Biosynthesis of substituted compounds and substituted cannabinoids

Abstract

Systems and methods for the production of isopentenylation and recombinant pathways of compounds, cannabinoids, cannabinoid precursors, and other isopentenylation chemicals in cell-free systems. The invention also includes enzymes and recombinant microorganisms that catalyze the reaction. The compounds, cannabinoids, cannabinoid precursors and other isopentenylation chemicals can 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

[0001] Cross - Reference to Related Applications

[0002] This application is related to and claims priority from the following U.S. patent applications. This application claims the priority and benefit of U.S. Application No. 18 / 227,719, filed on July 28, 2023, which claims the priority and benefit of U.S. Provisional Application No. 63 / 370,070, filed on August 1, 2022. The entire contents of both are incorporated herein by reference in their entirety. This application also claims the priority and benefit of U.S. Provisional Application No. 63 / 370,070.

[0003] Reference to Sequence Listing

[0004] A formal copy of the sequence listing is submitted via EFS - Web simultaneously with the specification as a file in XML format, having the file name "4482003.xml", a creation date of July 28, 2023, and a size of 133 kilobytes. The sequence listing submitted via EFS - Web is part of the specification and is incorporated herein by reference in its entirety. Background of the Invention 1. Field of the Invention

[0006] The present invention relates to cannabinoids, and more particularly to substituted compounds and substituted cannabinoids, and their biosynthesis.

[0007] 2. Description of the Related Art

[0008] Prenylation of natural compounds increases structural diversity, alters biological activity, and enhances therapeutic potential. Prenylated compounds are generally of low natural abundance or difficult to isolate. Some prenylated natural products include a large class of bioactive molecules with distinct medicinal properties. Examples include prenylated flavonoids, prenylated stilbenes, and cannabinoids.

[0009] Cannabinoids are a large class of bioactive plant - derived natural products that modulate the cannabinoid receptors (CB1 and CB2) of the human endocannabinoid system. Cannabinoids are promising pharmaceuticals, and currently over 100 clinical trials are studying their therapeutic benefits as anti - emetics, anti - convulsants, analgesics, and antidepressants. In addition, three cannabinoid therapies have been approved by the FDA for the treatment of chemotherapy - induced nausea, MS spasticity, and seizures associated with severe epilepsy.

[0010] Despite its therapeutic potential, the production of pharmaceutical-grade (>99%) cannabinoids still faces significant technical challenges. Cannabis plants, such as marijuana and hemp, produce high concentrations of tetrahydrocannabinolic acid (THCA) and cannabidiolic acid (CBDA), as well as a variety of less abundant cannabinoids. However, even highly expressed cannabinoids such as CBDA and THCA are difficult to isolate due to the high structural similarity of contaminating cannabinoids and the variable cannabinoid composition of each crop. These problems become even more severe when attempting to isolate rare cannabinoids. In addition, current cannabis cultivation practices pose serious environmental challenges. As a result, there is a strong interest in developing alternative methods for producing cannabinoids and cannabinoid analogs.

[0011] In addition, synthetic deuterated cannabinoids are generally known in the prior art.

[0012] The prior art literature includes the following:

[0013] U.S. Patent No. 5,036,014: Deuterated cannabinoids as standards for the analysis of tetrahydrocannabinol and its metabolites in biological fluids, inventors Elsohly et al., filed on January 31, 1989, and issued on July 30, 1991, which relates to a new internal standard for gas chromatography / mass spectrometry test methods, containing deuterated cannabinoids, which have been developed for the analysis of tetrahydrocannabinol and its metabolites in biological fluids.

[0014] U.S. Patent No. 5,633,357: Synthesis of carboxylic acid glucuronides, inventors Tius et al., filed on March 4, 1994, and issued on May 27, 1997, which relates to a method for producing carboxylic acid glucuronides by reacting a carboxylic acid precursor with a blocked sugar epoxide precursor. Also disclosed are: deuterated 11-nor-Δ8- or Δ9-THC carboxylic acid glucuronides having deuterated hydrocarbon chains; 5'-deuterated 11-nor-Δ8- or Δ9-THC carboxylic acids, or 5'-deuterated Δ8- or Δ9-THC glucuronides. The compositions can be used as GC-MS standards; in methods for preparing antibodies that react with THC glucuronides; and in GC-MS diagnostic methods for THC metabolites.

[0015] U.S. Patent No. 10,837,031: Recombinant production system for cannabinoid family prenylpolyketides, inventors Barr et al., filed on May 10, 2018, and issued on November 22, 2018, which relates to production methods, enzymes, and recombinant yeast strains for the biosynthesis of cannabinoid family prenylpolyketides of clinical importance. Using readily available starting materials, heterologous enzymes are used to direct cannabinoid biosynthesis in yeast.

[0016] WIPO Publication No. WO2021034403: Cannabinoid Acid Ester Compositions and Their Uses, inventors Swisa et al., filed on June 19, 2020, and published on February 25, 2021, which relates to pharmaceutical compositions comprising either a cannabinoid acid ester compound alone or a combination thereof with one or more additional cannabinoid compounds. The publication discloses that the cannabinoid acid ester compound is a tetrahydrocannabinolic acid (THCA) ester. The publication also discloses that the cannabinoid acid ester compound is a cannabigerolic (CBGA) acid ester. The publication also discloses that the cannabinoid acid ester compound is a cannabinol (CBNA) acid ester. Also provided are various therapeutic applications to which the cannabinoid acid ester compounds and the pharmaceutical compositions can be used, including combination therapies using the cannabinoid acid ester compounds and one or more additional therapeutic agents.

[0017] WIPO Publication No. WO2020186010: Cannabinoid Acid Ester Compositions and Their Uses, inventors Robinson et al., filed on March 12, 2020, and published on September 17, 2020, which relates to pharmaceutical compositions comprising either a cannabinoid acid ester compound alone or a combination thereof with one or more additional cannabinoid compounds. The publication discloses that the cannabinoid acid ester compound is a cannabidiol ester. Also provided are various therapeutic applications to which the cannabinoid acid ester compounds and the pharmaceutical compositions can be used, including combination therapies using the cannabinoid acid ester compounds and one or more additional therapeutic agents.

[0018] US Publication No. 20210403408: Cannabinoid Analogs and Methods of Preparation Thereof, inventors Barr et al., filed on April 29, 2021, and published on June 30, 2021, which relates to cannabinoid analogs, including halogenated cannabinoid analogs, hydroxylated cannabinoid analogs, deuterated cannabinoid analogs, and tritiated cannabinoid analogs. The cannabinoid analogs can be prepared by partial or total expression in a modified host cell (e.g., a recombinantly modified yeast cell), optionally in combination with chemical synthesis steps.

[0019] US Publication No. 20210230113: Cannabinoid Derivatives, inventor Filer, filed on January 22, 2021, and published on July 29, 2021, which relates to 8,9-dihydrocannabinoid derivatives, deuterated cannabinoid derivatives, and tritiated cannabinoid derivatives. The publication also provides compositions, methods of use, and preparation processes of the foregoing derivatives.

[0020] US Publication No. WO2021000053: Cannabinoid Derivatives, inventors Omeara et al., filed on July 3, 2020, and published on January 7, 2021, which relates to cannabinoid derivatives, pharmaceutical compositions comprising said derivatives, and methods of using said derivatives for treating or preventing diseases associated with cannabinoid receptors. The claimed cannabinoid derivatives are described by the following formula or its enantiomers, diastereomers, racemates, tautomers, or metabolites, or pharmaceutically acceptable salts, solvates, or hydrates of the compound.

[0021]

[0022] WIPO Publication No. WO2021046640: Cannabinoid Derivatives and Precursors and Their Asymmetric Synthesis, inventors Abdur-Rashid et al., filed on September 9, 2020, and published on March 18, 2021, which relates to novel cannabinoid derivatives and precursors and catalytic asymmetric methods for preparing them. The disclosure also relates to pharmaceutical compositions of the novel cannabinoid derivatives and pharmaceutical and analytical uses. For example, the disclosure relates to the preparation of novel precursors, and the use of such precursor compounds to prepare isotopically labeled cannabinoid products using chiral and achiral catalysts and catalytic processes. Compounds containing deuterium, carbon-13, and carbon-14 can be prepared and purified and then converted into the desired individual deuterated cannabinoid products.

[0023] WIPO Publication No. WO2021113669: Cannabinoids and Their Uses, inventors Deng et al., filed on December 4, 2020, and published on June 10, 2021, which relates to cannabinoid compounds, pharmaceutical compositions comprising one or more cannabinoid compounds, and the use of pharmaceutical compositions comprising one or more cannabinoid compounds for treating diseases or conditions (such as fibrotic diseases or inflammatory diseases) in a subject in need thereof.

[0024] WIPO Publication No. WO2022082313: Compositions and Methods for Treating Neuronal Diseases with Cannabinoids, inventors Hsu et al., filed on October 21, 2021, and published on April 28, 2022, which relates to methods and compositions comprising cannabinoid compounds for providing neuroprotection and / or stimulating neurite outgrowth. The cannabinoid compounds can be compounds as shown below (where R1 is COOH or H, R2 is C3H7 or C5H11, R3 is H or Me, R4 and R5 are Me or (CH2)2CH=C(CH3)2, such as CBGA), its derivatives, its prodrugs, or combinations thereof, and can be used to treat neurodegenerative diseases or to promote neurite elongation and / or restore neurite formation in a patient in need thereof.

[0025]

[0026] WIPO Publication No.: WO2021150636, Genetically Modified Yeast for the Production of Cannabigerolic Acid, Cannabichromenic Acid and Related Cannabinoids, inventors: Barr et al., filed on January 20, 2021, and published on July 29, 2021, which relates to production methods, enzymes and recombinant yeast strains for the biosynthesis of cannabinoid compounds of clinical importance.

[0027] US Publication No. 20100298579: Method for Preparing Synthetic Cannabinoids, inventors: Steup et al., filed on April 29, 2010, and published on November 25, 2010, which relates to organic synthesis, more specifically a method for preparing cannabinoids. This method is applicable to all stereoisomers and homologues of cannabinoids. For this purpose, this publication provides a method for preparing the above compounds through two or three chemical synthesis steps.

[0028] US Publication No. 20100152283: Tetrahydrocannabinol Modulators of Cannabinoid Receptors, inventors: Gant et al., filed on December 17, 2009, and published on June 17, 2010, which relates to tetrahydrocannabinol modulators of cannabinoid receptors, their pharmaceutical compositions and their use methods.

[0029] WIPO Publication No. WO2020102430: Use of Type I and Type II Polyketide Synthases for the Production of Cannabinoids and Cannabinoid Analogs, inventors: Barr et al., filed on November 13, 2019, and published on May 22, 2020, which relates to production methods, enzymes and recombinant yeast strains for the biosynthesis of prenylated polyketide cannabinoid compounds of clinical importance. Using readily available starting materials, heterologous enzymes are used to direct cannabinoid biosynthesis in yeast.

[0030] US Publication No. 20210040512: Recombinant Production System for Prenylated Polyketide Cannabinoid Compounds, inventors: Barr et al., filed on October 21, 2020, and published on February 11, 2021, which relates to production methods, enzymes and recombinant yeast strains for the biosynthesis of prenylated polyketide cannabinoid compounds of clinical importance. Using readily available starting materials, heterologous enzymes are used to direct cannabinoid biosynthesis in yeast.

[0031] US Publication No. WO2021133989: Preparation of Cannabichromene and Related Cannabinoids, inventors Marlowe, filed on December 23, 2020, and published on July 1, 2021, which relates to methods for producing cannabichromene and related cannabinoid compounds. The method includes: forming a reaction mixture comprising 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 the desired product. The method of the present disclosure may also include a one-pot conversion of a cannabichromene-type product to a cannabinol-type product.

[0032] WIPO Publication No. WO2021102567: Cannabigerol Derivatives and Their Use as Cannabinoid Receptor Modulators, inventors Ahmar et al., filed on November 25, 2020, and published on June 3, 2021, which relates to the synthesis of a series of pentylbenzene-1,3-diols compounds, the molecular formula of which is shown below. These compounds bind to cannabinoid 1 and 2 receptors (CB1 and CB2) and are thus contemplated to be useful for modulating the activity of such receptors. Accordingly, the use of such synthetic cannabinoids for treating various diseases mediated by CB1 and CB2 is considered.

[0033]

[0034] WIPO Publication No. WO2021050786: Cannabinoid Compositions with Improved Sensory and Therapeutic Properties, Their Production Methods and Uses, inventors Alarcon et al., filed on September 10, 2020, and published on March 18, 2021, which relates to compositions comprising cannabinoids, terpenoids, and other flavonoids. Also provided herein are methods for producing compositions comprising cannabinoids, terpenoids, and other flavonoids on an industrial scale. The compositions provided by this disclosure have desirable sensory properties and therapeutic effects upon ingestion or topical application. The disclosed compositions are used in inhalable, ingestible, or topical products for relieving and treating various acute or chronic diseases.

[0035] WIPO Publication No. WO2021222288: Compositions and Methods for Enhancing Cannabinoid Recombinant Biosynthesis, inventors Feng et al., filed on April 27, 2021, and published on November 4, 2021, which relates to recombinant host cells that contain a pathway capable of producing cannabinoids and a heterologous nucleic acid encoding a protein that is not in the pathway and enhances the ability of the host cell to produce cannabinoids. The disclosure also provides methods for producing cannabinoids using the host cells.

[0036] WIPO Publication No. WO2021195517: Compositions and Methods for the Recombinant Biosynthesis of Cannabinoids, inventors Schuetz, filed on March 26, 2021, and published on September 30, 2021, which relates to recombinant host cells comprising a pathway capable of producing cannabinoids and a nucleic acid derived from cannabis trichome mRNA that does not encode an enzyme in the pathway but enhances the ability of the host cell to produce cannabinoids. The disclosure also provides methods for producing cannabinoids using the host cells.

[0037] US Publication No. 20110311474: Novel Tricyclic Compounds, inventors Wishart et al., filed on December 1, 2010, and published on April 23, 2013, which relates to the compounds shown below, their pharmaceutically acceptable salts, prodrugs, bioactive metabolites, stereoisomers, and isomers, wherein the variables are defined in the disclosure. The disclosed compounds can be used to treat immunological and oncological conditions.

[0038]

[0039] WIPO Publication No. WO2021183448: Optimized Obtusifoliol Cyclase Polypeptides, inventors Horwitz et al., filed on March 8, 2021, and published on September 16, 2021, which relates to engineered variants of obtusifoliol cyclase polypeptides, wherein the engineered variants include: an amino acid sequence SEQ ID NO: 1 comprising at least one amino acid substitution, a nucleic acid comprising a nucleotide sequence encoding the engineered variant; a method for producing a modified host cell comprising the nucleic acid; a modified host cell expressing the engineered variant; a method for producing obtusifoliol, obtusifoliol derivatives, cannabinoids, or cannabinoid derivatives; and a method for screening engineered variants of obtusifoliol cyclase polypeptides.

[0040] US Publication No. 20200254041: Fast-acting and Long-lasting Phytocannabinoid and Synthetic Cannabinoid Formulations, inventors Leone-Bay et al., filed on October 5, 2018, and published on June 2, 2020, which relates to fast-acting and long-lasting phytocannabinoid pharmaceutical compounds or nutritional supplements and synthetic cannabinoid formulations. Fast-acting is provided by N-acylated fatty amino acids and / or permeation enhancers. Long-lasting can be provided by one or more sustained-release systems.

[0041] US Publication No. 20210251947: Stable Dronabinol Formulations, inventor Elkarim, filed on February 9, 2021, and published on August 19, 2021, which relates to formulations, methods of manufacture, and methods of treatment using cannabinoid formulations that are stable at room temperature for at least about one to two years. The disclosure discloses that the composition is an oxidation-stable formulation of dronabinol. Summary of the Invention

[0042] The present invention relates to substituted compounds and substituted cannabinoids and their biosynthesis.

[0043] The object of the present invention is to provide substituted compounds and substituted cannabinoids with more favorable pharmacokinetics, as well as systems and methods for their biosynthesis.

[0044] In one embodiment, the present invention provides a cannabinoid composition comprising a cannabinoid derived from a deuterated fatty acid, wherein the compound comprises a deuterated carbon chain, and wherein the deuterated carbon chain comprises at least one deuterated carbon.

[0045] In another embodiment, the present invention provides a method for synthesizing a cannabinoid compound or a derivative thereof, comprising: phosphorylating 3-methyl-2-buten-1-ol (prenol) and / or 3-methyl-3-buten-1-ol (isoprenol) using hydroxyethylthiazole kinase (ThiM) to produce isopentenyl phosphate; isomerizing the isopentenyl phosphate to produce isopentenyl diphosphate, wherein the isopentenyl diphosphate can 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 an acid to produce a second CoA thioester; synthesizing the deuterated CoA thioester and the second CoA thioester; cyclizing the synthesized product to produce deuterated olivetolic acid; isopentenylating 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.

[0046] In yet another embodiment, the present invention provides a method for cell-free synthesis of cannabinoids or their derivatives, comprising: 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 exhibits elevated expression of at least one enzyme compared to an unmodified parental 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 the cannabinoid biosynthetic pathway; and using the cannabinoid biosynthetic pathway to produce a cannabinoid or its derivative, including: activating a deuterated fatty acid in the presence of acyl-activating enzyme (AAE) 3 to produce a deuterated CoA thioester; activating an acid in the presence of a corresponding CoA synthase 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 synthesized product in the presence of olivetolic acid cyclase (OAC) to produce deuterated olivetolic acid; isoprenylating 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.

[0047] These and other aspects of the present invention will become apparent to those skilled in the art after reading the following description of the preferred embodiments and in conjunction with the drawings, as they support the claimed invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 Shows the metabolic pathway of cannabidiol (CBD).

[0049] Figure 2A Depicts an exemplary biosynthetic pathway for the production of prenylated natural products of the present disclosure.

[0050] Figure 2B Depicts an exemplary biosynthetic pathway for the production of prenylated natural products of the present disclosure, which continues from Figure 2A .

[0051] Figure 2C Shows Figures 2A to 2B a more detailed view of the pathway shown in

[0052] Figure 3A Shows the activity of pyruvate dehydrogenase (PDH) measured in the presence of various aromatic polyketides and 2% ethanol.

[0053] Figure 3B Shows a comparison of the final titers obtained using the complete pathway with the PDH and PDH bypass systems.

[0054] Figure 3C Shows the amounts of 5-isopentenyl-1,6-DHN and CBGA produced over time using the PDH bypass system with wild-type (WT) NphB.

[0055] Figure 3D Shows the results of pathways using various aromatic substrates with NphB, AtaPT, or NovQ prenyltransferases.

[0056] Figure 4A Shows a model of olivetolate in the active site of WT NphB.

[0057] Figure 4B Shows the results of activity assays to determine the approximate activity of NphB mutants with olivetolate as a substrate.

[0058] Figure 4D shows the gas chromatography - mass spectrometry (GC - MS) chromatograms of the full - pathway reaction products using the M23 mutant and WT NphB compared to the CBGA standard.

[0059] Figure 5A Shows the cell - free enzymatic production of cannabinoid precursors from glucose over time.

[0060] Figure 5B Shows a nonane - flow cannabigerolic acid (CBGA) capture system.

[0061] Figure 5C Shows cannabinoid production over time using cannabidiolic acid synthase (CBDAS).

[0062] Figure 6A Shows a schematic diagram of the MatB transferase pathway.

[0063] Figure 6B Shows a schematic diagram of the MdcA transferase pathway.

[0064] Figure 6C Shows Figures 6A to 6B more details of exemplary steps in the polyketide module of the pathway shown in

[0065] Figure 7 Shows a schematic diagram of the 3 - methyl - 2 - buten - 1 - ol / 3 - methyl - 3 - buten - 1 - ol ((iso)prenol) to geranyl pyrophosphate (GPP) pathway.

[0066] Figure 8 Shows various canonical (eukaryotic) and non - canonical (archaea I and II) mevalonate pathways that are capable of generating IPP / DMAPP from acetyl - CoA (or mevalonate).

[0067] Figure 9Probe electrospray ionization Fourier transform mass spectrometry (FTMS-pESI) spectral data of acetonitrile with 0.1% formic acid as a control are shown.

[0068] Figure 10 FTMS-pESI spectral data of cannabigerolic acid (CBGA) in positive mode are shown.

[0069] Figure 11 FTMS-pESI spectral data of CBGA in negative mode are shown.

[0070] Figure 12 FTMS-pESI spectral data of the control and CBGA in positive mode are shown.

[0071] Figure 13 FTMS-pESI spectral data of the control and CBGA in positive mode are shown.

[0072] Figure 14 FTMS-pESI spectral data of deuterated CBGA (dCBGA) in positive mode are shown.

[0073] Figure 15 FTMS-pESI spectral data of deuterated CBGA in negative mode are shown.

[0074] Figure 16 Additional FTMS-pESI spectral data of deuterated CBGA in positive mode are shown.

[0075] Figure 17 Additional FTMS-pESI spectral data of deuterated CBGA in negative mode are shown.

[0076] Figure 18 nLC-MS / MS data of CBGA are shown.

[0077] Figure 19 nLC-MS / MS data of dCBGA are shown.

[0078] Figure 20 nLC-MS / MS data of the first sample of CBGA after 1 hour are shown.

[0079] Figure 21 nLC-MS / MS data of the second sample of CBGA after 1 hour are shown.

[0080] Figure 22 nLC-MS / MS data of the third sample of CBGA after 1 hour are shown.

[0081] Figure 23 nLC-MS / MS data of the first sample of dCBGA after 1 hour are shown.

[0082] Figure 24 Shows the nLC-MS / MS data of the second sample of dCBGA after 1 hour.

[0083] Figure 25 Shows the nLC-MS / MS data of the third sample of dCBGA after 1 hour.

[0084] Figure 26 Shows a comparison chart of CBGA and dCBGA.

[0085] Figure 27A Shows an embodiment of the cell-free biosynthetic pathway of deuterated cannabinoids.

[0086] Figure 27B Shows an embodiment of the cell-free biosynthetic pathway of deuterated cannabinoids, which includes the GPP production pathway.

[0087] Figure 28A Shows an embodiment of a deuterated methyl compound.

[0088] Figure 28B Shows examples of methylation of various small molecules using d3-SAM and methyltransferase.

[0089] Figure 29 Shows an example of deuterated methylated psilocybin using a specific methyltransferase.

[0090] Figure 30 Shows an example of deuterated methylated chrysoeriol using a specific methyltransferase.

[0091] Figure 31 Shows an example of the synthesis of deuterated glucosides.

[0092] Figure 32 Shows the HPLC (High Performance Liquid Chromatography) traces of cell-free production of multiple cannabinoids.

[0093] Figure 33 Shows a bar chart of the progress made during the pathway optimization process.

[0094] Figure 34A Shows a bar chart depicting OLS variants and their corresponding CBGA production levels.

[0095] Figure 34B Shows a bar chart depicting OLS variants and their corresponding CBGA production levels at different enzyme loadings.

[0096] Figure 35 Shows the time course of the CBGA synthesis reaction according to an embodiment of the present invention.

[0097] Figure 36 Shows the HPLC traces of CBGA and THCA activities according to one embodiment of the present invention.

[0098] Figure 37 Shows the HPLC trace of THC in toluene according to one embodiment of the present invention.

[0099] Figure 38 Shows the comparison of the enzymatic activities of S-adenosyl-L-methionine-d3 and S-adenosyl-L-methionine. Detailed Description

[0100] The present invention generally relates to substituted compounds and substituted cannabinoids, and their biosynthesis.

[0101] In one embodiment, the present invention provides a cannabinoid composition comprising cannabinoids derived from deuterated fatty acids, wherein the compound comprises a deuterated carbon chain, and wherein the deuterated carbon chain comprises at least one deuterated carbon.

[0102] In another embodiment, the present invention provides a method for synthesizing a cannabinoid compound or a derivative thereof, comprising: phosphorylating 3-methyl-2-buten-1-ol (prenol) and / or 3-methyl-3-buten-1-ol (isoprenol) using hydroxythiazole kinase (ThiM) to produce isopentenyl phosphate; isomerizing isopentenyl phosphate to produce isopentenyl diphosphate, wherein isopentenyl diphosphate can 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 an acid to produce a second CoA thioester; synthesizing the deuterated CoA thioester and the second CoA thioester; cyclizing the synthesized product to produce deuterated olivetolic acid; isopentenylating deuterated olivetolic acid in the presence of geranyl pyrophosphate to produce deuterated cannabigerolic acid; and cyclizing deuterated cannabigerolic acid to produce a deuterated cannabinoid.

[0103] In yet another embodiment, the present invention provides a method for cell-free synthesis of cannabinoids or their derivatives, comprising: 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 exhibits elevated expression of at least one enzyme compared to the unmodified parental microorganism; lysing at least one cell of the modified microorganism to obtain at least one enzyme, wherein at least one enzyme is used in the cannabinoid biosynthetic pathway; utilizing the cannabinoid biosynthetic pathway to produce cannabinoids or their derivatives, including: activating a deuterated fatty acid in the presence of acyl-activating enzyme (AAE) 3 to produce a deuterated CoA thioester; activating an acid in the presence of a corresponding CoA synthase 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 synthesized product in the presence of olivetolic acid cyclase (OAC) to produce deuterated olivetolic acid; prenylating deuterated olivetolic acid in the presence of geranyl pyrophosphate to produce deuterated cannabigerolic acid; cyclizing deuterated cannabigerolic acid to produce deuterated cannabinoids.

[0104] The present invention includes substituted compounds and substituted cannabinoids which have reduced metabolism and similar pharmacokinetics compared to their unsubstituted analogs. The present invention also includes cell-free systems and methods for the biosynthesis of these substituted compounds and substituted cannabinoids. The present invention also includes high-purity substituted compounds and high-purity substituted cannabinoids synthesized by a cell-free method.

[0105] There is no fully cell-free synthesis system in the prior art that simultaneously provides highly isomerically pure and highly absolutely pure substituted compounds and substituted cannabinoids. The high isomeric purity is due to the regioselectivity of the prenylating enzymes of the present invention, while the high absolute purity is due to the fully cell-free synthesis and stoichiometric efficiency of the system of the present invention, which results in fewer foreign compounds to be removed and less residual reagent. Advantageously, higher isomeric purity is very important when producing substituted by-products that are not easily metabolized. In addition, none of the prior art provides a one-pot system for cell-free synthesis of highly pure substituted compounds and substituted cannabinoids.

[0106] Reference is now made generally to the drawings, which are for the purpose of describing one or more preferred embodiments of the present invention and are not intended to limit the present invention thereto.

[0107] Compound

[0108] The present invention includes compounds substituted with 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, 13N, 15 N, 18 O, 17 O, 15 O, 31 P, 32 P, 35 S, 18 F, 36 compounds and cannabinoids substituted with at least one tritium and / or at least one additional isotope (e.g., 3 H, 14 C). Advantageously, compounds substituted with at least one radioactive isotope (e.g., 3 H, 14 C) can be used for the determination of drug and / or substrate tissue distribution. An additional advantage is that compounds substituted with positron-emitting isotopes (e.g., 11 C, 18 F, 15 O, 13 N) can be used for positron emission tomography (PET) studies.

[0109] The present invention includes cannabinoids, cannabinoid precursors, and other prenylated chemicals substituted with 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 Cl). In one embodiment, at least one tritium and / or at least one additional isotope is radioactive (e.g., 3 H, 14 C). Advantageously, cannabinoids, cannabinoid precursors, or other prenylated chemicals substituted with at least one radioactive isotope (e.g., 3H, 14C) can be used for the determination of drug and / or substrate tissue distribution. An additional advantage is that compounds substituted with positron-emitting isotopes (e.g., 11 C, 18 F, 15 O, 13N) Substituted cannabinoids, cannabinoid precursors, or other prenylated chemicals can be used in positron emission topography (PET) studies.

[0110] Another advantage of cannabinoids, cannabinoid precursors, and other prenylated chemicals substituted with at least one deuterium is that deuterium is safe, stable, and non-radioactive. The bond formed by deuterium with carbon is stronger than the bond formed by hydrogen with carbon, which may have a positive impact on the absorption, distribution, metabolism, and / or excretion characteristics of cannabinoids, cannabinoid precursors, or other prenylated chemicals. In addition, since deuterium is similar to hydrogen, substituting hydrogen with deuterium is not expected to affect the synthetic selectivity of cannabinoid precursors or other prenylated chemicals.

[0111] In one embodiment, the cannabinoids, cannabinoid precursors, and / or other prenylated chemicals include cannabidiol (CBD), tetrahydrocannabinol (THC), cannabinol (CBN), cannabigerol (CBG), cannabichromene (CBC), cannabinol (CBL), cannabinodiol (CBND), cannabinol (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 can be found in U.S. Provisional Patent Application No. 63 / 333,670, filed on April 22, 2022, which is incorporated herein by reference in its entirety.

[0112] For example, but not limited to, in one embodiment, the cannabinoids, cannabinoid precursors, and / or other prenylated chemicals include at least one deuterium to form deuterated cannabinoids, deuterated cannabinoid precursors, or deuterated other prenylated chemicals. Compared with non-deuterated cannabinoids, cannabinoid precursors, or other prenylated chemicals, deuterated cannabinoids, deuterated cannabinoid precursors, or deuterated other prenylated chemicals can improve bioavailability and slow down metabolism.

[0113] In one embodiment, the cannabinoid, cannabinoid precursor, and / or other prenylated chemical includes at least 10% substitution (e.g., 15%) at a specified substitution site, at least 20% substitution (e.g., 25%) at a specified substitution site, at least 30% substitution (e.g., 35%) at a specified substitution site, at least 40% substitution (e.g., 45%) at a specified substitution site, at least 50% substitution (e.g., 55%) at a specified substitution site, at least 60% substitution (e.g., 65%) at a specified substitution site, at least 70% substitution (e.g., 75%) at a specified substitution site, at least 80% substitution (e.g., 85%) at a specified substitution site, at least 90% substitution (e.g., 95%) at a specified substitution site.

[0114] Figure 1 shows the metabolic pathway of CBD. As Figure 1 shown, CBD is hydroxylated under the action of metabolic enzymes, generally occurring at key metabolic sites. One of the key metabolic sites is the 5'-pentyl tail. Approximately 25% of the metabolites will undergo cleavage of the 5'-pentyl tail, resulting in inactive metabolites. The numbering systems of CBD and CBDA are shown below.

[0115]

[0116] In one embodiment, the present invention includes CBD molecules having at least one deuterium on the 5'-pentyl tail. Advantageously, substitution of hydrogen with deuterium, tritium, and / or halogen slows down cannabinoid metabolism by preventing Figure 1 the hydroxylation shown. In one embodiment, the 5'-pentyl tail is completely substituted with deuterium, tritium, and / or halogen. Alternatively, the 5'-pentyl tail is partially substituted with deuterium, tritium, and / or halogen.

[0117] However, substitution at the end of the group on the 5'-pentyl tail may affect the affinity of the pentyl for various cannabinoid receptors. In one embodiment, the end of the pentyl is unsubstituted. Thus, in one embodiment, the hydrogen on the pentyl chain at C1 to C4 is substituted with deuterium, tritium, and / or halogen. In another embodiment, the hydrogen on the pentyl chain at C1 to C3 is substituted with deuterium, tritium, and / or halogen. Alternatively, the hydrogen atoms on the pentyl chain at C1 to C2 are substituted with deuterium, tritium, and / or halogen. In yet another embodiment, the pentyl chain is substituted with deuterium, tritium, and / or halogen at C1.

[0118] Alternatively, the 5'-group is an alkyl group that is partially or fully substituted with deuterium, tritium, and / or halogen. Alkyl groups include, but are not limited to, methyl, ethyl, propyl (e.g., n-propyl, isopropyl), butyl (e.g., n-butyl, sec-butyl, tert-butyl), hexyl, heptyl, octyl, cycloalkane groups (e.g., cyclopropane, cyclobutane, cyclopentane, cyclohexane, etc.), branched groups (e.g., isopropyl, tert-butyl, etc.), and / or aromatic groups (e.g., benzyl, coumarinyl, etc.). In one embodiment, the alkyl group is unsubstituted at the terminus. There is no teaching in the prior art of an alkyl group that is unsubstituted at the terminus. In another embodiment, the alkyl group is unsubstituted at two terminal carbons, three terminal carbons, or four terminal carbons.

[0119] In another embodiment, C7 is substituted with deuterium, tritium, and / or halogen.

[0120] 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 add a boronic acid or ester group. See, for example, (1) Maslah H, Skarbek C, Pethe S, Labruère 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. Boronic 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, each of which is incorporated herein by reference in its entirety.

[0121] In one embodiment, the compound or cannabinoid includes a prodrug. In one embodiment, the prodrug is modified by 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, 36Cl) substitution. In one embodiment, the prodrug includes sedatives (such as codeine, diazepam, phenobarbital, hydrocodone, morphine, oxycodone, tramadol, ethylmorphine), cannabinoids (such as THC, THC-O-acetate, THC-O-phosphate, 11-hydroxy-THC), deliriants (such as ibotenic acid), dissociatives (such as dextromethorphan, ketamine), hallucinogens (such as 1A-LSD, 1B-LSD, 1P-ETH-LAD, 1P-LSD, bufotenin, ethocybin, MDMA, MDA, norpsilocin, noribogaine, psilocybin), nootropics (such as 5-HTP, adrafinil, mebracetam), stimulants (such as adrafinil, amphetamine, benzphetamine, droxidopa, fenfluramine, L-dopa, levamisole, lysineamphetamine, 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, cannaboside, 11-hydroxy-THC and / or cannabinoid glycoside. For example, see (1) Walker, L.A., Harland, E.C., Best, A.M., ElSohly, M.A. (1999). Δ9-THC Hemisuccinate in Suppository Form as an Alternative to Oral and Smoked THC. In: Nahas, G.G., Sutin, K.M., 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 PharmSci. 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 herein by reference in its entirety. See also, e.g., WIPO Publication No. WO2021173130 and U.S. Patent Publication Nos. 20220194916, 20220168428, and 20210379507, each of which is incorporated herein by reference in its entirety.

[0122] Synthesis

[0123] Prenylation (also known as isoprenylation or lipidation) is the addition of a hydrophobic molecule to a protein or chemical compound. Prenyl (3-methylbut-2-en-1-yl) is generally thought to contribute to attachment to the cell membrane, similar to lipid anchors such as GPI anchors. Prenylation has been shown to be important for protein-protein binding through specialized prenyl-binding domains.

[0124] Prenylated natural products are a large class of bioactive molecules with distinct medicinal properties. Examples include, but are not limited to, prenylated flavonoids, prenylated stilbenes, and cannabinoids. Plant-derived phenyl compounds are difficult to isolate due to the structural similarity of contaminating molecules and the compositional variability between crops. These challenges are further exacerbated when attempting to isolate low-abundance compounds. Many chemical syntheses have been developed to address the challenges associated with the manufacture of prenylated natural products, but they are generally not suitable for pharmaceutical manufacture due to their complexity and low yields.

[0125] Microbial production is a useful alternative to the natural extraction of prenylated natural products, but it also faces many challenges, such as the need to divert carbon flux from central metabolism and product toxicity, etc. For example, prenylated natural products such as prenylnaringenin, prenylresveratrol, and cannabidiolic acid (CBDA) are derived from a combination of metabolic pathways of fatty acid, isoprenoid, and polyketide biosynthesis. Therefore, high-level production requires the effective re-routing of long-distance, key, and tightly regulated pathways. Despite the challenges, many teams have engineered microbes to produce non-prenylated polyketides such as naringenin, resveratrol, and oleanolic acid, but at relatively low levels (110, 391, and 80 mg / L, respectively). Obtaining prenylated products is even more challenging because geranyl pyrophosphate (GPP) is an essential metabolite that is toxic to cells at moderate concentrations, thus posing a major obstacle to high-level microbial production.

[0126] Cannabinoids have shown great therapeutic potential, and currently more than 100 clinical trials are underway, being used as antiemetics, anticonvulsants, antidepressants, and analgesics. However, despite the therapeutic potential of prenylated natural products, their research and use are limited due to the lack of cost-effective production methods.

[0127] Two main alternative methods for plant-based cannabinoid production are organic synthesis and production in metabolically engineered hosts such as plants, yeast, or bacteria. Total syntheses for the production of certain cannabinoids such as THCA and CBDA have been elucidated, but they are generally not suitable for drug manufacturing. In addition, synthetic methods are not modular and require unique syntheses for each cannabinoid. Modular methods can be achieved by using natural biosynthetic pathways.

[0128] The three main cannabinoids (THCA, CBDA, and cannabichromene) are all derived from a single precursor, CBGA. In addition, three low-abundance cannabinoids are derived from cannabierovarinic acid (CBGVA)( Figures 2A to 2B ). Therefore, the ability to produce CBGA and CBGVA in heterologous hosts opens the door to the production of a range of cannabinoids. Unfortunately, engineering microorganisms to produce CBGA and CBGVA has proven to be extremely challenging.

[0129] Cannabinoids are derived from a combination of fatty acid, polyketide, and terpene biosynthetic pathways that produce the key building blocks geranyl pyrophosphate (GPP) and olivetolic acid (OA)( Figures 2A to 2B ). High levels of CBGA biosynthesis require reprogramming long-distance, essential, and highly regulated pathways. In addition, GPP is toxic to cells, presenting a significant obstacle to its high-level production in microorganisms. While Gagne et al. (incorporated herein by reference in its entirety) designed a pathway for the production of OA in yeast, but the titer was very low (0.5 mg L-1), indicating that the high-level production of intermediates in this pathway is not simple. In another study, Zirpel et al. produced THCA in yeast lysates containing the promiscuous prenyltransferase (NphB) and THCA synthase and supplemented with GPP and olivetolic acid (OA) (J. Biotechnol., 259:204-212, 2017, incorporated herein by reference in its entirety). However, there is currently no public report on the production of cannabinoids from low-cost raw materials in engineered living cells.

[0130] Synthetic biochemistry utilizes enzyme mixtures to perform complex biochemical conversions under cell-free conditions, offering potential advantages over traditional metabolic engineering, including: a higher level of flexibility in pathway design; stronger control over component optimization; a faster design-build-test cycle; and non-cytotoxic intermediates or products. Advantageously, the present invention provides a cell-free system for the production of cannabinoids.

[0131] The present invention provides enzyme variants and pathways comprising such variants for the prenylation of compounds, including the production of cannabinoids. Additionally, the biosynthetic pathways described herein use "scavenging valves" to regulate NAD(P)H levels. Such "scavenging valves" have been shown to produce monoterpenes at high levels from glucose, indicating that substantial amounts of GPP can be produced under cell-free conditions (see International Patent Publication WO2017 / 015429, which is incorporated herein by reference in its entirety). These scavenging valves are used to upgrade and diversify the original system for the production of complex natural products such as cannabinoids. Figures 2A to 2C The synthetic biochemistry methods are outlined in FIGS. 5, 6A, and 6B. In one embodiment, the present disclosure provides a cell-free system for prenylation using GPP derived from glucose (see FIGS. 5, 6A, 6B, and 8). In another embodiment, the present invention provides a cell-free system for prenylation using GPP derived from 3-methyl-3-buten-1-ol / 3-methyl-2-buten-1-ol ((iso)prenol) or 3-methyl-2-buten-1-ol (prenol) (see FIGS. 5, 6A, 6B, and 8). Figures 2A to 2C The pathways of FIGS. 5, 6A, 6B, and 8 are capable of being coupled to any ATP-generating system to produce the ATP required for the reaction. For example, the pathway can be coupled to a creatine kinase ATP-generating system, an acetate kinase system, a glycolysis system, etc. Figure 7 FIGS. 5, 6A, 6B, and 8 Figure 7 The enzymes (nucleic acid coding sequences and polypeptides) in FIGS. 5, 6A, 6B, and 8 are provided as SEQ ID NOs: 54-65 (for example, the PRK enzymes are provided as SEQ ID NOs: 54-57; the IPK enzymes are provided as SEQ ID NOs: 58-61; the IDI enzymes are provided as SEQ ID NOs: 62-63; the FPPS enzymes are provided as SEQ ID NOs: 64-65). Figure 7 FIGS. 5, 6A, 6B, and 8

[0132] NphB is an aromatic prenyltransferase that catalyzes the attachment of a 10-carbon geranyl moiety to aromatic substrates. NphB exhibits broad substrate selectivity and product regioselectivity. NphB was isolated from Streptomyces and catalyzes the addition of a 10-carbon geranyl moiety to a variety of small organic aromatic substrates. NphB has a spacious and solvent-accessible binding pocket that can accommodate two substrate molecules, geranyl diphosphate (GPP) and 1,6-dihydroxynaphthalene (1,6-DHN). GPP is stabilized by interactions between its negatively charged diphosphate moiety and several amino acid side chains (including Lys119, Thr171, Arg228, Tyr216, and Lys284, as well as Mg 2+ )). The activity of NphB requires Mg 2+ as a cofactor. NphB from Streptomyces has the sequence shown in SEQ ID NO: 30.

[0133] NovQ (accession number AAF67510, incorporated herein by reference) is a member of the CloQ / NphB class of prenyltransferases. The novQ gene can be cloned from Streptomyces niveus, a strain that produces the aminocoumarin antibiotic novobiocin. Recombinant NovQ can be expressed and purified to homogeneity in Escherichia coli. The purified enzyme is a soluble monomeric 40 kDa protein that catalyzes the transfer of dimethylallyl to 4-hydroxyphenylpyruvate (4-HPP) in the absence of divalent cations, generating the novobiocin intermediate 3-dimethylallyl-4-HPP. In addition to the isoprenylation of 4-HPP, NovQ also catalyzes various carbon-carbon and carbon-oxygen based isoprenylations of phenylpropanoids, flavonoids, and dihydroxynaphthalenes. Despite its broad catalytic scope, the isoprenylations catalyzed by NovQ occur in a regioselective manner. NovQ is the first reported prenyltransferase that can catalyze the transfer of dimethylallyl to the B-ring of phenylpropanoids (such as p-coumaric acid and caffeic acid) and flavonoids. NovQ can serve as a useful biocatalyst for the synthesis of isoprenyl phenylpropanoids and isoprenyl flavonoids.

[0134] The Aspergillus terreus aromatic prenyltransferase (AtaPT; accession number AMB20850, incorporated herein by reference) was recently discovered and characterized and is responsible for the isoprenylation of various aromatic compounds. Recombinant AtaPT can be overexpressed and purified in Escherichia coli. The Aspergillus terreus aromatic prenyltransferase (AtaPT) mainly catalyzes the C-monoisoprenylation of acylphloroglucinols in the presence of different isoprenyl diphosphates.

[0135] Oleic acid (OA) is a relatively weak substrate for wild-type NphB. Therefore, by using the more preferred NphB substrate 1,6-dihydroxynaphthalene (1,6-DHN), the ability of the cell-free system to isoprenylate the cosubstrate was tested. When starting with 2.5 mM 1,6-DHN and 500 mM glucose, an isoprenylated product of approximately 400 mg / L (1.3 mM) was obtained. However, when the starting 1,6-DHN concentration was increased from 2.5 mM to 5 mM, the final titer decreased to 1 / 2, indicating that 1,6-DHN inhibited one or more enzymes. Enzyme assays showed that Escherichia coli pyruvate dehydrogenase (EcPDH) was inhibited not only by 1,6-DHN but also by several other aromatic polyketides ( Figure 3B ). When the concentration of 1,6-DHN, oleanol, or resveratrol was 1 mM, the activity of PDH decreased to 1 / 2 ( Figure 3B ). Therefore, the experiment was designed to eliminate PDH by implementing a PDH bypass (see Figures 2A to 2B and 3B). In the PDH bypass, pyruvate is converted to acetyl-CoA using pyruvate oxidase (PyOx) and phosphotransacetylase (PTA), thereby eliminating PDH ( Figures 2A to 2B ). As shown in Figure 3A , the new system removed the inhibition observed at higher concentrations of 1,6-DHN and increased the titer of 5-isoprenyl-1,6-DHN to 4-fold that of the PDH system when starting from 5 mM 1,6-DHN ( Figure 3B ). Figure 3C shows the time course of 5-isoprenyl-1,6-DHN biosynthesis starting from 5 mM 1,6-DHN using the PDH bypass. Approximately 50% of 1,6-DHN was converted within the first 24 hours, and the final titer reached 705 ± 12 mg / L.

[0136] It is believed that the isoprenylation of aromatic polyketides by NphB proceeds through a carbocation intermediate, in which the first step is the dissociation of diphosphate from GPP to form a carbocation at the C1 carbon of GPP, followed by the attack of the carbocation on a nearby nucleophile. To improve the regioselectivity of isoprenyl transfer, starting from the crystal structure of NphB complexed with 1,6-DHN, Mg 2+ and a non-hydrolyzable analogue of GPP (geranyl S-thiodiphosphate), OA was modeled into the active site of NphB (PDBID 1ZB6; Protein Data Bank reference 1ZB6). In the design, OA was placed in the binding pocket guided by 1,6-DHN, thereby positioning the desired isoprenylation site (C3 carbon of OA) above the nascent geranyl C1 carbocation ( Figure 4A)。The selected distance is based on the distance between the C5 carbon of 1,6-DHN and the C1 carbon of GPP. Then the residues in contact with OA were altered using ROSETTA software to optimize the active site of NphB to bind OA. The side chains in contact with GPP or that may provide catalytic function were kept fixed. The result is a set of proposed NphB variants.

[0137] To reduce the number of variants that need to be experimentally tested, a scoring system was used to rank the changes that are likely to have the most significant impact on OA binding. A representative set of variants (Table 1) was selected, and each residue was systematically reverted to the wild-type side chain in the context of other mutations, and the change was evaluated in the energy score (Table 2). The Y288 substitution had the greatest impact on the energy score, so the Y288A or Y288N mutation was used in each experimentally evaluated construct. The frequency of mutations, how multiple mutations act in concert, and the computational energy scores for further shaping the NphB library were all taken into account. Taking these factors into consideration, a library containing 29 constructs was generated, each construct ranging from single-point mutations to up to 6 mutations, as shown in Table 1 (see also SEQ ID NO: 1-29; note that SEQ ID NO: 1-29 includes the hexahistidine leader sequence from the expression construct, i.e., amino acids 1-20, which are not essential for biological activity). Table 1 provides exemplary mutations and fold enhancements relative to the wild type (i.e., the polypeptide of SEQ ID NO: 30). NphB library constructs and mutations (amino acid positions refer to SEQ ID NO: 30).

[0138] Table 1

[0139]

[0140]

[0141] * Second-round focused library.

[0142] b Mutations predicted by Rosetta but not tested.

[0143] ND – Not determined

[0144] Table 2 illustrates the kinetic parameters of the NphB mutants.

[0145] Table 2

[0146]

[0147] b Kinetic parameters of 2,4-dihydroxy-6-propylbenzoic acid (divarinic acid)

[0148] The present disclosure describes recombinant methods for generating and isolating the modified NphB polypeptides of the present disclosure. In addition to recombinant production, polypeptides can also be produced by direct peptide synthesis using solid-phase techniques (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 in its entirety). Peptide synthesis can be carried out using manual techniques or automated techniques. Automated synthesis can be achieved, for example, by operating an Applied Biosystems 431A peptide synthesizer (Perkin Elmer, Foster City, Calif.) in accordance with the instructions provided by the manufacturer.

[0149] The crudely purified NphB mutants were obtained and used for a preliminary screening of CBGA production using GPP and OA at saturation concentrations for wild-type NphB. Six constructs were confirmed to have a significantly increased activity of >10-fold (M1, M2, M3, M6, M10, and M15), four constructs had a significantly increased activity of 2 to 10-fold (M5, M7, M12, and M20), and the activities of the remaining constructs were similar to those of WT NphB. The best hits (M1, M3, M10, and M15) in the initial screening were purified and characterized more carefully ( Figure 4B ). The following conclusions were apparent from the initial screening: (1) As predicted by the calculations, Y288A (M1) and Y288N (M2) alone significantly enhanced the activity; (2) The presence of Y288N in any construct decreased the purification yield, indicating that Y288N might be an unstable mutation, while Y288A was a more desirable mutation; (3) Relative to Y288N (M2), the addition of G286S (M10) in the Y288N background seemed to further increase the activity, indicating that G286S was another favorable mutation; (4) Although F213N had a neutral or detrimental effect in the Y288A / F213N (M5) construct, the activity of Y288A / F213N / A232S (M15) was slightly higher than that of Y288A (M1), indicating that A232S was also a favorable mutation.

[0150] Based on these preliminary conclusions, a focused library was designed, which included various combinations of the variants Y288A, GS86S, and A232S. The reason for adding other combinations with Y288V was that it could improve the stability while also reducing the size of the Y288 side chain. In the one-hour endpoint assay, all constructs in the second library, except for one, exhibited an activity that was at least 100-fold higher than that of WTNphB. Figure 4B Shows a comparison of the best mutants in the first round and the best mutants in the second round. Apparently, the combination of beneficial mutations in the first round increased the production of CBGA. In addition, compared with Y288N, the Y288A and Y288V constructs increased the expression of NphB without sacrificing activity.

[0151] The two best mutants obtained in the initial screening and the three best constructs in the focused library were further characterized. The kinetic parameters are summarized in Table 2. Although all mutants had relatively little effect on K m , a significant improvement in the k cat value was observed. M23 (NphB of SEQ ID NO: 23) in particular increased the k cat to 750-fold, from 0.0021 ± 0.00008 min-1 to 1.58 ± 0.05 min-1. Compared with the wild-type enzyme, the catalytic efficiency (k cat / K m ) of M23 and M31 was increased to more than 1000-fold. Although M31 had a higher k cat / K m than M23, M23 was adopted instead of M31 because M23 had a higher k cat , and synthetic biochemical systems usually operate under saturated OA conditions.

[0152] The designed mutant M23 not only showed a significant increase in the catalytic efficiency of OA prenylation, but also was very specific, producing only the correct CBGA product. WT NphB produced CBGA, but the main product was the prenylated isomer ( Figure 4C ). In contrast, the designed mutant M23 produced almost only CBGA. Overall, the designed enzyme is a more efficient CBGA synthase than the non-specific prenylating wild-type enzyme.

[0153] Accordingly, the present disclosure provides NphB variants, which comprise: (i) SEQ ID NO: 30 and having at least one Y288X mutation, wherein X is A, N, S, V or a non-natural amino acid; (ii) SEQ ID NO: 30, which has at least one Y288X mutation, wherein X is A, N, S, V or a non-natural amino acid, and at least one additional mutation selected from V49Z1, F213Z2, A232S, I234T, V271Z3 and / or G286S, wherein 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 one of (i), (ii) or (iii), which comprises 1-20 (e.g., 2, 5, 10, 15 or 20; or any value between 1 and 20) conservative amino acid substitutions and has NphB activity; (v) a sequence that is at least 85%, 90%, 95%, 98% or 99% identical to SEQ ID NOs: 1-29 or 30 and has at least the mutations described in (i), (ii) or (iii); (vi) an NphB mutation comprising any sequence recited starting from amino acid 21 in SEQ ID NOs: 1-28 or 29; or (vii) any sequence that is at least 99% identical to any one of SEQ ID NOs: 1-28 or 29 and has NphB activity. "NphB activity" refers to the ability of the enzyme to isoprenylate a substrate, more specifically the ability to generate CBGA from OA.

[0154] As used herein, a non-natural amino acid refers to an amino acid that does not exist in nature, such as an N-methyl amino acid (e.g., N-methyl-L-alanine, N-methyl-L-valine, etc.) or an α-methyl amino acid, a β-homo amino acid, a homo amino acid, and a D-amino acid. In certain embodiments, the non-natural amino acids useful in the present disclosure include small hydrophobic non-natural amino acids (e.g., N-methyl-L-alanine, N-methyl-L-valine, etc.).

[0155] In addition, the present disclosure provides polynucleotides encoding any of the foregoing NphB variants. Due to the degeneracy of the genetic code, the actual coding sequence can vary while still resulting in the polypeptides of the recited NphB mutants and variants. Exemplary polynucleotide sequences are provided in SEQ ID NOs: 66, 67 and 68 (corresponding to the polypeptide sequences of SEQ ID NOs: 23, 29 and 69, respectively). Again, it is obvious that the degeneracy of the genetic code allows for significant variation in the percent identity with SEQ ID NOs: 66, 67 and 68 while still encoding the polypeptides of SEQ ID NOs: 23, 29 and 69.

[0156] The present disclosure also provides recombinant host cells and cell-free systems comprising any of the NphB variant enzymes of the present disclosure. In some embodiments, the recombinant cells and cell-free systems are used for the prenylation process. In one embodiment, the system is preferably cell-free, without any living cells. Or rather, the system is completely cell-free, without micelles or other artificial cells, defined as the compartmentalization without any reagents, enzymes or other chemicals in the reaction environment. In one embodiment, the system comprises permeabilized cells (e.g., acetone-dried yeast). In one embodiment, permeabilized cells are used for ATP regeneration. In another embodiment, the system comprises engineered heat-treated cell lysates, where the biomass (e.g., growing cells) is collected, heated (with or without lysis) and then used. In one embodiment, ATP regeneration is carried out without using acetyl phosphate. In one embodiment, polyphosphate is used to regenerate ATP to simplify the biosynthetic pathway. Those of ordinary skill in the art will understand that the substrate acetyl phosphate can be used for two competing reactions, which will introduce equilibrium problems in the pathway. Using polyphosphate instead of acetyl phosphate solves and significantly reduces this risk.

[0157] For example, see (1) Valliere, M.A., Korman, T.P., Woodall, N.B. et al. A cell-free platform for the prenylation of natural products and application to cannabinoid production. Nat Commun 10, 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.

[0158] An object of the present disclosure is to produce the precursor GPP from glucose or 3-methyl-2-buten-1-ol (prenol) and / or 3-methyl-3-buten-1-ol (isoprenol), which can then be used to isoprenylate added OA with the mutant NphB of the present disclosure to produce CBGA.

[0159] Accordingly, the present disclosure provides a cell-free system that includes a plurality of enzymatic steps for converting glucose to geranyl pyrophosphate, wherein the pathway includes a scavenging valve and a PDH bypass enzymatic process.

[0160] As Figure 2C shown, one pathway of the present disclosure includes using hexokinase to convert glucose to glucose-6-phosphate. Hexokinase (EC 2.7.1.1) is an enzyme that phosphorylates hexose (six-carbon sugar) to form hexose phosphate. Hexokinase has the ability to transfer an inorganic phosphate group from ATP to the substrate. Multiple hexokinase proteins from various organisms have been cloned and expressed. In some embodiments, the hexokinase includes the sequence listed in UniProtKB accession number P04806 from Saccharomyces cerevisiae (Sc) (incorporated herein by reference) and sequences that are at least 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99% identical thereto and have hexokinase activity.

[0161] Then, glucose-6-phosphate is converted to fructose-6-phosphate under the action of phosphoglucose isomerase (Pgi) (EC5.3.1.9). Accordingly, in addition to the foregoing, the term "phosphoglucose isomerase" or "Pgi" refers to a protein capable of catalyzing the formation of fructose-6-phosphate from glucose-6-phosphate and having at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with SEQ ID NO: 31 or having at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or higher sequence similarity (calculated using default parameters by NCBI BLAST), and wherein the enzyme has phosphoglucose isomerase activity.

[0162] In another or further embodiment, the systems or recombinant microorganisms provided herein include the expression of phosphofructokinase (Pfk, polyphosphate-dependent Pfk or homologs or variants thereof). In one embodiment, such expression is capable of binding to other enzymes in a metabolic pathway. Pfk can be derived from Geobacillus stearothermophilus (SEQ ID NO: 32). In another embodiment, engineered variants of Pfk can be used as long as they have phosphofructokinase activity and are capable of converting fructose-6-phosphate to fructose-1,6-bisphosphate. Such engineered variants can be obtained by methods such as site-directed mutagenesis, directed evolution, etc. Accordingly, the present disclosure includes polypeptides that are at least 85-99% identical to the sequence shown in SEQ ID NO: 32 and have phosphofructokinase activity (see, for example, SEQ ID NOs: 33-34).

[0163] In addition to the foregoing, 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, and having at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher sequence identity or at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or higher sequence similarity (calculated using the default parameters of NCBI BLAST) with SEQ ID NO: 35. Other homologs include: Synechococcus elongatus PCC 6301 YP_170823.1, which has 26% identity with SEQ ID NO: 35; Vibrio nigripulchritudo ATCC 27043 ZP_08732298.1, which has 80% identity with SEQ ID NO: 35; Methylomicrobium album BG8 ZP_09865128.1, which has 76% identity with SEQ ID NO: 35; Pseudomonas fluorescens Pf0-1 YP_350990.1, which has 25% identity with SEQ ID NO: 35; and Methylobacterium nodulans ORS2060 YP_002502325.1, which has 24% identity with SEQ ID NO: 35. Accordingly, the present disclosure includes the use of a polypeptide having 26% to 100% identity with SEQ ID NO: 35, wherein the polypeptide has diisophosphoaldolase activity. The sequences associated with the foregoing accession numbers are incorporated herein by reference.

[0164] In addition to the foregoing, the term "triose phosphate isomerase" or "Tpi" refers to a protein capable of catalyzing the formation of glyceraldehyde-3-phosphate from dihydroxyacetone phosphate (DHAP), and having at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher sequence identity or at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or higher sequence similarity with SEQ ID NO: 36 (calculated using the default parameters of NCBI BLAST). Other homologs include: Rattus norvegicus AAA42278.1, which has 45% identity with SEQ ID NO: 36; Homo sapiens AAH17917.1, which has 45% identity with SEQ ID NO: 36; Bacillus subtilis BEST7613 NP_391272.1, which has 40% identity with SEQ ID NO: 36; Synechococcus elongatus PCC6301 YP_171000.1, which has 40% identity with SEQ ID NO: 36; and Salmonella enterica subsp. enterica serovar Typhi strain AG3 ZP_06540375.1, which has 98% identity with SEQ ID NO: 36. Accordingly, the present disclosure includes the use of polypeptides having 40% to 100% identity with SEQ ID NO: 36 and having triose phosphate isomerase activity. The sequences associated with the foregoing accession numbers are incorporated herein by reference.

[0165] In a further step of the pathway, glyceraldehyde-3-phosphate is capable of being converted to 1,3-bisphosphoglycerate. In one embodiment, the enzymatic step includes a "scavenging valve system" (discussed elsewhere herein). For example, glyceraldehyde-3-phosphate dehydrogenase (Gap, Tdh) converts glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate. In one embodiment, wild-type Gap using NAD + as a cofactor (see, for example, SEQ ID NO: 37) or mutant Gap containing the P191D mutation (relative to the sequence of SEQ ID NO: 37 and as shown in SEQ ID NO: 38) is used. In another embodiment, NADP +A mutant Gap (mGap) as a cofactor; for example, having D34A / L35R / T35K mutations; relative to the SEQ ID NO: 37 sequence and as shown in SEQ ID NO: 39). In yet another embodiment, a combination of Gap and mGap (GapM6) is used. The molecular scavenging valve includes a water-producing NADH oxidase (NoxE), which specifically oxidizes NADH but not NADPH and is capable of being used for recycling ("scavenging") NADH when using the wild-type gap or the P118D mutant gap that preferentially uses NAD + When used, it can be used for recycling.

[0166] In addition to the foregoing, the term "NADH oxidase" or "NoxE" refers to a protein capable of oxidizing NADH to NAD*, and having at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher sequence identity or at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or higher sequence similarity with SEQ ID NO: 18 (calculated using the default parameters of NCBI BLAST).

[0167] This pathway can further utilize phosphoglycerate kinase (EC 2.7.2.3) (PGK; for example, provided in SEQ ID NO: 40, or its homologs or variants having at least 80% identity therewith) to convert 1,3-bisphosphoglycerate to 3-phosphoglycerate. This kinase catalyzes the reversible transfer of a phosphate group from 1,3-bisphosphoglycerate (1,3-BPG) to ADP, thereby producing 3-phosphoglycerate (3-PG) and ATP. An ATP molecular scavenging valve can be present to recycle ADP (for example, using a GTPase or other enzyme or its homologs or variants).

[0168] Then, 3-phosphoglycerate can be converted to 2-phosphoglycerate by phosphoglycerate mutase (pgm; for example, as provided in SEQ ID NO: 41, or its homologs or variants having at least 80% identity therewith).

[0169] Then, enolase (eno; for example, provided in SEQ ID NO: 42, or its homologs or variants having at least 80% identity therewith) is capable of converting 2-phosphoglycerate to phosphoenolpyruvate (PEP).

[0170] Pyruvate kinase (pyk; for example, provided in SEQ ID NO: 43, 44, and 45, or its homologs or variants having at least 80% identity with any one of SEQ ID NO: 43, 44, or 45) converts PEP to pyruvate.

[0171] As described above, pyruvate dehydrogenase (PDH) is inhibited by the products of this pathway. Thus, the PDH bypass can be used to convert pyruvate to acetyl-CoA. The PDH bypass comprises two enzymatic steps: (i) pyruvate oxidase (e.g., PyOx from Aerococcus viridans; EC 1.2.3.3; see SEQ ID NO: 46) catalyzes pyruvate → acetyl phosphate; and (ii) acetyl phosphate transferase (also known as phosphoacetyltransferase) (e.g., PTA from Geobacillus stearothermophilus) catalyzes acetyl phosphate → acetyl-CoA.

[0172] As used herein, the 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.

[0173] Phosphotransacetylase (EC 2.3.1.8) is an enzyme that catalyzes the chemical reaction of acetyl-CoA + phosphate to CoA + acetyl phosphate and vice versa. Phosphotransacetylase is encoded by pta in Escherichia coli. PTA is involved in the conversion of acetate to acetyl-CoA. Specifically, PTA catalyzes the conversion of acetyl-CoA to acetyl phosphate. PTA homologs and variants are known. There are approximately 1,075 bacterial phosphotransacetylases on NCBI. For example, such homologs and variants include phosphotransacetylase Pta (Rickettsia felis URRWXCal2) gi|670040211|gb|AAY60947.11(67004021); phosphotransacetylase (Buchnera aphidicola Cc (Cinara cedri)) strain gi|116256910|gb|ABJ90592.1|(116256910); pta (Buchnera aphidicola Cc (Cinara cedri)) strain gi|116515056|ref|YP_802685.1|(116515056); pta (endosymbiont of Glossina brevipalpis - Wigglesworthia glossinidia) |25166135|dbj|Bac24326.1|(25166135); Pta (Pasteurella multocida subsp. multocida Pm70 strain) gi|12720993|gb|AAK02789.1|(12720993); Pta (Rhodospirillum rubrum) gi|25989720|gb|AAN75024.11(25989720); pta (Listeria welshimeri serotype 6b strain SLCC5334) gi|116742418|emb|CAK21542.1|(116742418); Pta (Mycobacterium avium subsp. paratuberculosis K-10) gi|41398816|gb|AAS06435.1|(41398816); phosphotransacetylase (pta) (Borrelia burgdorferi B31) gi|15594934|ref|NP_212723.1|(15594934); Phosphoacetyltransferase (pta) (Borrelia burgdorferi B31) gi|2688508|gb|AAB91518.1|(2688508); Phosphoacetyltransferase (pta) (Haemophilus influenzae Rd KW20) gi|1574131|gb|AAC22857.1|(1574131); Phosphoacetyltransferase Pta (Rickettsia bellii RML369-C) gi|91206026|ref|YP_538381.1|(91206026); Phosphoacetyltransferase Pta (Rickettsia bellii) RML369-C) gi|91206025|ref|YP_538380.1|(91206025); Phosphoacetyltransferase pta (Mycobacterium tuberculosis F11) gi|148720131|gb|ABR04756.1|(148720131); Phosphoacetyltransferase pta (Mycobacterium tuberculosis Haarlem strain) gi|134148886|gb|EBA40931.11(134148886); Phosphoacetyltransferase pta (Mycobacterium tuberculosis C) gi|124599819|gb|EAY58829.1|(124599819); Phosphoacetyltransferase Pta (Rickettsia bellii RML369-C) gi|91069570|gb|ABE05292.1|(91069570); Phosphoacetyltransferase Pta (Rickettsia bellii RML369-C) gi|91069569|gb|ABE05291.1|(91069569); Phosphoacetyltransferase (pta) (Treponema pallidum subsp. pallidum Nichols strain) gi|15639088|ref|NP_218534.1|(15639088); and Phosphoacetyltransferase (pta) (Treponema pallidum subsp. pallidum Nichols strain) gi|3322356|gb|AAC65090.11(3322356), each of the sequences associated with the accession numbers is incorporated herein by reference in its entirety.

[0174] Go back to Figure 2C, this pathway includes the conversion of acetyl-CoA to acetoacetyl-CoA. The conversion of acetyl-CoA to acetoacetyl-CoA is accomplished by acetyl-CoA acetyltransferase (such as PhaA). A variety of acetyl-CoA acetyltransferases are known in the art. For example, the acetyl-CoA acetyltransferase from Ralstonia eutropha. In another embodiment, the acetyl-CoA acetyltransferase has an amino acid sequence that is at least 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO: 47.

[0175] Acetoacetyl-CoA and acetyl-CoA can be converted to HMG-CoA by an enzyme HMG-CoA synthase with an A110G mutation (see, for example, SEQ ID NO: 48) or its homologs or variants having at least 85%, 90%, 95%, 98% or 99% (such as 85% to 95%) sequence identity thereto.

[0176] Then, HMG-CoA is reduced to mevalonic acid by the action of NADPH and HMG-CoA reductase (see, for example, SEQ ID NO: 49) or its homologs or variants having at least 85%, 90%, 95%, 98% or 99% (such as 85% to 95%) sequence identity thereto.

[0177] Then, mevalonic acid is phosphorylated by the action of ATP and mevalonate kinase (MVK) to produce mevalonate-5-phosphate and ADP. Mevalonate kinase is known in the art and includes sequences that are at least 85% to 100% (such as 85%, 90%, 95%, 98%, 99%) identical to the sequence of SEQ ID NO: 50 and have mevalonate kinase activity.

[0178] Mevalonate-5-phosphate is further phosphorylated by the action of ATP and phosphomevalonate kinase (PMVK) to produce mevalonate-5-diphosphate and ADP. Phosphomevalonate kinase is known in the art and includes sequences that are at least 85% to 100% (such as 85%, 90%, 95%, 98%, 99%) identical to the sequence of SEQ ID NO: 51 and have phosphomevalonate kinase activity.

[0179] Mevalonate-5-diphosphate is decarboxylated by the action of ATP and mevalonate diphosphate decarboxylase (MDC) to produce ADP, CO2 and isopentenyl pyrophosphate. Mevalonate diphosphate decarboxylase is known in the art and includes sequences that are at least 85% to 100% (such as 85%, 90%, 95%, 98%, 99%) identical to the sequence of SEQ ID NO: 52 and have mevalonate diphosphate kinase activity.

[0180] Any of a variety of other mevalonate pathways can be used (see, e.g., Figure 8 ).

[0181] Then, geranyl pyrophosphate (GPP) is formed from a combination of DMAPP and isopentenyl pyrophosphate in the presence of a 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 is capable of forming geranyl pyrophosphate from DMAPP and isopentenyl pyrophosphate.

[0182] Then, GPP can be used as a substrate for a variety of pathways to produce prenylflavonoids, geranylflavonoids, prenylstilbenes, geranylstilbenes, CBGA, CBGVA, CBDA, CBDVA, CBGVA, CBCVA, THCA and THCVA (see, e.g., Figures 2A to 2B ).

[0183] For example, using the available NphB mutants as described above (e.g., the M23 mutant), the ability to produce CBGA directly from glucose and OA was tested using a complete synthetic biochemical system including the PDH bypass (see Figures 2A to 2B and Figure 3C ). The initial productivity using M23 in this system was 67 mg L -1 hr -1 , and the final titer was 744 ± 34 mg L -1 CBGA. This is 100 times the rate of CBGA production using WT NphB and 21 times the titer. Notably, when using the mutant NphB enzyme, the maximum titer was reached within 24 hours and production stopped, but when using the wild-type enzyme, the system ran continuously for up to 4 days, indicating that the enzyme and cofactors remained active and viable for a longer period. Notably, once approximately 500 mg L -1 CBGA was produced, the reaction became turbid. The precipitate was collected and an enzyme mixture was identified in the precipitate by SDS-PAGE analysis, indicating that the high concentration of CBGA in the solution caused enzyme precipitation. We developed a more efficient system to remove the product during the reaction.

[0184] Although a nonane overlay was used in the reaction to extract CBGA, the solubility of CBGA in water is higher than that in nonane, which limits the amount of CBGA that can be extracted with a simple overlay. Therefore, a flow system was designed that captures CBGA from the nonane layer and traps it in a separate reservoir ( Figure 5B) By implementing this flow system, a lower CBGA concentration is maintained in the reaction vessel to mitigate enzyme precipitation. The flow system does increase the final titer to 1.2 g / L.

[0185] Then experiments were conducted to produce precursors of many rare cannabinoids CBGVA by replacing OA in the system with 2,4-dihydroxy-6-propylbenzoic acid (divirinic acid, DA) (for example, see Figure 2C ). The designed enzymes were first tested to determine whether they were active against the DA substrate. The ability of the two best mutants M23 and M31, as well as WT NphB, to produce CBGVA was tested. The kinetic data shown in Table 2 indicate that M31 performs more excellently, with a catalytic efficiency 15 times that of M23 and 650 times that of WT NphB. Therefore, further efforts were made to utilize M31 to produce CBGVA from glucose and 2,4-dihydroxy-6-propylbenzoic acid. As Figure 5A shown, the maximum productivity of CBGVA is approximately 107 mgL -1 hr -1 , and the final titer reaches 1.74 ± 0.09 gL -1 , converting 92% of the added 2,4-dihydroxy-6-propylbenzoic acid into CBGVA. Since the precipitation enzyme potency of CBGVA is weak, a nonane flow system is not required for the production of CBGVA.

[0186] To demonstrate that this method can ultimately be used to prepare other cannabinoids, CBDA synthase was used to convert CBGA to CBDA and CBGVA to CBDVA. For CBDA, the nonane overlay contained a large amount of CBGA, so simply transferring the nonane overlay to a solution containing CBDA synthase, CBGA would be converted to CBDA at a constant rate of 14.4 ± 0.8 mgL -1 hr -1 mg -1 total protein -1 in four days.

[0187] Due to the limited solubility of CBGVA in nonane, CBGVA was extracted and added to the reaction containing CBDA synthase. Using GC-MS, the product of CBDA synthase is actually CBDVA.

[0188] Accordingly, the present disclosure provides a cell-free system for the production of GPP. Additionally, the present disclosure provides cell-free methods for producing a range of pure cannabinoids and other prenylated natural products using the GPP pathway in combination with mutant NphB or using the substrates of the mutant NphB of the present disclosure. The success of this method lies in the utilization of the engineered prenyltransferase of the present disclosure (e.g., the NphB mutant described above), which is active, highly specific, and does not require a native transmembrane prenyltransferase. The modularity and flexibility of the synthetic biochemistry platform provided herein have the advantages of a biobased approach but eliminate the complexity of meeting the requirements of a living system. For example, GPP toxicity was not incorporated into the design process. Additionally, OA is not taken up by yeast, so methods of adding OA exogenously in cells may not necessarily be feasible. In fact, the flexibility of the cell-free system greatly facilitates the design-build-test cycles required for further optimization, additional pathway enzymes, and reagent and cofactor modifications.

[0189] Turning Figures 2A to 2C to the overall pathway in, the present disclosure provides multiple steps of enzyme-catalyzed conversion of a "substrate" to a product. In some cases, the steps may utilize cofactors, but some steps do not use cofactors (e.g., NAD(P)H, ATP / ADP, etc.). Table 3 provides a list of the enzymes, organisms, reaction amounts, and accession numbers used (the sequences associated with these accession numbers are incorporated herein by reference).

[0190] Table 3

[0191]

[0192]

[0193] As described above, the prenylation of OA by GPP is carried out by the activity of the mutant NphB polypeptide described herein and above.

[0194] In one embodiment, the pathway of the present 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 derived from Geobacillus stearothermophilus.

[0195] The present disclosure provides a method for in vitro production of prenylated compounds, and also provides a method for in vitro production of cannabinoids and cannabinoid precursors (e.g., CBGA, CBGVA or CBGXA, where "X" refers to any chemical group). In one embodiment, a cell-free preparation can be made by, for example, three methods. In one embodiment, enzymes of the pathways described herein are purchased and mixed in a suitable buffer, and then a suitable substrate is added and incubated under conditions suitable for producing prenylated compounds, cannabinoids or cannabinoid precursors. In some embodiments, the enzymes can be bound to a support or expressed in a phage display or other surface expression system, and, for example, immobilized in a fluid pathway corresponding to a point in the metabolic pathway cycle.

[0196] Figure 6A To B describes the 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 uses type III polyketide synthase (PKS) to convert hexanoyl-CoA and malonyl-CoA (derived from acetyl-CoA) into oleic acid (OA). The cannabinoid module uses the products of the isoprenoid module and the polyketide module to produce cannabigerolic acid, which is then converted into the final cannabinoid by cannabinoid synthase.

[0197] Figure 27A An embodiment of the cell-free biosynthetic pathway of deuterated cannabinoids is shown. Unlabeled malonic acid and deuterated fatty acids (e.g., fully deuterated, partially deuterated) are activated to the corresponding CoA thioesters. The deuterated fatty acid d11-hexanoic acid is activated to d11-hexanoyl-CoA in the presence of acyl-activating enzyme (AAE) 3, while malonic acid is activated to malonyl-CoA in the presence of malonyl-CoA synthetase. Then, the CoA thioesters are elongated and cyclized to produce d11-oleic acid (d11-OA). In Figure 27A the example shown, the deuterated fatty acid is d11-hexanoic acid. The present invention is compatible with alternative deuterated fatty acids. For example but not limited to, if d7-butyric acid is the deuterated fatty acid, d7-2,4-dihydroxy-6-propylbenzoic acid (d7-DA) can be produced using the same method. Thus, activating deuterated fatty acids to the corresponding CoA thioesters for incorporation into small molecules or for late-stage modification thereof is a viable general method.

[0198] After the formation of d11-OA, in the presence of geranyl pyrophosphate (GPP), prenylation can occur through the action of CsPT or engineered NphB enzymes to generate the deuterated cannabinoid d11-cannabigerolic acid (d11-CBGA). Then, d11-CBGA can be cyclized through the action of tetrahydrocannabinolic acid synthase (THCAS) to form d11-tetrahydrocannabinolic acid (d11-THCA). Alternative deuterated cannabinoids can be synthesized using alternative cannabinoid synthases (e.g., CBDAS, CBCAS). Those of ordinary skill in the art will understand that using cannabidiolic acid synthase (CBDAS) will result in the production of deuterated cannabinoids in the form of cannabidiolic acid (CBDA) (e.g., d11-CBDA) rather than in the form of tetrahydrocannabinolic acid. Similarly, using cannabichromenic acid synthase (CBCAS) will result in the production of deuterated cannabinoids in the form of cannabichromenic acid (CBCA) (e.g., d11-CBCA).

[0199] Figure 27B An embodiment of the cell-free biosynthetic pathway of deuterated cannabinoids according to one embodiment of the present invention is shown, including a pathway for deriving geranyl pyrophosphatase (GPP). Hydroxyethyl thiazole kinase (ThiM) is used to produce isopentenyl phosphate from 3-methyl-3-buten-1-ol (isoporenol). In the presence of isopentenyl diphosphate isomerase, isopentenyl phosphate is converted to isopentenyl diphosphate. Isopentenyl diphosphate can be converted to dimethylallyl pyrophosphate (DMAPP). In the presence of farnesyl pyrophosphate synthase (FPPS), both isopentenyl diphosphate and DMAPP can be converted to geranyl polyphosphate. Then, geranyl polyphosphate can be used in the cell-free biosynthetic pathway described herein. Those of ordinary skill in the art will understand that this process is described in further detail in Figure 7 although it is shown here to illustrate the steps of the cell-free biosynthetic pathway that utilizes geranyl polyphosphate during production.

[0200] In one embodiment, after the formation of the THCA compound of the present invention, the compound is purified to isolate the resulting THCA, as disclosed herein. After purification, THCA can be decarboxylated and purified to obtain THC.

[0201] Figure 28A An embodiment of the deuterated methyl compound is shown. Deuterated d3-S-adenosylmethionine (d3-SAM) can be generated from d3-L-methionine and ATP by the action of SAM synthase (MAT). As Figure 28B shown, d3-SAM can be used by methyltransferases to methylate various small molecules.

[0202] Figure 29Examples of deuteromethylated psilocybin using a specific methyltransferase (e.g., PsiM methyltransferase) are shown.

[0203] Figure 30 Examples of deuteromethylated chrysoeriol using a specific methyltransferase (e.g., CsOMT21 methyltransferase) are shown.

[0204] Figure 31 Examples of the synthesis of deuterated glucosides using UDP-glucose-d12 are shown.

[0205] Figure 32 HPLC (High Performance Liquid Chromatography) traces of cell-free production of deuterated cannabinoids (1), even-chain cannabinoids (2), branched-chain cannabinoids (3), and rare cannabinoids (4) (e.g., CBG derivatives) are shown.

[0206] Figure 33 A bar chart summarizing the progress made during pathway optimization according to one embodiment of the present invention is shown. All reactions were completed at a 200 μL scale, except for the 1 L scale reaction indicated by the bar farthest to the right of the figure (so labeled).

[0207] Figures 34A to 34B CBGA production of OLS variants is shown. Each variant was screened to determine the CBGA production level of the OLS variant. The OLS variants were observed at different enzyme loadings to determine the corresponding CBGA production levels.

[0208] Figure 35 The time course of the CBGA synthesis reaction according to one embodiment of the present invention is shown. Figure A depicts the time course of the CBGA synthesis reaction using OLS M187T at time points of 0 h, 2 h, 4 h, 6 h, and 20 h. Slight accumulation of oleic acid was observed after 4 h, indicating that SimplePath or NphB may limit the reaction. Figure B depicts the 20 h trace of the OLS variant, showing differences in byproduct formation between WT, M187T, and P131A M187T.

[0209] Figure 36 HPLC traces of CBGA and THCA activities according to one embodiment of the present invention are shown. HPLC analysis of the 1 L CBGA reaction, conversion of THCAS supernatant using Pichia pastoris to THCA, methanol extraction of the THCA reaction, ethanol precipitation of the methanol extract, and purified THCA trace. With increasing purity, there is a significant difference in the ABS at 216 nm, allowing visualization / quantification of impurities.

[0210] Figure 37HPLC traces of THC in toluene according to one embodiment of the present invention are shown. 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 ten minutes is shown by the HPLC trace below it. The reaction after thirty-five minutes is shown below the ten-minute reaction, with the recovered THC product indicated by the bottommost HPLC trace.

[0211] One of ordinary skill in the art will appreciate that alternative starting materials and enzymes can be used to produce alternative products.The present invention is not limited to the examples provided herein.

[0212] In another embodiment, one or more polynucleotides encoding one or more enzymes of the pathway are cloned into one or more microorganisms under conditions where the enzymes are expressed. Subsequently, the cells are lysed, and a lysis preparation comprising one or more enzymes derived from the cells is combined with a suitable buffer and substrate (and one or more additional enzymes in the pathway, if desired) to produce a prenylated compound, cannabinoid, or cannabinoid precursor. Alternatively, the enzyme can be isolated from the lysis preparation and then recombined in a suitable buffer. In another embodiment, a combination of purchased and expressed enzymes is used to provide the pathway in a suitable buffer. In one embodiment, the thermostable polypeptide / enzyme of the pathway is cloned and expressed. In one embodiment, the enzymes of the pathway are derived from thermophilic microorganisms. The microorganism is then lysed and the preparation is heated to a temperature where the thermostable polypeptides of the pathway are active, while other polypeptides (not of interest) are denatured and become inactive. The preparation therefore comprises a subset of all enzymes in the microorganism and comprises an active thermostable enzyme. The preparation can then be used to execute a pathway that produces a prenylated compound, cannabinoid, or cannabinoid precursor.

[0213] For example, to construct an in vitro system, all enzymes can be purchased commercially or purified by affinity chromatography, tested for activity, and mixed together in an appropriately chosen reaction buffer.

[0214] Also contemplated are in vivo systems utilizing all or part of the aforementioned enzymes in a biosynthetic pathway and engineering the aforementioned enzymes into a microorganism to obtain a recombinant microorganism.

[0215] The present disclosure also provides recombinant organisms comprising a metabolically engineered biosynthetic pathway including a mutant nphB for producing a prenylated compound, and optionally further comprises one or more additional organisms expressing enzymes for producing a cannabinoid (e.g., a co-culture of one group of microorganisms expressing a portion of a pathway with a second group of microorganisms expressing another portion or remainder of the pathway, etc.).

[0216] In one embodiment, the present disclosure provides a recombinant microorganism that includes at least one target enzyme with increased expression compared to a parental microorganism or an enzyme not found in the parental organism. In another or further embodiment, the microorganism provides for a reduction, disruption, or knockout of at least one gene encoding such an enzyme that competes with a metabolite necessary for the production of a desired metabolite or produces an unwanted product. In one embodiment, the recombinant microorganism expresses such an enzyme that produces at least one metabolite involved in a biosynthetic pathway for the production of, for example, an isoprenylated compound or a cannabinoid or a cannabinoid precursor. Generally, the recombinant microorganism includes at least one recombinant metabolic pathway that includes the target enzyme and optionally also includes a reduction in the activity or expression of an enzyme in a competing biosynthetic pathway. The role of the pathway is to modify a substrate or a metabolic intermediate in the production of, for example, an isoprenylated compound, a cannabinoid, or a cannabinoid precursor. The target enzyme is encoded and expressed by a polynucleotide from a suitable biological source. In some embodiments, the polynucleotide includes a gene from a bacterial or yeast source and is recombinantly engineered into the microorganism of the present disclosure. In another embodiment, the polynucleotide encoding the desired target enzyme is naturally present in an organism but is recombinantly engineered to be overexpressed compared to the native expression level.

[0217] The term "microorganism" includes prokaryotic and eukaryotic microbial species from the domains Archaea, Bacteria, and Eukarya, the latter including yeasts and filamentous fungi, protozoa, algae, or more advanced protists. The terms "microbial cell" and "microbial type" are used interchangeably with the term microorganism.

[0218] The term "prokaryote" is well known in the art and refers to cells that do not contain a nucleus or other organelles. Prokaryotes are generally divided into two domains: the domain Bacteria and the domain Archaea. The defining difference between organisms in the domains Archaea and Bacteria is based on fundamental differences in the nucleotide base sequences in 16S ribosomal RNA.

[0219] "Bacteria" or "eubacteria" refers to the domain Bacteria. Bacteria include at least the following 11 different groups: (1) Gram-positive bacteria (gram+), which can be mainly divided into two major categories: (1) the high G+C group (actinomycetes, mycobacteria, micrococci, etc.) (2) the low G+C group (bacilli, clostridia, lactobacilli, staphylococci, streptococci, mycoplasmas); (2) proteobacteria, such as purple photosynthetic + non-photosynthetic Gram-negative bacteria (including the most "common" Gram-negative bacteria); (3) cyanobacteria, such as oxygenic phototrophs; (4) spirochetes and related species; (5) planctomycetes; (6) bacteroides, flavobacteria; (7) chlamydiae; (8) green sulfur bacteria; (9) green non-sulfur bacteria (also including anaerobic phototrophs); (10) radiation-resistant micrococci and their relatives; and (11) Thermotoga and Thermosipho thermophile.

[0220] "Gram-negative bacteria" include cocci, non-enteric bacilli, and enteric bacilli. Gram-negative genera include, for example, Neisseria, Spirillum, Pasteurella, Brucella, Yersinia, Francisella, Haemophilus, Bordetella, Escherichia, Salmonella, Shigella, Klebsiella, Proteus, Vibrio, Pseudomonas, Bacteroides, Acetobacter, Aerobacter, Agrobacterium, Azotobacter, Spirillum, Serratia, Vibrio, Rhizobium, Chlamydia, Rickettsia, Treponema, and Fusobacterium.

[0221] "Gram-positive bacteria" include cocci, non-spore-forming bacilli, and spore-forming bacilli. Gram-positive genera include, for example, Actinomyces, Bacillus, Clostridium, Corynebacterium, Erysipelothrix, Lactobacillus, Listeria, Mycobacterium, Myxococcus, Nocardia, Staphylococcus, Streptococcus, and Streptomyces.

[0222] As used herein, the "activity" of an enzyme is a measure of its ability to catalyze a reaction that produces a metabolite (i.e., "function"), and can be expressed as the production rate of the reaction metabolite. For example, enzyme activity can be expressed as the amount of metabolite produced per unit time or per unit enzyme (e.g., concentration or weight), or as an affinity or dissociation constant.

[0223] The term "biosynthetic pathway", also known as "metabolic pathway", refers to a set of anabolic or catabolic biochemical reactions used to convert (transmute) one chemical substance into another (see, for example Figures 2A to 2C ). If gene products act on the same substrate in parallel or in series, produce the same product, or act on or produce a metabolite that is an intermediate between the same substrate and the metabolic end product (i.e., metabolite), then these gene products belong to the same "metabolic pathway". The present disclosure provides recombinant microorganisms having metabolically engineered pathways for producing desired products or intermediates.

[0224] Thus, metabolically "engineered" or "modified" microorganisms are produced by introducing genetic material into a selected host or parental microorganism, thereby modifying or altering the cellular physiology and biochemistry of the microorganism. By introducing the genetic material, the parental 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 illustrative embodiment, introducing the genetic material into the parental microorganism results in a new or modified ability to produce acetyl phosphate and / or acetyl-CoA via the PDH bypass using pyruvate oxidase and acetyl phosphate transferase. The genetic material introduced into the parental microorganism comprises a gene or portion of a gene that encodes one or more enzymes involved in the biosynthetic pathway for producing isoprenoid compounds or cannabinoids or cannabinoid precursors, and the genetic material can further include additional elements for expressing and / or regulating the expression of these genes, such as promoter sequences.

[0225] Instead of or in addition to introducing genetic material into a host or parental microorganism, engineered or modified microorganisms can also include disrupting, deleting, or knocking out genes or polynucleotides to alter the cellular physiology and biochemistry of the microorganism. By reducing, disrupting, or knocking out genes or polynucleotides, the microorganism acquires new or enhanced properties (e.g., the ability to produce new or increased amounts of intracellular metabolites, enhanced flux of metabolites along a desired pathway, and / or reduced production of unwanted by-products) or eliminates enzymes from cell-free preparations that can compete with the biosynthetic pathway formed by the lysis preparation.

[0226] "Enzyme" refers to any substance that is generally composed of amino acids that make up a protein or polypeptide, and that more or less specifically catalyzes or facilitates one or more chemical or biochemical reactions.

[0227] The terms "protein" or "polypeptide" are used interchangeably herein and include a chain of one or more chemical building blocks called amino acids that are joined together by a chemical bond called a peptide bond. A protein or polypeptide can function as an enzyme.

[0228] As used herein, the term "metabolic engineering" or "metabolic engineering modification" involves rational pathway design and the assembly of biosynthetic genes, genes associated with operons, and control elements of such polynucleotides for the production of desired metabolites in a microorganism, such as acetyl phosphate and / or acetyl-CoA, higher alcohols, or other chemicals. "Metabolic engineering" can further include the regulation and optimization of metabolic flux by using genetic engineering and appropriate culture conditions (including reducing, disrupting, or knocking out competing metabolic pathways that compete with intermediates leading to the desired pathway) to regulate and optimize transcription, translation, protein stability, and protein function. Biosynthetic genes can be modified by being foreign to the host or by mutagenesis, recombination, and / or binding to heterologous expression control sequences in the endogenous host cell, such that they are heterologous to the host microorganism. In one embodiment, when a polynucleotide is xenogeneic to a host organism, the polynucleotide can be codon-optimized.

[0229] "Metabolite" refers to any substance produced by metabolism, or a substance that is necessary for or participates in a specific metabolic process that produces a desired metabolite, chemical, alcohol, or ketone. A metabolite can be an organic compound, such as a starting material for metabolism (e.g., glucose, etc.), an intermediate (e.g., acetyl-CoA), or a final product (e.g., CBDA). Metabolites can be used to build more complex molecules or can be broken down into simpler molecules. Intermediate metabolites can be synthesized from other metabolites, may be used to make more complex substances, or can be broken down into simpler compounds, usually with the release of chemical energy.

[0230] "Mutation" refers to any process or mechanism that results in a mutated protein, enzyme, polynucleotide, gene, or cell. This includes any mutation in which the sequence of a protein, enzyme, polynucleotide, or gene is altered, as well as any detectable change in a cell caused by such a mutation. Generally, mutations occur in a polynucleotide or gene sequence by point mutation, deletion, or insertion of single or multiple nucleotide residues. 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 sequences or promoter sequences. A gene mutation can be "silent", i.e., it does not reflect an amino acid change upon expression, resulting in a "sequence-conserved" variant of the gene. This typically occurs when more than one codon corresponds to an amino acid. Mutations that result in a different primary sequence of a protein can be referred to as mutant proteins or protein variants.

[0231] A "native" or "wild-type" protein, enzyme, polynucleotide, gene, or cell refers to a protein, enzyme, polynucleotide, gene, or cell that exists in nature.

[0232] "Parental microorganism" refers to the cell used to generate a recombinant microorganism. In one embodiment, the term "parental microorganism" describes a cell existing in nature, i.e., a "wild-type" cell that has not been genetically modified. The term "parental microorganism" further describes a cell that serves as a "parent" for further engineering. In the latter embodiment, the cell may have been genetically engineered and serves as a source for further genetic engineering.

[0233] For example, a wild-type microorganism can be genetically modified to express or overexpress a first target enzyme, such as hexokinase. This microorganism can serve as a parental microorganism for generating a microorganism modified to express or overexpress a second target enzyme (e.g., fructose-1,6-bisphosphate aldolase). Subsequently, this microorganism can be modified to express or overexpress, for example, NADH oxidase and Gald-3-phosphate dehydrogenase (and its mutants), which can be further modified to express or overexpress a third target enzyme, such as phosphoglycerate kinase, etc. As used herein, "express" or "overexpress" refers to the phenotypic expression of the desired gene product. In one embodiment, a gene naturally present in an organism can be engineered such that it is linked to a heterologous promoter or regulatory domain, where the regulatory domain causes gene expression, thereby altering its normal expression relative to the wild-type organism. Alternatively, an organism can be engineered to remove or reduce a repressive function on a gene, thereby modifying its expression. In another embodiment, a cassette containing a gene sequence capable of being linked to the desired expression control / regulatory elements is engineered into the microorganism.

[0234] Thus, the parental microorganism serves as a reference cell for successive genetic modification events. Each modification event can be accomplished by introducing one or more nucleic acid molecules into the reference cell. The introduction promotes the expression or overexpression of one or more target enzymes, or facilitates the reduction or elimination of one or more target enzymes. It should be understood that the term "promote" encompasses activating an endogenous polynucleotide encoding a target enzyme through genetic modification (e.g., promoter sequence) in the parental microorganism. It is further understood that the term "promote" encompasses introducing an exogenous polynucleotide encoding a target enzyme into the parental microorganism.

[0235] Polynucleotides encoding enzymes for producing metabolites (including homologs, variants, fragments, related fusion proteins, or functional equivalents thereof) are used in recombinant nucleic acid molecules for directing the expression of such polypeptides in a suitable host cell (e.g., a bacterial or yeast cell). The sequences and accession numbers provided herein enable those skilled in the art to obtain and acquire the coding sequences of the various enzymes of the present disclosure using readily available software and basic biological knowledge.

[0236] The accompanying Sequence Listing provides exemplary polypeptides for the methods described herein. It should be understood that adding sequences that do not alter the activity of the polypeptide molecule, such as adding non-functional or non-coding sequences (e.g., polyHIS tags), are conservative variations of the underlying molecule.

[0237] It should be understood that the polynucleotides described herein include "genes", and the above nucleic acid molecules include "vectors" or "plasmids".

[0238] The terms "polynucleotide", "nucleic acid", or "recombinant nucleic acid" refer to polynucleotides, such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA).

[0239] For a gene or polynucleotide, the term "expression" refers to the transcription of the gene or polynucleotide, and, where appropriate, the translation of the resulting mRNA transcript into a protein or polypeptide. Thus, it will be clear from the context that the expression of a protein or polypeptide is caused by the transcription and translation of an open reading frame.

[0240] Those skilled in the art will recognize that due to the degenerate nature of the genetic code, a variety of nucleotide sequence-different codons can be used to encode a given amino acid. The specific polynucleotide or gene sequences cited herein encoding the above biosynthetic enzymes or polypeptides are merely for illustration of the embodiments of the present disclosure, and the present disclosure includes polynucleotides of any sequence encoding polypeptides having the same amino acid sequences as the polypeptides and proteins of the enzymes used in the methods of the present disclosure. In a similar manner, a polypeptide can generally tolerate one or more amino acid substitutions, deletions, and insertions in its amino acid sequence without losing or significantly losing the required activity. The present disclosure includes such polypeptides having alternative amino acid sequences, and the amino acid sequences encoded by the DNA sequences shown herein merely illustrate exemplary embodiments of the present disclosure.

[0241] 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 replicate either in the cytoplasm of the host microorganism or can integrate into the chromosomal DNA of the host microorganism. In either case, the vector can be a stable vector (i.e., the vector remains present after multiple cell divisions even only under selective pressure) or a transient vector (i.e., the vector is gradually lost by the host microorganism as the number of cell divisions increases). The present disclosure provides DNA molecules in isolated form (i.e., not pure, but present in a preparation at an abundance and / or concentration not found in nature) and in purified form (i.e., substantially free of contaminants or substantially free of the substances in which the corresponding DNA exists in nature).

[0242] The polynucleotides of the present disclosure can be amplified using cDNA, mRNA, or alternatively 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 acids so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. In addition, oligonucleotides corresponding to the nucleotide sequences can be prepared by standard synthetic techniques, such as using an automated DNA synthesizer.

[0243] The present disclosure provides a variety of polypeptide sequences in the attached sequence listing of the present application, which 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 publicly available databases.

[0244] It should also be 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 in the nucleotide sequence encoding a particular polypeptide, thereby introducing one or more amino acid substitutions, additions, or deletions in the encoded protein. Mutations can be introduced into the polynucleotide by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. In certain positions, conservative amino acid substitutions are preferred, as opposed to positions where non-conservative amino acid substitutions are required.

[0245] Those skilled in the art will understand that it can be advantageous to modify the coding sequence to enhance its expression in a particular host. The genetic code is redundant, with 64 possible codons, but most organisms typically use a subset of these codons. The most commonly used codons in a species are referred to as optimal codons, while less commonly used codons are classified as rare codons or low-usage codons. Codons can be substituted to reflect the codon usage preferred by the host, a process sometimes referred to as "codon optimization" or "controlling codon bias of the species."

[0246] Optimized coding sequences can be prepared that contain codons preferred by a particular prokaryotic or eukaryotic host (see also Murray et al. (1989) Nucl. Acids Res. 17:477-508, which is incorporated herein by reference in its entirety), for example, to increase translation rates or to produce recombinant RNA transcripts with desired properties (such as a longer half-life compared to transcripts produced from non-optimized sequences). Translation termination codons can also be modified to reflect host preferences. For example, the typical termination codons for Saccharomyces cerevisiae and mammals are UAA and UGA, respectively. The typical termination codon for monocotyledonous plants is UGA, while insects and Escherichia coli typically use UAA as the termination codon (Dalphin et al. (1996) Nucl. Acids Res. 24:216-218, which is incorporated herein by reference in its entirety). For example, U.S. Patent No. 6,015,891 and the references cited therein provide methods for optimizing nucleotide sequences for expression in plants. The entire content of U.S. Patent No. 6,015,891 is incorporated herein by reference.

[0247] The term "substrate" or "suitable substrate" refers to any 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, and other materials containing at least one substrate, or derivatives thereof. In addition, the term "substrate" encompasses not only the compound that provides the starting material, but also intermediates and end-product metabolites used in the pathways associated with the metabolic engineering microorganisms described herein.

[0248] "Transformation" refers to the process of introducing a vector into a host cell. Transformation (or transduction, transfection) can be achieved by any of a variety of means, including electroporation, microinjection, gene gun (or particle bombardment-mediated delivery), or Agrobacterium-mediated transformation.

[0249] A "vector" generally refers to a polynucleotide that is capable of replicating and / or transferring between organisms, cells, or cell 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), etc., which are "episomes", i.e., they replicate autonomously or are capable of integrating into the chromosome of the host cell. A vector can also be a naked RNA polynucleotide, a naked DNA polynucleotide, a polynucleotide consisting of DNA and RNA within the same strand, a polylysine-conjugated DNA or RNA, a peptide-conjugated DNA or RNA, a liposome-conjugated DNA, etc., which are not episomal in nature, or can be an organism that includes one or more of the above polynucleotide constructs, such as Agrobacterium or bacteria.

[0250] The various components of an expression vector can vary widely, depending on the intended use of the vector and the host cell in which the vector is intended to replicate or drive expression. Expression vector components suitable for gene expression and vector maintenance in Escherichia coli, yeast, Streptomyces, and other common cells are well known and commercially available. For example, promoters suitable for inclusion in the expression vectors of the present disclosure include promoters that function in eukaryotic or prokaryotic host microorganisms. The promoter can include regulatory sequences that allow regulation of expression relative to the growth of the host microorganism, or that cause the expression of the gene to be turned on or off in response to chemical or physical stimuli. For Escherichia coli and certain other bacterial host cells, promoters derived from biosynthetic enzymes, antibiotic resistance-conferring enzymes, and phage protein genes can be used, such as the galactose, lactose (lac), maltose, tryptophan (trp), beta-lactamase (bla), phage lambda PL, and T5 promoters. In addition, synthetic promoters, such as the tac promoter (U.S. Patent No. 4,551,433, the entire content of which is incorporated herein by reference), can also be used. For Escherichia coli expression vectors, the inclusion of an Escherichia coli origin of replication (e.g., pUC, plP, pl, or pBR) is available.

[0251] Thus, a recombinant expression vector contains at least one expression system, which in turn consists of at least a portion of a gene coding sequence operably linked to a promoter and optionally a termination sequence capable of effecting the expression of the coding sequence in a compatible host cell. A host cell is modified by transformation with the recombinant DNA expression vector of the present disclosure to contain the expression system sequence as an extrachromosomal element or integrated into the chromosome.

[0252] In addition, as described above, the microorganisms and methods provided herein encompass homologs of enzymes for producing metabolites. The term "homolog" as used with respect to the original enzyme or gene of a first family or species refers to a different enzyme or gene of a second family or species that is determined by functional, structural, or genomic analysis to correspond to the original enzyme or gene of the first family or species. Homologs generally have functional, structural, or genomic similarities. Techniques for readily cloning homologs of enzymes or genes using gene probes and PCR are known. The homology of a cloned sequence can be confirmed using functional analysis and / or by genomic mapping of the gene.

[0253] A protein is "homologous" or "is homologous to" a second protein if the nucleic acid sequence encoding the first protein has a sequence similar to the nucleic acid sequence encoding the second protein. Alternatively, if two proteins have "similar" amino acid sequences, the first protein is homologous to the second protein (thus, the term "homologous proteins" is defined as two proteins having similar amino acid sequences).

[0254] As used herein, two proteins (or a region of a protein) are substantially homologous when their amino acid sequences have 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, the sequences are aligned for optimal comparison purposes (e.g., for optimal alignment, gaps can be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences can be disregarded for comparison purposes). In one embodiment, the length of the reference sequence aligned for comparison purposes is at least 30% of the length of the reference sequence, usually at least 40%, more usually at least 50%, even more usually at least 60%, and even more usually at least 70%, 80%, 90% or 100%. 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 (the amino acid or nucleic acid "identity" used herein 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 introduced for optimal alignment of the two sequences and the length of each gap.

[0255] When "homologous" is used to refer to proteins or peptides, it is recognized that the positions of non-identical residues typically differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions do not significantly alter the functional properties of the protein. When two or more amino acid sequences differ from each other by conservative substitutions, the degree of percent sequence identity or homology can be adjusted upward to correct for the conservative nature of the substitution. Methods for making such adjustments are well known to those of skill in the art (see, e.g., Pearson et al., 1994, which is incorporated herein by reference).

[0256] In some cases, "isozymes" can be used to perform the same functional conversion / reaction, but are generally determined to be non-"homologous" due to significant structural differences.

[0257] "Conservative amino acid substitutions" are those 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 have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). 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), tryptophan (W).

[0258] Sequence homology of polypeptides (which can also be referred to as percent sequence identity) is generally measured using sequence analysis software. See, for example, the sequence analysis software package of the Genetics Computer Group (GCG) of the Biotechnology Center of the University of Wisconsin (910 University Avenue, Madison, Wisconsin 53705). Protein analysis software uses homology measures assigned to various substitutions, deletions, and other modifications (including conservative amino acid substitutions) to match similar sequences. For example, GCG contains programs such as "Gap" and "Bestfit" which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides (e.g., homologous polypeptides from different organism species), or between a wild-type protein and its mutant protein. See, for example, GCG version 6.1.

[0259] A typical algorithm for comparing a molecular sequence to a database containing a large number of sequences from different organisms is the computer program BLAST (Altschul, 1990; Gish, 1993; Madden, 1996; Altschul, 1997; Zhang, 1997), particularly blastp or tblastn (Altschul, 1997). Typical parameters for BLASTp are: Expect value: 10 (default); Filter: seg (default); Open gap penalty: 11 (default); Extension gap penalty: 1 (default); Max matches: 100 (default); Word size: 11 (default); Number of descriptions: 100 (default); Penalty matrix: BLOWSUM62.

[0260] When searching a database containing a large number of sequences from different organisms, amino acid sequences are typically compared. Database searches using amino acid sequences can be performed using algorithms other than BLASTp known in the art. For example, the program FASTA in GCG version 6.1 can be used to compare polypeptide sequences. FASTA provides an alignment of the best overlapping regions between the query sequence and the search sequence and the percent sequence identity (Pearson, 1990, incorporated herein by reference). For example, the percent sequence identity between amino acid sequences can be determined using FASTA with its default parameters (word size of 2, PAM250 scoring matrix) as provided in GCG version 6.1, which is incorporated herein by reference in its entirety.

[0261] The present disclosure provides accession numbers and sequences of various genes, homologs, and variants for generating recombinant microorganisms and proteins for in vitro systems. It should be understood that the homologs and variants described herein are exemplary and not limiting. Those skilled in the art can utilize various databases to obtain additional homologs, variants, and sequences, including, for example, the National Center for Biotechnology Information (NCBI) accessible via the World Wide Web.

[0262] Those skilled in the art are fully capable of using the sequences and accession numbers described herein to identify homologs and isozymes that can be used in place of or to replace any polypeptide used herein. In fact, performing a BLAST search on any one of the sequences provided herein will identify multiple related homologs.

[0263] Culture conditions suitable for the growth and maintenance of the recombinant microorganisms provided herein are known (see, for example, “Culture of Animal Cells—A Manual of Basic Technique”, Freshney, Wiley-Liss, N.Y., 3rd ed., 1994). Those skilled in the art will recognize that these conditions can be modified to accommodate the requirements of various microorganisms.

[0264] It should be understood that a series of microorganisms can be modified to include all or part of a recombinant metabolic pathway suitable for the production of prenyl compounds, cannabinoids, or cannabinoid precursors. It should also be understood that various microorganisms can be used as a "source" of genetic material encoding the target enzymes suitable for the recombinant microorganisms provided herein.

[0265] As mentioned above, general texts describing molecular biology techniques useful herein (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., Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989 ("Sambrook"), and Current Protocols in Molecular Biology, F.M. 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.

[0266] Examples of protocols sufficient to guide one of ordinary skill in the art, e.g., for generating the homologous nucleic acids of the present disclosure, through in vitro amplification methods including polymerase chain reaction (PCR), ligase chain reaction (LCR), Qβ-replicase amplification, and other RNA polymerase-mediated techniques (e.g., NASBA), can be found in the following references: Berger, Sambrook, and Ausubel, as well as 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 (Oct. 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.

[0267] Improved methods for cloning in vitro amplified nucleic acids are described by Wallace et al. in U.S. Patent No. 5,426,039, which is incorporated herein by reference in its entirety.

[0268] Cheng et al. (1994) Nature 369:684-685 and the references cited therein summarize improved methods for amplifying large nucleic acids by PCR, in which PCR amplicons up to 40 kb in length were generated. The entire content of Cheng et al. (1994) Nature 369:684-685 is incorporated herein by reference. Those skilled in the art will understand that, using reverse transcriptase and polymerase, essentially any RNA can be converted into double-stranded DNA suitable for restriction digestion, PCR amplification, and sequencing. See, for example, Ausubel, Sambrook, and Berger (all as above).

[0269] The present invention is illustrated by the following examples, which are for illustrative purposes only and are not intended to limit the present invention.

[0270] Examples

[0271] Chemicals and reagents. Yeast hexokinase and Corynebacterium glutamicum catalase were both purchased from Sigma Aldrich. Aerococcus viridans pyruvate oxidase was purchased from A.G. scientific. All cofactors and reagents were purchased from Sigma Aldrich or Thermo Fisher Scientific, except for oleanolic acid, which was purchased from Santa Cruz Biotechnology, and 2,4-dihydroxy-6-propylbenzoic acid, which was purchased from Toronto Research Chemicals.

[0272] Cloning and purification of enzymes. The NphB gene was purchased as a gene block from IDT DNA and cloned into the pET28(+) vector using the Gibson assembly method. The remaining enzymes were amplified from genomic DNA or plasmids and cloned into pET28(+) using the same 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 2 mL saturated culture in the same medium was inoculated into 1 L of culture and grown at 37 °C until OD 600It was 0.5 - 0.8. The culture was induced with 1 mM IPTG and expressed at 18 °C for 16 hours. 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, and 10 mM imidazole). Cells were lysed using an Emulsiflex instrument. The lysate was clarified by centrifugation at 20,000×g, and the supernatant was batch-bound to 1 mL of NiNTA resin at 4 °C for 30 minutes. The resin was transferred to a gravity flow column. The resin was washed with 10 column volumes of wash buffer: 50 mM Tris [pH 8.0], 150 mM NaCl, and 10 mM imidazole. Then the protein was eluted with 2 column volumes of elution buffer: 50 mM Tris [pH 8.0], 150 mM NaCl, 250 mM imidazole, and 30% (v / v) glycerol. The enzyme was quickly frozen in the elution buffer using liquid nitrogen, and the stock solution of the enzyme was stored at -80 °C.

[0273] PDH cell-free reaction. The PDH reaction was divided into two parts. First, the cofactors and substrates were combined in one tube, and then the enzyme was combined in another tube. The reaction was initiated by mixing the cofactors and the enzyme in a solution with a final volume of 200 μL. The final substrate and cofactor concentrations were as follows: 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, 1.5 mM NADP + , 0.5 mM TPP, 6 mM MgCl2, 10 mM KCl, 50 mM Tris [pH 8.0], and 20 mM phosphate buffer [pH 8.0], 5 mM glutathione, and 0.5 mM - 5 mM 1,6-DHN. The reaction was terminated at 24 hours.

[0274] PDH activity assay. The activity of PDH was assayed in the presence of several aromatic polyketides. The solvent control was 1% ethanol, and the activity was compared to the assay without aromatic polyketides. The final reaction volume was 200 μL and contained 2 mM NAD +, 2 mM CoA, 1 mM TPP, 5 mM MgCl2, 5 mM KCl, 50 mM Tris pH 8.0, and 5 μL of 1.25 mg / mL PDH. The reaction was carried out in a 96-well plate. Aromatic polyketide was 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 then the reaction was initiated with 10 μL of 100 mM pyruvate. Absorbance at 340 nm was monitored for 10 minutes using a M200 spectrometer. Since aromatic molecules have background absorbance at 340 nm, the reaction was blanked with the reaction mixture and aromatic molecules, but instead of initiating the reaction with pyruvate, water was added. The initial rate was determined using the initial slope of a linear fit. The amount of NADH produced per unit time was calculated using Beer's law, with an extinction coefficient of 6.22×103 M -1 cm -1 . The reactions were performed in triplicate, and the mean and standard error were calculated.

[0275] PyOx / PTA cell-free reaction. The PyOx / PTA reaction was divided into two parts. First, the cofactors and substrates were combined in one tube, and then the enzymes were combined in another tube. The final cofactor and substrate concentrations in a 200 μL reaction were as follows: 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, 3 mM NADP + , 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 cofactors and enzymes were mixed to initiate the reaction, and a 500 μL n-nonane overlay was added on top. The reaction was incubated at room temperature and gently shaken on a gel shaker.

[0276] For 1,6-DHN / 5-p-1,6-DHN: When the aromatic substrate was the variable part, 0.5 to 5 mM of the aromatic substrate was added to the reaction, and the reaction was quenched at 24 hours. When time was the variable part, 5 mM 1,6-DHN was added, and the reaction was quenched at approximately 12, 24, 48, and 72 hours, respectively.

[0277] For oleic acid / CBGA: Optimization of the cannabinoid pathway showed that the same titer could be achieved using less glucose, so the glucose concentration was reduced to 150 mM. In addition, increasing the NADP + concentration to 6 mM and reducing the ATP concentration to 1 mM resulted in a higher CBGA titer. The oleic acid concentration was set at 5 mM. The amount of NphB added to the reaction was variable. Figure 3C The data shown were obtained using 1.5 mg / mL NphB, and the reactions were quenched at approximately 4, 8, 14, 24, 48, 72, and 96 hours. Figure 5A The data shown were obtained using 0.5 mg / mL WT NphB and M23, and the reactions were quenched at approximately 6, 9, 12, 24, 48, 72, and 96 hours.

[0278] For 2,4-dihydroxy-6-propylbenzoic acid / CBGVA: The conditions were very similar to the general method described above, except that 150 mM glucose, 1 mM ATP, and 6 mM NADP were used + , and the reactions were quenched at approximately 6, 9, 12, 24, and 48 hours. Additionally, the final concentration of isopentenyltransferase was 1 mg / mL, and AtaPT, NovQ, and NphB were tested with apigenin, daidzein, genistein, naringenin, and resveratrol. NphB was also tested with oleanol, oleanic acid, and 1,6-DHN. The reactions were quenched at 24 hours.

[0279] Quenching the reactions. To quench the reactions, the aqueous and organic layers were transferred to a 1.5 mL microcentrifuge tube. The reaction vial was rinsed with 200 μL of ethyl acetate and then the rinsate was pooled with the reactants in the microcentrifuge tube. The sample was vortexed for 5 - 10 seconds and then centrifuged at 13,000 rpm for 3 minutes. The organic layer was removed, and the remaining aqueous layer was extracted an additional 2 times with 200 μL of ethyl acetate. The organic extracts from each sample were pooled and then evaporated using a vacuum centrifuge. The sample was redissolved in methanol and subjected to HPLC analysis.

[0280] For oleanic acid / CBGA: Due to protein precipitation being observed, Figure 5A the CBGA reactions shown were extracted in the presence of 0.12 g of urea (solid) to facilitate the extraction of CBGA. This was not necessary for Figure 3C the WT NphB CBGA data shown, as no protein precipitation occurred.

[0281] Product quantification. The reactions were fractionated by reverse-phase chromatography on a C18 column (4.6 × 100 mm) using a Thermo Ultimate 3000 HPLC. The column oven temperature was set to 40 °C and the flow rate was 1 mL / min. Gradient elution was performed using water + 0.1% TFA (solvent A) and acetonitrile + 0.1% TFA (solvent B) as the mobile phase to separate the compounds. Solvent B was held at 20% for the first minute. Then solvent B was increased to 95% B over 4 minutes and then 95% B was held for 3 minutes. Then the column was re-equilibrated to 20% B for three minutes, with a total run time of 11 minutes.

[0282] Cannabinoids (CBGA, CBDA, and CBDVA) were quantified using an external calibration curve obtained with analytical standards purchased from Sigma Aldrich. Since there were no authoritative standards, an external calibration curve was generated using 5-p-1,6-DHN and CBGVA nuclear magnetic resonance (NMR) samples. Standards of known concentration were dissolved in water and then extracted using the method detailed above.

[0283] Prenylated products were quantified in the absence of authoritative standards. Due to the lack of authoritative standards for prenylated products such as prenylapigenin, prenyl daidzein, prenylnaringenin, prenyl genistein, prenyl resveratrol, and prenyl oleanol, prenylated products were quantified based on substrate consumption. To generate the standard curve, serial dilutions of each aromatic substrate were placed in the reaction mixture, but the prenyltransferase was omitted to prevent product formation. The amount of substrate consumed in the reaction was quantified according to the standard curve using liquid chromatography–mass spectrometry.

[0284] Electrospray ionization time-of-flight measurements were performed on a Waters LCT-Premier XE time-of-flight instrument, controlled by MassLynx 4.1 software (Waters Corporation, Milford, MA). The instrument was equipped with a multimode ionization source operating in the electrospray mode. A leucine enkephalin (Sigma Chemical, L9133) solution was used in Lock-Spray to obtain accurate mass measurements. Samples were injected using direct loop injection on a Waters Acquity UPLC system. An Acquity BEH C18 1.7 column (50×2.1 mm) was used, and the sample was eluted in 10 minutes with a gradient of 30–95% solvent B (solvent A: water, solvent B: acetonitrile, both containing 0.2% formic acid (vol / vol)) to separate the sample on the Waters Acquity UPLC system. Mass spectra from 300–2000 Da mass were recorded.

[0285] NMR spectroscopy. NMR spectroscopy was used to identify prenylated products and to quantify 5-p-1,6-DHN.

[0286] For 1,6-DHN / 5-p-1,6-DHN: The PyOx / PTA cell-free system was used to produce prenylated-DHN. 200 μL of the reaction mixture was pooled and extracted three times with an equal volume of nonane, and then the nonane was evaporated. The reaction product was suspended in 500 μL of deuterated methanol (CD3OD) with 2 mM of 1,3,5-trimethoxybenzene (TMB) as an internal standard. The spectra were collected on an AV400 Bruker NMR spectrometer. The amount of prenylated product in the sample was determined with reference to the TMB internal standard. The proton signal of TMB (3H, s) at 6.05 ppm was compared with the aromatic proton corresponding to 5-p-1,6-DHN (1H, d) at 7.27 ppm.

[0287] For 2,4-dihydroxy-6-propylbenzoic acid / CBGVA: NMR was also used to identify the products of the enzymatic system with 2,4-dihydroxy-6-propylbenzoic acid as the aromatic substrate. The PyOx / PTA system was set up as detailed above and the reaction was quenched at 24 h. The reaction was extracted as described above and analyzed on HPLC. There was a new main peak at 6.7 min, predicted to be prenylated 2,4-dihydroxy-6-propylbenzoic acid. The HPLC peak was purified, the solvent was removed, and the pure component was redissolved in 600 μL of CD3OD. The proton spectra collected on an AV500 Bruker NMR spectrometer were compared with the proton spectra of CBGVA published by Shoyama et al. to confirm that CBGVA was the main product. Based on the papers of Shoyama et al. and Bohlman et al., it was concluded that the prenylation of 2,4-dihydroxy-6-propylbenzoic acid occurred at the C3 carbon of 2,4-dihydroxy-6-propylbenzoic acid.

[0288] Rosetta Design modified the binding pocket of NphB to accept oleanolic acid. Oleanolic acid was placed at six different starting positions in the active site of NphB, labeled as oleanolic acid P1-6 in Table 4. ROSETTA was run 5 times at each oleanolic acid position, for a total of 30 designs. The predicted mutations in each design are listed in Table 4. For each oleanolic acid position, a set of consensus mutations (i.e., the most frequently selected residues) were chosen for further evaluation: consensus groups A to F (Table 4). Then the relative importance of each mutation proposed by ROSSETTA was evaluated. For each consensus group, the mutations were set back to the WT residues one by one, and the change in the energy score was calculated using ROSETTA (see Table 5). Those mutants that caused the largest change in energy were considered the most important mutants and incorporated into the library for experimental testing.

[0289] Table 4

[0290]

[0291]

[0292] Table 5

[0293]

[0294]

[0295] Initial screening of the NphB mutant library. To screen the initial library, small-scale expression and purification were performed. A 25 μL 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 the OD 600 reached 0.4 - 0.6. Expression of the NphB construct was induced by adding 1 mM IPTG, and then the culture was incubated at 18 °C for 18 h. 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, and 5 mM imidazole) and lysed by sonication. The cell lysate was centrifuged at 20,000 × g for 10 min at 4 °C to clarify, and the supernatant was incubated with 50 μL of NiNTA resin at 4 °C. A 96-well spin column plate was used to purify the NphB construct. The supernatant / resin was added to the column and centrifuged at 500 × g for 2 min. Then 500 μL of lysis buffer was added, and the plate was centrifuged again at 500 × g for 1 min. The protein was eluted with 200 μL of elution buffer (50 mM Tris [pH 8.0], 150 mM NaCl, 250 mM imidazole, and 30% (v / v) glycerol).

[0296] The enzyme was assayed under the following conditions: 2.5 mM geranyl pyrophosphate, 5 mM oleic acid, 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 equalize the final concentration of imidazole in each reaction. The reactions were incubated at room temperature for 12 h and then extracted three times with 100 μL of ethyl acetate. The organic extracts of each reaction were pooled and then the solvent was removed using a vacuum centrifuge. The samples were redissolved in 100 μL of methanol and analyzed by HPLC.

[0297] Screening of the focused NphB mutant library. For the focused library, expression and purification of the NphB construct on a 1 L scale were performed as described above. The enzyme was assayed under the following conditions: 2.5 mM GPP, 5 mM oleanic acid, 5 mM MgCl2, 50 mM Tris pH 8.0, and approximately 1 mg / mL NphB enzyme, in a final volume of 100 μL. The reaction was incubated at room temperature for 1 hour. 40 μL of each reaction was added to 80 μL of acetonitrile to quench. The samples were centrifuged at 13,000 rpm for 5 minutes to remove precipitated protein. The supernatant was analyzed by HPLC as described above.

[0298] Enzyme kinetic parameters. The reaction was carried out under the following conditions: 50 mM Tris [pH 8.0], 2.5 mM GPP, 5 mM MgCl2, approximately 27 μM enzyme, with oleanic acid or 2,4-dihydroxy-6-propylbenzoic acid varying from 0.1 mM to 6 mM, in a final volume of 200 μL. At time intervals detailed below, 40 μL of the reaction was added to 80 μL of acetonitrile + 0.1% TFA to quench. The reaction was centrifuged at 13,000 - 16,060 × g for 5 minutes to precipitate the protein, and then the supernatant was analyzed using the HPLC method detailed above. A plot of the initial rate versus substrate concentration was made and fitted with the Michaelis-Menten equation to determine the kinetic parameters k cat and K M (OriginPro). Each Michaelis-Menten curve was performed in triplicate. The mean and standard deviation of the kinetic parameters were reported.

[0299] For oleanic acid / CBGA: For WT, M1, M10, and M30, the time courses were 3, 6, 9, and 12 minutes. For mutant 25, the reaction was quenched at 1, 2, 4, and 8 minutes, and for M31, the reaction was quenched at 1, 2, 4, and 6 minutes.

[0300] For 2,4-dihydroxy-6-propylbenzoic acid / CBGVA: For M31, the time courses were 0.5, 1, 1.5, and 2 minutes. For M23, the time courses were 5, 10, 15, and 20 minutes, and for WT NphB, the time courses were 8, 16, 24, and 32 minutes. The enzyme concentration of the mutants was approximately 27 μM, and the concentration of WT NphB was approximately 35 μM.

[0301] GC-MS Characterization of the Isomer Profiles of WT NphB and M23. The samples were dissolved in 200 μL of ethyl acetate. GC-MS measurements were carried out in electron ionization mode using an Agilent 7693 autosampler, a 7890B gas chromatograph, and a 7250Q-TOF mass selective detector. Sample injection was performed in split mode, and the inlet temperature was set at 280 °C. Separation was carried out on an Agilent HP5-MS column with dimensions 30 m × 250 μm × 0.25 μm. Ultra-high purity grade helium (Airgas) was used as the carrier gas, and the flow rate was set at 1.1 mL / min in constant flow mode. The initial oven temperature was set at 120 °C for 1 minute and then ramped up to the final temperature of 300 °C at a rate of 20 °C / minute and held for 4 minutes. A 3.0-minute solvent delay was used. The EI energy was set at 15 eV. The MSD was set to scan the 50 - 500 m / z range. Data collection and analysis were performed using Mass Hunter acquisition and qualitative analysis software (Agilent).

[0302] Due to the elevated temperature at the GC inlet, CBGA undergoes spontaneous decarboxylation (as described by Radwan et al.), resulting in the formation of the M+ ion at 316 m / z. The retention time corresponding to the 316 m / z ion of the CBGA standard is 10.48 minutes.

[0303] A nonane flow system for extracting CBGA from solution. The PyOx / PTA reaction was set up as detailed above. A 500 μL nonane overlay was added to the reaction in a 2 ml glass vial covered with two layers of breathable cell culture membranes. Two needles were inserted at the approximately 750 μL and 3.5 mL marks of a 15 mL Falcon tube. A Luer lock connected to a tubing connector was attached to the needles, and a Viton tubing was connected to the other end of the Luer lock. The needles were connected to the other end of the tubing through a Luer lock connector and inserted through the mesh overlay so that they only contacted the nonane layer and not the reaction. 2 mL of Tris buffer [pH 8.5] was added to a 15 mL conical tube, and then 6 mL of nonane was added. The nonane was pumped into the system using a peristaltic pump such that the nonane flowed over the buffer solution from the top of the reaction. The nonane pumped into the reservoir separated into the top layer of the 15 mL conical tube. The nonane at the top of the 15 mL conical tube was pumped into the top of the reaction vial. This substantially diluted the CBGA in the entire system, prompting the diffusion of CBGA into the nonane layer and out of the reaction.

[0304] Cloning CBDAS. The codon-optimized CBDAS gene block for Pichia pastoris was ordered from IDT. The signal peptide sequence was removed by PCR amplifying the protein terminus from the 28th residue of the protein sequence (NPREN...) with overhangs compatible with the pPICZa vector. The PCR product was cloned into the pPICZa vector digested with EcoRI and XbaI using Gibson cloning. The product of the assembly reaction was transformed into BL21 Gold(DE3) cells, and clones with the correct sequence were isolated. The plasmid was digested with Pmei for 2 hours and then purified using the Qiagen PCR purification protocol. The plasmid was transformed into Pichia pastoris X33 by electroporation. Immediately after electroporation, the cells were incubated in 1 mL of cold 1 M sorbitol and 1 mL of YPD medium for 2 hours without shaking. The cells were plated onto YPDS plates containing 500 μg / mL zeocin. Colonies were screened by PCR to determine the presence of the CBDAS gene between the AOX1 promoter and terminator. For screening, the colonies were resuspended in 15 μL of sterile water, and 5 μL of the resuspended colonies was transferred to a PCR tube containing 0.2% SDS. The samples were heated at 99 °C for 10 minutes and then used 1 μL as the PCR template. Six colonies with positive colony PCR hits were screened for CBDAS expression.

[0305] CBDAS expression test. The six colonies were grown overnight at 30 °C to obtain saturated cultures. The overnight cultures were inoculated into 25 mL cultures in BMGY medium and grown to an OD of approximately 2. The cells were harvested by centrifugation at 2,000×g for 10 minutes. The cell pellet was resuspended in 90 mL of BMMY medium and incubated at 30 °C for 5 days. 1 mL of the culture was taken out every day for SDS-PAGE analysis, and 500 μL of methanol was added. The cultures were screened for CBDAS activity on the 3rd day. The detection conditions were as follows: 100 μL of 200 mM citrate buffer, 100 μM CBGA, 5 mM MgCl2, 5 mM KCl, 1 mM FAD, and 50 μL of the expression medium, with a final volume of 200 μL. The reaction was incubated overnight at room temperature and then extracted 3 times with 200 μL of ethyl acetate. For each sample, the ethyl acetate extracts were pooled and then removed using a vacuum centrifuge. The samples were resuspended in 200 μL of methanol and analyzed by HPLC. All clones produced active CBDAS.

[0306] Collect three cloned cultures (total of approximately 300 mL) to obtain CBDAS activity. Centrifuge at approximately 3,000×g for 20 minutes at 4 °C to pellet the cells. Then filter the supernatant through a 0.22 μm filter. Concentrate and buffer exchange the medium to 100 mM citrate buffer pH 5.0 using a 50,000 MWCO protein concentrator from Millipore. Determine the total protein in the medium concentrate to be 0.4 mg / mL using the Bradford assay, with a total protein yield of approximately 5 mg / L.

[0307] Production of CBDVA and CBDA. To convert the precursors CBGA and CBGVA to CBDA and CBGVA, respectively, a secondary reaction was established using CBDAS synthase.

[0308] For CBGA / CBDA: A PyOx / PTA enzyme system was established as described above to produce CBGA. After 24 hours, transfer 200 μL of the nonane overlay from the CBGA reaction to the CBDAS reaction vessel. In 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. Incubate the reaction with gentle shaking at 30 °C. Quench the reaction at 12, 24, 48, 72, and 96 hours.

[0309] For CBGVA / CBDVA: HPLC-purified CBGVA was converted to CBDVA. The final reaction volume was 200 μL and contained 50 mM Hepes [pH 7.0], 5 mM MgCl2, 5 mM KCl, 25 μM FAD, and 0.1 mg / mL (total protein) CBDAS concentrate. Add 200 μL of nonane overlay and incubate the reaction with gentle shaking at 30 °C. Quench the reaction at approximately 24, 48, 72, and 96 hours.

[0310] MatB Activity Assay. Coupled enzymatic assays were used to determine the activity of malonyl-CoA synthetase (MatB) of Rhodopseudomonas palustris (see, e.g., SEQ ID NO: 82 - 83) in the presence of OA and DA. The reaction conditions were: 2.5 mM malonic acid, 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 (malonic acid), and 1% ethanol, 250 μM or 5 mM OA or 5 mM DA was added to the remaining reactions. MatB activity was determined by monitoring the decrease in absorbance at 340 nm due to NADH consumption using an M2 SpectraMax. To ensure MatB was limited to 5 mM OA or DA, MatB was doubled to 1.5 μg / mL. A doubling of the reaction rate indicated that MatB was the limiting component in the system. The NADH consumption rates at 5 mM OA and 5 mM DA were normalized to the 1% ethanol control.

[0311] Met and d3-Met Activity Assays. Enzyme activities were determined to compare the activities of methionine adenosyltransferase (MAT) from Thermococcus kodakarensis (tk) and Methanocaldococcus jannaschii (mj) towards methionine (met), d3-met, and the production of S-adenosyl-L-methionine-d3 (SAM-d3). The reaction conditions were: 200 μl reactions contained 200 mM Tris-HCl (pH = 8), 20 mM MgCl2, 50 mM KCl, 5 mM ATP, 5 mM met or d3-met. The enzyme concentration of each MAT was 10 μM in four samples, for a total of eight samples. PPase (2.5 μM) was added to two samples of each MAT type. The reaction mixtures were incubated at room temperature for 1 hour, quenched with freshly prepared malachite green working solution (Sigma-Aldrich Malachite Green Phosphate Assay Kit), and measured at 620 nm on a microplate reader with phosphate standards according to the manufacturer's protocol. Enzyme activity was quantified as the inorganic phosphate (Pi) released per hour.

[0312] The results of this activity assay are as Figure 38As shown, it indicates that methionine adenosyltransferase (MAT) from Thermococcus kodakarensis (tk) and Methanococcus jannaschii (mj) has the same activity towards met and d3-met. As expected, the addition of PPase increases the amount of P released per hour. i This overall confirms the production of SAM-d3.

[0313] AAE3 activity assay. The activity of acyl-activating enzyme 3 (AAE3) (see, for example, SEQ ID NO: 70-71 and homologs - SEQ ID NO: 72-75) in the presence of OA and DA was determined using a coupled enzymatic assay similar to that described above for MatB. The conditions were the same as 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 limiting, AAE3 was doubled in the presence of 5 mM OA or DA. Doubling of the reaction rate indicated that AAE3 was limiting.

[0314] ADK activity assay. The activity of adenylate kinase (ADK) in the presence of OA and DA was determined using a coupled enzymatic assay (see, for example, SEQ ID NO: 81). The conditions were similar to the MatB assay, with the following modifications: 2 mM AMP was added instead of malonic acid, CoA was not added, and 0.001 mg / mL ADK was added. To ensure that ADK was the limiting reagent in the presence of 5 mM OA and DA, the amount of ADK was doubled. An increase in the rate to 2-fold indicated that ADK was the limiting factor.

[0315] CPK activity assay. A coupled enzymatic assay was used to determine 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, 2 mM NADP + , 5 mM MgCl2, 5 mM KCl, 0.3 mg / mL Zwf, 0.1 mg / mL ScHex, and 0.08 units of CPK. The positive control reaction contained 1% ethanol, and 5 mM OA or DA was added to the remaining reactions. The absorbance of NADPH at 340 nm was monitored. To ensure that CPK was limiting, it was doubled in the presence of 5 mM OA or DA. The resulting rate doubled, indicating that CPK was limiting even at high OA and DA.

[0316] OLS activity assay. The oleanol synthase (OLS) (see, for example, SEQ ID NO: 76 - 77) was assayed by setting the following conditions: 200 μM malonyl CoA, 100 μM hexanoyl CoA, 0.65 mg / mL OAS, dissolved in 50 mM citrate buffer (pH 5.5) or 50 mM Tris buffer (pH 8.0). The reaction was initiated by adding OAS and then quenched at 30 minutes by adding 150 μL of methanol to 50 μL of the reaction. The samples were centrifuged at approximately 16,000×g for 2 minutes to precipitate the protein. The supernatant was analyzed by HPLC.

[0317] For the inhibition experiment, the conditions were changed to: 1 mM malonyl CoA, 400 μM hexanoyl CoA, in 50 mM citrate buffer, pH 5.5, final volume of 200 μL. 1% ethanol, 250 μM OA or 1 mM DA was added to the reaction and then the reaction was initiated by adding 0.65 mg / mL OLS. 50 μL aliquots were quenched in 150 μL of methanol at 2, 4, 6, and 8 minutes, respectively. The reaction mixtures were vortexed briefly and centrifuged at 16,000×g for 2 minutes to precipitate the protein. The supernatant was analyzed by HPLC. The raw peak areas of HTAL, PDAL, and oleanol were added together and plotted against time to determine the rate. The rates of the OA - supplemented reaction and the DA - supplemented reaction were normalized to the ethanol control.

[0318] OLS / OAC activity assay. To produce OA, the same OLS conditions as specified above were used, but 0.6 mg / mL of oleanic acid cyclase (OAC) (see, for example, SEQ ID NO: 78 - 79) was added to the reaction. The reaction was quenched and analyzed in the same manner as 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.

[0319] Complete pathway setup. The enzymes used in this study and their final concentrations (mg / mL) are shown in Table 6 (MatB pathway) and Table 7 (MdcA pathway). For the MatB pathway, the addition concentrations of cofactors are as follows: 150 mM glucose, 1 mM fructose diphosphate, 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 caproic acid, 15 mM malonic acid, 5 mM creatine phosphate, and 50 mM Tris pH 8.0. The reaction was initiated by adding the enzymes listed in Table 6. The reaction was incubated overnight at room temperature, then quenched with 200 μL of ethyl acetate and extracted 3 times. The ethyl acetate was removed using a vacuum centrifuge. The sample was dissolved in 200 μL of methanol and analyzed by HPLC.

[0320] Table 6

[0321]

[0322] Table 7

[0323]

[0324]

[0325] Both the MatB and MdcA pathways are as Figure 6A shown in B.

[0326] Figure 9 The probe electrospray ionization Fourier transform mass spectrometry (FTMS-pESI) spectral data of acetonitrile with 0.1% formic acid as a control are shown.

[0327] Figure 10 The FTMS-pESI spectral data of CBGA in positive mode are shown.

[0328] Figure 11 The FTMS-pESI spectral data of CBGA in negative mode are shown.

[0329] Figure 12 The FTMS-pESI spectral data of the control and CBGA in positive mode are shown.

[0330] Figure 13 The FTMS-pESI spectral data of the control and CBGA in positive mode are shown.

[0331] Figure 14 The FTMS-pESI spectral data of deuterated CBGA in positive mode are shown.

[0332] Figure 15The FTMS-pESI spectral data of deuterated CBGA in negative mode is shown.

[0333] Figure 16 Additional FTMS-pESI spectral data of deuterated CBGA in positive mode is shown.

[0334] Figure 17 Additional FTMS-pESI spectral data of deuterated CBGA in negative mode is shown.

[0335] Table 8 shows the results of FTMS-pESI spectral analysis, which shows that the compounds can be identified in both positive and negative modes.

[0336] Table 8

[0337] just burden CBGA 361.2372 359.2209 dCBGA 372.3064 370.2899

[0338] Nano liquid chromatography tandem mass spectrometry (nLC-MS / MS) was performed on additional samples. Samples were prepared using 2 μL of standard and 198 μL of 50:50 water:acetonitrile (in 0.1% formic acid solution). 1 μL of the sample was injected.

[0339] Figure 18 The nLC-MS / MS data of CBGA is shown.

[0340] Figure 19 The nLC-MS / MS data of dCBGA is shown.

[0341] Table 9 compares the nLC-MS / MS data of CBGA and dCBGA.

[0342] Table 9

[0343] just burden Retention time (min) CBGA 361.2372 359.2209 41.5-43.0 dCBGA 372.3064 370.2899 41.97±0.5

[0344] Figure 20 The nLC-MS / MS data of the first sample of CBGA after 1 hour is shown.

[0345] Figure 21 The nLC-MS / MS data of the second sample of CBGA after 1 hour is shown.

[0346] Figure 22 The nLC-MS / MS data of the third sample of CBGA after 1 hour is shown.

[0347] Figure 23 The nLC-MS / MS data of the first sample of dCBGA after 1 hour is shown.

[0348] Figure 24 The nLC-MS / MS data of the second sample of dCBGA after 1 hour is shown.

[0349] Figure 25 Shows the nLC-MS / MS data of the third sample of dCBGA after 1 hour.

[0350] Table 10 compares the nLC-MS / MS data of CBGA samples and dCBGA samples after one hour.

[0351] Table 10

[0352] CBGA dCBGA 9365619 90416704 10768244 162639303 8328277 91712604

[0353] Figure 26 Shows a comparison chart of CBGA and dCBGA. Samples 1, 2, and 3 of CBGA and dCBGA are shown from left to right. The results show that the degree of degradation of CBGA after one hour is significantly higher than that of dCBGA.

[0354] In one embodiment, after forming the THCA compound of the present invention, the compound is purified to isolate the resulting THCA. CBGA and THCA are extracted into ethyl acetate to form an emulsion (the emulsion can only be partially disrupted by adding hexane). Bovine serum albumin (BSA) binds tightly to CBGA and THCA, so not all products can be recovered in the organic phase during the purification process. However, acid precipitation of BSA by adding 1% HCl can denature BSA, and subsequently dissociate CBGA and THCA, enabling extraction and purification. In one embodiment, HCl is directly added to the reaction mixture and then incubated for about 10 minutes to ensure complete denaturation of BSA. Then the mixture is centrifuged at a speed of 4000g for about 30 minutes. After centrifuging the mixture, CBGA and THCA co-precipitate in particulate form. Then the particles are washed with water until the pH of the water reaches neutral. Then the particles are resuspended in methanol using vigorous vortexing. The methanol mixture is rotary evaporated on a water bath at 40 °C, and the resulting oil is resuspended in ethanol. Then THCA precipitates as a brown oil. Then the reaction mixture is filtered, washed with water, and then resuspended in hexane. Then hexane is rotary evaporated to remove the mass of excess methane added to the product. The resulting oil is resuspended in methanol for storage. Table 11 below shows a summary of the product recovery in the entire process starting from 67.5 mg of the CBGA reaction mixture. In the experiment detailed in Table 11, 41 mg of purified product was obtained using this purification process, which is 60% of the theoretical yield. In one embodiment, implementing the crystallization step avoids ethanol precipitation, thereby improving the purity and recovery rate of THCA.

[0355] Table 11

[0356]

[0357]

[0358] After THCA was purified, the purified THCA was further decarboxylated to obtain THC. In the continuation of the above experiment according to Table 11, 23 mg of purified THCA was used for decarboxylation. Methanol in the product was removed by rotary evaporation, and then the product was resuspended in 2 mL of toluene. Next, 100 μL of saturated bicarbonate was added to the mixture, and the mixture was heated to 95 °C on a rotary evaporator for reflux and condensation. Samples were collected 10 minutes and 35 minutes after the reaction stopped. The reaction mixture was washed with water to remove excess salts. Then ethanol was added to the toluene to further assist in evaporating the excess reactants, and the mixture was dried by rotary evaporation three times to remove toluene. This process produced 13.4 mg of THC with a purity of 90%.

[0359] In one embodiment, impurities in the compound that cause an increase in retention time can be removed before decarboxylation by crystallization of THCA in an ethanol / hexane mixture.

[0360] One-pot synthesis

[0361] In one embodiment, the synthesis is a one-pot synthesis. In a one-pot synthesis, the synthesis reaction is carried out by mixing all the reagents, catalysts, enzymes, buffers and other necessary chemicals in one reaction vessel. In one embodiment, the one-pot synthesis includes at least one deuterated compound (e.g., deuterated fatty acid). Advantageously, the one-pot synthesis improves the efficiency of the chemical reaction, thereby increasing the chemical yield.

[0362] Formulation

[0363] 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 other isotope are incorporated into a pharmaceutical composition. In one embodiment, the pharmaceutical composition contains about 1% (w / w) or more of 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 other isotope (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).

[0364] 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).

[0365] In one embodiment, the pharmaceutical composition further comprises a lipid. Lipids include but are not limited to phospholipids (such as soy lecithin, egg lecithin, phosphocholine, phosphoglycerol), fats, oils (such as olive oil, vegetable oil), and / or fatty acids. In one embodiment, the lipid is capable of forming micelles, emulsions, or liposomes.

[0366] In one embodiment, a 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 other isotope is microencapsulated or nanoencapsulated.

[0367] In one embodiment, the pharmaceutical composition further comprises at least one masking agent (e.g., taste masking agent, odor masking agent). In a preferred embodiment, the at least one masking agent includes but is not limited to at least one sweetening agent and / or at least one flavoring agent. The at least one sweetening agent includes but is not limited to saccharin (e.g., sodium salt, calcium salt), fructose, glucose, aspartame, acesulfame potassium, glycerol, sucralose, maltodextrin, sucrose, glucose, maltose, xylitol, sorbitol, erythritol, and / or mannitol. In one embodiment, the at least one masking agent includes phenethyl alcohol, vanilla, cherry, cinnamon, lavender, lemon, menthol, orange, peppermint, spearmint, raspberry, strawberry, grape, ethyl vanillin, coriander, ginger, nutmeg, cardamom, butterscotch, cocoa, gum arabic syrup, anethole, fennel oil, benzaldehyde, ethyl acetate, methyl salicylate, and / or balsam of tolu. In one embodiment, the at least one masking agent accounts for about 0.001% to about 1% w / w of the pharmaceutical composition, e.g., based on the weight of the pharmaceutical composition, 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. In one embodiment, based on the weight of the pharmaceutical composition, the at least one masking agent is about 0.01% to 0.5%, 0.02% to 0.2%, or 0.015% to 0.15% w / w.

[0368] In one embodiment, the pharmaceutical composition further comprises a vitamin or a mineral. Vitamins include but are not limited to vitamin A, vitamin C, vitamin D (e.g., vitamin D1, D2, D3, D4, D5, D6, and / or D7), vitamin E, vitamin B (e.g., B1, B2, B3, B5, B9, and / or B12), vitamin K (e.g., K1, K2, K3, K4, and / or K5). Minerals include but are not limited to magnesium, calcium, iron, zinc, chromium, selenium, and / or potassium.

[0369] In one embodiment, the pharmaceutical composition further comprises at least one anti-caking agent. The at least one anti-caking agent includes but is not limited to tricalcium phosphate, cellulose, microcrystalline cellulose, silica, sodium chloride, magnesium stearate, magnesium carbonate, and / or sodium bicarbonate. In one embodiment, the at least one anti-caking agent accounts for about 0.5% to about 5% w / w of the pharmaceutical composition.

[0370] In one embodiment, the pharmaceutical composition comprises at least one preservative. The at least one preservative includes, but is not limited to, parabens, benzalkonium chloride, phenethyl 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 the composition in an amount of about 0.01% to about 5% w / w, for example, about 0.01%, 0.05%, 0.1%, 0.2%, 0.5%, 1%, 2%, 3%, 4%, or 5% w / w based on the weight of the pharmaceutical composition. In one embodiment, based on the weight of the pharmaceutical composition, the at least one preservative is about 0.01% to 5%, 0.02% to 4%, or 0.05% to 2.5% w / w. In a preferred embodiment, the at least one preservative is free of sulfites.

[0371] In one embodiment, the pharmaceutical composition is in the form of a crystalline agent, powder, granule, capsule, tablet, syrup, solution, emulsion, topical agent, thin film, aerosol, oil, patch (e.g., transdermal patch), parenteral agent, suppository, intravenous agent, or suspension. In one embodiment, the pharmaceutical composition is delivered via oral administration, sublingual route, buccal route, rectal route, intravenous injection, intramuscular administration, subcutaneous injection, intranasal route, inhalation route, ocular route, or vaginal route. In one embodiment, the pharmaceutical composition is provided in an edible form. Edible forms include, but are not limited to, confections, baked goods (e.g., brownies, cookies, muffins), beverages, breads, cereals, or pastas. Topical forms include, but are not limited to, creams, ointments, lotions, pastes, or gels.

[0372] Treatment

[0373] In one embodiment, the pharmaceutical composition is used for treating cancer (e.g., gastric cancer, colon cancer, pancreatic ductal adenocarcinoma), glaucoma, fibromyalgia, peripheral neuropathy, nausea, diabetes, obesity, liver diseases, 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), skin diseases (e.g., psoriasis, atopic dermatitis (AD), eczema, acne, contact dermatitis, herpes simplex, herpes zoster, actinic keratosis, ichthyosis, Bowen's disease, keratoacanthoma, lichen sclerosus, hidradenitis suppurativa, seborrheic keratosis, rosacea, pityriasis lichenoides, seborrhea), joint diseases (e.g., osteoarthritis), reproductive diseases (e.g., endometriosis, dysmenorrhea, irregular menstrual bleeding, dyspareunia), bacterial infections, and / or mental diseases (e.g., anxiety, depression, stress, panic disorder, attention deficit hyperactivity disorder (ADHD), post-traumatic stress disorder (PTSD), bipolar disorder, obsessive-compulsive disorder, schizophrenia, personality disorder). For example, see U.S. Patent Publication No. 20210315837, which is incorporated herein by reference in its entirety.

[0374] In one embodiment, the pharmaceutical composition is administered once a day, twice a day, three times a day, or four times a day. 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.

[0375] Those skilled in the art will think of certain modifications and improvements after reading the above description. The purpose of providing the above examples is to illustrate various aspects of the present invention, and it will be apparent to those skilled in the art that they do not limit the scope of the present invention. For the sake of brevity and readability, all modifications and improvements are omitted herein, but these modifications and improvements are still within the scope of the present invention.

Claims

1. A cannabinoid composition, comprising: A cannabinoid derived from a deuterated fatty acid, wherein the cannabinoid is a compound of formula I: Formula I wherein R is a deuterated carbon chain; and wherein the deuterated carbon chain comprises at least one deuterated carbon.

2. The composition according to claim 1, wherein the deuterated carbon chain is C5D 11 .

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

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

5. The composition according to claim 1, wherein the composition further comprises phospholipids, fats, oils and / or fatty acids.

6. The composition according to claim 1, wherein the composition further comprises at least one sweetening agent and / or at least one flavoring agent.

7. The composition according to claim 1, wherein the composition further comprises vitamins or minerals.

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

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

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

11. A method for synthesizing a cannabinoid compound or its derivative, comprising: Phosphorylating 3-methyl-2-buten-1-ol and / or 3-methyl-3-buten-1-ol using hydroxethylthiazole kinase (ThiM) to produce isopentenyl phosphate; Isomerizing the isopentenyl phosphate to produce isopentenyl diphosphate, wherein the isopentenyl diphosphate can be phosphorylated in the presence of inositol polyphosphate kinase to produce dimethylallyl diphosphate (DMAPP); Synthesizing geranyl diphosphate from isopentenyl diphosphate and / or DMAPP in the presence of farnesyl pyrophosphate synthase; Activating the deuterated fatty acid to produce deuterated CoA thioester; Activating the acid to produce a second CoA thioester; Synthesizing the deuterated CoA thioester and the second CoA thioester; Cyclizing the synthesized product to produce deuterated olivetolic acid; Isopentenylating the deuterated olivetolic acid in the presence of geranyl diphosphate to produce deuterated cannabigerolic acid; Cyclizing the deuterated cannabigerolic acid to produce a deuterated cannabinoid.

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

13. The method according to claim 11, wherein the method for synthesizing a cannabinoid compound or its derivative is a one-pot synthesis method.

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

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

16. A method for cell-free synthesis of cannabinoids or their derivatives, comprising: 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 exhibits increased expression of the at least one enzyme compared to the unmodified parental microorganism; lysing at least one cell of the modified microorganism to obtain the at least one enzyme, wherein the at least one enzyme is used in the cannabinoid biosynthesis pathway; utilizing the cannabinoid biosynthesis pathway to produce cannabinoids or their derivatives, comprising: 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; Formula II: activating an acid in the presence of a corresponding CoA synthase 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 synthesized product in the presence of olivetolic acid 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 deuterated cannabinoids.

17. The method according to claim 16, wherein the cannabinoid biosynthesis pathway is a one-pot synthesis method.

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

19. The method according to claim 16, wherein tetrahydrocannabinolic acid synthase (THCAS), cannabidiolic acid synthase (CBDAS), and / or cannabichromenic acid synthase (CBCAS) are used to cyclize the deuterated cannabigerolic acid.

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

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