a method for producing a phospholipid-containing enzyme comprising the steps of phosphorylating prenol and / or isoprenol using hydroxyethylthiazole kinase (ThiM) to produce isopentenyl phosphate, isomerizing the isopentenyl phosphate to produce isopentenyl diphosphate, which can be phosphorylated in the presence of inositol polyphosphate kinase to produce dimethylallyl diphosphate (DMAPP), and synthesizing geranyl pyrophosphate from the isopentenyl diphosphate and / or DMAPP in the presence of farnesyl pyrophosphate synthase. The present invention provides a method for synthesizing a cannabinoid compound or a derivative thereof, the method comprising the steps of: activating a deuterated fatty acid to produce a deuterated CoA thioester; activating an acid to produce a second CoA thioester; synthesizing the deuterated CoA thioester and the second CoA thioester; cyclizing the synthesized products to produce deuterated olivetolic acid; prenylating the deuterated olivetolic acid in the presence of geranyl pyrophosphate to produce deuterated cannabigerolic acid; and cyclizing the deuterated cannabigerolic acid to produce a deuterated cannabinoid.

[0050] In yet another embodiment, the present invention provides a method for producing a cannabinoid or derivative thereof by cloning at least one polynucleotide sequence encoding at least one enzyme into at least one microorganism to produce a modified microorganism, wherein the modified microorganism has increased expression of at least one enzyme compared to an unmodified parent microorganism; lysing at least one cell of the modified microorganism to obtain at least one enzyme, wherein the at least one enzyme is used in a cannabinoid biosynthetic pathway; and producing a cannabinoid or derivative thereof using the cannabinoid biosynthetic pathway. The method further provides a method for producing a cannabinoid or derivative thereof by activating a deuterated fatty acid in the presence of an acyl-activating enzyme (AAE)3 to produce a deuterated CoA thiol. The present invention provides a cell-free method for synthesizing a cannabinoid or a derivative thereof, the method comprising the steps of: producing an ester; activating the acid in the presence of a corresponding CoA synthetase to produce a second CoA thioester; synthesizing the deuterated CoA thioester and the second CoA thioester in the presence of olivetol synthase (OLS); cyclizing the synthesis product in the presence of olivetolate cyclase (OAC) to produce deuterated olivetolic acid; prenylating the deuterated olivetolic acid in the presence of geranyl pyrophosphate to produce deuterated cannabigerolic acid; and cyclizing the deuterated cannabigerolic acid to produce a deuterated cannabinoid.

[0051] The present invention includes pharmacologically similar substituted compounds and cannabinoids that have reduced metabolism compared to their unsubstituted analogs. Additionally, the present invention includes cell-free systems and methods for biosynthesizing these substituted compounds and cannabinoids. The present invention also includes highly pure substituted compounds and cannabinoids synthesized by cell-free methods.

[0052] None of the prior art discloses a complete extracellular synthesis system for substituted compounds and cannabinoids that provides both high isomeric purity and high absolute purity. The high isomeric purity is due to the positional specificity of the prenylation enzymes of the present invention, and the high absolute purity is due to the completely cell-free synthesis and stoichiometric efficiency of the system of the present invention, which results in fewer contaminants to remove and fewer residual reagents. Advantageously, high isomeric purity is important when producing substitution by-products that are not easily metabolized. Furthermore, none of the prior art discloses a one-pot cell-free synthesis system that provides highly pure substituted compounds and cannabinoids.

[0053] Reference will now be made generally to the drawings, which are for the purpose of illustrating one or more preferred embodiments of the present invention and are not intended to limit the invention thereto.

[0054] compound

[0055] The present invention provides a compound that contains at least one deuterium, at least one tritium, at least one halogen (e.g., fluorine, chlorine, bromine, iodine), at least one hydroxyl group, and / or at least one additional isotope (e.g., 11 C. 13 C. 14 C. 13 N, 15 N, 18 O. 17 O. 15 O. 31 P, 32 P, 35 S, 18 F, 36 In one embodiment, at least one tritium and / or at least one additional isotope is radioactive (e.g., 3 H, 14 C). Advantageously, at least one radioisotope (e.g. 3 H, 14 C) can be used in drug and / or substrate tissue distribution assays.11 C. 18 F, 15 O. 13 N) substituted compounds can be used in positron emission topography (PET) studies.

[0056] The present invention provides a compound that contains at least one deuterium, at least one tritium, at least one halogen (e.g., fluorine, chlorine, bromine, iodine), at least one hydroxyl group, and / or at least one additional isotope (e.g., 11 C. 13 C. 14 C. 13 N, 15 N, 18 O. 17 O. 15 O. 31 P, 32 P, 35 S, 18 F, 36 In one embodiment, at least one tritium and / or at least one additional isotope is radioactive (e.g., 3 H, 14 C). Advantageously, at least one radioisotope (e.g. 3 H, 14 C)-substituted cannabinoids, cannabinoid precursors, or other prenylated chemicals can be used in drug and / or substrate tissue distribution assays. As an added advantage, positron-emitting isotopes (e.g., 11 C. 18 F, 15 O. 13 N)-substituted cannabinoids, cannabinoid precursors, or other prenylated chemicals can be used in positron emission topography (PET) studies.

[0057] An additional advantage of cannabinoids, cannabinoid precursors, and other prenylated chemicals substituted with at least one deuterium is that deuterium is safe, stable, and non-radioactive. Because deuterium forms stronger bonds with carbon than hydrogen, it may positively influence the absorption, distribution, metabolism, and / or excretion properties of cannabinoids, cannabinoid precursors, or other prenylated chemicals. Furthermore, because deuterium is similar to hydrogen, replacing hydrogen with deuterium is not expected to affect the synthetic selectivity of cannabinoid precursors or other prenylated chemicals.

[0058] In one embodiment, cannabinoids, cannabinoid precursors, and / or other prenylated chemicals include cannabidiol (CBD), tetrahydrocannabinol (THC), cannabinol (CBN), cannabigerol (CBG), cannabichromene (CBC), cannabicyclol (CBL), cannabinodiol (CBND), cannabiditriol (CBT), tetrahydrocannabivarin (THCV), cannabidivarin (CBDV), cannabigerovarin (CBGV), cannabigerophorol (CBGP), tetrahydrocannabiphorol (THCP), cannabidiphorol (CBDP), cyclolavandulyl pyrophosphate (CLPP), derivatives thereof, acids thereof, and / or esters of acids thereof. Examples of cyclolavandulyl derivatives are described in U.S. Provisional Patent Application No. 63 / 333,670, filed April 22, 2022, the entire contents of which are incorporated herein by reference.

[0059] For example, and not by way of limitation, in one embodiment, the cannabinoid, cannabinoid precursor, and / or other prenylated chemical includes at least one deuterium to form a deuterated cannabinoid, cannabinoid precursor, or other prenylated chemical, which acts to improve bioavailability and slow metabolism relative to non-deuterated cannabinoids, cannabinoid precursors, or other prenylated chemicals.

[0060] In one embodiment, the cannabinoid, cannabinoid precursor, and / or other prenylated chemical comprises at least 10% substitutions (e.g., 15%) at the designated substitution sites, at least 20% substitutions (e.g., 25%) at the designated substitution sites, at least 30% substitutions (e.g., 35%) at the designated substitution sites, at least 40% substitutions (e.g., 45%) at the designated substitution sites, at least 50% substitutions (e.g., 55%) at the designated substitution sites, at least 60% substitutions (e.g., 65%) at the designated substitution sites, at least 70% substitutions (e.g., 75%) at the designated substitution sites, at least 80% substitutions (e.g., 85%) at the designated substitution sites, or at least 90% substitutions (e.g., 95%) at the designated substitution sites.

[0061] Figure 1 shows the metabolic pathway of CBD. As shown in Figure 1, CBD undergoes hydroxylation by metabolic enzymes, primarily at major metabolic sites. One of the major metabolic sites is the 5' pentyl terminus. Approximately 25% of metabolites undergo cleavage of the 5' pentyl terminus, resulting in inactive metabolites. The numbering system for CBD and CBDA is shown below.

[0062] [ka]

[0063] In one embodiment, the present invention includes a CBD molecule having at least one deuterium atom at the 5' pentyl terminus. Advantageously, the substitution of hydrogen atoms with deuterium, tritium, and / or halogens inhibits hydroxylation, as shown in Figure 1, slowing cannabinoid metabolism. In one embodiment, the 5' pentyl terminus is fully substituted with deuterium, tritium, and / or halogen. Alternatively, the 5' pentyl terminus is partially substituted with deuterium, tritium, and / or halogen.

[0064] However, substitution of the substituent at the 5' pentyl end may affect the affinity of the pentyl group for various cannabinoid receptors. In one embodiment, the end of the pentyl group is unsubstituted. Thus, in one embodiment, the hydrogens at C1 to C4 of the pentyl chain are substituted with deuterium, tritium, and / or halogen. In another embodiment, the hydrogens at C1 to C3 of the pentyl chain are substituted with deuterium, tritium, and / or halogen. Alternatively, the hydrogens at C1 to C2 of the pentyl chain are substituted with deuterium, tritium, and / or halogen. In yet another embodiment, the hydrogen at C1 of the pentyl chain is substituted with deuterium, tritium, and / or halogen.

[0065] Alternatively, the 5' group is an alkyl group partially or fully substituted with deuterium, tritium, and / or a halogen. Alkyl groups include, but are not limited to, methyl, ethyl, propyl (e.g., n-propyl, isopropyl), butyl (e.g., n-butyl, sec-butyl, t-butyl), hexyl, heptyl, octyl, cycloalkane (e.g., cyclopropane, cyclobutane, cyclopentane, cyclohexane, etc.), branched chain (e.g., isopropyl, t-butyl, etc.), and / or aromatic (e.g., benzyl, coumaryl, etc.) groups. In one embodiment, the alkyl group is not terminally substituted. None of the prior art teaches unsubstituting terminal alkyl groups. In another embodiment, the alkyl group is not substituted at the two, three, or four terminal carbons.

[0066] In another embodiment, C7 is substituted with deuterium, tritium, and / or halogen.

[0067] In one embodiment, the compound or cannabinoid is substituted with at least one hydroxyl group. In one embodiment, at least one hydroxyl group is used to attach a boronic acid or ester group. For example, see (1) Maslah H, Skarbek C, Pethe S, Labruere R. Anticancer boron-containing prodrugs responsive to oxidative stress from the tumor microenvironment. Eur J Med Chem. 2020 Dec 1;207:112670. doi:10.1016 / j.ejmech.2020.112670. Epub 2020 Aug 5. PMID:32858470; (2) Silva MP, Saraiva L, Pinto M, Sousa ME. Acids and Their Derivatives in Medicinal Chemistry: Synthesis and Biological Applications. Molecules. 2020 Sep 21;25(18):4323. doi:10.3390 / molecules25184323. PMID:32967170; PMCID:PMC7571202; and (3) Liederer BM, Borchardt RT. Enzymes involved in the bioconversion of ester-based prodrugs. J Pharm Sci. 2006 Jun;95(6):1177-95. doi:10.1002 / jps.20542. PMID:16639719, which are incorporated herein by reference in their entireties.

[0068] In one embodiment, the compound or cannabinoid comprises a prodrug. In one embodiment, the prodrug contains at least one deuterium, at least one tritium, at least one halogen (e.g., fluorine, chlorine, bromine, iodine), at least one hydroxyl group, and / or at least one additional isotope (e.g., 11 C. 13 C. 14 C. 13 N, 15 N, 18 O. 17 O. 15 O. 31 P, 32 P, 35 S, 18 F, 36In one embodiment, the prodrug is selected from the group consisting of sedatives (e.g., codeine, diazepam, benzobarbital, hydrocodone, morphine, oxycodone, tramadol, ethymorphine), cannabinoids (e.g., THC, THC-O-acetate, THC-O-phosphate, 11-hydroxy-THC), delirious agents (e.g., ibotenic acid), dissociatives (e.g., dextromethorphan, ketamine), hallucinogens (e.g., 1A-LSD, 1B-LSD, 1P-ETH-LAD, 1 P-LSD, bufotenin, etosibine, MDMA, MDA, norpsilocin, norbogaine, psilocybin), nootropics (e.g., 5-HTP, adrafinil, moveracetam), stimulants (e.g., adrafinil, amphetamine, benzphetamine, droxidopa, fencamine, L-DOPA, levamisole, lisdexamfetamine, nicotine), naloxone, risperidone, sertraline, trazodone, etoperidone, and / or 5-MT. In one embodiment, the prodrug includes Δ9-THC hemisuccinate, Δ9-tetrahydrocannabinol-valine-hemisuccinate, THC-O-phosphate, THC-O-acetate, cannabinoids, 11-hydroxy-THC, and / or cannabinoid glycosides. For example, (1) Walker, LA, Harland, EC, Best, AM, ElSohly, MA (1999). Δ9-THC Hemisuccinate in Suppository Form as an Alternative to Oral and Smoked THC. In: Nahas, GG, Sutin, KM, Harvey, D., Agurell, S., Pace, N., Cancro, R. (eds) Marihuana and Medicine. Humana Press, Totowa, NJ. https: / / doi.org / 10.1007 / 978-1-59259-710-9_13;(2)ElSohly MA, Stanford DF, Harland EC, Hikal AH, Walker LA, Little TL Jr, Rider JN, Jones AB.Rectal bioavailability of delta-9-tetrahydrocannabinol from the hemisuccinate ester in monkeys.J Pharm Sci.1991 Oct;80(10):942-5.doi:10.1002 / jps.2600801008.PMID:1664466;(3)ElSohly MA,Gul W,Walker LA:Pharmacokinetics and Tolerability of Δ9-THC-Hemisuccinate in a Suppository Formulation as an Alternative to Capsules for the Systemic Delivery of Δ9-THC.Med Cannabis Cannabinoids 2018;1:44-53.doi:10.1159 / 000489037;(4)Upadhye SB,Kulkarni SJ,Majumdar S,Avery MA,Gul W,ElSohly MA,Repka MA.Preparation and characterization of inclusion complexes of a hemisuccinate ester prodrug of delta9-tetrahydrocannabinol with modified beta-cyclodextrins.AAPS PharmSciTech.2010 Jun;11(2):509-17.doi:10.1208 / s12249-010-9401-4.Epub 2010 Mar 24.PMID:20333489;PMCID:PMC2902337;and(5)Adelli GR,Bhagav P,Taskar P,Hingorani T,Pettaway S,Gul W,ElSohly MA,Repka MA,Majumdar S.Development of a Δ9-Tetrahydrocannabinol Amino Acid-Dicarboxylate Prodrug With Improved Ocular Bioavailability.Invest Ophthalmol Vis Sci.2017 Apr 1;58(4):2167-2179. doi:10.1167 / iovs.16-20757. PMID:28399267; PMCID:PMC5389743, each of which is incorporated by reference in its entirety. See also, e.g., WIPO Publication No. 2021173130, U.S. Patent Publication Nos. 20220194916, 20220168428, and 20210379507, each of which is incorporated by reference in its entirety.

[0069] synthesis

[0070] Prenylation (also known as isoprenylation or lipidation) is the addition of hydrophobic molecules to proteins or compounds. The prenyl group (3-methylbut-2-en-1-yl group) is thought to facilitate attachment to cell membranes, similar to lipid anchors such as GPI anchors. Prenyl groups have been shown to be important for protein-protein interactions via specialized prenyl-binding domains.

[0071] Prenylated natural products are a broad class of bioactive molecules with proven medicinal properties. Examples include, but are not limited to, prenylflavonoids, prenylstilbenoids, and cannabinoids. Plant-derived phenyl compounds are difficult to separate due to structural similarities of contaminating molecules and compositional variability between crops. These challenges are exacerbated when attempting to isolate compounds with low abundance. Many chemical synthesis methods have been developed to address the challenges associated with producing prenylated natural products, but their complexity and low yields make them generally impractical for pharmaceutical manufacturing.

[0072] Microbial production of prenylated natural products offers a useful alternative to natural extraction, but it faces many challenges, including the need to divert carbon flux from central metabolism and product toxicity, to name a few. For example, prenylated natural products such as prenylnaringenin, prenylresveratrol, and cannabidiolic acid (CBDA) are generated from a combination of metabolic pathways for fatty acid, isoprenoid, and polyketide biosynthesis. Therefore, high-level production requires efficient rerouting of long, essential, and highly regulated pathways. Despite these challenges, many research groups have developed microorganisms that produce non-prenylated polyketides, such as naringenin, resveratrol, and olivetolate, at relatively low concentrations (110, 391, and 80 mg / L, respectively). Obtaining prenylated products is even more challenging because geranyl pyrophosphate (GPP), an essential metabolite that is toxic to cells at moderate concentrations, poses a significant barrier to high-level microbial production.

[0073] Cannabinoids, in particular, have shown great therapeutic potential, with over 100 clinical trials underway as antiemetics, anticonvulsants, antidepressants, and analgesics. However, despite the therapeutic potential of prenylated natural products, their research and utilization is limited by the lack of cost-effective production methods.

[0074] There are two main alternatives for the production of plant-derived cannabinoids: organic synthesis and production using metabolically engineered hosts (e.g., plants, yeast, or bacteria). While total synthesis methods have been established for some cannabinoids, such as THCA and CBDA, these methods are often impractical for pharmaceutical manufacturing. Furthermore, these synthetic approaches are not modular, requiring different synthetic methods for each cannabinoid. A modular approach could be achieved by utilizing natural biosynthetic pathways.

[0075] The three major cannabinoids (THCA, CBDA, and cannibichromene) are derived from a single precursor, CBGA. Additionally, three low-abundance cannabinoids are derived from cannabierovarinic acid (CBGVA) (Figures 2A-2B). Thus, the ability to produce CBGA and CBGVA in heterologous hosts opens the door to the production of multiple cannabinoids. However, engineering microorganisms to produce CBGA and CBGVA has proven extremely challenging.

[0076] Cannabinoids are produced from a combination of fatty acid, polyketide, and terpene biosynthetic pathways, generating the major components geranyl pyrophosphate (GPP) and olivetolic acid (OA) (Figures 2A-2B). High-level biosynthesis of CBGA requires rerouting of a long, essential, and highly regulated pathway. Furthermore, GPP is cytotoxic, creating a significant barrier to high-level production in microorganisms. Gagne et al. (Proc. Natl. Acad. Sci., 109:12811, 2012, which is incorporated by reference in its entirety) engineered a pathway to produce OA in yeast, but the titer was very low (0.5 mg L -1 ), suggesting that high-level production of intermediates in this pathway is not easy. In another study, Zirpel et al. produced THCA by adding GPP and olivetolic acid (OA) to a yeast lysate containing promiscuous prenyltransferase (NphB) and THCA synthase (J. Biotechnol., 259:204-212, 2017, which is incorporated by reference in its entirety). However, there have been no published reports of cannabinoid production in engineered living cells from low-cost feedstocks.

[0077] Synthetic biochemistry, which uses mixtures of enzymes to perform complex biochemical transformations in cell-free systems, offers potential advantages over traditional metabolic engineering, including greater flexibility in pathway design, greater control over component optimization, faster design-build-test cycles, and elimination of cytotoxicity of intermediates or products. Advantageously, the present invention provides a cell-free system for cannabinoid production.

[0078] The present invention provides enzyme variants and pathways containing such variants for the prenylation of compounds, including the production of cannabinoids. Additionally, the biosynthetic pathways described herein use a "purge valve" to regulate NAD(P)H levels. Such "purge valves" have been demonstrated to produce high levels of monoterpenes from glucose, indicating that significant GPP production is possible in cell-free systems (see International Patent Publication WO 2017 / 015429, which is incorporated herein by reference in its entirety). These purge valves were used to refine and diversify the original system to produce complex natural products, such as cannabinoids. Synthetic biochemistry approaches are outlined in Figures 2A-2C, 6A, and 6B. In one embodiment, the present disclosure provides a cell-free system for prenylation using glucose-derived GPP (see Figures 2A-2C, 6A, 6B, and 8). In another embodiment, the present disclosure provides a cell-free system for prenylation using (iso)prenol or prenol-derived GPP (see Figure 7). The pathway in Figure 7 can be coupled to any ATP-generating system to generate the ATP required for the reaction. For example, this pathway can be coupled to the creatine kinase ATP-generating system, acetate kinase system, glycolysis system, and others. The enzymes (nucleic acid coding sequences and polypeptides) in Figure 7 are shown in SEQ ID NOs:54-65 (e.g., PRK enzymes are provided in SEQ ID NOs:54-57, IPK enzymes are provided in SEQ ID NOs:58-61, IDI enzymes are provided in SEQ ID NOs:62-63, and FPPS enzymes are provided in SEQ ID NOs:64-65).

[0079] NphB is an aromatic prenyltransferase that catalyzes the addition of a 10-carbon geranyl group to aromatic substrates. NphB exhibits excellent substrate selectivity and product regioselectivity. NphB, identified from the genus Streptomyces, catalyzes the addition of a 10-carbon geranyl group to various small organic aromatic substrates. NphB has a large, solvent-accessible binding pocket where two substrate molecules, geranyl diphosphate (GPP) and 1,6-dihydroxynaphthalene (1,6-DHN), bind. GPP binds a negatively charged diphosphate group and two substrates, Lys119, Thr171, Arg228, Tyr216, Lys284, and Mg. 2+ It is stabilized by interactions with several amino acid side chains, including Mg 2+ A cofactor is required for the activity of NphB. NphB from Streptomyces has the sequence shown in SEQ ID NO:30.

[0080] NovQ (Accession No. AAF67510, incorporated herein by reference) belongs to the CloQ / NphB class of prenyltransferases. The novQ gene can be cloned from Streptomyces nibeus, which produces the aminocoumarin antibiotic novobiocin. Recombinant NovQ was expressed in Escherichia coli and engineered to be purified to homogeneity. The purified enzyme is a soluble, 40 kDa, monomeric protein that catalyzes the divalent cation-independent transfer of a dimethylallyl group to 4-hydroxyphenylpyruvate (4-HPP), yielding 3-dimethylallyl-4-HPP, an intermediate in novobiocin synthesis. In addition to 4-HPP prenylation, NovQ catalyzed the carbon-carbon and carbon-oxygen prenylation of a variety of compounds, including phenylpropanoids, flavonoids, and dihydroxynaphthalenes. Despite its diverse catalytic activity, NovQ performs prenylation reactions regiospecifically. NovQ was the first reported prenyltransferase capable of transferring dimethylallyl groups to both the B-rings of phenylpropanoids, such as p-coumaric acid and caffeic acid, and flavonoids. NovQ functions as a useful biocatalyst for the synthesis of prenylated phenylpropanoids and prenylated flavonoids.

[0081] The recently discovered and characterized Aspergillus terreus aromatic prenyltransferase (AtaPT; accession number AMB20850, incorporated herein by reference) is responsible for the prenylation of various aromatic compounds. Recombinant AtaPT has been overexpressed in Escherichia coli and can be purified. Aspergillus terreus aromatic prenyltransferase (AtaPT) catalyzes primarily the C-monoprenylation of acylphloroglucinols in the presence of various prenyl diphosphates.

[0082] Olivetolic acid (OA) is a relatively inactive substrate for wild-type NphB. Consequently, we tested the prenylation potential of the cosubstrate in a cell-free system using 1,6-dihydroxynaphthalene (1,6-DHN), a more preferred NphB substrate. Starting with 2.5 mM 1,6-DHN and 500 mM glucose, approximately 400 mg / L (1.3 mM) of prenylated product was obtained. However, when the initial 1,6-DHN concentration was increased from 2.5 mM to 5 mM, the final titer decreased by half. This suggests that 1,6-DHN inhibits one or more enzymes. Enzyme assays revealed that E. coli pyruvate dehydrogenase (EcPDH) is inhibited not only by 1,6-DHN but also by several other aromatic polyketides (Figure 3B). At 1 mM concentrations of 1,6-DHN, olivetol, or resveratrol, PDH activity was reduced by two-fold (Figure 3B). Therefore, an experiment to remove PDH by implementing the PDH bypass was designed (see Figures 2A-2B and 3B). In the PDH bypass, pyruvate is converted to acetyl-CoA by pyruvate oxidase (PyOx) and acetyl phosphate transferase (PTA), and PDH is removed (Figures 2A-2B). As shown in Figure 3A, this new system eliminated the inhibition seen at high concentrations of 1,6-DHN and increased the titer of 5-prenyl-1,6-DHN fourfold compared to the PDH system when starting from 5 mM 1,6-DHN (Figure 3B). Figure 3C shows the time course of 5-prenyl-1,6-DHN biosynthesis starting from 5 mM 1,6-DHN using the PDH bypass. Approximately 50% of the 1,6-DHN was converted in the first 24 h, ultimately reaching a titer of 705 ± 12 mg / L.

[0083] Prenylation of aromatic polyketides by NphB is thought to proceed via a carbocation intermediate. In the first step, the diphosphate is released from GPP to generate a carbocation at the C1 carbon of GPP, which then attacks a nearby nucleophile. To improve the regiospecificity of the prenylation, 1,6-DHN, Mg 2+Starting from the crystal structure of NphB complexed with OA and a non-hydrolyzable analog of GPP (geranyl S-thiolodiphosphate) (PDBID 1ZB6; Protein Data Bank reference number 1ZB6), we modeled OA into the active site of NphB. To design, we used 1,6-DHN as a guide to place OA in the binding pocket. The C3 carbon of OA, the desired prenylation site, was positioned 3.7 Å above the nascent geranyl C1 carbocation (Figure 4A). The selected distance was based on the distance from the C5 carbon of 1,6-DHN to the C1 carbon of GPP. Next, residues in contact with OA were varied using ROSETTA software to optimize the active site of NphB for OA binding. Side chains that contact GPP or potentially provide catalytic function were left fixed. This resulted in an ensemble of candidate NphB variants.

[0084] To reduce the number of variants to be experimentally tested, we used a scoring system to rank the changes most likely to affect OA binding. A representative set of variants (Table 1) was selected, and each residue was systematically reverted to its wild-type side chain under the influence of other mutations. The changes were then evaluated by energy score (Table 2). Because the Y288 substitution had the greatest impact on energy scores, the Y288A or Y288N mutation was used in all experimentally evaluated constructs. The frequency of mutations, how multiple mutations function together, and the calculated energy scores were all considered to further shape the NphB library. Taking these factors into account, a library consisting of 29 constructs was generated, ranging from a single point mutation to up to six mutations per construct, as listed in Table 1 (see also SEQ ID Nos: 1-29. Note that SEQ ID Nos: 1-29 include the hexahistidine leader from the expression construct, i.e., amino acids 1-20, which are not required for biological activity). Table 1 shows exemplary mutations and the degree of improvement over the wild type (i.e., the polypeptide of SEQ ID NO: 30). NphB library constructs and mutations (amino acid positions with reference to SEQ ID NO: 30).

[0085] [Table 1-1] [Table 1-2]

[0086] Table 2 shows the kinetic parameters of the NphB mutants.

[0087] [Table 2]

[0088] Recombinant methods for producing and isolating the modified NphB polypeptides of the present disclosure are described herein. In addition to recombinant production, the polypeptides can also be engineered to be produced by direct peptide synthesis using solid-phase methods (e.g., Stewart et al. (1969) Solid-Phase Peptide Synthesis (WH Freeman Co, San Francisco); and Merrifield (1963) J. Am. Chem. Soc. 85:2149-2154, each of which is incorporated herein by reference). Peptide synthesis can be performed manually or automated, for example, using an Applied Biosystems 431A peptide synthesizer (Perkin Elmer, Foster City, Calif.) according to the manufacturer's instructions.

[0089] After obtaining crudely purified NphB mutants, we performed an initial screening for CBGA production using GPP and OA at concentrations that saturate wild-type NphB. Six constructs (M1, M2, M3, M6, M10, and M15) exhibited activity greater than 10-fold compared to WT NphB, and four constructs (M5, M7, M12, and M20) exhibited activity improvements of 2- to 10-fold. The remaining constructs exhibited activity comparable to that of WT NphB. The top constructs from the initial screening (M1, M3, M10, and M15) were purified and further characterized (Figure 4B). Several observations emerged from the initial screening: (1) As predicted by computation, Y288A (M1) and Y288N (M2) dramatically improved activity on their own. (2) The presence of Y288N reduced the purification yield in all constructs, suggesting that Y288N may be a destabilizing mutation and that Y288A is a more desirable mutation. (3) Addition of G286S to the Y288N(M10) background appeared to further improve activity compared to Y288N(M2), suggesting that G286S is another desirable mutation. (4) Although F213N had only a neutral or deleterious effect in the Y288A / F213N(M5) construct, Y288A / F213N / A232S(M15) slightly improved activity compared to Y288A(M1). This suggests that A232S is also a desirable mutation.

[0090] From these initial observations, a focused library was designed containing various combinations of the Y288A, GS86S, and A232S variants. Other combinations with Y288V were added based on the rationale that they could improve stability while reducing the size of the Y288 side chain. All but one of the constructs in the second library exhibited over 100-fold greater activity than WT NphB in a 1-hour endpoint assay. A comparison of the best mutations obtained in the first round with those obtained in the second round is shown in Figure 4B. It is clear that the combination of beneficial mutations obtained in the first round improved CBGA production. Furthermore, the Y288A and Y288V constructs improved NphB expression compared to Y288N without sacrificing activity.

[0091] The best two mutations from the initial screen and the best three constructs from the focused library were further analyzed in detail. Kinetic parameters are summarized in Table 2. All mutations exhibited K m Although the effect on the value was relatively small, k cat The values were dramatically improved. In particular, M23 (NphB of SEQ ID NO: 23) showed a k cat 0.0021±0.00008min -1 to 1.58±0.05min -1 The catalytic efficiency (k cat / K m ) was improved by more than 1000-fold compared to the wild-type enzyme. M31 had a higher k cat / K m was high, but M23 has a higher k cat M23 was chosen rather than M31 because it has a saturating OA and synthetic biochemical systems generally operate under saturating OA conditions.

[0092] The engineered mutant M23 not only dramatically improves catalytic efficiency for OA prenylation, but is also highly specific, producing only the correct CBGA product. While WT NphB produces CBGA, the major product is the prenylated isomer (Figure 4C). In contrast, the engineered mutant M23 produces almost exclusively CBGA. Overall, the engineered enzyme is a much more effective CBGA synthase than the nonspecifically prenylated wild-type enzyme.

[0093] Accordingly, the present disclosure provides a method for the preparation of NphB polypeptides comprising: (i) SEQ ID NO:30 and at least a Y288X mutation, where X is A, N, S, V, or an unnatural amino acid; (ii) SEQ ID NO:30 and at least a Y288X mutation, where X is A, N, S, V, or an unnatural amino acid, and at least one other mutation selected from V49Z1, F213Z2, A232S, I234T, V271Z3, and / or G286S, where Z1 is S, N, T, or G, Z2 is H, N, or G, and Z3 is N or H; (iii) any combination of mutations listed in Table 1; (iv) any of (i), (ii), or (iii) comprising 1 to 20 (e.g., 2, 5, 10, 15, or 20, or any value between 1 and 20) conservative amino acid substitutions, and having NphB activity; (vi) any of the sequences set forth in SEQ ID NOs: 1-28 or 29, wherein the NphB mutation begins at amino acid 21; or (vii) any sequence at least 99% identical to any of SEQ ID NOs: 1-28 or 29, and wherein the NphB mutation begins at amino acid 21. By "NphB activity" is meant the ability of the enzyme to prenylate a substrate, more specifically to produce CBGA from OA.

[0094] As used herein, unnatural amino acids refer to amino acids that do not occur in nature, such as N-methyl amino acids (e.g., N-methyl L-alanine, N-methyl L-valine, etc.), or α-methyl amino acids, β-homo amino acids, homo amino acids, and D-amino acids. In certain embodiments, unnatural amino acids useful in the present disclosure include small hydrophobic unnatural amino acids (e.g., N-methyl L-alanine, N-methyl L-valine, etc.).

[0095] Additionally, the present disclosure provides polynucleotides encoding any of the above-described NphB variants. Due to the degeneracy of the genetic code, the actual coding sequence may vary, but still result in the described polypeptides of NphB mutations and variants. Exemplary polynucleotide sequences are shown in SEQ ID NOs:66, 67, and 68 (corresponding to the polypeptide sequences of SEQ ID NOs:23, 29, and 69, respectively). Due to the degeneracy of the genetic code, there may be significant variations in the percent identity of SEQ ID NOs:66, 67, and 68, but it will be readily apparent that they still encode the polypeptides of SEQ ID NOs:23, 29, and 69.

[0096] The present disclosure also provides recombinant host cells and cell-free systems comprising any of the disclosed NphB variant enzymes. In some embodiments, recombinant cells and cell-free systems are used to carry out the prenylation process. In one embodiment, the system is preferably cell-free, preferably without viable cells. Alternatively, the system is completely cell-free, without micelles or other artificial cells, defined as the absence of compartments for reagents, enzymes, or other chemical species in the reaction environment. In one embodiment, the system comprises permeabilized cells (e.g., acetone-dried yeast). In one embodiment, ATP regeneration is performed using permeabilized cells. In another embodiment, the system comprises artificially heat-treated cell lysate, where biomass (e.g., growing cells) is harvested, heated (with or without lysis), and then used. In one embodiment, ATP is regenerated without the use of acetyl phosphate. In one embodiment, ATP is regenerated using polyphosphate to simplify the biosynthetic pathway. Those skilled in the art will recognize that the substrate, acetyl phosphate, can be used in two competing reactions, which creates a balancing issue for the pathway. Using polyphosphate instead of acetyl phosphate eliminates or significantly reduces this risk.

[0097] (1) Valliere, MA, Korman, TP, Woodall, NB et al., A cell-free platform for the prenylation of natural products and application to cannabinoid production. Nat Commun10, 565 (2019). https: / / doi.org / 10.1038 / s41467-019-08448-y; (2) Bloemendal VRLJ, van Hest JCM, Rutjes FPJT.Synthetic pathways to tetrahydrocannabinol(THC):an overview.Org Biomol Chem.2020 May 6;18(17):3203-3215.doi:10.1039 / d0ob00464b.PMID:32259175;(3)Masanori Asada, Kazuhiro Morimoto, Kazuhiro Nakanishi, Ryuichi Matsuno,Atsuo Tanaka,Akira Kimura,Tadashi Kamikubo,Continuous ATP Regeneration Using Immobilized Yeast Cells,Agricultural and Biological Chemistry,Volume 43,Issue 8,1 August 1979,Pages 1773-1774,https: / / doi.org / 10.1080 / 00021369.1979.10863703;and(4)Alissandratos A,Caron K,Loan TD,Hennessy JE,Easton CJ.ATP Recycling with Cell Lysate for Enzyme-Catalyzed Chemical Synthesis,Protein Expression and PCR.ACS Chem Biol.2016 Dec 16;11(12):3289-3293.doi:10.1021 / acschembio.6b00838.Epub 2016 Nov 10. PMID:27978706, each of which is incorporated herein by reference in its entirety.

[0098] One goal of the present disclosure is to generate precursor GPP from glucose or prenol and / or isoprenol, which can then be used to prenylate added OA with the mutant NphB of the present disclosure, thereby producing CBGA.

[0099] Thus, the present disclosure provides a cell-free system that includes multiple enzymatic steps to convert glucose to geranyl pyrophosphate, the pathway including a purge valve and a PDH bypass enzymatic process.

[0100] As shown in Figure 2C, one pathway of the present disclosure involves converting glucose to glucose-6-phosphate using hexokinase. Hexokinase (EC 2.7.1.1) is an enzyme that phosphorylates hexoses (six-carbon sugars) to produce hexose phosphate. Hexokinases have the ability to transfer an inorganic phosphate group from ATP to a substrate. Numerous hexokinase proteins from various organisms have been cloned and expressed. In some embodiments, hexokinases include sequences set forth in UniProtKB Accession No. P04806 from Saccharomyces cerevisiae (Sc) (incorporated herein by reference), as well as sequences at least 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99% identical thereto and having hexokinase activity.

[0101] Glucose-6-phosphate is then converted to fructose-6-phosphate by phosphoglucose isomerase (Pgi) (EC 5.3.1.9). Thus, in addition to the above, the term "phosphoglucoisomerase" or "Pgi" refers to a protein capable of catalyzing the formation of fructose-6-phosphate from glucose-6-phosphate, which shares at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity or at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence similarity with SEQ ID NO: 31, as calculated by NCBI BLAST using default parameters, and the enzyme has phosphoglucoisomerase activity.

[0102] In another or further embodiment, the system or recombinant microorganism provided herein includes expression of phosphofructokinase (Pfk, polyphosphate-dependent Pfk, or a homologous gene or variant thereof). In one embodiment, this expression can be combined with other enzymes in a metabolic pathway. Pfk can be derived from G. stearothermophilus (SEQ ID NO: 32). In another embodiment, an engineered variant of Pfk can be used, so long as it has phosphofructokinase activity and is capable of converting fructose-6-phosphate to fructose-1,6-bisphosphate. Such engineered variants can be obtained by site-directed mutagenesis, directed evolution, and the like. Thus, the present disclosure includes polypeptides that are at least 85-99% identical to the sequence set forth in SEQ ID NO: 32 and have phosphofructokinase activity (see, e.g., SEQ ID NOs: 33-34).

[0103] In addition to the above, the term "fructose 1,6-bisphosphate aldolase" or "Fba" refers to a protein capable of catalyzing the formation of dihydroxyacetone phosphate and glyceraldehyde-3-phosphate from fructose 1,6-bisphosphate, which shares at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity or at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence similarity to SEQ ID NO: 35, as calculated by NCBI BLAST using default parameters. Additional homologous genes include Synechococcus elongatus PCC 6301 YP_170823.1, which has 26% identity to SEQ ID NO:35; Vibrio nigripulchritudo ATCC 27043 ZP_08732298.1, which has 80% identity to SEQ ID NO:35; Mmethylomicrobium album BG8 ZP_09865128.1, which has 76% identity to SEQ ID NO:35; Pseudomonas fluorescens Pf0-1 YP 350990.1, which has 25% identity to SEQ ID NO:35; and Mmethylobacterium nodulans ORS 2060 YP_002502325.1, which has 24% identity to SEQ ID NO:35. Thus, the present disclosure includes the use of polypeptides having 26% to 100% identity to SEQ ID NO: 35, wherein the polypeptide comprises bisphosphate aldolase activity. The sequences associated with the above accession numbers are incorporated herein by reference.

[0104] In addition to the above, the term "triosephosphate isomerase" or "Tpi" refers to a protein capable of catalyzing the formation of glyceraldehyde-3-phosphate from dihydroxyacetone phosphate (DHAP) and sharing at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity or at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence similarity to SEQ ID NO:36 as calculated by NCBI BLAST using default parameters. Additional homologous genes include Rattus norvegicus AAA42278.1, which shares 45% identity with SEQ ID NO:36; Homo sapiens AAH17917.1, which shares 45% identity with SEQ ID NO:36; Bacillus subtilis BEST7613 NP_391272.1, which shares 40% identity with SEQ ID NO:36; Synechococcus elongatus PCC 6301 YP_171000.1, which shares 40% identity with SEQ ID NO:36; and Salmonella enterica subsp. enterica serovar Typhi str. AG3 ZP_06540375.1, which shares 98% identity with SEQ ID NO:36. Thus, the present disclosure includes the use of polypeptides having 40% to 100% identity to SEQ ID NO: 36 and having triosephosphate isomerase activity. The sequences associated with the above accession numbers are incorporated herein by reference.

[0105] In a further step of the pathway, glyceraldehyde-3-phosphate can be converted to 1,3-bisphosphoglycerate. In one embodiment, this enzymatic step includes a "purge valve system" (described elsewhere herein). For example, glyceraldehyde-3-phosphate dehydrogenase (Gap, Tdh) converts glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate. In one embodiment, wild-type Gap (see, e.g., SEQ ID NO:37) that uses NAD+ as a cofactor, or mutant Gap with a P191D mutation (related to the sequence of SEQ ID NO:37 and shown in SEQ ID NO:38) is used. In another embodiment, NADP + A mutant Gap (mGap; e.g., with D34A / L35R / T35K mutations; related to the sequence of SEQ ID NO:37 and shown in SEQ ID NO:39) using NADH as a cofactor is used. In yet another embodiment, a combination of Gap and mGap (GapM6) is used. A molecular purge valve containing a water-producing NADH oxidase (NoxE) that specifically oxidizes NADH but not NADPH is used to oxidize NAD. + It can be used to recycle ("purge") NADH when wild-type or P118D mutant GAPs are used, which preferentially use NADH.

[0106] In addition to the above, the term "NADH oxidase" or "NoxE" refers to a protein capable of oxidizing NADH to NAD* and sharing at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity or at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence similarity to SEQ ID NO: 18 as calculated by NCBI BLAST using default parameters.

[0107] This pathway can further convert 1,3-bisphosphoglycerate to 3-phosphoglycerate by the use of phosphoglycerate kinase (EC 2.7.2.3) (PGK, e.g., as provided in SEQ ID NO:40, or a homolog or variant thereof that is at least 80% identical thereto), which catalyzes the reversible transfer of a phosphate group from 1,3-bisphosphoglycerate (1,3-BPG) to ADP, generating 3-phosphoglycerate (3-PG) and ATP. It is possible that a molecular purge valve for ATP can exist to recycle ADP, for example, using a GTPase or other enzyme (or a homolog or variant thereof).

[0108] 3-phosphoglycerate can then be converted to 2-phosphoglycerate by phosphoglycerate mutase (pgm; for example, as provided in SEQ ID NO:41, or a homolog or variant thereof that is at least 80% identical thereto).

[0109] Enolase (eno; e.g., that provided in SEQ ID NO:42, or a homolog or variant thereof that is at least 80% identical thereto) is capable of converting 2-phosphoglycerate to phosphophenolpyruvate (PEP).

[0110] Pyruvate kinase (pyk; e.g., those provided in SEQ ID NOs:43, 44, and 45, or homologs or variants thereof that are at least 80% identical to any of SEQ ID NOs:43, 44, or 45) converts PEP to pyruvate.

[0111] As mentioned above, pyruvate dehydrogenase (PDH) is inhibited by the product of this pathway. Therefore, the PDH bypass can be used to convert pyruvate to acetyl-CoA. The PDH bypass involves two enzymatic reaction steps: (i) pyruvate → acetyl phosphate by pyruvate oxidase (e.g., PyOx from Aerococcus viridans, EC 1.2.3.3, see SEQ ID NO: 46), and (ii) acetyl phosphate → acetyl-CoA by acetyl phosphate transferase (also known as phosphate acetyltransferase) (e.g., PTA from G. stearothermophilus).

[0112] As used herein, PyOx used in the compositions and methods of the present disclosure includes sequences that are at least 85%, 90%, 95%, 98%, or 99% identical to SEQ ID NO:46 and have pyruvate oxidase activity.

[0113] Phosphate acetyltransferase (EC 2.3.1.8) is an enzyme that catalyzes the chemical reaction from acetyl-CoA + phosphate to CoA + acetyl phosphate and vice versa. Phosphate acetyltransferase is encoded by pta in E. coli. PTA is involved in the conversion of acetate to acetyl-CoA. Specifically, PTA catalyzes the conversion of acetyl-CoA to acetyl phosphate. Homologs and variants of PTA are known. Approximately 1075 bacterial phosphate acetyltransferases are available in NCBI. For example, such homologous genes and variants include phosphate acetyltransferase Pta(Rickettsia felis URRWXCal2)gi|670040211|gb|AAY60947.11(67004021), phosphate acetyltransferase(Buchnera aphidicola str.Cc(Cinara cedri))gi|116256910|gb|ABJ90592.1|(116256910), pta(Buchnera aphidicola str.Cc(Cinara cedri))gi|116515056|ref|YP_802685.1|(116515056), pta(Glossina brevipalpis Wigglesworthia glowsinidia) endosymbiotic bacteria)gi|25166135|dbj|BAC24326.1|(25166135);Pta(Pasteurella multocida subsp.multocida str.Pm70)gi|12720993|gb|AAK02789.1|(12720993);Pta(Rhodospirillum rubrum)gi|25989720|gb|AAN75024.11(25989720);pta(Listeria welshimeri serovar 6b str.SLCC5334)gi|116742418|emb|CAK21542.1|(116742418);Pta(Mycobacterium avium subsp.paratuberculosis K-10)gi|41398816|gb|AAS06435.1|(41398816);Phosphate acetyltransferase pta(Borrelia burgdorferi B31)gi|15594934|ref|NP_212723.1|(15594934);Phosphate acetyltransferase pta(Borrelia burgdorferi B31)gi|2688508|gb|AAB91518.1|(2688508);Phosphate acetyltransferase pta(Haemophilus influenzae Rd KW20)gi|1574131|gb|AAC22857.1|(1574131);Phosphate acetyltransferase Pta(Rickettsia bellii RML369-C)gi|91206026|ref|YP_538381.1|(91206026);Phosphate acetyltransferase Pta(Rickettsia bellii RML369-C)gi|91206025|ref|YP_538380.1|(91206025);Phosphate acetyltransferase pta(Mycobacterium tuberculosis F11)gi|148720131|gb|ABR04756.1|(148720131);Phosphate acetyltransferase PTA(Mycobacterium tuberculosis str. Haarlem)gi|134148886|gb|EBA40931.11(134148886);Phosphate acetyltransferase PTA(Mycobacterium tuberculosis C)gi|124599819|gb|EAY58829.1|(124599819);Phosphate acetyltransferase Pta(Rickettsia bellii RML369-C)gi|91069570|gb|ABE05292.1|(91069570);Phosphate acetyltransferase Pta(Rickettsia bellii RML369-C)gi|91069569|gb|ABE05291.1|(91069569);Phosphate acetyltransferase (pta)(Treponema pallidum subsp. pallidum str. Nichols)gi|15639088|ref|NP_218534.1|(15639088); and phosphate acetyltransferase (pta) (Treponema pallidum subsp. pallidum str. Nichols)gi|3322356|gb|AAC65090.11(3322356), each of the sequences associated with the accession numbers is incorporated herein by reference in its entirety.

[0114] Returning to Figure 2C, the pathway involves the conversion of acetyl-CoA to acetoacetyl-CoA. The conversion of acetyl-CoA to acetoacetyl-CoA is carried out by an acetyl-CoA acetyltransferase (e.g., PhaA). Numerous acetyl-CoA acetyltransferases are known in the art, including the acetyl-CoA acetyltransferase from R. eutropha. In another embodiment, the acetyl-CoA acetyltransferase has an amino acid sequence at least 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:47.

[0115] Acetoacetyl-CoA and acetyl-CoA can be converted to HMG-CoA by the enzyme HMG-CoA synthase having an A110G mutation (see, e.g., SEQ ID NO:48), or a homolog or variant thereof having at least 85%, 90%, 95%, 98%, or 99% (e.g., 85%-95%) sequence identity thereto.

[0116] HMG-CoA is then reduced to mevalonate by the action of NADPH and HMG-CoA reductase (see, e.g., SEQ ID NO:49), or a homolog or variant thereof having at least 85%, 90%, 95%, 98%, or 99% (e.g., 85%-95%) sequence identity thereto.

[0117] Mevalonate is then phosphorylated by ATP and the action of mevalonate kinase (MVK) to produce mevalonate-5-phosphate and ADP. Mevalonate kinases are known in the art and include sequences having at least 85-100% (e.g., 85%, 90%, 95%, 98%, 99%) identity to the sequence of SEQ ID NO:50, which have mevalonate kinase activity.

[0118] Mevalonate-5-phosphate is further phosphorylated by ATP and the action of phosphomevalonate kinase (PMVK) to produce mevalonate-5-diphosphate and ADP. Phosphomevalonate kinases are known in the art and include sequences having at least 85-100% (e.g., 85%, 90%, 95%, 98%, 99%) identity to the sequence of SEQ ID NO:51, which have phosphomevalonate kinase activity.

[0119] Mevalonate-5-diphosphate is decarboxylated by ATP and diphosphomevalonate decarboxylase (MDC) to produce ADP, CO, and isopentyl pyrophosphate. Diphosphomevalonate decarboxylases are known in the art and are enzymes with at least 85-100% (e.g., 85%, 90%, 95%, 98%, 99%) identity to the sequence of SEQ ID NO:52 and with diphosphomevalonate kinase activity.

[0120] A variety of other mevalonate pathways can also be used (see, for example, Figure 8).

[0121] Geranyl pyrophosphate (GPP) is then formed from the combination of DMAPP and isopentyl pyrophosphate in the presence of farnesyl PP synthase having an S82F mutation relative to SEQ ID NO: 53. In one embodiment, the farnesyl diphosphate synthase has a sequence that is at least 95%, 98%, 99%, or 100% identical to SEQ ID NO: 53 having an S82F mutation and that is capable of forming geranyl pyrophosphate from DMAPP and isopentyl pyrophosphate.

[0122] GPP is used as a substrate for many pathways leading to prenylflavonoids, geranylflavonoids, prenylstilbenoids, geranylstilbenoids, CBGA, CBGVA, CBDA, CBDVA, CBGVA, CBCVA, THCA, and THCVA (e.g., see Figure 2A-2B).

[0123] For example, we used the NphB mutations described above (e.g., the M23 mutation) to test their ability to produce CBGA directly from glucose and OA using a fully synthetic biochemical system containing the PDH bypass (see Figures 2A-2B and 3C). The initial productivity using M23 in this system was 67 mg L−1. -1 hr -1 The final CBGA titer was 744±34 mg / L. -1 This was 100 times faster than CBGA production using WT NphB, and the titer was 21 times higher. The mutant NphB enzyme reached maximum titer within 24 hours and production ceased, whereas the wild-type enzyme allowed the system to run continuously for up to 4 days, suggesting that the enzyme and cofactors remained active and viable for a longer period. CBGA was produced at approximately 500 mg L−1. -1 Upon formation, the reaction mixture became cloudy. The precipitate was collected and analyzed by SDS-PAGE, which confirmed that it contained the enzyme mixture. This suggests that the enzyme precipitates at high concentrations of CBGA in solution. A more efficient system for removing the product during the reaction was developed.

[0124] The nonane overlayer was used in the reaction to extract CBGA, but because CBGA is more soluble in water than nonane, this limits the amount of CBGA that can be extracted with a simple overlay. Therefore, we designed a flow system to capture CBGA from the nonane layer and encapsulate it in a separate water reservoir (Figure 5B). By introducing this flow system, we were able to maintain a low CBGA concentration in the reaction vessel and reduce enzyme precipitation. This flow system improved the final titer to 1.2 g / L.

[0125] Next, we performed experiments to produce CBGVA, a precursor of many rare cannabinoids, by substituting dibyrinic acid (DA) for OA in the system (see Figure 2C, for example). The engineered enzyme was first tested for activity toward the DA substrate. The two most effective mutants, M23 and M31, were tested for their ability to produce CBGVA, similar to that of WT NphB. The kinetic data shown in Table 2 indicated that M31 was far superior, with catalytic efficiency 15-fold higher than M23 and 650-fold higher than WT NphB. Thus, further attempts were made to produce CBGVA from glucose and dibyrinic acid using M31. As shown in Figure 5A, CBGVA was obtained with a maximum productivity of approximately 107 mg L−1. -1 hr -1 The final titer was 1.74±0.09g L -1 The nonane flow system was not necessary for the production of CBGVA because CBGVA was less effective as a precipitating enzyme.

[0126] To demonstrate that this method could ultimately be used to prepare other cannabinoids, we converted CBGA to CBDA and CBGVA to CBDVA using CBDA synthase. In the case of CBDA, the nonane top layer contained a significant amount of CBGA, so simply transferring the nonane top layer to a solution containing CBDA synthase produced 14.4 ± 0.8 mg L−1 of CBGA over a 4-day period. -1 hr -1 mg -1 Total protein -1 It was converted to CBDA at a constant rate.

[0127] Because CBGVA has low solubility in nonane, CBGVA was extracted and added to the reaction mixture containing CBDA synthase. GC-MS analysis confirmed that the product of CBDA synthase was CBDVA.

[0128] Thus, the present disclosure provides a cell-free system for producing GPP. Furthermore, the present disclosure provides a cell-free approach for combining the GPP pathway with mutant NphB or substrates of the mutant NphB disclosed herein to produce numerous pure cannabinoids and other prenylated natural products. This method is successful by using the modified prenyltransferases disclosed herein (e.g., the NphB mutants described above), which are active, highly specific, and do not require native transmembrane prenyltransferases. The modularity and flexibility of the synthetic biochemistry platform provided herein combines the advantages of a biobased approach with the elimination of the complexities of living systems. For example, the toxicity of GPP was not considered in the design process. Furthermore, because OA cannot be incorporated into yeast, an exogenous addition approach is not necessarily possible within cells. Indeed, the flexibility of the cell-free system greatly facilitates the design-build-test cycles required for further optimization and modification of additional pathway enzymes, reagents, and cofactors.

[0129] Referring to the overall pathway in Figures 2A-2C, the present disclosure shows multiple steps that are catalyzed by enzymes to convert a "substrate" into a product. In some cases, the steps may utilize a cofactor, but in some instances, the steps do not use a cofactor (e.g., NAD(P)H, ATP / ADP, etc.). Table 3 lists the enzymes, organisms, reaction volumes, and accession numbers used (the sequences associated with these accession numbers are incorporated herein by reference).

[0130] [Table 3-1] [Table 3-2] [Table 3-3]

[0131] As noted above, prenylation of olivetolate by GPP is carried out by the activity of the mutant NphB polypeptides described herein and above.

[0132] In one embodiment, the pathway of the invention utilizes polyphosphate kinase (PPK), malonyl-CoA synthetase (MatB), and pyrophosphatase (PPase) in the ATP regeneration pathway. In one embodiment, the polyphosphate kinase is MBP-AaPPK. In one embodiment, the pyrophosphatase is from G. stearothermophilus.

[0133] The present disclosure provides methods for the in vitro production of prenylated compounds, as well as cannabinoids and cannabinoid precursors (e.g., CBGA, CBGVA, or CBGXA, where "X" represents any chemical group). In one embodiment, cell-free preparations can be engineered for production by, for example, three methods. In one embodiment, enzymes of a pathway described herein are purchased, mixed in an appropriate buffer, and appropriate substrates are added and incubated under conditions suitable for the production of a prenylated compound, cannabinoid, or cannabinoid precursor. In some embodiments, enzymes can be bound to a support or expressed in a phage display or other surface expression system, e.g., immobilized in a fluid pathway corresponding to a point in the cycle of a metabolic pathway.

[0134] Figure 6A-B shows this pathway as various "modules" (e.g., glycolysis module, mevalonate / isoprenoid module, cannabinoid module, polyketide module). For example, the isoprenoid module produces the isoprenoid geranyl pyrophosphate (GPP) from acetyl-CoA via the mevalonate pathway. The aromatic polyketide module utilizes a type III polyketide synthase (PKS) to convert hexanoyl-CoA and malonyl-CoA (derived from acetyl-CoA) to olivetolic acid (OA). The cannabinoid module utilizes the products from the isoprenoid and polyketide modules to produce cannabigerolic acid, which is converted to the final cannabinoid by cannabinoid synthase.

[0135] Figure 27A shows one embodiment of a cell-free biosynthetic pathway for deuterated cannabinoids. Unlabeled malonic acid and a deuterated fatty acid (e.g., fully deuterated or partially deuterated) are activated to produce the corresponding CoA thioester. The deuterated fatty acid, d11-hexanoic acid, is activated to d11-hexanoyl-CoA in the presence of acyl-activating enzyme (AAE) 3, and malonic acid is activated to malonyl-CoA in the presence of malonyl-CoA synthetase. The CoA thioester then undergoes elongation and cyclization reactions to produce d11-olivetolic acid (d11-OA). In the example shown in Figure 27A, the deuterated fatty acid is d11-hexanoic acid. The present invention is also applicable to other deuterated fatty acids. For example, but not limited to, when d7-butyric acid is the deuterated fatty acid, d7-divalic acid (d7-DA) can be produced in a similar manner. Therefore, a general approach is feasible to activate deuterated fatty acids to the corresponding CoA thioesters for incorporation into small molecules or for later-stage modifications.

[0136] Following the formation of d11-OA, prenylation can occur through the action of CsPT or modified NphB enzymes in the presence of geranyl pyrophosphate (GPP), producing the deuterated cannabinoid d11-cannabigerolic acid (d11-CBGA). d11-CBGA is then cyclized through the action of tetrahydrocannabidiolic acid synthase (THCAS) to form d11-tetrahydrocannabidiolic acid (d11-THCA). Alternative deuterated cannabinoids can be synthesized using alternative cannabinoid synthases (e.g., CBDAS, CBCAS). Those skilled in the art will understand that using cannabidiolic acid synthase (CBDAS) produces deuterated cannabinoids in the form of cannabidiolic acid (CBDA) (e.g., d11-CBDA), rather than tetrahydrocannabidiolic acid. Similarly, the use of cannabichromene acid synthase (CBCAS) produces deuterated cannabinoids in the form of cannabichromene acid (CBCA) (e.g., d11-CBCA).

[0137] Figure 27B shows an embodiment of a cell-free biosynthetic pathway for deuterated cannabinoids, including a pathway for inducing geranyl pyrophosphatase (GPP), according to one embodiment of the present invention. Hydroxyethylthiazole kinase (ThiM) is used to generate isopentenyl phosphate from isopolenol. In the presence of isopentenyl diphosphate isomerase, isopentenyl phosphate is converted to isopentenyl diphosphate. Isopentenyl diphosphate can be converted to dimethylallyl pyrophosphate (DMAPP). Both isopentenyl diphosphate and DMAPP can be converted to geranyl polyphosphate in the presence of farnesyl pyrophosphate synthase (FPPS). Geranyl polyphosphate can be used in the cell-free biosynthetic pathways described herein. Those skilled in the art will understand that this process is also shown in detail in Figure 7, but is shown here to illustrate the steps in the cell-free biosynthetic pathways that utilize geranyl polyphosphate for production.

[0138] In one embodiment, once the THCA compounds of the present invention are produced, the compounds are purified and the produced THCA is isolated as disclosed herein. After purification of the THCA, the THCA can be decarboxylated and purified to obtain THC.

[0139] Figure 28A shows one embodiment of a deuterated methyl compound. Deuterated d3-S-adenosylmethionine (d3-SAM) is generated from d3-L-methionine and ATP by the action of SAM synthase (MAT). As shown in Figure 28B, d3-SAM is utilized by methyltransferases to methylate various small molecules.

[0140] FIG. 29 shows examples of deuterated methylated psilocybin using specific methyltransferases (e.g., PsiM methyltransferase).

[0141] FIG. 30 shows examples of deuterated methylated chrysoeriol using specific methyltransferases (e.g., CsOMT21 methyltransferase).

[0142] FIG. 31 shows an example of the synthesis of a deuterated glucoside produced using UDP-glucose-d12.

[0143] Figure 32 shows HPLC (high performance liquid chromatography) traces of cell-free production of deuterated (1), even-chain (2), branched (3), and rare (4) cannabinoids (e.g., CBG derivatives).

[0144] 33 is a bar graph outlining the progress of a route optimization according to one embodiment of the present invention. All reactions were completed at the 200 uL scale, except for the 1 L scale reaction indicated by the bar on the far right of the graph.

[0145] Figures 34A-34B show the CBGA production by OLS variants. Each variant was screened and the CBGA production from the OLS variants was measured. The OLS variants were observed with various enzyme loadings, and the corresponding CBGA production was measured.

[0146] Figure 35 shows the time course of a CBGA synthesis reaction according to one embodiment of the present invention. The time course of a CBGA synthesis reaction using OLS M187T is shown in graph A at 0, 2, 4, 6, and 20 hours. After 4 hours, a slight accumulation of olivetolic acid was observed, indicating that SimplePath or NphB may be limiting the reaction. Graph B shows the traces for the OLS variants after 20 hours, revealing differences in by-product formation between WT, M187T, and P131A M187T.

[0147] Figure 36 shows HPLC traces of CBGA and THCA activity according to one embodiment of the present invention. The results show a 1 L CBGA reaction, conversion to THCA using P. pastoris THCA supernatant, methanol extraction of the THCA reaction, ethanol precipitation of the methanol extract, and HPLC analysis of the purified THCA trace. As purity increased, significant differences in ABS at 216 nm were observed, allowing visualization and quantification of impurities.

[0148] Figure 37 shows an HPLC trace of THC in toluene according to one embodiment of the present invention. This is an HPLC trace of the decarboxylation reaction of THCA in toluene at 95°C. The starting material in toluene is shown at the top. The reaction after 10 minutes is displayed in the HPLC below. The reaction after 35 minutes is displayed below the reaction after 10 minutes, and the recovered THC product is shown at the bottom of the HPLC.

[0149] Those skilled in the art will appreciate that alternative starting materials and enzymes may be used to produce alternative products. The present invention is not limited to the examples presented herein.

[0150] In another embodiment, one or more polynucleotides encoding one or more enzymes of the pathway are cloned into one or more microorganisms under conditions in which the enzymes are expressed. The cells are then lysed, and a lysed preparation containing one or more enzymes from the cells is mixed with an appropriate buffer and substrates (and one or more additional enzymes of the pathway, if necessary) to produce a prenylated compound, cannabinoid, or cannabinoid precursor. Alternatively, the enzymes can be isolated from the lysed preparation and recombined in an appropriate buffer. In yet another embodiment, purchased enzymes are combined with expressed enzymes to provide the pathway in an appropriate buffer. In one embodiment, thermostabilizing polypeptides / enzymes of the pathway are cloned and expressed. In one embodiment, the pathway enzymes are derived from a thermophilic microorganism. The microorganism is then lysed, and the preparation is heated to a temperature at which the thermostabilizing polypeptides of the pathway become active and other polypeptides (non-desired) become denatured and inactive. This results in a preparation containing a subset of all enzymes of the microorganism, including active thermostable enzymes. The preparation can then be used to implement the pathway to produce a prenylated compound, cannabinoid, or cannabinoid precursor.

[0151] For example, to construct an in vitro system, all enzymes are obtained commercially or purified by affinity chromatography, activity tested, and used in combination in an appropriately selected reaction buffer.

[0152] Also contemplated are in vivo systems that use all or some of the above enzymes in biosynthetic pathways engineered into a microorganism to yield a recombinant microorganism.

[0153] The present disclosure also provides recombinant organisms comprising a metabolically engineered biosynthetic pathway comprising a mutated nphB for producing a prenylated compound, and optionally further comprising one or more additional organisms expressing enzymes for producing cannabinoids (e.g., co-culture of a set of microorganisms expressing part of the pathway with a second set of microorganisms expressing a further or final part of the pathway).

[0154] In one embodiment, the present disclosure provides a recombinant microorganism that has elevated expression of at least one target enzyme compared to the parent microorganism, or encodes an enzyme not found in the parent organism. In another or further embodiment, the microorganism provides a reduction, disruption, or knockout of at least one gene encoding an enzyme that competes with a metabolite required for the production of a desired metabolite or produces an undesirable product. In one embodiment, the recombinant microorganism expresses an enzyme that produces at least one metabolite involved in a biosynthetic pathway for, for example, the production of a prenylated compound or a cannabinoid or cannabinoid precursor. Generally, the recombinant microorganism contains at least one engineered metabolic pathway that includes the target enzyme, and optionally further includes reduced activity or expression of an enzyme in a competing biosynthetic pathway. This pathway acts to modify a substrate or metabolic intermediate in the production of, for example, a prenylated compound, cannabinoid, or cannabinoid precursor. The target enzyme is encoded and expressed by a polynucleotide from an appropriate organism. In some embodiments, the polynucleotide comprises a gene from bacteria or yeast and is incorporated into and integrated into the microorganism of the present disclosure. In another embodiment, the polynucleotide encoding the desired target enzyme occurs naturally in the organism, but is recombinantly modified to be overexpressed compared to native expression levels.

[0155] The term "microorganism" includes prokaryotic and eukaryotic microbial species belonging to the archaea, bacteria, and eukaryotes, the latter of which includes yeast, filamentous fungi, protozoa, algae, or higher protists. The terms "microbial cell" and "microorganism" are used interchangeably with the term microorganism.

[0156] The term "prokaryote" is technically accepted and refers to cells that lack a nucleus or other organelles. Prokaryotes are broadly classified into two domains: Bacteria and Archaea. The defining difference between organisms in the Archaea and Bacteria domains is based on fundamental differences in the nucleotide base sequences of the 16S ribosomal RNA.

[0157] "Bacteria" or "eubacteria" refers to the domain of prokaryotes. Bacteria include at least 11 different groups: (1) Gram-positive (gram+) bacteria, which have two major divisions: (1) the high G+C group (Actinomycetes, Mycobacteria, Micrococcus, etc.) and (2) the low G+C group (Bacillus, Clostridium, Lactobacillus, Staphylococcus, Streptococcus, Mycoplasma); (2) Proteobacteria, e.g., purple photosynthetic and nonphotosynthetic Gram-negative bacteria (including the most "common" Gram-negative bacteria); (3) Cyanobacteria, e.g., oxygenic phototrophs; (4) Spirochetes and related species; (5) Planctomycetes; (6) Bacteroidetes and Flavobacteria; (7) Chlamydia; (8) Green sulfur bacteria; (9) Green nonsulfur bacteria (also anaerobic photosynthetic organisms); (10) Radioresistant Cocci and related species; and (11) Thermophiles Thermotoga and Hermosiphos.

[0158] "Gram-negative bacteria" include cocci, non-enteric bacilli, and enteric bacilli. Examples of genera of Gram-negative bacteria include the genera Neisseria, Spirillum, Pasteurella, Brucella, Yersinia, Francisella, Haemophilus, Bordetella, Escherichia, Salmonella, Shigella, Klebsiella, Proteus, Vibrio, Pseudomonas, Bacteroides, Acetobacter, Aerobacter, Agrobacterium, Azotobacter, Spirilla, Serratia, Vibrio, Rhizobium, Chlamydia, Rickettsia, Treponema, and Fusobacterium.

[0159] "Gram-positive bacteria" include cocci, non-spore-forming bacilli, and spore-forming bacilli. Genera of Gram-positive bacteria include, for example, Actinomycetes, Bacillus, Clostridium, Corynebacterium, Erysipelothricus, Lactobacillus, Listeria, Mycobacterium, Myxococcus, Nocardia, Staphylococcus, Streptococcus, Streptomyces, etc.

[0160] As used herein, the "activity" of an enzyme is a measure of its ability to catalyze a reaction that produces a metabolite, i.e., its ability to "function," and can be expressed as the rate at which the metabolite of the reaction is produced. For example, enzyme activity can be expressed as the amount of metabolite produced per unit time or per enzyme unit (e.g., concentration or weight), or as an affinity constant or dissociation constant.

[0161] The term "biosynthetic pathway," also known as a "metabolic pathway," refers to a series of anabolic or catabolic biochemical reactions that convert (transform) one chemical species into another (see, e.g., Figures 2A-2C). Gene products are considered to belong to the same "metabolic pathway" if they act, in parallel or series, on the same substrate and produce the same product, or act on or produce a metabolic intermediate (i.e., metabolite) between the same substrate and the metabolic end product. The present disclosure provides recombinant microorganisms with metabolically engineered pathways to produce desired products or intermediates.

[0162] Thus, metabolically "engineered" or "modified" microorganisms are produced by introducing genetic material into a selected host or parent microorganism, thereby modifying or altering the microorganism's cellular physiology and biochemistry. Through the introduction of the genetic material, the parent microorganism acquires new properties, such as the ability to produce new or increased amounts of intracellular metabolites or the ability to express polypeptides that are not normally expressed. In an exemplary embodiment, introducing the genetic material into the parent microorganism results in a new or modified ability to produce acetyl phosphate and / or acetyl-CoA through the PDH bypass using pyruvate oxidase and acetyl phosphate transferase. The genetic material introduced into the parent microorganism contains gene(s) or portions of gene(s) encoding one or more enzymes involved in biosynthetic pathways for producing prenylated compounds or cannabinoids or cannabinoid precursors, and is also operable to include additional elements for expression and / or regulation of expression of these genes, such as promoter sequences.

[0163] Alternative to or in addition to introducing genetic material into a host or parent microorganism, engineered or modified microorganisms can also be operable to disrupt, delete, or knock out genes or polynucleotides to alter the cellular physiology and biochemistry of the microorganism. Through gene or polynucleotide reduction, disruption, or knockout, the microorganism may acquire new or improved properties (e.g., the ability to produce new or greater amounts of intracellular metabolites, improve the flux of metabolites through desired pathways, and / or reduce the production of undesirable by-products) or eliminate enzymes from cell-free preparations that are operable to compete with biosynthetic pathways developed from lysed preparations.

[0164] By "enzyme" is meant any substance, usually composed entirely or mostly of protein or polypeptide amino acids, that catalyzes or promotes, more or less specifically, one or more chemical or biochemical reactions.

[0165] The terms "protein" and "polypeptide" are used interchangeably herein and include one or more chains of chemical building blocks called amino acids joined by chemical bonds called peptide bonds. A protein or polypeptide can function as an enzyme.

[0166] As used herein, the term "metabolically engineered" or "metabolic engineering" includes the rational pathway design and assembly of biosynthetic genes, operon-related genes, and regulatory elements of such polynucleotides to produce desired metabolites, such as acetyl phosphate and / or acetyl-CoA, higher alcohols, or other chemicals, in a microorganism. "Metabolically engineered" can further include the optimization of metabolic fluxes through the modulation and optimization of transcription, translation, protein stability, and protein functionality using genetic manipulation and appropriate culture conditions, including the reduction, disruption, or knockout of competing metabolic pathways that compete with intermediates leading to the desired pathway. Biosynthetic genes can be heterologous to the host microorganism either by being exogenous to the host or by modification through mutagenesis, recombination, and / or association with heterologous expression control sequences in the endogenous host cell. In one embodiment, when a polynucleotide is heterologous to the host organism, the polynucleotide can be codon-optimized.

[0167] "Metabolite" refers to a substance produced by metabolism or a substance necessary for or involved in a specific metabolic process that produces a metabolite, chemical, alcohol, or ketone of interest. Metabolites are organic compounds that serve as starting materials (e.g., glucose), intermediates (e.g., acetyl-CoA), or end products (e.g., CBDA) in metabolism. Metabolites can be used to build more complex molecules, or they can be broken down into simpler ones. Intermediate metabolites can be synthesized from other metabolites, used to make more complex substances, or broken down into simpler compounds, often releasing chemical energy.

[0168] "Mutation" refers to any process or mechanism that results in a change in a protein, enzyme, polynucleotide, gene, or cell. This includes any mutation that changes the sequence of a protein, enzyme, polynucleotide, or gene, and any detectable change that occurs in a cell due to such a mutation. Mutations typically occur through point mutations, deletions, or insertions of single or multiple nucleotide residues in the sequence of a polynucleotide or gene. Mutations include polynucleotide changes that occur within the protein-coding region of a gene, as well as changes in regions outside the protein-coding sequence, such as, but not limited to, regulatory or promoter sequences. Gene mutations are "silent," meaning they are not reflected in a change in the amino acid sequence during expression, thus resulting in a "sequence-conservative" variant of the gene. This generally occurs when one amino acid corresponds to multiple codons. Mutations that result in a different primary protein sequence can be referred to as mutant proteins or protein variants.

[0169] A "native" or "wild-type" protein, enzyme, polynucleotide, gene, or cell means a protein, enzyme, polynucleotide, gene, or cell that is found in nature.

[0170] "Parent microorganism" refers to a cell used to generate a recombinant microorganism. The term "parent microorganism" refers, in one embodiment, to a cell that occurs in nature, i.e., a "wild-type" cell that has not been genetically modified. Additionally, the term "parent microorganism" refers to a cell that serves as a "parent" for further modification. In this latter embodiment, the cell may have been genetically engineered, but serves as a source for further genetic manipulation.

[0171] For example, a wild-type microorganism can be genetically engineered to express or overexpress a first target enzyme, such as hexokinase. This microorganism can serve as a parent microorganism in the generation of a microorganism engineered to express or overexpress a second target enzyme (e.g., fructose-1,6-bisphosphate aldolase). The microorganism can then be modified to express or overexpress, for example, NADH oxidase and Gald-3 phosphate dehydrogenase (and variants thereof), which can be further modified to express or overexpress a third target enzyme, such as phosphoglycerate kinase. As used herein, "express" or "overexpress" refers to the phenotypic expression of a desired gene product. In one embodiment, a naturally occurring gene in an organism can be engineered to be linked to a heterologous promoter or regulatory domain, which drives expression of the gene, thereby altering its normal expression relative to a wild-type organism. Alternatively, the organism can be engineered to remove or reduce a repressor function of the gene, thereby altering its expression. In yet another embodiment, a cassette containing the gene sequence operably linked to desired expression control / regulatory elements is incorporated into the microorganism.

[0172] Thus, the parental microorganism serves as a reference cell in successive genetic modification events. Each modification event is achieved by introducing one or more nucleic acid molecules into the reference cell. This introduction promotes the expression or overexpression, or the reduction or elimination, of one or more target enzymes. It should be understood that the term "promoting" includes the activation of an endogenous polynucleotide encoding a target enzyme, for example, through genetic modification of a promoter sequence in the parental microorganism. It should also be understood that the term "promoting" includes the introduction of an exogenous polynucleotide encoding a target enzyme into the parental microorganism.

[0173] Polynucleotides encoding enzymes useful for producing metabolic products, including homologs, variants, fragments, related fusion proteins, or functional equivalents thereof, are used in recombinant nucleic acid molecules that direct the expression of such polypeptides in suitable host cells, such as bacterial or yeast cells. The sequences and accession numbers provided herein provide one of skill in the art with the ability to obtain the coding sequences for the various enzymes of the present disclosure using readily available software and basic biological knowledge.

[0174] The sequence listings accompanying this specification provide exemplary polypeptides useful in the methods described herein. It is understood that the addition of sequences that do not alter the activity of the polypeptide molecule, such as the addition of non-functional or non-coding sequences (e.g., poly-HIS tags), is a conservative variation of the base molecule.

[0175] It is to be understood that the polynucleotides described herein include "genes," and the nucleic acid molecules described above include "vectors" or "plasmids."

[0176] The terms "polynucleotide," "nucleic acid," or "recombinant nucleic acid" refer to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA).

[0177] The term "expression," with respect to a gene or polynucleotide, refers to the transcription of the gene or polynucleotide and, if necessary, the translation of the resulting mRNA transcript into a protein or polypeptide. Thus, as will be clear from the context, protein or polypeptide expression occurs through the transcription and translation of an open reading frame.

[0178] Those skilled in the art will understand that due to the degenerate nature of the genetic code, various codons with different nucleotide sequences can be used to encode a given amino acid. The specific polynucleotide or gene sequences encoding the biosynthetic enzymes or polypeptides described above are referenced herein solely to illustrate embodiments of the present disclosure, and the present disclosure includes polynucleotides of any sequence that encode polypeptides containing the same amino acid sequences as the enzyme polypeptides and proteins used in the methods of the present disclosure. Similarly, polypeptides can generally tolerate substitutions, deletions, and insertions of one or more amino acids in their amino acid sequences without loss or significant loss of the desired activity. The present disclosure includes polypeptides with such alternative amino acid sequences, and the amino acid sequences encoded by the DNA sequences set forth herein merely exemplify exemplary embodiments of the present disclosure.

[0179] The present disclosure provides polynucleotides in the form of recombinant DNA expression vectors or plasmids encoding one or more target enzymes, as described in more detail elsewhere herein. Generally, such vectors can either replicate within the cytoplasm of the host microorganism or integrate into the chromosomal DNA of the host microorganism. In either case, the vector can be operated as a stable vector (i.e., the vector remains present over many cell divisions, even under selective pressure alone) or as a transient vector (i.e., the vector is gradually lost by the host microorganism over increasing numbers of cell divisions). The present disclosure provides DNA molecules in isolated (i.e., not pure, but present in a preparation at an abundance and / or concentration not found in nature) and purified (i.e., substantially free from contaminants or from materials with which the corresponding DNA is found in nature) forms.

[0180] Polynucleotides of the present disclosure can be amplified using cDNA, mRNA, or genomic DNA as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques and the procedures described in the Examples section below. The nucleic acid thus amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to the nucleotide sequence can be prepared by standard synthetic techniques, for example, using an automated DNA synthesizer.

[0181] The present disclosure provides in the sequence listing accompanying this application a number of polypeptide sequences that can be used to design, synthesize and / or isolate polynucleotide sequences by taking advantage of the degeneracy of the genetic code or by searching for coding sequences using public databases.

[0182] It is also understood that isolated polynucleotide molecules encoding polypeptides homologous to the enzymes described herein can be generated by introducing one or more nucleotide substitutions, additions, or deletions into the nucleotide sequence encoding a particular polypeptide, resulting in one or more amino acid substitutions, additions, or deletions in the encoded protein. Mutations can be introduced into polynucleotides by standard techniques, such as site-directed mutagenesis or PCR. Conservative amino acid substitutions are preferred at some positions, as opposed to positions where non-conservative amino acid substitutions are desirable.

[0183] As those skilled in the art can appreciate, modifying a coding sequence to enhance expression in a particular host can be advantageous. Although the genetic code is redundant with 64 possible codons, most organisms typically use a subset of these codons. The most frequently used codons in a species are called optimal codons, while less frequently used codons are classified as rare codons or low-usage codons. Codons can be substituted to reflect the preferred codon usage of the host; this process is also called "codon optimization" or "control of species codon bias."

[0184] Optimized coding sequences containing codons preferred by a particular prokaryotic or eukaryotic host (see also Murray et al. (1989) Nucl. Acids Res. 17:477-508, incorporated herein by reference in its entirety) can be prepared to, for example, increase the translation rate or to generate recombinant RNA transcripts with desirable properties, such as a longer half-life, compared to transcripts produced from non-optimized sequences. Translation stop codons can also be modified depending on host preference. For example, typical stop codons for S. cerevisiae and mammals are UAA and UGA, respectively. While the typical stop codon for monocotyledonous plants is UGA, UAA is commonly used as the stop codon in insects and E. coli (Dalphin et al. (1996) Nucl. Acids Res. 24:216-218, incorporated herein by reference). Methods for optimizing nucleotide sequences for expression in plants are described, for example, in U.S. Patent No. 6,015,891 and the references cited therein, which is incorporated herein by reference in its entirety.

[0185] The term "substrate" or "suitable substrate" refers to a substance or compound that is converted, or is intended to be converted, into another compound by the action of an enzyme. The term includes not only single compounds but also combinations of compounds, such as solutions, mixtures, or other materials containing at least one substrate or its derivatives. Furthermore, the term "substrate" includes not only compounds that provide starting materials, but also intermediate and end-product metabolites used in pathways associated with the metabolically engineered microorganisms described herein.

[0186] "Transformation" refers to the process of introducing a vector into a host cell. Transformation (or transduction, or transfection) can be accomplished by any of a variety of means, including electroporation, microinjection, biolistics (or particle bombardment), and Agrobacterium-mediated transformation.

[0187] A "vector" generally refers to a polynucleotide capable of propagation and / or transmission between organisms, cells, or cellular components. Vectors include viruses, bacteriophages, proviruses, plasmids, phagemids, transposons, and artificial chromosomes, such as YACs (yeast artificial chromosomes), BACs (bacterial artificial chromosomes), and PLACs (plant artificial chromosomes). They function as "episomes," meaning they replicate autonomously or integrate into the host cell's chromosomes. Vectors can be non-episomal in nature, such as naked RNA polynucleotides, naked DNA polynucleotides, polynucleotides composed of both DNA and RNA in the same strand, polylysine-linked DNA or RNA, peptide-linked DNA or RNA, or liposome-linked DNA, or they can operate as organisms containing one or more of the above polynucleotide constructs, such as Agrobacterium or bacteria.

[0188] The various components of expression vectors operate in a wide variety of ways, depending on the use of the vector and the host cell in which it will replicate or express. Expression vector components suitable for gene expression and vector maintenance in E. coli, yeast, actinomycetes, and other commonly used cells are widely known and commercially available. For example, promoters suitable for incorporation into the expression vectors of the present disclosure include those that function in eukaryotic or prokaryotic host microorganisms. Promoters can include regulatory sequences that control expression in response to growth of the host microorganism or that turn gene expression on or off in response to chemical or physical stimuli. For E. coli and certain other bacterial host cells, promoters derived from genes for biosynthetic enzymes, antibiotic resistance-conferring enzymes, and phage proteins can be used, including, for example, the galactose, lactose (lac), maltose, tryptophan (trp), beta-lactamase (bla), bacteriophage lambda PL, and T5 promoters. In addition, synthetic promoters, such as the tac promoter (U.S. Patent No. 4,551,433, incorporated herein by reference in its entirety), can also be used. For E. coli expression vectors, it is useful to include an E. coli origin of replication such as pUC, plP, pl, or pBR.

[0189] Thus, a recombinant expression vector contains at least one expression system, which is comprised of at least a portion of a gene coding sequence operably linked to a promoter and, optionally, a termination sequence, which operates to effect expression of the coding sequence in a compatible host cell. The host cell is modified by transformation with a recombinant DNA expression vector of the invention to contain the expression system sequences, either as an extrachromosomal element or integrated into the chromosome.

[0190] Furthermore, as described above, homologs of enzymes useful for producing metabolic products are also encompassed by the microorganisms and methods provided herein. The term "homolog," when used with reference to an original enzyme or gene of a first family or species, refers to a different enzyme or gene of a second family or species that has been determined by functional, structural, or genomic analysis to be an enzyme or gene of the second family or species that corresponds to the original enzyme or gene of the first family or species. In many cases, homologous genes are functionally, structurally, or genomically similar. Techniques are known that allow homologous genes of an enzyme or gene to be easily cloned using gene probes and PCR. The identity of the cloned sequence as a homologous gene can be confirmed by functional assays and / or genomic mapping of the gene.

[0191] A protein is said to be "homologous" or "homologous" to another protein if the nucleic acid sequence encoding that protein has a similar sequence to the nucleic acid sequence encoding that protein. Alternatively, a protein is homologous to another protein if the two proteins have "similar" amino acid sequences. (Thus, the term "homologous proteins" is defined to mean that two proteins have similar amino acid sequences.)

[0192] As used herein, two proteins (or protein regions) are considered substantially homologous when their amino acid sequences share at least about 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. To determine the percent identity of two amino acid sequences or two nucleic acid sequences, these sequences are aligned for optimal comparison (e.g., for optimal alignment, gaps can be introduced into one or both of the first and second amino acid or nucleic acid sequences, and non-homologous sequences can be ignored for comparison). In one embodiment, the length of the reference sequence aligned for comparison is at least 30%, typically at least 40%, more typically at least 50%, even more typically at least 60%, and even more typically at least 70%, 80%, 90%, or 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position (as used herein, amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced to optimally align the two sequences.

[0193] When the term "homologous" is used in reference to proteins or peptides, it is known that non-identical residue positions often differ by conservative amino acid substitutions. A "conservative amino acid substitution" is the replacement of one amino acid residue with another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially change the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percentage of sequence identity or degree of homology can be adjusted upward to correct for the conservative nature of the substitution. Means for making this adjustment are known to those skilled in the art (e.g., Pearson et al., 1994, incorporated herein by reference).

[0194] In some instances, "isoenzymes" are available that perform the same functional transformation / reaction, but are so structurally distinct that they are not usually considered "homologous."

[0195] "Conservative amino acid substitution" refers to a substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the present technology. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine, etc.), acidic side chains (e.g., aspartic acid, glutamic acid, etc.), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, etc.), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, etc.), beta-branched side chains (e.g., threonine, valine, isoleucine, etc.), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine, etc.). The following six groups each contain amino acids that are conservative substitutions for one another: (1) serine (S), threonine (T), (2) aspartic acid (D), glutamic acid (E), (3) asparagine (N), glutamine (Q), (4) arginine (R), lysine (K), (5) isoleucine (I), leucine (L), methionine (M), alanine (A), valine (V), and (6) phenylalanine (F), tyrosine (Y), and tryptophan (W).

[0196] Sequence homology of polypeptides, also known as percent sequence identity, is typically measured using sequence analysis software. See, for example, the sequence analysis software package from the Genetics Computer Group (GCG), University of Wisconsin Biotechnology Center, 910 University Avenue, Madison, Wis. 53705. Protein analysis software matches similar sequences using measures of homology assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG includes programs such as "Gap" and "Bestfit," which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species or between wild-type proteins and their mutant proteins. See, for example, GCG version 6.1.

[0197] A common algorithm used to compare molecular sequences to databases containing numerous sequences from various organisms is the computer program BLAST (Altschul, 1990; Gish, 1993; Madden, 1996; Altschul, 1997; Zhang, 1997), specifically blastp or tblastn (Altschul, 1997). Typical parameters for BLASTp are: expectation: 10 (default); filter: seg (default); gap opening cost: 11 (default); gap extension cost: 1 (default); maximum alignment: 100 (default); word size: 11 (default); number of descriptions: 100 (default); penalty matrix: BLOWSUM62.

[0198] When searching a database containing sequences from many different organisms, it is common to compare amino acid sequences. Database searches using amino acid sequences can be measured by known algorithms other than BLASTp. For example, polypeptide sequences can be compared using FASTA, a program in GCG version 6.1. FASTA provides alignment and percent sequence identity of the most overlapping regions between the query sequence and the search sequence (Pearson, 1990, incorporated herein by reference). For example, the percent sequence identity between amino acid sequences is determined using the default FASTA parameters (word size 2 and PAM250 score matrix) as specified in GCG version 6.1. GCG version 6.1 is incorporated herein by reference in its entirety.

[0199] The present disclosure provides the accession numbers and sequences of various genes, homologous genes and variants that are useful for producing recombinant microorganisms and proteins for use in in vitro systems.It should be understood that the homologous genes and variants described herein are exemplary and are not limited thereto.Those skilled in the art can obtain additional homologous genes, variants and sequences by using various databases, such as the National Center for Biotechnology Information (NCBI), which can be accessed on the World Wide Web.

[0200] It is within the level of skill of one in the art to use the sequences and accession numbers provided herein to identify homologous genes and isozymes that can be used or substituted for any of the polypeptides described herein. Indeed, a BLAST search of any one of the sequences provided herein will identify multiple related homologous genes.

[0201] Culture conditions suitable for the growth and maintenance of the recombinant microorganisms provided herein are known (see, e.g., Animal Cell Culture - A Basic Techniques Manual, Freshney, Wiley-Liss, New York (1994), 3rd ed.). Those skilled in the art will understand that such conditions can be modified to suit the requirements of each microorganism.

[0202] It is understood that there are numerous microorganisms that can be modified to contain all or part of a recombinant metabolic pathway suitable for the production of prenylated compounds, cannabinoids, or cannabinoid precursors. It is also understood that a variety of microorganisms can function to act as "sources" of genetic material encoding target enzymes suitable for use in the recombinant microorganisms described herein.

[0203] As noted above, general texts describing molecular biology techniques useful in the present invention, including the use of vectors, promoters, and many other related topics, include Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology Volume 152, (Academic Press, Inc., San Diego, Calif.) (“Berger”), Sambrook et al., Molecular Cloning—A Laboratory Manual, 2nd ed., Vols. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989 (“Sambrook”), and Current Protocols in Molecular Biology, FM Ausubel et al. (eds.), Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (supplemented through 1999) (“Ausubel”), each of which is incorporated herein by reference in its entirety.

[0204] For example, exemplary protocols sufficient to instruct one of skill in in vitro amplification methods, including polymerase chain reaction (PCR), ligase chain reaction (LCR), Qp replicase amplification, and other RNA polymerase-mediated techniques (e.g., NASBA), for the generation of homologous nucleic acids of the present disclosure can be found in Berger, Sambrook, and Ausubel, as well as in Mullis et al. (1987) U.S. Pat. No. 4,683,202; Innis et al., eds. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press Inc. San Diego, Calif.) (“Innis”); Arnheim & Levinson (October 1, 1990) C&EN 36-47; The Journal of NIH Research (1991) 3:81-94; Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173; Guatelli et al. (1990) Proc. Nat'l. Acad. Sci. USA 87:1874; Lomell et al. (1989) J. Clin. Chem 35:1826; Landegren et al. (1988) Science 241:1077-1080; Van Brunt (1990) Biotechnology 8:291-294; Wu and Wallace (1989) Gene 4:560; Barringer et al. (1990) Gene 89:117; and Sooknanan and Malek (1995) Biotechnology 13:563-564, each of which is incorporated herein by reference in its entirety.

[0205] An improved method for cloning in vitro amplified nucleic acids is described in US Pat. No. 5,426,039 to Wallace et al., which is incorporated herein by reference in its entirety.

[0206] Improved methods for amplifying large nucleic acids by PCR are summarized in Cheng et al. (1994) Nature 369:684-685 and the references cited therein, which generate PCR amplicons up to 40 kb. Cheng et al. (1994) Nature 369:684-685 is incorporated herein by reference in its entirety. Those skilled in the art will appreciate that virtually any RNA can be converted into double-stranded DNA suitable for restriction digestion using reverse transcriptase and polymerase, PCR amplification, and sequencing. See, e.g., Ausubel, Sambrook, and Berger, supra.

[0207] The present invention is described in the following examples, which are offered by way of illustration and are not intended to be limiting. [Example]

[0208] Example

[0209] Chemicals and Reagents. Yeast hexokinase and Corynebacterium glutamicum catalase were purchased from Sigma-Aldrich. Aerococcus viridians pyruvate oxidase was purchased from AGScientific. All cofactors and reagents were purchased from either Sigma-Aldrich or Thermo Fisher Scientific, except for olivetolic acid, which was purchased from Santa Cruz Biotechnology, and divalanic acid, which was purchased from Toronto Research Chemicals.

[0210] Enzyme cloning and purification. The NphB gene was purchased as a gene block from IDT DNA and cloned into the pET 28(+) vector using the Gibson assembly method. The remaining enzymes were amplified from genomic DNA or plasmids and cloned into the pET28(+) vector using a similar Gibson assembly method. All plasmids were transformed into BL21(DE3)Gold, and the enzymes were expressed in LB medium containing 50 μg / mL kanamycin. A 1 L culture was inoculated with 2 mL of a saturated culture in the same medium and grown at 37 °C until OD 600 The culture was grown until the chromatin concentration reached 0.5–0.8. The culture was induced with 1 mM IPTG and expressed for 16 hours at 18°C. Cells were harvested by centrifugation at 2,500 × g and resuspended in approximately 20 mL of lysis buffer (50 mM Tris [pH 8.0], 150 mM NaCl, 10 mM imidazole). Cells were lysed using an Emulsiflex tube. The lysate was centrifuged at 20,000 × g, and the supernatant was batch-bound to 1 mL of NiNTA resin for 30 minutes at 4°C. The resin was transferred to a gravity-flow column and washed with 10 column volumes of wash buffer (50 mM Tris [pH 8.0], 150 mM NaCl, 10 mM imidazole). The protein was then eluted with two column volumes of elution buffer (50 mM Tris [pH 8.0], 150 mM NaCl, 250 mM imidazole, 30% (v / v) glycerol). The enzyme was flash-frozen in the elution buffer using liquid nitrogen, and the enzyme stock was stored at -80°C.

[0211] PDH cell-free reaction. The PDH reaction was performed in two parts. First, the cofactors and substrate were mixed in one tube, and the enzyme was mixed in another. The reaction was initiated by combining the cofactors and enzyme in a final volume of 200 μL. The final substrate and cofactor concentrations were 500 mM glucose, 1 mM 1,6-fructose bisphosphate, 4 mM ATP, 0.5 mM 2,3-bisphosphoglycerate, 0.5 mM NAD*, 1.5 mM CoA, and 1.5 mM NADP. +The buffer was 0.5 mM TPP, 6 mM MgCl, 10 mM KCl, 50 mM Tris (pH 8.0), 20 mM phosphate buffer (pH 8.0), 5 mM glutathione, and 0.5-5 mM 1,6-DHN. The reaction was stopped after 24 hours.

[0212] PDH activity assay. PDH activity was measured in the presence of several aromatic polyketides. 1% ethanol was used as a solvent control, and activity was compared to assays without aromatic polyketides. The final reaction volume was 200 μL, containing 2 mM NAD. + The reaction mixture contained 2 mM CoA, 1 mM TPP, 5 mM Cl2, 5 mM KCl, 50 mM Tris pH 8.0, and 5 μL of 1.25 mg / mL PDH. Reactions were prepared in a 96-well plate. Aromatic polyketides were added to a final concentration of 1 mM, and an ethanol control was added to a final concentration of 1% (v / v). The plate was incubated at room temperature for 10 minutes, and the reaction was initiated with 10 μL of 100 mM pyruvate. The absorbance at 340 nm was measured for 10 minutes using an M200 spectrometer. Because aromatic molecules exhibited background absorption at 340 nm, reactions were blanked using the reaction mixture and aromatic molecules, but water was added instead of initiating the reaction with pyruvate. The initial rate was determined using the initial slope of the linear fit. The amount of NADH produced per unit time was calculated using Beer's law and an extinction coefficient of 6.22 × 103 M -1 cm -1 The reaction was repeated three times, and the mean and standard error were calculated.

[0213] PyOx / PTA cell-free reaction. The PyOx / PTA reaction was performed in two parts. First, the cofactors and substrate were mixed in one tube, and the enzyme was mixed in another. The final cofactor and substrate concentrations in the 200 μL reaction were 500 mM glucose, 1 mM 1,6 fructose bisphosphate, 4 mM ATP, 0.5 mM 2,3 bisphosphoglycerate, 0.5 mM NAD*, 1.5 mM CoA, and 3 mM NADP. +The buffer consisted of 0.5 mM TPP, 6 mM MgCl2, 10 mM KCl, 50 mM Tris pH 8.0, and 50 mM phosphate buffer [pH 8.0]. The amount of enzyme added to each reaction is detailed in Table 3. The reaction was initiated by mixing the cofactors and enzyme, and 500 μL of nonane overlay was added on top. The reaction was incubated at room temperature with gentle shaking on a gel shaker.

[0214] For 1,6-DHN / 5-p-1,6-DHN: When aromatic substrate was used as the variable, 0.5–5 mM aromatic substrate was added to the reaction and the reaction was stopped after 24 hours. When time was used as the variable, 5 mM 1,6-DHN was added and the reaction was stopped after approximately 12, 24, 48, and 72 hours, respectively.

[0215] In the case of olivetrate / CBGA, the glucose concentration was lowered to 150 mM because optimization of the cannabinoid pathway showed that the same titer could be achieved with a lower glucose concentration. + The titer of CBGA was increased by increasing the concentration to 6 mM and decreasing the ATP concentration to 1 mM. The olivetrate concentration was set at 5 mM. The amount of NphB added to the reaction was variable. Data shown in Figure 3C used 1.5 mg / mL NphB, and the reaction stopped at approximately 4, 8, 14, 24, 48, 72, and 96 hours. Data shown in Figure 5A used 0.5 mg / mL WT NphB and M23, and the reaction stopped at approximately 6, 9, 12, 24, 48, 72, and 96 hours.

[0216] In the case of divaleric acid / CBGVA, the conditions were almost the same as in the general method above, except that 150 mM glucose, 1 mM ATP, and 6 mM NADP were used. + The reactions were terminated at approximately 6, 9, 12, 24, and 48 hours. AtaPT, NovQ, and NphB were also tested with apigenin, daidzein, genistein, naringenin, and resveratrol at a final prenyltransferase concentration of 1 mg / mL. NphB was also tested with olivetol, olivetolate, and 1,6-dihydro-N-nitrosamine. The reactions were terminated at 24 hours.

[0217] The reaction was terminated by transferring the aqueous and organic layers to a 1.5 mL microcentrifuge tube. The reaction vial was washed with 200 μL of ethyl acetate and combined with the reaction mixture in the microcentrifuge tube. The sample was vortexed for 5–10 seconds and centrifuged at 13,000 rpm for 3 minutes. The organic layer was removed, and the remaining aqueous layer was extracted twice more with 200 μL of ethyl acetate. The organic extracts from each sample were pooled and evaporated in a vacuum centrifuge. The samples were redissolved in methanol for HPLC analysis.

[0218] For the olivetrate / CBGA case: Because protein precipitation was observed, the CBGA reaction shown in Figure 5A was extracted in the presence of 0.12 g of urea (solid) to facilitate CBGA extraction. This step was unnecessary for the WT NphB CBGA data in Figure 3C, as no protein precipitated.

[0219] Product quantification. The reaction was fractionated by reversed-phase chromatography using a Thermo Ultimate 3000 HPLC with a C18 column (4.6 × 100 mm). The column compartment temperature was 40 °C, and the flow rate was 1 mL / min. The compounds were separated by gradient elution using water + 0.1% TFA (solvent A) and acetonitrile + 0.1% TFA (solvent B) as mobile phases. The solvent B concentration was maintained at 20% for the first minute. The solvent B concentration was then increased to 95% B over 4 minutes, and then maintained at 95% B for 3 minutes. The column was then re-equilibrated to 20% B over 3 minutes, resulting in a total run time of 11 minutes.

[0220] Cannabinoids (CBGA, CBDA, and CBDVA) were quantified using external calibration curves generated from analytical standards purchased from Sigma-Aldrich. Because standards were unavailable, external calibration curves were generated using nuclear magnetic resonance (NMR) samples of 5-p-1,6-DHN and CBGVA. Standards of known concentrations were dissolved in water and extracted using the method described above.

[0221] Quantification of prenylation products without standards. Due to the lack of standards for the prenylation products prenylapigenin, prenyldaidzein, prenylnaringenin, prenylgenistein, prenylresveratrol, and prenylolivetol, prenylation products were quantified based on substrate consumption. To generate a standard curve, serial dilutions of each aromatic substrate were added to the reaction mixture, but the prenyltransferase was omitted to prevent product formation. Liquid chromatography-mass spectrometry was used to quantify the amount of substrate consumed by the reaction by comparison with the standard curve.

[0222] Electrospray ionization time-of-flight measurements were performed using a Waters LCT-Premier XE time-of-flight ionizer equipped with MassLynx 4.1 software (Waters Corporation, Milford, MA). The instrument was equipped with a multimode ionization source operating in electrospray mode. For accurate mass measurements, leucine enkephalin solution (Sigma Chemical, L9133) was used for lock-spray. Samples were injected via direct loop injection using a Waters Acquity UPLC system. Samples were separated on a Waters Acquity UPLC system using an Acquity BEH C18 1.7 μM column (50 × 2.1 mm) and eluted with a 30–95% solvent B gradient over 10 min (solvent A: water, solvent B: acetonitrile, both containing 0.2% formic acid (vol / vol)). Mass spectra were recorded from 300 to 2000 Da.

[0223] NMR spectroscopy. NMR spectroscopy was used to identify the prenyl products and to quantify 5-p-1,6-DHN.

[0224] For 1,6-DHN / 5-p-1,6-DHN: Prenyl-DHN was produced using the PyOx / PTA cell-free system. 200 μL of the reaction mixture was pooled and extracted three times with an equal volume of nonane, followed by evaporation of the nonane. The reaction product was suspended in 500 μL of deuterated methanol (CD3OD) and 2 mM 1,3,5-trimethoxybenzene (TMB) was added as an internal standard. Spectra were acquired on an AV400 Bruker NMR spectrometer. The amount of prenylated compound in the sample was determined relative to the TMB internal standard. The proton signal (3H,s) from TMB at 6.05 ppm was compared to the aromatic proton corresponding to 5-p-1,6-DHN (1H,d) at 7.27 ppm.

[0225] Regarding divalinic acid / CBGVA: NMR was also used to identify the products of the enzymatic reaction using divalinic acid as an aromatic substrate. The PyOx / PTA system was set up as described above, and the reaction was stopped after 24 hours. The reaction mixture was extracted as described above and analyzed by HPLC. A new major peak was observed at 6.7 minutes, which was predicted to be prenylated divalinic acid. After purifying the HPLC peak and removing the solvent, the pure component was redissolved in 600 μL of CD3OD. The proton spectrum acquired on an AV500 Bruker NMR spectrometer was compared with the proton spectrum published by Shoyama et al. for CBGVA, confirming that CBGVA was the major product. Based on the papers by Shoyama et al. and Bohlman et al., it was concluded that prenylation of divalinic acid occurs at the C3 carbon of divalinic acid.

[0226] Rosetta designs for modifying the binding pocket of NphB to accept olivetrate. Olivetrate was placed in the active site of NphB at six different starting positions, shown as olivetrate P1–P6 in Table 4. ROSETTA was run five times for each olivetrate position, resulting in a total of 30 designs. The predicted mutations for each design are shown in Table 4. For each olivetrate position, mutations from the consensus set (the most frequently selected residues) were selected and further evaluated in consensus groups A–F (Table 4). Next, the relative importance of each mutation suggested by ROSSETTA was evaluated. For each consensus group, mutations were reverted one by one to the WT residue, and the change in energy score was calculated using ROSETTA (see Table 5). The mutations that resulted in the largest energy change were determined to be the most important mutations to include in the library for experimental validation.

[0227] [Table 4-1] [Table 4-2]

[0228] [Table 5-1] [Table 5-2] [Table 5-3]

[0229] Initial screening of the NphB mutant library. For the initial library screening, small-scale expression and purification were performed. 25 μL of a saturated culture of BL21 DE3 Gold containing the NphB expression plasmid was inoculated into 25 mL of LB medium. The culture was incubated at 37°C until OD 600The cells were incubated until a pH of 0.4–0.6 was reached. Expression of the NphB construct was induced by the addition of 1 mM IPTG and incubated at 18°C for 18 hours. The cells were harvested by centrifugation at 2500 × g. The pellet was resuspended in 500 μL of lysis buffer (50 mM Tris pH 8.0, 150 mM NaCl, 5 mM imidazole) and lysed by sonication. The cell lysate was centrifuged at 20,000 × g for 10 minutes at 4°C, and the supernatant was incubated with 50 μL of NiNTA resin at 4°C. Purification of the NphB construct was performed using a 96-well spin column plate. The supernatant / resin mixture was applied to the column and centrifuged at 500 × g for 2 minutes. 500 μL of lysis buffer was then added, and the plate was centrifuged again at 500 × g for 1 minute. The protein was eluted with 200 μL of elution buffer (50 mM Tris [pH 8.0], 150 mM NaCl, 250 mM imidazole, 30% (v / v) glycerol).

[0230] The enzymes were analyzed in 2.5 mM geranyl pyrophosphate, 5 mM olivetrate, 5 mM MgCl2, 50 mM Tris pH 8.0, approximately 0.1 mg / mL NphB mutant, and a final volume of 100 μL. All enzymes were first diluted to 0.5 mg / mL with elution buffer to maintain a constant final concentration of imidazole in each reaction. Reactions were incubated at room temperature for 12 hours and then extracted three times with 100 μL of ethyl acetate. The organic extracts from each reaction were pooled and the solvent removed using a vacuum centrifuge. Samples were redissolved in 100 μL of methanol and subjected to HPLC analysis.

[0231] Screening of NphB Mutation Library. For the focused library, NphB constructs were expressed and purified at a 1 L scale as described above. Enzyme assays were performed in 2.5 mM GPP, 5 mM olivetrate, 5 mM mgCl2, 50 mM Tris pH 8.0, and approximately 1 mg / mL NphB enzyme in a final volume of 100 μL. Reactions were incubated at room temperature for 1 hour. 40 μL of each reaction was quenched with 80 μL of acetonitrile. Samples were centrifuged at 13,000 rpm for 5 minutes to remove precipitated protein. The supernatants were analyzed by HPLC as described above.

[0232] Enzyme kinetic parameters. Reactions were carried out in 50 mM Tris (pH 8.0), 2.5 mM GPP, 5 mM MgCl2, and approximately 27 μM enzyme, with varying concentrations of olivetrate or divalerate ranging from 0.1 mM to 6 mM, in a final volume of 200 μL. 40 μL of the reaction was stopped with 80 μL acetonitrile + 0.1% TFA at the following time intervals: The reaction was centrifuged at 13,000–16,060 × g for 5 minutes to pellet the proteins, and the supernatant was analyzed by the HPLC method described above. Initial velocities were plotted against substrate concentration and fitted to a Michaelis-Menten curve to determine the kinetic parameter k. cat and K. M (OriginPro) was used to determine the Michaelis-Menten curves. Each Michaelis-Menten curve was measured in triplicate. The mean and standard deviation of the kinetic parameters are reported.

[0233] For olivetrate / CBGA: For WT, M1, M10, and M30, the reaction times were 3, 6, 9, and 12 minutes. For mutant 25, the reaction stopped at 1, 2, 4, and 8 minutes, and for M31, it stopped at 1, 2, 4, and 6 minutes.

[0234] For divalinic acid / CBGVA: M31: 0.5, 1, 1.5, and 2 minutes. M23: 5, 10, 15, and 20 minutes. WT NphB: 8, 16, 24, and 32 minutes. The mutant enzyme concentrations were approximately 27 μM, and WT NphB was approximately 35 μM.

[0235] GC-MS characterization of the isomer profiles of WT NphB and M23. Samples were dissolved in 200 μL of ethyl acetate. GC-MS measurements were performed using an Agilent Model 7693 autosampler, a 7890B gas chromatograph, and a 7250Q-TOF mass selective detector in electron ionization mode. Sample injection was performed in split mode, with the inlet temperature set to 280 °C. Separation was performed using a 30 m x 250 μM x 0.25 μM Agilent HP5-MS column. Ultra-high-purity grade He (Airgas) was used as the carrier gas, with a flow rate set to 1.1 mL / min in constant flow mode. The oven temperature was initially set to 120 °C for 1 min, then increased at 20 °C / min to a final temperature of 300 °C, which was held for 4 min. The solvent delay time was 3.0 min. The EI energy was set to 15 eV. The MSD was set to scan the m / z range from 50 to 500. Data collection and analysis were performed using Mass Hunter Acquisition and Qualitative Analysis software (Agilent).

[0236] Due to the temperature increase at the GC inlet, CBGA spontaneously undergoes decarboxylation, as reported by R. adwan et al., producing an M+ ion at 316 m / z. In a CBGA standard sample, the retention time corresponding to this 316 m / z ion was 10.48 min.

[0237] A nonane flow system for extracting CBGA from solution was used. The PyOx / PTA reaction was set up as described above. 500 μL of the nonane top layer was added to the reaction mixture in a 2 mL glass vial covered with two layers of breathable cell culture film. Two needles were inserted into a 15 mL Falcon tube at approximately the 750 μL and 3.5 mL marks. A Luer lock with a tubing connector was attached to the needle, and the other end of the Luer lock was connected to a Viton tubing. The needle was then connected to the other end of the tubing via the Luer lock connector and inserted through the mesh cover, ensuring contact only with the nonane layer and not with the reaction mixture. 2 mL of Tris buffer (pH 8.5) was added to a 15 mL conical tube, followed by 6 mL of nonane. The nonane was pumped throughout the system using a peristaltic pump, flowing out of the top of the reaction mixture through the buffer. The nonane, injected into the reservoir, separated into the top layer of the 15 mL conical tube. Nonane was injected into the top of the reaction vial from the top of a 15 mL conical tube, which diluted the CBGA throughout the system and facilitated its diffusion into the nonane layer and out of the reaction.

[0238] Cloning of CBDAS. A codon-optimized CBDAS gene block for Pichia pastoris was ordered from IDT. The signal sequence was removed by PCR amplification from the 28th residue (NPREN...) of the protein sequence to the end of the protein, leaving an overhang compatible with the pPICZa vector. The PCR product was cloned into pPICZa vector digested with EcoRI and XbaI using the Gibson cloning method. The assembly reaction product was transformed into BL21 Gold (DE3) cells, and a clone with the correct sequence was isolated. The plasmid was digested with PmeI for 2 hours and then purified using the Qiagen PCR purification protocol. This plasmid was transformed into Pichia pastoris X33 using electroporation. Immediately after electroporation, the cells were incubated in 1 mL of cold 1 M sorbitol and 1 mL of YPD medium without shaking for 2 hours. The cells were plated onto YPDS plates supplemented with 500 μg / mL Zeocin. Colonies were screened by PCR for the presence of the CBDAS gene between the AOX1 promoter and terminator. For screening, colonies were resuspended in 15 μL of sterile water, and 5 μL was transferred to a PCR tube containing 0.2% SDS. The sample was heated at 99°C for 10 minutes, and then 1 μL was used as a PCR template. Six colonies that yielded positive colony PCR hits were screened for CBDAS expression.

[0239] CBDAS expression test. Six colonies were grown overnight at 30°C to obtain a saturated culture. 25 mL of the overnight culture was inoculated into BMGY medium and grown to an OD of approximately 2. Cells were harvested by centrifugation at 2,000 x g for 10 minutes. The cell pellet was resuspended in 90 mL of BMGY medium and grown at 30°C for 5 days. Each day, 1 mL of culture was taken for SDS-PAGE analysis, and 500 μL of methanol was added. On day 3, the cultures were screened for CBDAS activity. The assay conditions were 100 μL of 200 mM citrate buffer, 100 μM CBGA, 5 mM MgCl2, 5 mM KCl, 1 mM FAD, and 50 μL of expression medium (final volume 200 μL). The reaction was incubated overnight at room temperature and extracted three times with 200 μL of ethyl acetate. The ethyl acetate extracts for each sample were pooled and removed using a vacuum centrifuge. The samples were resuspended in 200 μL of methanol and analyzed by HPLC. Active CBDAS was detected in all clones.

[0240] Cultures (approximately 300 mL total) from three clones were harvested for CBDAS activity. Cells were pelleted by centrifugation at approximately 3,000 × g for 20 minutes at 4°C. The supernatant was filtered through a 0.22 μM filter. The medium was concentrated using a Millipore 50,000 MWCO protein concentrator and buffer-exchanged into 100 mM citrate buffer (pH 5.0). The total protein concentration in the medium concentrate was determined to be 0.4 mg / mL using a Bradford assay, resulting in a total protein yield of approximately 5 mg / L.

[0241] Production of CBDVA and CBDA. A secondary reaction using CBDAS synthase was set up to convert the precursors CBGA and CBGVA to CBDA and CBGVA, respectively.

[0242] For CBGA / CBDA: The PyOx / PTA enzyme system was constructed as described above to produce CBGA. After 24 hours, 200 μL of the nonane top layer from the CBGA reaction was transferred to a CBDAS reaction vessel. To the aqueous layer, 50 mM Hepes [pH 7.0], 5 mM MgCl2, 5 mM KCl, 25 μM FAD, and 0.1 mg / mL CBDAS concentrate were added. The reaction was incubated at 30°C with gentle shaking. The reaction was terminated after 12, 24, 48, 72, and 96 hours.

[0243] For CBGVA / CBDVA: HPLC-purified CBGVA was converted to CBDVA. The final reaction volume was 200 μL, containing 50 mM Hepes [pH 7.0], 5 mM MgCl2, 5 mM KCl, 25 μM FAD, and 0.1 mg / mL (total protein) CBDAS concentrate. 200 μL of nonane overlay was added and the mixture was incubated at 30°C with gentle shaking. The reaction was terminated after approximately 24, 48, 72, and 96 hours.

[0244] MatB activity assay. An enzyme-coupled assay was used to measure the activity of malonyl-CoA synthetase (MatB) from R. palustris (see, e.g., SEQ ID NOs:82-83) in the presence of OA and DA. Reaction conditions were 2.5 mM malonate, 2 mM ATP, 1 mM CoA, 2.5 mM phosphoenolpyruvate (PEP), 1 mM NADH, 5 mM MgCl2, 10 mM KCl, 0.35 mg / mL ADK, 0.75 μg / mL MatB, 1.6 units of PK and 2.5 units of LDH, and 50 mM Tris [pH 8.0]. Background ATPase activity was controlled by omitting the substrate (malonate), and the remaining reactions were supplemented with 1% ethanol, 250 μM or 5 mM OA, or 5 mM DA. MatB activity was measured using an M2 SpectraMax by monitoring the decrease in absorbance at 340 nm due to NADH consumption. To confirm that MatB was the limiting enzyme at 5 mM OA or DA, the MatB concentration was doubled to 1.5 μg / mL. The reaction rate doubled, suggesting that MatB was the limiting enzyme in the system. NADH consumption rates at 5 mM OA and 5 mM DA were normalized to 1% ethanol as a control.

[0245] Met and d3-Met Activity Assay. Enzyme activity was performed to compare the activity of methionine adenosyltransferase (MAT) from Thermococcus kodamarensis (tk) and Methanocaldococcus jannaschii (mj) for the production of methionine (met), d3-met, and S-adenosyl-L-methionine-d3 (SAM-d3). The reaction conditions were 200 μl reactions containing 200 mM Tris-HCl (pH = 8), 20 mM MgCl2, 50 mM KCl, 5 mM ATP, and 5 mM met or d3-met. The enzyme concentration of each MAT was 10 μM in each of four samples, for a total of eight samples. PPase (2.5 μM) was added to two samples of each MAT. The reaction mixture was incubated for 1 hour at room temperature, stopped with a prepared malachite green standard (Sigma-Aldrich Malachite Green Phosphate Assay Kit), and measured at 620 nm on a plate reader in conjunction with a phosphate standard according to the manufacturer's protocol. Enzyme activity was quantified as inorganic phosphate (Pi) liberated per hour.

[0246] The results of this activity assay, shown in Figure 38, indicate that methionine adenosyltransferases (MAT) from Thermococcus kodakarensis (tk) and Methanocaldococcus jannaschii (mj) exhibit comparable activity toward met and d3-met. As expected, the addition of PPase increased the amount of Pi released per hour, providing overall support for the generation of SAM-d3.

[0247] AAE3 Activity Assay. Using a coupled enzyme assay similar to the assay described above for MatB, the activity of acyl-activating enzyme 3 (AAE3) (see, e.g., SEQ ID NOS: 70-71 and homologous genes 72-75) was measured in the presence of OA and DA. Conditions were identical to those for the MatB assay, with the following modifications: 2.5 mM hexanoic acid was added instead of malonic acid, and 15 μg / mL AAE3 was added instead of MatB. To ensure that AAE3 was the restriction enzyme, AAE3 was doubled in the presence of 5 mM OA or DA. The doubling of the reaction rate indicated that AAE3 was the restriction enzyme.

[0248] ADK Activity Assay. An enzyme-linked assay was used to measure the activity of adenylate kinase (ADK) (see, e.g., SEQ ID NO:81) in the presence of OA and DA. Conditions were similar to those for the MatB assay, with the following modifications: 2 mM AMP was added instead of malonate, no CoA was added, and 0.001 mg / mL ADK was added. The amount of ADK was doubled to ensure that ADK was the limiting enzyme under conditions of 5 mM OA and DA. A two-fold increase in activity suggested that ADK was the limiting factor.

[0249] CPK activity assay. An enzyme-linked assay was used to measure the activity of creatine kinase (CPK) in the presence of OA or DA. The reaction conditions were 5 mM phosphocreatine, 2 mM ADP, 5 mM glucose, and NADP. + The buffer was 2 mM, MgCl2 5 mM, KCl 5 mM, Zwf 0.3 mg / mL, ScHex 0.1 mg / mL, and CPK 0.08 units. 1% ethanol was added to the positive control reaction, and 5 mM OA or DA was added to the remaining reactions. The absorbance of NADPH was measured at 340 nm. To confirm that CPK was the limiting enzyme, we doubled the CPK activity to 5 mM OA and 5 mM DA. The resulting activity doubled, indicating that CPK was the limiting enzyme even at high OA and DA concentrations.

[0250] OLS Activity Assay. Olivetol synthase (OLS) (see, e.g., SEQ ID NOs:76-77) was assayed in 200 μM malonyl-CoA, 100 μM hexanoyl-CoA, 0.65 mg / mL OAS, 50 mM citrate buffer (pH 5.5) or 50 mM Tris buffer (pH 8.0). Reactions were initiated by the addition of OAS and stopped 30 minutes later by adding 150 μL of methanol to a 50 μL reaction. Samples were centrifuged at approximately 16,000 × g for 2 minutes to pellet the protein. The supernatant was analyzed by HPLC.

[0251] For inhibition experiments, the conditions were modified as follows: 1 mM malonyl-CoA and 400 μM hexanoyl-CoA were dissolved in 50 mM citrate buffer (pH 5.5) to a final volume of 200 μL. Reactions were initiated by adding 1% ethanol, 250 μM OA, or 1 mM DA, followed by the addition of 0.65 mg / mL OLS. Fifty-five μL aliquots were stopped with 150 μL of methanol after 2, 4, 6, and 8 minutes. Reactions were briefly vortexed and centrifuged at 16,000 × g for 2 minutes to pellet the proteins. The supernatants were analyzed by HPLC. The raw peak areas of HTAL, PDAL, and olivetol were summed and plotted against time to determine the reaction rates. The reaction rates for OA- and DA-added reactions were normalized to the ethanol control.

[0252] OLS / OAC activity assay. To generate OA, the same OLS conditions as above were used, except that olivetol acid cyclase (OAC) (see, for example, SEQ ID NOs: 78-79) was added to the reaction at a concentration of 0.6 mg / mL. The reaction was stopped and analyzed as in the OLS assay. Acetyl phosphate and BSA were added to the assay at final concentrations of 5 mM–40 mM AcP and 10–30 mg / mL BSA, respectively.

[0253] Complete Pathway Setup. The enzymes and final concentrations (mg / mL) used in this study are listed in Table 6 for the MatB pathway and Table 7 for the MdcA pathway. For the MatB pathway, cofactors were added at the following concentrations: 150 mM glucose, 1 mM fructose bisphosphate, 2 mM ATP, 0.25 mM NAD+, 3 mM NADP+, 2 mM CoA, 0.25 mM 2,3-bisphosphoglycerate, 6 mM MgCl2, 10 mM KCl, 0.5 mM thiamine pyrophosphate, 50 mM phosphate (pH 8.0), 5 mM hexanoate, 15 mM malonate, 5 mM creatine phosphate, and 50 mM Tris (pH 8.0). The reaction was initiated by the addition of the enzymes listed in Table 6. The reaction was incubated overnight at room temperature and then terminated by extraction with 200 μL of ethyl acetate three times. The ethyl acetate was removed using a vacuum centrifuge. The samples were dissolved in 200 μL of methanol and analyzed by HPLC.

[0254] [Table 6-1] [Table 6-2]

[0255] [Table 7-1] [Table 7-2]

[0256] Both the MatB and MdcA pathways are shown in Figure 6A-B.

[0257] FIG. 9 shows Fourier transform mass spectrometry by probe electrospray ionization (FTMS-pESI) spectral data of acetonitrile with 0.1% formic acid added as a control.

[0258] FIG. 10 shows the FTMS-pESI spectral data of CBGA in the positive mode.

[0259] FIG. 11 shows the FTMS-pESI spectral data of CBGA in the negative mode.

[0260] FIG. 12 shows the FTMS-pESI spectral data of CBGA in control and positive modes.

[0261] FIG. 13 shows the FTMS-pESI spectral data of CBGA in control and positive modes.

[0262] FIG. 14 shows the FTMS-pESI spectral data of deuterated CBGA in the positive mode.

[0263] FIG. 15 shows FTMS-pESI spectral data in the negative mode for deuterated CBGA.

[0264] FIG. 16 shows additional FTMS-pESI spectral data of deuterated CBGA in the positive mode.

[0265] FIG. 17 shows additional FTMS-pESI spectral data of deuterated CBGA in negative mode.

[0266] Table 8 shows the results of the FTMS-pESI spectral analysis, which shows that the compounds are distinguishable in positive and negative modes.

[0267] [Table 8]

[0268] Additional samples were subjected to nanoscale liquid chromatography-tandem mass spectrometry (nLC-MS / MS). Samples were prepared using 2 μL of the standard solution and 198 μL of 50:50 water:acetonitrile in 0.1% formic acid. 1 μL of sample was injected.

[0269] FIG. 18 shows the nLC-MS / MS data for CBGA.

[0270] FIG. 19 shows the nLC-MS / MS data for dCBGA.

[0271] Table 9 compares the nLC-MS / MS data for CBGA and dCBGA.

[0272] [Table 9]

[0273] FIG. 20 shows the nLC-MS / MS data after 1 hour of the first sample of CBGA.

[0274] FIG. 21 shows the nLC-MS / MS data of the second sample of CBGA after 1 hour.

[0275] FIG. 22 shows the nLC-MS / MS data for the third sample of CBGA after 1 hour.

[0276] FIG. 23 shows the nLC-MS / MS data after 1 hour of the first sample of dCBGA.

[0277] FIG. 24 shows the nLC-MS / MS data after 1 hour of the second sample of dCBGA.

[0278] FIG. 25 shows the nLC-MS / MS data after 1 hour of the third sample of dCBGA.

[0279] Table 10 compares the nLC-MS / MS data for the CBGA and dCBGA samples after 1 hour.

[0280] [Table 10]

[0281] Figure 26 is a graph comparing CBGA and dCBGA. From left to right, Sample 1, Sample 2, and Sample 3 are shown for both CBGA and dCBGA. The results show that CBGA is significantly more degraded than dCBGA after 1 hour.

[0282] In one embodiment, once the THCA compound of the present invention is produced, the compound is purified and the resulting THCA is isolated. Extraction of CBGA and THCA into ethyl acetate resulted in the formation of an emulsion, which was only partially broken down by the addition of hexane. Because bovine serum albumin (BSA) strongly binds both CBGA and THCA, not all of the product can be recovered in the organic phase of the purification process. However, acid precipitation of BSA with 1% HCl denatures the BSA, dissociating the CBGA and THCA bonds and enabling extraction and purification. In one embodiment, HCl is added directly to the reaction mixture and incubated for approximately 10 minutes to completely denature the BSA. The mixture is then centrifuged at 4000 g for approximately 30 minutes. Upon centrifugation, CBGA and THCA co-precipitate and form a pellet. The pellet is then washed with water until the water is pH neutral. The pellet is then resuspended in methanol and vortexed vigorously. The methanol mixture is then rotary evaporated in a 40°C water bath, and the resulting oil is resuspended in ethanol. THCA precipitates as a brown oil. The reaction mixture is filtered, washed with water, and then resuspended in hexane. The hexane is evaporated on a rotary evaporator to remove the mass added to the product by excess methane. The resulting oil is resuspended in methanol and stored. Table 11 below provides an overview of the recovered product over the entire process, starting with 67.5 mg of CBGA reaction mixture. In the experiment detailed in Table 11, this purification process yielded 41 mg of purified product, corresponding to 60% of the theoretical yield. In one embodiment, a crystallization step is performed to avoid ethanol precipitation, thereby improving the purity and recovery of THCA.

[0283] [Table 11]

[0284] After purifying THCA, the purified THCA can be further decarboxylated to obtain THC. Continuing the experiment shown in Table 11, 23 mg of purified THCA was used in the decarboxylation reaction. Methanol was removed from the product using a rotary evaporator, and the product was then resuspended in 2 mL of toluene. Next, 100 μL of saturated bicarbonate was added to the mixture, which was then heated to 95 °C on a rotary evaporator, refluxed, and condensed. Samples were taken after 10 and 35 minutes to terminate the reaction. The reaction mixture was washed with water to remove excess salts. Next, ethanol was added to the toluene to facilitate evaporation of excess reactants, and the mixture was dried three times on a rotary evaporator to remove the toluene. This process yielded 13.4 mg of THC with a purity of 90%.

[0285] In one embodiment, impurities in the compound that result in increased retention time can be removed prior to decarboxylation by crystallizing THCA in an ethanol / hexane mixture.

[0286] One-pot synthesis

[0287] In one embodiment, the synthesis is a one-pot synthesis. In one-pot synthesis, the synthesis reaction is carried out by mixing all reagents, catalysts, enzymes, buffers, and other necessary chemicals in a single reaction vessel. In one embodiment, the one-pot synthesis includes at least one deuterated compound (e.g., a deuterated fatty acid). An advantage of one-pot synthesis is that it improves the efficiency of the chemical reaction, thereby improving the chemical yield.

[0288] formulation

[0289] In one embodiment, cannabinoids, cannabinoid precursors, and / or other prenylated chemicals substituted with at least one deuterium, at least one tritium, at least one halogen, at least one hydroxyl group, and / or at least one additional isotope are incorporated into the pharmaceutical composition. In one embodiment, the pharmaceutical composition comprises about 1% (w / w) or more (e.g., about 2% (w / w) or more, about 3% (w / w) or more, about 4% (w / w) or more, about 5% (w / w) or more, about 6% (w / w) or more, about 7% (w / w) or more, about 8% (w / w) or more, about 9% (w / w) or more, about 10% (w / w), about 15% (w / w), about 20% (w / w) or more, or about 25% (w / w) or more) cannabinoid, cannabinoid precursor, and / or other prenylated chemical substituted with at least one deuterium, at least one tritium, at least one halogen, at least one hydroxyl group, and / or at least one additional isotope.

[0290] In one embodiment, the pharmaceutical composition further comprises an unsubstituted cannabinoid, an unsubstituted cannabinoid precursor, and / or an unsubstituted other prenylated chemical. In one embodiment, the pharmaceutical composition comprises about 1% (w / w) or more of an unsubstituted cannabinoid, an unsubstituted cannabinoid precursor, and / or an unsubstituted other prenylated chemical (e.g., about 2% (w / w) or more, about 3% (w / w) or more, about 4% (w / w) or more, about 5% (w / w) or more, about 6% (w / w) or more, about 7% (w / w) or more, about 8% (w / w) or more, about 9% (w / w) or more, about 10% (w / w), about 15% (w / w), about 20% (w / w) or more, or about 25% (w / w) or more).

[0291] In one embodiment, the pharmaceutical composition further comprises a lipid. The lipid includes, but is not limited to, a phospholipid (e.g., soybean lecithin, egg lecithin, phosphocholine, phosphoglycerol), a fat, an oil (e.g., olive oil, vegetable oil), and / or a fatty acid. In one embodiment, the lipid can form a micelle, an emulsion, or a liposome.

[0292] In one embodiment, cannabinoids, cannabinoid precursors, and / or other prenylated chemicals substituted with at least one deuterium, at least one tritium, at least one halogen, at least one hydroxyl group, and / or at least one additional isotope are microencapsulated or nanoencapsulated.

[0293] In one embodiment, the pharmaceutical composition further comprises at least one masking agent (e.g., taste masking agent, olfactory masking agent). In a preferred embodiment, the at least one masking agent includes, but is not limited to, at least one sweetener and / or at least one flavoring. The at least one sweetener includes, but is not limited to, saccharin (e.g., sodium salt, calcium salt), fructose, dextrose, aspartame, acesulfame potassium, glycerin, sucralose, maltodextrin, sucrose, glucose, maltose, xylitol, sorbitol, erythritol, and / or mannitol. In one embodiment, the at least one masking agent comprises phenethyl alcohol, vanilla, cherry, cinnamon, lavender, lemon, menthol, orange, peppermint, spearmint, raspberry, strawberry, grape, ethyl vanillin, coriander, ginger, nutmeg, cardamom, butterscotch, cocoa, acacia syrup, anethole, aniseed oil, benzaldehyde, ethyl acetate, methyl salicylate, and / or toluol. In one embodiment, the at least one masking agent is present in an amount of about 0.001% to about 1% w / w of the weight of the pharmaceutical composition, e.g., about 0.001%, 0.005%, 0.01%, 0.02%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% w / w based on the weight of the pharmaceutical composition. In one embodiment, the at least one masking agent is about 0.01%-0.5%, 0.02%-0.2%, or 0.015%-0.15% w / w based on the weight of the pharmaceutical composition.

[0294] In one embodiment, the pharmaceutical composition further comprises a vitamin or mineral. Vitamins include, but are not limited to, vitamin A, vitamin C, vitamin D (e.g., vitamins D1, D2, D3, D4, D5, D6, and / or D7), vitamin E, vitamin B (e.g., vitamins B1, B2, B3, B5, B9, and / or B12), and vitamin K (e.g., vitamins K1, K2, K3, K4, and / or K5). Minerals include, but are not limited to, magnesium, calcium, iron, zinc, chromium, selenium, and / or potassium.

[0295] In one embodiment, the pharmaceutical composition further comprises at least one anti-caking agent, including, but not limited to, tribasic calcium phosphate, cellulose, microcrystalline cellulose, silicon dioxide, sodium chloride, magnesium stearate, magnesium carbonate, and / or sodium bicarbonate. In one embodiment, the at least one anti-caking agent is present in an amount of about 0.5% to about 5% w / w of the pharmaceutical composition.

[0296] In one embodiment, the pharmaceutical composition includes at least one preservative, including, but not limited to, parabens, benzalkonium chloride, phenylethyl alcohol, ethylenediaminetetraacetic acid (EDTA), benzoyl alcohol, sulfur dioxide, sulfites, thiols, propionic acid, benzoic acid, sorbic acid, sodium sorbate, calcium sorbate, potassium sorbate, sodium benzoate, potassium benzoate, lactic acid, and / or sodium propionate. In one embodiment, the at least one preservative is present in an amount of about 0.01% to about 5% w / w of the composition, e.g., about 0.01%, 0.05%, 0.1%, 0.2%, 0.5%, 1%, 2%, 3%, 4%, or 5% w / w of the pharmaceutical composition by weight. In one embodiment, the at least one preservative is about 0.01%-5%, 0.02%-4%, or 0.05%-2.5% w / w based on the weight of the pharmaceutical composition. In a preferred embodiment, the at least one preservative does not contain sulfites.

[0297] In one embodiment, the pharmaceutical composition is in the form of a crystal, powder, granule, capsule, tablet, syrup, solution, emulsion, topical, wafer, aerosol, oil, patch (e.g., transdermal patch), parenteral, suppository, intravenous, or suspension. In one embodiment, the pharmaceutical composition is delivered via oral administration, sublingual, buccal, rectal, intravenous injection, intramuscular, subcutaneous injection, intranasal, inhalation, ocular, or vaginal route. In one embodiment, the pharmaceutical composition is provided in an edible form. Edible forms include, but are not limited to, candies, pastries (e.g., brownies, cookies, muffins), beverages, bread, cereal, pasta, etc. Topical forms include, but are not limited to, creams, ointments, lotions, pastes, gels, etc.

[0298] treatment

[0299] In one embodiment, the pharmaceutical composition is used to treat cancer (e.g., gastric cancer, colon cancer, pancreatic ductal adenocarcinoma), glaucoma, fibromyalgia, peripheral neuropathy, nausea, diabetes, obesity, liver disease, neurological diseases (e.g., seizures, epilepsy, multiple sclerosis, stroke, Parkinson's disease, vascular dementia, senile dementia, Alzheimer's disease, mild cognitive impairment, Huntington's disease, amyotrophic lateral sclerosis (ALS), migraine), autoimmune diseases (e.g., type 1 diabetes, Crohn's disease, celiac disease, ulcerative colitis, inflammatory bowel disease (IBD), lupus, rheumatoid arthritis, psoriatic arthritis, Addison's disease, Graves' disease, vasculitis, pernicious anemia), or skin diseases. (e.g., psoriasis, atopic dermatitis (AD), eczema, acne, contact dermatitis, herpes simplex, shingles, actinic keratosis, ichthyosis, Bowen's disease, keratoacanthoma, lichen sclerosus, hidradenitis suppurativa, seborrheic keratosis, rosacea, pityriasis lichenoides, seborrhea), joint diseases (e.g., osteoarthritis), reproductive disorders (e.g., endometriosis, dysmenorrhea, menstrual irregularities, dyspareunia), bacterial infections, and / or psychiatric disorders (e.g., anxiety, depression, stress, panic attacks, attention deficit hyperactivity disorder (ADHD, post-traumatic stress disorder (PTSD)), bipolar disorder, obsessive-compulsive disorder, schizophrenia, personality disorders). See, e.g., U.S. Patent Publication No. 20210315837, which is incorporated herein by reference in its entirety.

[0300] In one embodiment, the pharmaceutical composition is administered once daily, twice daily, three times daily, or four times daily. In another embodiment, the pharmaceutical composition is administered once every two days, once every three days, once every four days, once every five days, once every six days, or once every seven days. In yet another embodiment, the pharmaceutical composition is administered once every two weeks, once every three weeks, once every four weeks, once every six weeks, once every two months, once every three months, or once every four months.

[0301] Those skilled in the art will recognize certain modifications and improvements upon reading the above description. It will be apparent to those skilled in the art that the above examples are provided for the purpose of clarifying aspects of the present invention, and are not intended to limit the scope of the present invention. For the sake of brevity and readability, all changes and improvements have been omitted from this specification, but they are nonetheless within the scope of the present invention.

Claims

1. 1. A cannabinoid composition comprising: It contains cannabinoids derived from deuterated fatty acids, the deuterated fatty acid is activated to produce a deuterated CoA thioester; the deuterated CoA thioester is combined with a second CoA thioester and cyclized to form deuterated olivetolic acid; the deuterated olivetolic acid is prenylated and cyclized to produce the cannabinoid; The cannabinoid is a compound of formula I, 【Chemical 1】 R is a deuterated carbon chain containing at least one deuterated carbon; Cannabinoid composition.

2. The deuterated carbon chain is C5D １１ 2. The composition of claim 1, wherein:

3. 10. The composition of claim 1, wherein the composition further comprises an unsubstituted cannabinoid and / or an unsubstituted cannabinoid precursor.

4. The composition of claim 1 , wherein the composition comprises a prodrug.

5. The composition of claim 1 , wherein the composition further comprises a phospholipid, a fat, an oil, and / or a fatty acid.

6. The composition of claim 1 , wherein the composition further comprises at least one sweetener and / or at least one flavoring agent.

7. The composition of claim 1 , wherein the composition further comprises a vitamin or mineral.

8. The composition of claim 1 , wherein the composition further comprises at least one anti-caking agent.

9. The composition of claim 1 , wherein the composition further comprises at least one preservative.

10. 10. The composition of claim 1, wherein the composition is in the form of a crystalline, powder, granule, capsule, tablet, syrup, solution, emulsion, topical, wafer, aerosol, oil, patch, parenteral, suppository, intravenous, or suspension.

11. 1. A method for synthesizing a cannabinoid compound or a derivative thereof, comprising: phosphorylating prenol and / or isoprenol with hydroxyethylthiazole kinase (ThiM) to produce isopentenyl phosphate; isomerizing isopentenyl phosphate to produce isopentenyl diphosphate, the isopentenyl diphosphate being operable to be phosphorylated in the presence of inositol polyphosphate kinase to produce dimethylallyl diphosphate (DMAPP); synthesizing geranyl pyrophosphate from isopentenyl diphosphate and / or DMAPP in the presence of farnesyl pyrophosphate synthase; activating a deuterated fatty acid to produce a deuterated CoA thioester; activating the acid to form a second CoA thioester; combining the deuterated CoA thioester with the second CoA thioester; cyclizing the synthesis product to produce deuterated olivetolic acid; prenylation of the deuterated olivetolic acid in the presence of geranyl pyrophosphate to form deuterated cannabigerolic acid; cyclizing the deuterated cannabigerolic acid to produce a deuterated cannabinoid; 1. A method for synthesizing a cannabinoid compound or a derivative thereof, comprising:

12. 12. The method of claim 11, further comprising recovering the deuterated cannabinoid from the reaction mixture, wherein the cannabinoid is isolated from the reaction mixture using acid precipitation, centrifugation, washing, rotary evaporation, and precipitation.

13. 12. The method of claim 11, wherein the method for synthesizing the cannabinoid compound or derivative thereof is a one-pot synthesis.

14. 12. The method of claim 11, wherein the deuterated fatty acid is a fully deuterated acid or a partially deuterated fatty acid.

15. 12. The method of claim 11, wherein the deuterated cannabigerolic acid is cyclized using tetrahydrocannabidiolic acid synthase (THCAS), cannabidiolic acid synthase (CBDAS), and / or cannabichromenic acid synthase (CBCAS).

16. 1. A method for cell-free synthesis of a cannabinoid or a derivative thereof, comprising: cloning at least one polynucleotide sequence encoding at least one enzyme into at least one microorganism to generate an engineered microorganism, wherein the engineered microorganism exhibits increased expression of at least one enzyme compared to an unmodified parent microorganism; Lysing at least one cell of the modified microorganism to obtain at least one enzyme, wherein the at least one enzyme is used in a cannabinoid biosynthetic pathway; Producing a cannabinoid or derivative thereof using the cannabinoid biosynthetic pathway, activating a deuterated fatty acid in the presence of acyl-activating enzyme (AAE) 3 to produce a deuterated CoA thioester, wherein the deuterated fatty acid is a compound of formula II; 【Chemistry 2】 activating a deuterated fatty acid in the presence of acyl-activating enzyme (AAE) 3 to produce a deuterated CoA thioester; activating the acid in the presence of a corresponding CoA synthetase to form a second CoA thioester; synthesizing the deuterated CoA thioester and the second CoA thioester in the presence of olivetol synthase (OLS); cyclizing the synthesis product in the presence of olivetolic acid cyclase (OAC) to produce the deuterated olivetolic acid; prenylation of said deuterated olivetolic acid in the presence of geranyl pyrophosphate to form deuterated cannabigerolic acid; cyclizing the deuterated cannabigerolic acid to produce a deuterated cannabinoid; producing a cannabinoid or derivative thereof using said cannabinoid biosynthetic pathway, comprising: A method for cell-free synthesis of cannabinoids or derivatives thereof, comprising:

17. 17. The method of claim 16, wherein the cannabinoid biosynthetic pathway is a one-pot synthesis.

18. 17. The method of claim 16, wherein the deuterated fatty acid is a fully deuterated acid or a partially deuterated fatty acid.

19. 17. The method of claim 16, wherein the deuterated cannabigerolic acid is cyclized using a tetrahydrocannabidiolic acid synthase (THCAS), a cannabidiolic acid synthase (CBDAS), and / or a cannabichromene synthase enzyme (CBCAS).

20. 17. The method of claim 16, wherein the at least one enzyme is AAE3, OLS, OAC, THCAS, CBDAS and / or CBCAS.

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