Biosynthesis of cyclolavandulyl derivatives of aromatic compounds.

Abstract

The present disclosure relates to compositions comprising cyclolavandulyl derivatives of aromatic compounds, such as cannabinoids, flavonoids, and alkaloids, and methods of making and using the compositions.

Description

[Technical field]

[0001] [CROSS REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 333,670, filed April 22, 2022, the entirety of which is incorporated herein by reference.

[0002] The present disclosure relates to cyclolavandulyl derivatives of aromatic compounds, including cannabinoid, alkaloid, and flavonoid compounds, as well as compositions and biosynthetic methods for making and using these compounds.

[0003] [Reference to sequence table] An official copy of the sequence listing has been filed contemporaneously herewith as an XML file in WIPO standard ST.26 format with filename "15041-001PV1.xml", created on April 19, 2023, and 27,126 bytes in size. This sequence listing, submitted electronically via USPTO EFS-Web, is incorporated herein by reference in its entirety. This sequence listing corresponds to the sequence listing in ST.25 format filed with priority U.S. Provisional Application No. 63 / 333,670 on April 22, 2022 with filename "15041-001PV1_SeqList_ST25.txt", created on February 25, 2022, and 44,691 bytes in size. [Background technology]

[0004] Cannabinoids are a large and well-known class of plant-derived bioactive compounds that regulate the cannabinoid receptors (CB1 and CB2) of the human endocannabinoid system. Cannabinoids are promising pharmaceutical agents, with over 100 clinical trials underway investigating their therapeutic effects as antiemetics, anticonvulsants, analgesics, and antidepressants. In addition, three cannabinoid therapies are FDA-approved for the treatment of chemotherapy-induced nausea, MS spasticity, and seizures associated with severe epilepsy.

[0005] The Cannabis sativa plant is known to produce over 100 different cannabinoid compounds, but the best known and most studied cannabinoids include tetrahydrocannabidiolic acid (THCA), tetrahydrocannabidivalic acid (THCVA), cannabidiolic acid (CBDA), cannabidivalic acid (CBDVA), and their decarboxylated analogs (e.g., THC, THCV, CBD, CBDV). All cannabinoids produced by plants are generated by enzymatic prenylation of aromatic polyketide substrates such as olivetolic acid (OA) or divaleric acid (DA) with geranyl pyrophosphate (GPP). The enzymatic prenylation is carried out by the membrane protein GPP:OA transferase (GOT), also called prenyltransferase. Naturally occurring prenyltransferases found in C. sativa (eg, PT4, UniProt: A0A455ZJC3) are membrane-bound proteins.

[0006] Soluble prenyltransferase NphB (UniProt: A0A2Z4JFA9) has been isolated from Streptomyces sp. CL190. See, for example, U.S. Patent No. 5,399,663. NphB has further been engineered to provide soluble prenyltransferase mutants capable of prenylating aromatic polyketides OA or DA with GPP under a range of biosynthetic conditions to form cannabinoid compounds CBGA or CBGVA, respectively. See, for example, U.S. Patent No. 5,399,663; U.S. Patent No. 5,399,671; U.S. Patent No. 5,399,671; and U.S. Patent No. 5,433,636. The engineered NphB mutants can be used in cell-free biosynthetic systems and methods for preparing cannabinoid compounds. See, for example, U.S. Patent No. 5,433,671 and U.S. Patent No. 5,433,671.

[0007] It has also been shown that NphB can transfer a range of alkyl groups from pyrophosphate donor substrates (other than GPP) to aromatic substrates such as 1,6-dihydroxynaphthalene.

[0008] Flavonoids are another well-known large class of plant-derived bioactive aromatic compounds that exhibit biological activity. Furthermore, prenylated derivatives of flavonoid compounds exhibit enhanced biological activity. For example, see Non-Patent Document 2.

[0009] Cyclolavanduryl is a branched and cyclic carbon C10 monoterpene chemical moiety represented in the "forward" configuration by the following chemical structure: [ka]

[0010] The biosynthesis of the cyclolavandulyl group occurs in several natural product compounds (e.g., Kallistein A, Sesserinolonol, Lavanduquinocin, and Lavanducyanin) via an enzyme-catalyzed "non-head-to-tail" condensation and cyclization of two molecules of C5 dimethylallyl pyrophosphate (DMAPP). See, for example, Non-Patent Document 3. The structure of cyclolavandulyl diphosphate synthase (CLDS) from Streptomyces sp. CL190 has been structurally characterized and shown to catalyze the conversion of two molecules of dimethylallyl pyrophosphate (DMAPP) to the product compound, cyclolavandulyl pyrophosphate. See, for example, Non-Patent Document 4.

[0011] There remains a need for new cannabinoid derivatives, flavonoid derivatives, alkaloid derivatives, and other aromatic derivative compounds, as well as efficient methods for producing such compounds. [Prior art documents] [Patent documents]

[0012] [Patent Document 1] U.S. Patent No. 7,361,483 [Patent Document 2] International Publication No. 2019173770 [Patent Document 3] International Publication No. 2019183152 [Patent Document 4] International Publication No. 2020028722 [Patent Document 5] International Publication No. 2021134024 [Non-patent literature]

[0013] [Non-Patent Document 1] Johnson et al., "Acceptor substrate determination donor specificity of an aromatic prenyltransferase: expanding the biocatalytic potential of NphB," Appl. Microbiol. Biotechnol. (2020)104:4383-4395 [Non-Patent Document 2] Mukai, "Prenylation enhances the biological activity of dietary flavonoids by altering their bioavailability," Bioscience, Biotechnology and Biochemistry (2018) 82(2);207-215 [Non-Patent Document 3] Ozaki et al., "Cyclolavandulyl SkeletonBiosynthesis via Both Condensation and Cyclization Catalyzed by anUnprecedented Member of the cis-Isoprenyl Diphosphate SynthaseSuperfamily," J. Am. Chem. Soc. 2014, 136, 4837-4840 [Non-Patent Document 4] Tomita et al., "Structure and mechanismof the monoterpene cyclolavandulyl diphosphate synthase that catalysesconsecutive condensation and cyclisation," Angew. Chemie. Int'l Ed. 2017,10.1002 / anie201708474 Summary of the Invention

[0014] The present disclosure generally relates to cyclolavanduryl derivatives of aromatic compounds, including cannabinoids, alkaloids, and flavonoids, and in vitro biosynthetic methods and compositions for producing such compounds. This summary is intended to introduce the subject matter of the present disclosure, but does not exhaustively describe every embodiment, combination, or variation contemplated and described within the present disclosure. Further embodiments are contemplated and described by the disclosure of the detailed description, drawings, and claims.

[0015] In at least one embodiment, the present disclosure provides a compound of structural formula (Ia) or (Ib): [ka] (In the formula, R 1 teeth, [ka] (In the formula, R 2 is -H or -OH, R 3 is -H or -COOH, R 4 is selected from -H, -OH, linear or branched C1-C10 alkyl, linear or branched C1-C10 alkylamide, linear or branched C1-C10 alkylamine, linear or branched C1-C10 alkylalkylene, linear or branched C1-C10 alkylalkoxy, or C1-C10 alkylaryl, wherein any of the C1-C10 groups are optionally substituted with -OH, -OCH3, or halogen.

[0016] In at least one embodiment of the compounds of the present disclosure, R 1 but, [ka] (In the formula, R 2 is -H or -OH, R 3 is -H or -COOH, R 4 is selected from linear or branched C1-C10 alkyl, linear or branched C1-C10 alkylamide, linear or branched C1-C10 alkylamine, linear or branched C1-C10 alkylalkylene, linear or branched C1-C10 alkylalkoxy, or C1-C10 alkylaryl, wherein any of the C1-C10 groups are optionally substituted with -OH, -OCH3, or halogen. 2 is -OH and R 3 In at least one embodiment of the compound, R 4 is selected from CH3, CH2CH3, (CH2)2CH3, (CH2)3CH3, (CH2)4CH3, (CH2)5CH3, and (CH2)6CH3, and optionally, R 2 is selected from (CH2)2CH3, (CH2)4CH3, and (CH2)6CH3.

[0017] In at least one embodiment of the compound of the present disclosure, the compound has structural formula (IIa), (IIb), (IIc), or (IId): [ka] (In the formula, R 3 is -H or -COOH, R 4is selected from linear or branched C1-C10 alkyl, linear or branched C1-C10 alkylamide, linear or branched C1-C10 alkylamine, linear or branched C1-C10 alkylalkylene, linear or branched C1-C10 alkylalkoxy, or C1-C10 alkylaryl, wherein any of the C1-C10 groups are optionally substituted with -OH, -OCH3, or halogen. 4 is selected from CH3, CH2CH3, (CH2)2CH3, (CH2)3CH3, (CH2)4CH3, (CH2)5CH3, (CH2)6CH3, and (CH2)7CH3, and optionally, R 4 is selected from (CH2)2CH3, (CH2)4CH3, and (CH2)6CH3.

[0018] In at least one embodiment of the compound of the present disclosure, the compound is selected from the group consisting of compounds (2a), (2b), (2c), (2d), (2e), (2f), (2g), (2h), (2i), (2j), (2k), (2l), (2m), (2n), (2o), (2p), (2q), (2r), (2s), (2t), (2u), (2v), (2w), (2x), (2y), (2z), (2aa), (2bb), (2cc), (2dd), (2ee), (2ff), (2gg), (2hh), (2ii), (2jj), (2kk), (2 ll), (2mm), (2nn), (2oo), (2pp), (2qq), (2rr), (2ss), (2tt), (2uu), (2vv), (2ww), and (2xx): [ka] JPEG2025513329000008.jpg233150JPEG2025513329000009.jpg210150JPEG2025513329000010.jpg210150JPEG2025513329000011.jpg210150 are selected.

[0019] In at least one embodiment of the compounds of the present disclosure, R 1 but, [ka] Selected from JPEG2025513329000013.jpg76154.

[0020] In at least one embodiment, the disclosure provides a method for producing a cyclolavandulyl-substituted aromatic compound, comprising: (a) reacting, under suitable reaction conditions, a prenyltransferase with cyclolavandulyl pyrophosphate (CLPP) of compound (1). [ka] and compounds of structural formulas (III), (IV), (V), and (VI) [ka] (In the formula, R 2 is -H or -OH, R 3 is -H or -COOH, R 4 is -H, -OH, linear or branched C1-C10 alkyl, linear or branched C1-C10 alkylamide, linear or branched C1-C10 alkylamine, linear or branched C1-C10 alkylalkylene, linear or branched C1-C10 alkylalkoxy, or C1-C10 alkylaryl, wherein any of the C1-C10 groups are optionally substituted with -OH, -OCH3, or halogen; and (b) recovering a cyclolavandulyl-substituted aromatic compound from the reaction mixture.

[0021] In another embodiment, the disclosure provides a method for producing a cyclolavandulyl-substituted aromatic compound, the method comprising: (a) reacting, under suitable reaction conditions, cyclolavandulyl diphosphate synthase (CLDS), dimethylallyl pyrophosphate, a prenyltransferase, and compounds of structural formulas (III), (IV), (V), and (VI), where R 2 is -H or -OH, R 3 is -H or -COOH, R 4is -H, -OH, linear or branched C1-C10 alkyl, linear or branched C1-C10 alkylamide, linear or branched C1-C10 alkylamine, linear or branched C1-C10 alkylalkylene, or linear or branched C1-C10 alkylalkoxy, where any of the linear or branched C1-C10 chains are optionally substituted with a halogen; and (b) recovering a cyclolavandulyl-substituted aromatic compound from the reaction mixture. In at least one embodiment of the method, the cyclolavandulyl diphosphate synthase (CLDS) is a polypeptide comprising an amino acid sequence of SEQ ID NO:2 or 4, or a variant of SEQ ID NO:2 or 4, optionally wherein the variant of SEQ ID NO:2 comprises an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:2 or 4.

[0022] In at least one embodiment of any of the methods of producing a cyclolavanduryl-substituted aromatic compound of the present disclosure, the prenyltransferase is selected from the group consisting of: (i) NphB (SEQ ID NO: 8), or a variant of NphB comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NO: 8; (ii) NphBM31s (SEQ ID NO: 6), or a variant of NphB comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NO: 6; (iii) a prenyltransferase comprising an amino acid sequence having at least 99% identity to any one of SEQ ID NOs: 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19; and (iv) a variant prenyltransferase comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NOs: 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19.

[0023] In at least one embodiment of the method for preparing a cyclolavandulyl-substituted aromatic compound, the cyclolavandulyl-substituted aromatic compound is selected from the group consisting of compounds (4a), (4b), (5a), (5b), (6a), (6b), (7a), (7b), (8a), and (8b): [ka] is selected from.

[0024] In at least one embodiment of the method for producing a cyclolavandulyl-substituted aromatic compound, the aromatic compound is a compound of structural formula (III), 2 is -H or -OH, R 3 is -H or -COOH, R 4 is a linear or branched C1-C10 alkyl, a linear or branched C1-C10 alkylamide, a linear or branched C1-C10 alkylamine, a linear or branched C1-C10 alkylalkylene, a linear or branched C1-C10 alkylalkoxy, or a C1-C10 alkylaryl, wherein any of the C1-C10 groups are optionally substituted with -OH, -OCH3, or halogen. 2 is -OH and R 3 In at least one embodiment, R 4 is selected from CH3, CH2CH3, (CH2)2CH3, (CH2)3CH3, (CH2)4CH3, (CH2)5CH3, (CH2)6CH3, and (CH2)7CH3, and optionally, R 4 is selected from (CH2)2CH3, (CH2)4CH3, and (CH2)6CH3. In at least one embodiment, the aromatic compound is a cannabinoid precursor compound selected from divalanic acid (DA), olivetolic acid (OA), and forolic acid (PA).

[0025] In at least one embodiment of the method for preparing a cyclolavanduryl-substituted aromatic compound, the aromatic compound of structural formula (III) is selected from compounds (3a), (3b), (3c), (3d), (3e), (3f), (3g), and (3h): [ka] is selected from one of the following:

[0026] In at least one embodiment of the method for producing a cyclolavandulyl-substituted aromatic compound, the recovered cyclolavandulyl-substituted aromatic compound has structural formula (IIa), (IIb), (IIc), or (IId): [ka] (In the formula, R 3 is -H or -COOH, R 4 is a linear or branched C1-C10 alkyl, a linear or branched C1-C10 alkylamide, a linear or branched C1-C10 alkylamine, a linear or branched C1-C10 alkylalkylene, a linear or branched C1-C10 alkylalkoxy, or a C1-C10 alkylaryl, wherein any of the C1-C10 groups are optionally substituted with -OH, -OCH3, or halogen.

[0027] In at least one embodiment of the method, the recovered cyclolavandulyl-substituted compound is a cyclolavandulyl-substituted cannabinoid, optionally the cyclolavandulyl-substituted cannabinoid is selected from the group consisting of compounds (2a), (2b), (2c), (2d), (2e), (2f), (2g), (2h), (2i), (2j), (2k), (2l), (2m), (2n), Selected from (2o), (2p), (2q), (2r), (2s), (2t), (2u), (2v), (2w), (2x), (2y), (2z), (2aa), (2bb), (2cc), (2dd), (2ee), (2ff), (2gg), (2hh), (2ii), (2jj), (2kk), (2ll), (2mm), (2nn), (2oo), and (2pp).

[0028] In at least one embodiment of any of the methods of producing a cyclolavandulyl-substituted cannabinoid compound of the present disclosure, the method may further comprise the step of decarboxylating the recovered cyclolavandulyl-substituted compound. For example, the carboxylated cyclolavandulyl-substituted cannabinoid of compound (2a) may be decarboxylated to produce a compound that is a cyclolavandulyl-substituted cannabinoid of compound (2b).

[0029] In another embodiment, the present disclosure also provides a composition of reactants used in the method. Thus, in at least one embodiment, the present disclosure provides a method for producing a cyclolavandulyl pyrophosphate of compound (1) by reacting a prenyltransferase with a cyclolavandulyl pyrophosphate of compound (1). [ka] and a compound of structural formula (III), (IV), (V), or (VI), 2 is -H or -OH, R 3 is -H or -COOH, R 4is -H, -OH, linear or branched C1-C10 alkyl, linear or branched C1-C10 alkylamide, linear or branched C1-C10 alkylamine, linear or branched C1-C10 alkylalkylene, linear or branched C1-C10 alkylalkoxy, or C1-C10 alkylaryl, wherein any of the C1-C10 groups are optionally substituted with -OH, -OCH3, or halogen.

[0030] In at least one embodiment, the present disclosure provides a method for the preparation of a cyclolavandulyl diphosphate synthase (CLDS), a dimethylallyl pyrophosphate, a prenyltransferase, and a compound of structural formulas (III), (IV), (V), and (VI), 2 is -H or -OH, R 3 is -H or -COOH, R 4 is -H, -OH, linear or branched C1-C10 alkyl, linear or branched C1-C10 alkylamide, linear or branched C1-C10 alkylamine, linear or branched C1-C10 alkylalkylene, linear or branched C1-C10 alkylalkoxy, or C1-C10 alkylaryl, where any of the C1-C10 groups are optionally substituted with -OH, -OCH3, or halogen. In at least one embodiment, the cyclolavandulyl diphosphate synthase (CLDS) is a polypeptide comprising an amino acid sequence of SEQ ID NO:2 or 4, or a variant of SEQ ID NO:2 or 4, optionally wherein the variant of SEQ ID NO:2 or 4 comprises an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:2 or 4.

[0031] In at least one embodiment of the composition, the prenyltransferase is selected from the group consisting of: (i) NphB (SEQ ID NO: 8), or a variant of NphB comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NO: 8; (ii) NphBM31s (SEQ ID NO: 6), or a variant of NphB comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NO: 6. (iii) a prenyltransferase comprising an amino acid sequence or any one of SEQ ID NOs: 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19; and (iv) a variant prenyltransferase comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NOs: 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19.

[0032] In at least one embodiment of the composition, the aromatic compound is a compound of structural formula (III), 2 is -H or -OH, R 3 is -H or -COOH, R 4 is -H, -OH, linear or branched C1-C10 alkyl, linear or branched C1-C10 alkylamide, linear or branched C1-C10 alkylamine, linear or branched C1-C10 alkylalkylene, linear or branched C1-C10 alkylalkoxy, or C1-C10 alkylaryl, wherein any of the C1-C10 groups are optionally substituted with -OH, -OCH3, or halogen. 4 is selected from CH3, CH2CH3, (CH2)2CH3, (CH2)3CH3, (CH2)4CH3, (CH2)5CH3, (CH2)6CH3, and (CH2)7CH3, and optionally, R 4is selected from (CH2)2CH3, (CH2)4CH3, and (CH2)6CH3. In at least one embodiment, the aromatic compound is a cannabinoid precursor compound selected from divalanic acid (DA), olivetolic acid (OA), and phorolic acid (PA).

[0033] In at least one embodiment of the composition, the aromatic compound having structural formula (III) is selected from any one of compounds (3a), (3b), (3c), (3d), (3e), (3f), (3g), and (3h).

[0034] In at least one embodiment of the composition, the composition further comprises a cyclolavandulyl-substituted aromatic compound selected from compounds (3i), (3j), (3k), (3l), (4a), (5a), (6a), and (6b). In at least one embodiment, the cyclolavandulyl-substituted compound is a cyclolavandulyl-substituted cannabinoid, and optionally, the cyclolavandulyl-substituted cannabinoid is selected from compounds (2a), (2b), (2c), (2d), (2e), (2f), (2g), (2h), (2i), (2j), (2k), (2l), (2m), (2n), (2o), (2p), (2q), (2r), (2s), (2t). , (2u), (2v), (2w), (2x), (2y), (2z), (2aa), (2bb), (2cc), (2dd), (2ee), (2ff), (2gg), (2hh), (2ii), (2jj), (2kk), (2ll), (2mm), (2nn), (2oo), (2pp), (2qq), (2rr), (2ss), (2tt), (2uu), (2vv), (2ww), and (2xx).

[0035] The novel features and advantages of the present disclosure will be better understood by reference to the following detailed description that sets forth illustrative embodiments in which the principles of the disclosure are utilized and the accompanying drawings (also referred to herein as "Figure" and "FIG.")). [Brief description of the drawings]

[0036] [Figure 1] FIG. 1 is a schematic diagram of the steps involved in the biosynthetic methods and compositions for producing cyclolavanduryl derivatives of cannabinoids of the present disclosure, molecular inputs / outputs, and enzymes involved in the biosynthesis of cannabinoids. [Diagram 2] FIG. 1 shows exemplary prenylation reactions to produce the standard cannabinoids CBGVA, CBGA and CBGPA, as well as an alternative "cyclolavandulylation" reaction using cyclolavandulyl pyrophosphate (CLPP) as the cyclolavandulyl group donor to an acceptor cannabinoid precursor substrate to produce the cyclolavandulyl-cannabinoid compounds CBCLVA and CBCLA. [Diagram 3] FIG. 1 shows the LC-MS results of the in vitro biosynthesis of cyclolavanduryl-CBCLA, as described in Example 1. [Figure 4] FIG. 1 shows exemplary LC-MS results of the in vitro biosynthesis of standard cannabinoids CBGVA, CBGA and CBGPA, and cyclolavandurylated cannabinoid compounds CBCLVA and CBCLA. [Figure 5A] Figures 5A, 5B, 5C, 5D, and 5E show the LC-MS results of the cell-free biosynthesis of five different cyclolavandulyl-substituted aromatic compounds prepared as described in Example 2. Figure 5A: HPLC results for cyclolavandulyl-resveratrol. [Figure 5B] FIG. 5B: HPLC results for cyclolavanduryl-luteolin. [Figure 5C] FIG. 5C: HPLC results for cyclolavanduryl-naringenin. [Figure 5D] FIG. 5D: HPLC results for cyclolavanduryl-tryptophan. [Figure 5E] FIG. 5E: HPLC results for cyclolavandulyl-olivetolic acid. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "a protein" includes two or more proteins, and a reference to "a compound" refers to two or more compounds. It is further noted that the claims may be drafted to exclude any optional element. Thus, this statement is intended to serve as a prelude to using exclusive terminology, such as "solely," "only," or using a "negative" limitation in connection with the recitation of claim elements. "Comprise," "comprises," "comprising," "include," "includes," and "including" are used interchangeably and are not intended to be limiting. It is further understood that when the term "comprising" is used in describing various embodiments, those of ordinary skill in the art will understand that in some specific cases, the embodiments can be alternatively described using the language "consisting essentially of" or "consisting of."

[0038] Where a range of values ​​is stated, it is understood that each intervening integer between the upper and lower limits of that range, and each tenth of each intervening integer, unless the context clearly dictates otherwise, as well as any other stated or intervening value within the stated range, are encompassed within the invention, unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may be independently included within the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where a stated range includes one or both of the limits, ranges excluding either (i) or both of (ii) those included limits are also encompassed within the invention. For example, "1 to 50" includes "2 to 25," "5 to 20," "25 to 50," "1 to 10," etc.

[0039] In general, the nomenclature used herein, and the techniques and procedures described herein, include those well understood and commonly used by those of skill in the art, such as the conventional techniques and methodologies described in, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual (Fourth Edition), Vols. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 2012 (hereinafter "Sambrook"), and Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., first published as a book in 1987 by Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., supplemented periodically until 2011, and now available online in journal format as Current Protocols in Molecular Biology, Vols. 00 - 130, (1987-2020), published by Wiley & Sons, Inc. in the Wiley Online Library) (hereinafter "Ausubel").

[0040] All publications, patents, patent applications, and other documents referred to in this disclosure are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document was individually indicated to be incorporated by reference for all purposes.

[0041] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. It is to be understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting. For the purposes of interpreting this disclosure, the following explanation of terms shall apply, and where appropriate, terms used in the singular shall include the plural and vice versa.

[0042] definition "Cyclolavandulyl group" refers to a branched and cyclic carbon C10 monoterpene group in either the "forward" or "reverse" form as represented by the following chemical substructures: [ka]

[0043] Cyclolabandulyl groups are known to occur in several natural product compounds, including callistein A, seserinolonol, lavanduskinosin, and lavandusyanin. The biosynthesis of the cyclolavandulyl structure occurs via an enzyme-catalyzed "non-head-to-tail" condensation and cyclization of two molecules of C5 dimethylallyl pyrophosphate (DMAPP). See, for example, Non-Patent Document 3.

[0044] "Cyclolavandulyl diphosphate synthase" or "CLDS" refers to an enzyme capable of catalyzing the conversion of two molecules of dimethylallyl pyrophosphate (DMAPP) to the product compound cyclolavandulyl pyrophosphate (CLPP) (compound (1)). A "CLDS" polypeptide can include any naturally occurring, recombinant and / or engineered (mutant) polypeptide having the CLDS activity of Scheme 1, and is intended to include enzymes such as wild-type CLDS from Streptomyces sp. CL190 (GenBank accession: BAO66170.1, PDB: 5YGJ_A, UniProt entry X5IYJ5), and recombinant or engineered polypeptides derived from the wild-type CL190 enzyme, as well as any other enzymes having CLDS activity.

[0045] As used herein, "prenyltransferase" or "aromatic prenyltransferase" or "PT" refers to an enzyme capable of catalyzing the transfer of a prenyl pyrophosphate donor substrate (e.g., geranyl pyrophosphate) to an aromatic acceptor compound (e.g., olivetolic acid). Aromatic prenyltransferase (PT) polypeptides can include naturally occurring and recombinant polypeptides having the PT activity of Scheme 2, and are intended to include enzymes such as wild-type NphB from Streptomyces sp. CL190, and recombinant or engineered polypeptide variants derived from the wild-type NphB enzyme, such as NphBM31s, and others disclosed in U.S. Patent No. 5,399,233, which is incorporated herein by reference, as well as other naturally occurring enzymes having PT activity, including those disclosed in the Examples. The mutant NphBM31s has the following mutations relative to the wild-type M14I, Y31W, T69P, T77I, T98I, S136A, E222D, G224S, A232S, N236T, Y288V, and G297K. As disclosed elsewhere herein, it is a surprising discovery of the present disclosure that enzymes with PT activity can also catalyze the transfer of a cyclolavanduryl group from a pyrophosphate donor substrate, such as CLPP (compound (1)), to an aromatic compound.

[0046] "Cannabinoid" refers to compounds that act on cannabinoid receptors and is intended to include endocannabinoid compounds that are naturally produced in animals, phytocannabinoid compounds that are naturally produced in the cannabis plant, and synthetic cannabinoid compounds. Exemplary cannabinoids are provided in Table 1. Exemplary cannabinoids include, but are not limited to, cannabigerolic acid (CBGA) and cannabigerovarinic acid (CBGVA).

[0047] Table 1: Exemplary cannabinoid compounds [Table 1] JPEG2025513329000022.jpg207154JPEG2025513329000023.jpg221155JPEG20255133290 00024.jpg222153JPEG2025513329000025.jpg234153JPEG2025513329000026.jpg180154

[0048] As used herein, a "cannabinoid precursor compound" or "cannabinoid precursor substrate" refers to a compound or molecule that is acted upon by an enzyme in the biosynthetic process to produce a cannabinoid. Exemplary cannabinoid precursors are provided in Table 2. Exemplary cannabinoid precursors include, but are not limited to, the aromatic polyketides olivetolic acid (OA) and divalic acid (DA), which are enzymatically prenylated with a geranyl group to form the cannabinoids, CBGA and CBGVA, respectively.

[0049] Table 2: Exemplary cannabinoid precursor compounds [Table 2]

[0050] "Cyclolavandulyl-substituted cannabinoid" refers to a cannabinoid precursor compound modified with a cyclolavandulyl group. Typically, the cyclolavandulyl group modifies the cannabinoid precursor compound at the position where a prenyltransferase (e.g., NphB) enzymatically prenylates the compound (e.g., with a geranyl group derived from GPP). Exemplary cyclolavandulyl-substituted cannabinoids of the present disclosure include compounds (2a), (2b), (2c), (2d), (2e), (2f), (2g), (2h), (2i), (2j), (2k), (2l), (2m), (2n), (2o), (2p), (2q), (2r), (2s), (2t), (2u), (2v), (2w), (2x), (2y), (2z) shown in Table 3. , (2aa), (2bb), (2cc), (2dd), (2ee), (2ff), (2gg), (2hh), (2ii), (2jj), (2kk), (2ll), (2mm), (2nn), (2oo), and (2pp) (including CBCLA, CBCL, CBCLVA, CBCLV, CBCLPA, and CPCLP).

[0051] Table 3: Exemplary cyclolavanduryl-substituted cannabinoids [Table 3] JPEG2025513329000029.jpg212155JPEG2025513329000030.jpg204155JPEG2025513329000031.jpg230154JPEG2025513329 000032.jpg231155JPEG2025513329000033.jpg209154JPEG2025513329000034.jpg236153JPEG2025513329000035.jpg54153

[0052] "Flavonoid compounds" refers to compounds of the class of polyphenol secondary metabolism compounds found in plants, including, but not limited to, compounds from the flavone, flavonol, flavanone, flavanonol, flavan, and isoflavonoid families. Exemplary flavonoids useful in the compositions and methods of the present disclosure include, but are not limited to, luteolin, apigenin, tangeritin, quercetin, kaempferol, myricetin, fisetin, galangin, isorhamnetin, pachypodol, rhamnagin, pyranoflavonol, furanoflavonol, hesperetin, naringenin, eriodictyol, and homoeriodictyol.

[0053] "Aromatic compound" refers to a compound having at least one aromatic group that can accept the transfer of a cyclolavandulyl group from cyclolavandulyl pyrophosphate mediated by a prenyltransferase (e.g., NphB). Typically, such aromatic compounds can also accept the transfer of a prenyl group (e.g., a geranyl group from GPP) mediated by a prenyltransferase. Exemplary aromatic compounds of the present disclosure include, but are not limited to, compounds of structural formulas III, IV, V, and VI disclosed elsewhere herein. Also included are compounds of the chemical structures shown in Table 4.

[0054] Table 4: Exemplary aromatic compounds [Table 4] JPEG2025513329000037.jpg232151JPEG2025513329000038.jpg53151

[0055] "Cyclolavandulyl-substituted aromatic compounds" refers to aromatic compounds (e.g., cannabinoid precursor alkaloid or flavonoid compounds) modified with a cyclolavandulyl group. Typically, the cyclolavandulyl group modifies the aromatic compound at the position where a prenyltransferase (e.g., NphB) enzymatically prenylates the compound (e.g., with a geranyl group derived from GPP). Exemplary cyclolavandulyl-substituted aromatic compounds of the present disclosure include, but are not limited to, the exemplary cyclolavandulyl-substituted cannabinoids of Table 3, and cyclolavandulyl-substituted aromatic compounds (4a), (4b), (5a), (5b), (6a), (6b), (7a), (7b), (8a), (8b), (2qq), (2rr), (2ss), (2tt), (2uu), (2vv), (2ww), and (2xx) having the chemical structures shown in Table 5.

[0056] Table 5: Exemplary cyclolavanduryl-substituted aromatic compounds [Table 5] JPEG2025513329000040.jpg189152JPEG2025513329000041.jpg191152

[0057] As used herein, "conversion" refers to the enzymatic conversion of a substrate(s) to the corresponding product(s). "Percent conversion" refers to the percent of a substrate that is converted to a product under specified conditions within a given period of time. Thus, "enzyme activity" or "activity" of an enzymatic conversion can be expressed as the "percent conversion" of substrate to product.

[0058] As used herein in the context of an enzyme-mediated process, "product" refers to a compound or molecule resulting from the activity of an enzyme. In the context of an enzyme with CLDS activity useful in the methods and compositions of the present disclosure, an exemplary product is cyclolavandulyl pyrophosphate (compound (1)) shown in Scheme 1. In the context of an enzyme with PT activity useful in the methods and compositions of the present disclosure, an exemplary product is the cyclolavandulyl-substituted aromatic compounds listed in Tables 3 and 5.

[0059] As used herein in the context of an enzyme-mediated process, a "substrate" refers to a compound(s) or molecule(s) that is / are acted upon by an enzyme. In the context of an enzyme having CLDS activity useful in the compositions and methods of the present disclosure, the substrate that is acted upon by the enzyme may include dimethylallyl pyrophosphate (DMAPP) (see, e.g., Scheme 1) and its derivatives. Similarly, in the context of an enzyme having PT activity, the substrate that is acted upon by the enzyme may include the cyclolavandulyl group donor cyclolavandulyl pyrophosphate (compound (1)), and a series of cyclolavandulyl acceptor aromatic compounds, including but not limited to aromatic polyketides, such as the cannabinoid precursor compounds of Table 2 (e.g., olivetolic acid), alkaloid compounds, flavonoid compounds, and other aromatic compounds of Table 4 (including the exemplary aromatic compounds described elsewhere herein, including in this Example).

[0060] As used herein, "host cell" refers to a cell that can be functionally modified with a recombinant nucleic acid and function to express recombinant products, including polypeptides and compounds produced by the activity of the polypeptide.

[0061] As used interchangeably herein, "nucleic acid" or "polynucleotide" refers to two or more covalently linked nucleosides. A nucleic acid may be composed entirely of ribonucleosides (e.g., RNA), entirely of 2'-deoxyribonucleotides (e.g., DNA), or may be composed of a mixture of ribonucleosides and 2'-deoxyribonucleosides. The nucleoside units of a nucleic acid may be linked via phosphodiester bonds (e.g., in the case of naturally occurring nucleic acids), or the nucleic acid may contain one or more non-natural bonds (e.g., phosphorothioester bonds). Nucleic acid or polynucleotide is intended to include single-stranded or double-stranded molecules, or molecules having both single-stranded and double-stranded regions. Nucleic acid or polynucleotide is intended to include molecules composed of naturally occurring nucleobases (i.e., adenine, guanine, uracil, thymine, and cytosine) or molecules that contain one or more modified and / or synthetic nucleobases, such as, for example, inosine, xanthine, hypoxanthine, etc.

[0062] "Protein," "polypeptide," and "peptide" are used interchangeably herein to mean a polymer of at least two amino acids covalently joined by an amide bond, regardless of length or post-translational modification (e.g., glycosylation, phosphorylation, lipidation, myristylation, ubiquitination, etc.). As used herein, a "protein" or "polypeptide" or "peptide" polymer can include D- and L-amino acids, and mixtures of D- and L-amino acids.

[0063] As used herein, "naturally occurring" or "wild-type" refers to a form found in nature. For example, a naturally occurring nucleic acid sequence is a sequence present in an organism that can be isolated from a source in nature and has not been intentionally modified by human manipulation.

[0064] As used herein, for example with respect to a cell, nucleic acid, or polypeptide, "recombinant," "engineered," or "non-naturally occurring" refers to material that has been modified in a way that does not occur in nature, or that is identical to, but has been produced or obtained from synthetic material and / or by manipulation using recombinant techniques, i.e., material that corresponds to the native or naturally occurring form of the material. Non-limiting examples include, among others, recombinant cells that express genes that are not present in the native (non-recombinant) form of the cell, or that express native genes that are otherwise expressed at different levels.

[0065] As used herein, a "derived nucleic acid" refers to a nucleic acid having a sequence at least substantially identical to a sequence naturally occurring in an organism, such as a cDNA molecule prepared by reverse transcription of mRNA isolated from an organism, or a nucleic acid molecule prepared synthetically to have a sequence at least substantially identical to a nucleic acid sequence present in an organism, or which hybridizes to a sequence at least substantially identical to a nucleic acid sequence present in an organism.

[0066] "Coding sequence" refers to a nucleic acid segment (eg, a gene) that codes for the amino acid sequence of a protein.

[0067] As used herein, "heterologous nucleic acid" refers to any polynucleotide that is introduced into a host cell by laboratory techniques, and includes polynucleotides that have been removed from a host cell, subjected to laboratory manipulation, and then reintroduced into the host cell.

[0068] "Codon optimization" refers to changing the codons of a polynucleotide encoding a protein to those codons preferentially used in a particular organism so that the encoded protein is efficiently expressed in the organism of interest. Although the genetic code is degenerate in that most amino acids are represented by multiple codons, termed "synonymous" or "synonymous" codons, it is well known that codon usage by a particular organism is not random, but is biased toward certain codon triplets. This bias in codon usage may be greater for a given gene, genes of common function or common ancestral origin, highly expressed proteins versus low copy number proteins, and aggregated protein-coding regions of the organism's genome. In some embodiments, a polynucleotide encoding an imine reductase enzyme may be codon optimized for optimal production from the host organism selected for expression.

[0069] "Preferred optimal high codon usage biased codons" refer to codons that are used more frequently in protein coding regions than other codons that code for the same amino acid. Preferred codons can be determined with respect to a single gene, a set of genes of common function or common origin, the frequency of codon usage in highly expressed genes, the frequency of the codon in aggregate protein coding regions across organisms, the frequency of the codon in aggregate protein coding regions of related organisms, or combinations thereof. Codons whose frequency increases with the level of gene expression are typically optimal codons for expression. Various methods are known for determining the frequency (e.g., codon usage, relative synonymous codon usage) and codon preference in a particular organism, including multivariate analysis using, for example, cluster analysis or correspondence analysis, as well as for determining the effective number of codons used in a gene (see GCG CodonPreference, Genetics Computer Group Wisconsin Package; CodonW, John Peden, University of Nottingham; McInerney, J. O, 1998, Bioinformatics 14:372-73; Stenico et al., 1994, NucleicAcids Res. 222437-46; Wright, F., 1990, Gene 87:23-29). Codon usage tables are available for a growing list of organisms (see, e.g., Wada et al., 1992, Nucleic Acids Res. 20:2111-2118; Nakamura et al., 2000, Nucl. Acids Res. 28:292; Duret et al., supra; Henaut and Danchin, "Escherichia coli and Salmonella," 1996, Neidhardt,et al. Eds., ASM Press, Washington DC, p. 2047-2066). The data source for obtaining codon usage can be based on any available nucleotide sequence capable of encoding a protein.These datasets include nucleic acid sequences that are actually known to encode expressed proteins (e.g., complete protein-coding sequences (CDS)), expressed sequence tags (ESTs), or predicted coding regions of genomic sequences (see, e.g., Mount, D., Bioinformatics: Sequence and Genome Analysis, Chapter 8, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001; Uberbacher, EC, 1996, Methods Enzymol. 266:259-281; ​​Tiwari et al., 1997, Comput. Appl. Biosci. 13:263-270).

[0070] As used herein, "control sequences" refers to all sequences necessary or advantageous for the expression of polynucleotides and / or polypeptides used in the present disclosure. Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, a leader, a promoter, a polyadenylation sequence, a propeptide sequence, a signal peptide sequence, and a transcription terminator. At a minimum, control sequences typically include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites that facilitate ligation of the control sequences with the coding region of the nucleic acid sequence encoding the polypeptide.

[0071] As used herein, "operably linked" refers to a configuration in which a control sequence is suitably positioned (e.g., in a functional relationship) relative to a polynucleotide or polypeptide sequence of interest such that the control sequence directs or regulates the expression of the sequence of interest.

[0072] "Promoter sequence" refers to a nucleic acid sequence recognized by a host cell for expression of a polynucleotide of interest, such as a coding sequence. The promoter sequence contains transcriptional control sequences that mediate expression of the polynucleotide of interest. The promoter may be any nucleic acid sequence that exhibits transcriptional activity in a selected host cell, including mutant promoters, truncated promoters, and hybrid promoters, and may be derived from a gene encoding an extracellular or intracellular polypeptide that is either homologous or heterologous to the host cell.

[0073] "Percentage of sequence identity", "percent sequence identity", "percentage of homology" or "percent homology" are used interchangeably herein to refer to values ​​that quantify the comparison of polynucleotide or polypeptide sequences, and are determined by comparing two optimally aligned sequences over a comparison window, where a portion of the polynucleotide or polypeptide sequence within the comparison window may contain additions or deletions (i.e., gaps) compared to the reference sequence due to optimal alignment of the two sequences. The percentage value can be calculated by determining the number of positions where the same nucleic acid base or amino acid residue is present in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Alternatively, the percentage can be calculated by determining the number of positions where either the same nucleic acid base or amino acid residue is present in both sequences, or aligning the nucleic acid base or amino acid residue with gaps to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Those skilled in the art appreciate that there are many established algorithms available to align two sequences.Optimal alignment of sequences for comparison can be carried out, for example, by the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482, by the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, by the similarity search method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444, by computer implementations of these algorithms (GAP, BESTFIT, FASTA and TFASTA in the GCG Wisconsin Software Package), or by visual inspection (see generally Current Protocols in Molecular Biology, FM Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (supplemented in 1995) (Ausubel)). Examples of algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, described in Altschul et al., 1990, J. Mol. Biol. 215: 403-410, and Altschul et al., 1977, Nucleic Acids Res. 3389-3402, respectively. Software for performing BLAST analyses is publicly available on the National Center for Biotechnology Information website. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short word lengths W in the query sequence that match or meet some positive threshold score T when aligned with words of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them.The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. For nucleotide sequences, the parameters M (reward score for a pair of matching residues, always >0) and N (penalty score for mismatching residues, always <0) are used to calculate the cumulative score. For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hits in each direction is stopped when the cumulative alignment score falls off its maximum achieved value by an amount X, when the accumulation of one or more negative scoring residue alignments causes the cumulative score to fall below 0, or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, 1989, Proc Natl Acad Sci USA 89:10915). For exemplary determination of sequence alignment and percent sequence identity, the BESTFIT or GAP programs in the GCG Wisconsin Software package (Accelrys, Madison Wis.) can be employed using the default parameters provided.

[0074] "Reference sequence" refers to a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset of a larger sequence, for example, a segment of a full-length nucleic acid or polypeptide sequence. A reference sequence is typically at least 20 nucleotide or amino acid residue units in length, and may be the full-length nucleic acid or polypeptide. Since two polynucleotides or polypeptides each may (1) contain similar sequences between the two sequences (i.e., a portion of the complete sequence) and (2) further contain sequences that differ between the two sequences, sequence comparison between two (or more) polynucleotides or polypeptides is typically performed by comparing the sequences of the two polynucleotides or polypeptides over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of at least about 20 contiguous nucleotide positions or amino acid residues, where a sequence can be compared to a reference sequence of at least 20 contiguous nucleotides or amino acids, and the portion of the sequence in the comparison window can contain no more than 20 percent additions or deletions (i.e., gaps) when compared to the reference sequence (which does not include additions or deletions) with respect to optimal alignment of the two sequences.

[0075] "Substantial identity" or "substantially identical" refers to a polynucleotide or polypeptide sequence having at least 70 percent sequence identity, at least 80 percent sequence identity, at least 85 percent sequence identity, at least 90 percent sequence identity, at least 95 percent sequence identity, or at least 99 percent sequence identity when compared to a reference sequence over a comparison window of at least 20 nucleoside or amino acid residue positions, often over a window of at least 30-50 positions, where the percentage of sequence identity is calculated by comparing the reference sequence to a sequence that contains deletions or additions that total no more than 20 percent of the reference sequence over the comparison window.

[0076] When used in the context of numbering of a given amino acid or polynucleotide sequence, "corresponding to," "with respect to," or "compared to" refers to the numbering of residues in a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. In other words, the residue numbers or residue positions of a given polymer are specified with respect to the reference sequence, not according to the actual numerical position of the residues in the given amino acid or polynucleotide sequence. For example, a given amino acid sequence, such as the amino acid sequence of an engineered imine reductase, can be aligned to a reference sequence by introducing gaps to optimize the residue matches between the two sequences. In these cases, even though gaps exist, the numbering of the residues in the given amino acid or polynucleotide sequence is done with respect to the reference sequence to which it is aligned.

[0077] "Isolated," as used herein with respect to a molecule, means that the molecule (e.g., cannabinoid, polynucleotide, polypeptide) is substantially separated from other compounds with which it is naturally associated, such as proteins, lipids, and polynucleotides. The term includes nucleic acids that have been removed or purified from their naturally occurring environment or expression system (e.g., a host cell or in vitro synthesis).

[0078] "Substantially pure" refers to a composition in which the desired molecule is the predominant species present (i.e., on a molar or weight basis, it is more abundant than any other individual macromolecular species in the composition); generally, a composition is substantially purified when the desired species constitutes at least about 50 mole % or 50 weight % of the macromolecular species present.

[0079] "Recovered" as used herein with respect to an enzyme, protein or cannabinoid compound refers to a more or less pure form of the enzyme, protein or cannabinoid.

[0080] Biosynthetic methods and compositions for producing cyclolavandulyl-substituted aromatic compounds Enzymes with prenyltransferase (PT) activity can transfer prenyl groups from certain prenyl donor substrates (e.g., geranyl pyrophosphate, or "GPP") to compounds that can act as prenyl acceptor substrates, including a wide range of aromatic compounds such as flavonoids, alkaloids, and cannabinoid precursors. See, for example, J. Am. Chem. Soc. 1999, 143:1311-1326 (2002). Scheme 1 (below) shows an example of the prenyltransferase-catalyzed transfer of the geranyl group of GPP to the aromatic polyketide cannabinoid precursor olivetolic acid (OA) to form the cannabinoid CBGA. [ka]

[0081] FIG. 1 shows an illustration of the molecular input / output and enzymatic pathways in cannabinoid biosynthesis leading to the enzymatic prenylation step, in which a prenyltransferase (or "GOT") (e.g., NphB) catalyzes the transfer of the geranyl group of GPP to an aromatic polyketide (e.g., olivetolic acid), resulting in the formation of a prenylated aromatic cannabinoid product (e.g., CBGA).

[0082] An enzyme with cyclolavandulyl diphosphate synthase (CLDS) activity can catalyze the conversion of two molecules of dimethylallyl pyrophosphate (DMAPP) to the product compound cyclolavandulyl pyrophosphate (CLPP) (compound (1)), as shown in Scheme 2 below. [ka]

[0083] A naturally occurring enzyme with CLDS activity from Streptomyces sp. CL190 has been isolated and structurally characterized. See, for example, Non-Patent Document 3 and Non-Patent Document 4. The polypeptide sequence of CLDS from Streptomyces sp. CL190 (GenBank Accession: BAO66170.1, PDB: 5YGJ_A) has 217 amino acids and is presented herein as SEQ ID NO: 2: MTTLMLLPDGMRRWSEKNGVSLDDGYAAMGDKIIEFMGWAKEEGVKTLYITASSAANHGRPEAAVNTFMEAFTEVIRRCHSQFKFDFSGSLDLVSEDYLTELSALRDKSDSESDFTLHYILGMSLSHEVVGIFNKLNGKIPEMTEEILAENAYVPTQVDYIIRTGGAIRMSSFFPLMSPYAELHFSPVLFPDTTRADFDAALKDLRARDRRFGGYPA (SEQ ID NO: 2)

[0084] It is a surprising discovery of the present disclosure that a cyclolavandulyl group donor compound, such as CLPP (compound (1)), can be used as a donor substrate by an enzyme with PT activity (e.g., NphB) in the biosynthesis of various cyclolavandulyl-substituted aromatic compounds, such as the cyclolavandulylated cannabinoid CBCLA of compound (2e), as shown in Scheme 3 below: [ka]

[0085] The discovery that enzymes with PT activity can catalyze the transfer of cyclolavandulyl to aromatic compounds provides novel biosynthetic methods and compositions useful for the production of cyclolavandulyl-substituted aromatic compounds by the use of enzymes. In at least one embodiment, the present disclosure contemplates a method for producing cyclolavandulyl-substituted aromatic compounds as shown in Scheme 3. Such a method includes a biocatalytic step of contacting a cyclolavandulyl donor compound CLPP (compound (1)) with an aromatic compound (e.g., OA) and a prenyltransferase under suitable conditions, and then recovering the enzymatic product of a cyclolavandulyl-substituted aromatic compound (e.g., compound (2e)) from the reaction mixture. In such a method, the reaction may be carried out using in vitro enzymatic reaction conditions similar to those typically used for prenyltransferase reactions, using GPP as the prenyl donor rather than the cyclolavandulyl donor compound CLPP (compound (1)).

[0086] By way of comparison, FIG. 2 shows the biosynthetic prenylation reactions that produce the standard cannabinoids CBGVA, CBGA and CBGPA, and the alternative biosynthetic cyclolavandulylation reactions that can be carried out using CLPP as a cyclolavandulyl group donor to a precursor cannabinoid substrate, resulting in the cyclolavandulyl-cannabinoid compounds CBCLVA and CBCLA.

[0087] While not intending to be bound by mechanism, it is well established that prenylation catalyzed by prenyltransferases occurs in two steps: (1) formation of a resonance-stabilized allylic cation, and (2) nucleophilic attack by an activated aromatic substrate. See, e.g., Tanner, "Mechanistic studies on the indole prenyltransferases," Natural Product Reports, Issue 1, 2015; doi.org / 10.1039 / C4NP00099D. After dissociation of the prenyl donor pyrophosphate (e.g., GPP) at the active site of the prenyltransferase, the resulting primary allylic carbocation is stabilized by resonance with a tertiary carbocation. This allylic cation is susceptible to nucleophilic attack at both the primary (C1) and tertiary (C3) positions, depending on the configuration of the active site of the prenyltransferase. When the nucleophile attacks the C1 position, it is said to undergo "forward" prenylation, whereas when the nucleophile attacks the C3 position, it is said to undergo "reverse" prenylation. See, for example, Walsh "Biological Matching of Chemical Reactivity: Pairing Indole Nucleophilicity with Electrophilic Isoprenoids," ACS Chem. Biol.2014, 9, 12, 2718-2728; doi.org / 10.1021 / cb500695k. In general, the nucleophile must be either an indole or an activated benzene. Stereoselectivity and regioselectivity (either -C, -O, or -N atom of the activated substrate) are determined by the configuration of the active site and can be easily tuned through protein engineering.For example, Yang et al., "Catalytic Mechanism of Aromatic Prenylation byNphB," Biochemistry 2012, 51, 12, 2606-2618, Fan et al., "Site-directed Mutagenesis Switching a Dimethylallyl TryptophanSynthase to a Specific Tyrosine C3-Prenylating Enzyme," J. Biol. Chem. 290(3), 16 January 2015, 1364-1373, see Valliere et al., "A cell-free platform for the prenylation of natural products and application to cannabinoid production," Nature Communications 10, 565(2019); DOI: 10.1038 / s41467-019-08448-y. Due to the electron-rich nature of aromatic hydroxyls, both forward and reverse prenylation at the hydroxyl position has been widely observed in natural products (Ref 6, Ref 7, Ref 8).

[0088] Thus, it is contemplated that aromatic cyclolavandurylation catalyzed by the aromatic prenyltransferases disclosed herein can provide both a "forward" product (as shown in Scheme 3) or a "reverse" product as shown in exemplary Scheme 4 below. [ka]

[0089] It is also contemplated that cyclolavandulylation of aromatic compounds catalyzed by the aromatic prenyltransferases disclosed herein can provide both "forward" and "reverse" products at the hydroxyl position to give cyclolavandulylated aromatic products, as shown in exemplary Schemes 5 and 6 below. [ka]

[0090] Thus, in at least one embodiment, the present disclosure provides a method for the biosynthesis of a cyclolavandulyl-substituted aromatic compound, the method comprising: (a) contacting in a reaction mixture under suitable reaction conditions a cyclolavandulyl donor compound, CLPP (compound (1)), an enzyme having aromatic prenyltransferase activity (e.g., NphB or an NphB mutant), and an aromatic compound capable of acting as a cyclolavandulyl acceptor substrate; and (b) recovering the cyclolavandulyl-substituted aromatic compound from the reaction mixture.

[0091] As described elsewhere herein, it is also contemplated that the enzymatic reactions of Schemes 3, 4, 5 and 6 which produce the exemplary carboxylated forms of cyclolavandulyl-substituted cannabinoid compounds (2a), (2q), (2ee) and (2mm) may be extended to include a further step of decarboxylation, thereby producing the corresponding decarboxylated cannabinoid compounds. For example, the reaction of Scheme 3 produces a carboxylated cyclolavandulyl-substituted cannabinoid compound (2a), which can be further decarboxylated to produce compound (2b).

[0092] In an alternative method contemplated by the present disclosure, rather than adding CLPP (compound (1)) as a reagent in the enzymatic reaction with PT, this cyclolavandulyl donor compound can be prepared in situ through an enzymatic reaction between DMAPP and an enzyme having CLDS activity (e.g., the reaction of Scheme 2). In other words, the enzymatic reaction to produce CLPP in Scheme 2 can be combined with a PT-catalyzed cyclolavandulyl group transfer reaction, as shown in Scheme 3. In such a method, two enzymes, one with CLDS activity and the other with PT activity, are combined in a reaction mixture with DMAPP and an aromatic compound that is a cyclolavandulyl group acceptor. Under appropriate reaction conditions, the cyclolavandulyl donor substrate CLPP (compound (1)) is catalyzed in situ by CLDS and then transferred to an aromatic compound acting as a substrate by an enzyme having PT activity.

[0093] Thus, in at least one embodiment, the disclosure provides a method for the biosynthesis of a cyclolavandulyl-substituted aromatic compound, the method comprising: (a) contacting in a reaction mixture under suitable reaction conditions an enzyme having cyclolavandulyl diphosphate synthase (CLDS) activity, dimethylallyl pyrophosphate (DMAPP), an enzyme having aromatic prenyltransferase activity, and an aromatic compound capable of acting as a cyclolavandulyl acceptor substrate; and (b) recovering the cyclolavandulyl-substituted aromatic compound from the reaction mixture.

[0094] Also provided herein are compositions of enzymes and reactants used in enzymatic reaction methods.Thus, in at least one embodiment, the present disclosure provides a composition comprising cyclolavandulyl donor compound CLPP (compound (1)), an enzyme with aromatic prenyltransferase activity (e.g., NphB or NphB mutant), and an aromatic compound that can act as a cyclolavandulyl acceptor substrate.In another embodiment, the present disclosure provides a composition comprising an enzyme with cyclolavandulyl diphosphate synthase (CLDS) activity, dimethylallyl pyrophosphate (DMAPP), an enzyme with aromatic prenyltransferase activity, and an aromatic compound that can act as a cyclolavandulyl acceptor substrate.

[0095] As noted elsewhere herein, enzymes with PT activity are known to transfer prenyl groups to a range of aromatic compounds, including aromatic polyketides, flavonoids, alkaloids, and aromatic amino acid analogs. The present disclosure contemplates that the promiscuity of enzymes with PT activity (e.g., NphB and its variants) with aromatic prenyl group acceptor compounds will enable the transfer of cyclolavandulyl to a range of such compounds in a similar manner. Thus, aromatic compounds that can act as cyclolavandulyl group acceptor substrates for the biosynthesis of cyclolavandulyl-substituted aromatic products in the methods and compositions of the present disclosure include compounds of structural formula (III): [ka] (In the formula, R 2 is -H or -OH, R 3 is -H or -COOH, R 4is a linear or branched C1-C10 alkyl, linear or branched C1-C10 alkylamide, linear or branched C1-C10 alkylamine, linear or branched C1-C10 alkylalkylene, linear or branched C1-C10 alkylalkoxy, or C1-C10 alkylaryl, where any of the C1-C10 groups are optionally substituted with -OH, -OCH3, or halogen. Exemplary aromatic polyketides that can act as cyclolavanduryl group acceptor substrates, as represented by structural formula (III), include, but are not limited to, the cannabinoid precursor compounds of Table 2.

[0096] Cannabinoid precursor compounds (3a), (3b), (3c), (3d), (3e), (3f), (3g), and (3h) (see Table 2) are compounds (2a), (2b), (2c), (2d), (2e), (2f), (2g), (2h), ( 2i), (2j), (2k), (2l), (2m), (2n), (2o), (2p), (2q), (2r), (2s), (2t), (2u), (2v), (2w), (2x), (2y), (2z), (2aa), (2bb), (2cc), A series of cannabinoid producing compounds can be formed by cyclolavandulylation via PT activity (e.g., NphB) with a suitable cyclolavandulyl donor such as compound (1) to form a series of cyclolavandulyl substituted cannabinoid products: (2dd), (2ee), (2ff), (2gg), (2hh), (2ii), (2jj), (2kk), (2ll), (2mm), (2nn), (2oo), and (2pp) (see, for example, compounds in Table 3).

[0097] It is further contemplated that the preparation of the carboxylated cyclolavandulyl-substituted cannabinoid compounds of Table 3 can include the additional step of decarboxylation to produce the corresponding decarboxylated cyclolavandulyl-substituted cannabinoid compounds. For example, the carboxylated cyclolavandulyl-substituted cannabinoids of compounds (2a) and (2c) can be decarboxylated to produce the cyclolavandulyl-substituted cannabinoids of compounds (2b) and (2d), respectively.

[0098] Other aromatic compounds capable of acting as cyclolavanduryl group acceptor substrates for enzymes having PT activity include compounds of structural formulas (IV), (V), and (VI): [ka] (In the formula, R 2 is -H or -OH, R 3 is -H or -COOH, R 4 is -H, -OH, linear or branched C1-C10 alkyl, linear or branched C1-C10 alkylamide, linear or branched C1-C10 alkylamine, linear or branched C1-C10 alkylalkylene, linear or branched C1-C10 alkylalkoxy, or C1-C10 alkylaryl, wherein any of the C1-C10 groups are optionally substituted with -OH, -OCH3, or halogen.

[0099] Compounds represented by structural formulas (IV), (V), and (VI) that can act as cyclolavandulyl group acceptor substrates include, but are not limited to, flavonoid compounds, alkaloid compounds, and other compounds listed in Table 4. The aromatic compounds in Table 4 can be cyclolavandulylated to form a series of product compounds via PT activity (e.g., NphB) with a suitable cyclolavandulyl donor, such as compound (1), to form a series of cyclolavandulyl-substituted aromatic products, including compounds (3i), (3j), (3k), (3l), (4a), (5a), (6a), and (6b) (see, e.g., compounds in Table 5).

[0100] In another embodiment, the disclosure provides a method for producing a cyclolavandulyl-substituted aromatic compound, the method comprising: (a) reacting, under suitable reaction conditions, cyclolavandulyl diphosphate synthase (CLDS), dimethylallyl pyrophosphate, a prenyltransferase, and compounds of structural formulas (III), (IV), (V), and (VI), where R 2 is -H or -OH, R 3 is -H or -COOH, R 4 is -H, -OH, linear or branched C1-C10 alkyl, linear or branched C1-C10 alkylamide, linear or branched C1-C10 alkylamine, linear or branched C1-C10 alkylalkylene, linear or branched C1-C10 alkylalkoxy, or C1-C10 alkylaryl, wherein any of the C1-C10 groups are optionally substituted with -OH, -OCH3, or halogen; and (b) recovering a cyclolavandulyl-substituted aromatic compound from the reaction mixture.

[0101] As described elsewhere herein, naturally occurring cyclolavandulyl diphosphate synthase (CLDS) from Streptomyces CL190 can convert two molecules of DMAPP to CLPP (compound (1)). In at least one embodiment of the method, the cyclolavandulyl diphosphate synthase (CLDS) is a polypeptide comprising the amino acid sequence of CLDS in SEQ ID NO:2. It is also contemplated that mutants of the naturally occurring CLDS in SEQ ID NO:2 can be prepared with improved properties for use in the methods and compositions of the present disclosure, such as CLDS mutants with improved thermostability and / or solubility for use in cell-free reaction methods. Thus, in at least one embodiment of the method or composition, an enzyme with CLDS activity can be used, such as a mutant of the naturally occurring CLDS in SEQ ID NO:2, which mutant comprises an amino acid sequence with at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:2. In at least one embodiment, the CLDS is modified with a HIS tag, such as the CLDS in SEQ ID NO:4.

[0102] Naturally occurring prenyltransferase NphB from Streptomyces sp. CL190, having the amino acid sequence of SEQ ID NO:8, has been engineered to provide soluble mutants such as NphBM31s (SEQ ID NO:6) with PT activity capable of prenylating aromatic polyketides OA or DA with GPP to form cannabinoid compounds CBGA or CBGVA, respectively. See, e.g., U.S. Patent No. 5,399,411; U.S. Patent No. 5,399,421; U.S. Patent No. 5,399,433; and U.S. Patent No. 5,499,511, each of which is incorporated herein by reference. These engineered NphB mutants can be used in cell-free biosynthetic systems and methods for the production of cannabinoid compounds. See, e.g., U.S. Patent No. 5,399,421 and U.S. Patent No. 5,499,511. Mutant NphBM31s (SEQ ID NO:6) is an exemplary enzyme with PT activity useful in the methods and compositions of the present disclosure. Specific protocols and conditions for the use of NphB, NphBM31s, other NphB mutants, and other PTs are provided in the Examples and elsewhere herein. Thus, in at least one embodiment of any of the methods or compositions for producing cyclolavandulyl-substituted aromatic compounds disclosed herein, the enzyme having prenyltransferase activity used is NphB or a variant of NphB, optionally wherein the NphB or variant of NphB comprises an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to NphB (SEQ ID NO: 8) or NphBM31s (SEQ ID NO: 6), or another variant of NphB or NphBM31s, such as the variants disclosed in WO 2006 / 023361, which is incorporated herein by reference.

[0103] Aromatic prenyltransferases other than NphB from Streptomyces sp. CL190 are known and are contemplated to be used in the method and composition of the present disclosure for producing cyclolavandulyl-substituted aromatic compounds.Useful prenyltransferases can be identified using screening as described in the examples of the present disclosure.Thus, in at least one embodiment, the method and composition of the present disclosure can be carried out using naturally occurring prenyltransferases that comprise amino acids selected from SEQ ID NOs: 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18.In another embodiment, it is contemplated that mutants of these prenyltransferases can be engineered and screened for the activity of transferring cyclolavandulyl groups to aromatic compound acceptors. Thus, in at least one embodiment, the methods and compositions of the disclosure can be practiced using a variant of a prenyltransferase comprising amino acids selected from SEQ ID NOs: 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18, where the variant comprises an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NOs: 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18. In at least one embodiment, the prenyltransferase comprises an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NOs: 6, 8, 9, 14, 17, 18, or 19.

[0104] As mentioned elsewhere herein, the transfer of a prenyl group from a donor substrate, such as geranyl pyrophosphate (GPP), to an aromatic polyketide compound is a key enzymatic step in the biosynthesis of many compounds of interest, including cannabinoids. Therefore, it is contemplated that the disclosed methods and compositions, which use prenyltransferase activity to catalyze the transfer of a cyclolavanduryl group to an aromatic acceptor compound, may be used in a range of in vitro, cell-free, or in vivo, recombinant host cell systems for the biosynthesis of cyclolavandurylated aromatic compounds that are normally synthesized via a prenyltransferase step. Figure 1 shows a schematic diagram of the molecular inputs / outputs and enzymes involved in an exemplary system that utilizes prenyltransferases in the biosynthesis of cannabinoid compounds. On the right side of the scheme in Figure 1, the input molecule glucose is converted to the precursor compounds hexanoyl-CoA and malonyl-CoA via fatty acid biosynthesis enzymes. Alternatively, it is converted to hexanoyl-CoA, butyryl-CoA, or octanoyl-CoA. The precursors hexanoyl-CoA and malonyl-CoA are converted to the cannabinoid precursor compound olivetolic acid (OA) via polyketide chalcone biosynthetic enzymes. Alternatively, butyryl-CoA is converted to divaleric acid (DA) or the precursor octanoyl-CoA is converted to sphaerophorolic acid (PA). The cannabinoid precursors OA, DA or PA in this scheme can act as cannabinoid precursor substrate compounds or aromatic cyclolavandulyl group acceptors in the disclosed methods. The left side of this scheme in FIG. 1 shows a terpene biosynthetic pathway that converts glucose input molecules to geranyl pyrophosphate (GPP), which is a co-substrate used by engineered NphB to convert the cannabinoid precursors OA, DA or PA to the corresponding cannabinoid-producing compounds CBGA, CBGVA or CBGPA. It is contemplated that this terpene biosynthetic pathway can be shortened with the production of the cyclolavanduryl precursor DMAPP, thereby incorporating the methods of the present disclosure.As shown in the scheme in FIG. 1, the cannabinoid products are themselves precursor substrate compounds that can be converted by cannabinoid synthases to the cannabinoids, THCA, CBDA, CBCA, and other structural analogs.

[0105] In at least one embodiment, the disclosed methods and compositions for biosynthesis of cyclolavandulyl-substituted aromatic compounds can be used for cell-free in vitro biosynthesis of cyclolavandulyl-substituted cannabinoid compounds. Cell-free cannabinoid biosynthesis methods utilizing soluble prenyltransferase NphB are described in Valliere et al. "A bio-inspired cell-free system for cannabinoid production from inexpensive inputs," Nature Chemical Biology Vol. 16, Dec. 2020, 1427-1433, U.S. Patent No. 5,333,511,1427-1433, U.S. Patent No. 5,333,1427-1433, and U.S. Patent No. 5,333,511,1427-1433, each of which is incorporated herein by reference. For example, some of the engineered NphB mutant polypeptides disclosed in U.S. Patent No. 5,511,142, have improved thermostability and can therefore be directly incorporated into the cell-free cyclolavandulyl-substituted cannabinoid biosynthesis methods of the present disclosure. Moreover, these engineered NphB mutant polypeptides allow for higher temperatures and improved conversion rates.

[0106] In at least one embodiment, the present disclosure provides a cell-free biosynthetic reaction scheme and system for producing a series of cyclolavandulyl-substituted aromatic compounds, including cannabinoids, flavonoids, alkaloids, and other cyclolavandulylation products, using the methods and compositions of the present disclosure. In at least one embodiment, the cell-free biosynthetic reaction scheme provides a pathway for the production of DMAPP and an enzyme with CLDS activity that converts DMAPP to CLPP. In at least one embodiment, the cell-free biosynthetic reaction scheme also provides a pathway for the production of aromatic cyclolavandulyl acceptor substrates, such as cannabinoid precursors, such as OA, DA, or PA. In at least one embodiment, the cell-free biosynthetic reaction scheme also incorporates soluble prenyltransferase NphB or one of its variants as an enzyme with PT activity for transferring the cyclolavandulyl group to the aromatic acceptor in the reaction mixture. The use of cell-free biosynthetic schemes and systems can simplify biosynthesis optimization by allowing for easy modification or addition of pathway enzymes and modification of reagents or cofactors.

[0107] As shown by the exemplary cannabinoid biosynthesis reaction scheme in FIG. 1, the input compounds hexanoyl-CoA and malonyl-CoA can be used as substrates in a cell-free biosynthetic pathway to produce the cannabinoid precursor compound olivetolic acid (OA). The biosynthesis begins with the condensation of hexanoyl-CoA and malonyl-CoA catalyzed by olivetolic acid synthase (OLS) (BAG14339.1 from C. sativa) to produce 3,5,7-trioxododecanoyl-CoA. The enzyme olivetolic acid cyclase (OAC) (AFN42527.1 from C. sativa) cyclizes 3,5,7-trioxododecanoyl-CoA to OA. A similar biosynthetic pathway can produce the OA analogs DA and PA. The prenyl donor substrate GPP is produced via the terpene biosynthetic enzyme pathway. In some cases, enzymatic pathway steps may utilize cofactors (e.g., NAD(P)H, ATP / ADP, etc.). Table 6 provides a list of exemplary enzymes that may be used in cell-free biosynthetic systems incorporating the recombinant prenyltransferase polypeptides of the present disclosure.

[0108] Table 6: Enzymes useful in cell-free enzyme systems [Table 6]

[0109] The cell-free biosynthetic reaction using the recombinant polypeptide of the present disclosure can be carried out using a series of biocatalytic reaction methods. For example, pathway enzymes are commercially available and can be mixed with the recombinant prenyltransferase polypeptide of the present disclosure in a suitable buffer, and then the solution is exposed to a suitable substrate and incubated under suitable conditions for the production of the desired cannabinoid compound. It is contemplated that in some embodiments, one or more of the pathway enzymes can be bound to a solid support. It is also contemplated that one or more of the pathway enzymes can be expressed using phage display or other surface expression systems and immobilized, for example, in a fluid pathway corresponding to a cycle point of a metabolic pathway.

[0110] It is also contemplated that one or more polynucleotides encoding one or more pathway enzymes can be cloned into one or more host cells under conditions that result in expression of the pathway enzymes. The host cells can then be lysed, and the lysate containing one or more enzymes (including recombinant prenyltransferase polypeptides) can be combined with an appropriate buffer and substrate (and optionally one or more additional enzymes of the pathway) to produce the desired cannabinoids. Alternatively, the enzymes can be isolated from the lysate preparation, with or without heat treatment, and then recombined in an appropriate buffer.

[0111] In one embodiment, pathway enzymes other than the PT and CLDS polypeptides of the disclosure may be derived from a thermophilic microorganism. The microorganism is cultured to express the thermostable enzymes, then lysed and the culture lysate is heated to a temperature at which the thermostable enzymes of the pathway remain active while other enzymes are inactive. Such heat-purified lysate preparations can then be used in cell-free biosynthetic reactions with the PT and / or CLDS polypeptides of the disclosure to produce the desired cyclolavandulyl-substituted aromatic compounds.

[0112] In at least one embodiment of the method for producing cyclolavandulyl-substituted aromatic compounds (e.g., cannabinoids), a heterologous nucleic acid encoding a recombinant polypeptide having CLDS activity and improved thermostability (e.g., a thermostable CLDS mutant) may be introduced into a recombinant host cell. The recombinant host cell may then be used to produce the polypeptide or may be incorporated into a biocatalytic process that utilizes the CLDS activity of the recombinant polypeptide expressed by the host cell to catalytically prepare a cyclolavandulyl group donor substrate CLPP, which is used by PT to produce the cyclolavandulyl-substituted cannabinoid CBCLA. In at least one embodiment, the recombinant host cell may further comprise an enzymatic pathway capable of producing a cannabinoid (e.g., CBGA) in addition to the recombinant polypeptide having CLDS activity. It is contemplated that a recombinant host cell comprising a heterologous nucleic acid encoding a recombinant polypeptide of the present disclosure may improve the biosynthesis of CBCLA in terms of titer, yield, and production rate due to the improved thermostability of the expressed activity.

[0113] Thus, in at least one embodiment, the present disclosure provides a method for producing cyclolavandulyl-substituted aromatic compounds, the method comprising: (a) culturing the recombinant host cell of the present disclosure in an appropriate medium; and (b) recovering the produced cyclolavandulyl-substituted derivatives. In at least one embodiment, the method for producing cyclolavandulyl-substituted derivatives further comprises contacting a cell-free extract of the culture containing the produced cyclolavandulyl-substituted aromatic compounds with a biocatalytic or chemical reagent capable of converting the compound into a further derivative compound. In at least one embodiment, the biocatalytic reagent is an enzyme capable of converting the produced cyclolavandulyl-substituted cannabinoid (e.g., CBCLA) into a different cyclolavandulyl-substituted cannabinoid compound (e.g., CBCL). In at least one embodiment, the chemical reagent can chemically modify the produced cyclolavandulyl-substituted cannabinoid to produce a different cyclolavandulyl-substituted cannabinoid. In at least one embodiment of the method of producing a cyclolavanduryl-substituted cannabinoid, the method may further comprise contacting the cell-free extract of the culture containing the produced cyclolavanduryl-substituted cannabinoid with a biocatalytic or chemical reagent.

[0114] It is contemplated that the cyclolavandulyl-substituted cannabinoids, flavonoids, alkaloids, or other aromatic derivatives produced using the methods and compositions of the present disclosure can be produced and / or recovered from the reaction in the form of a salt.In at least one embodiment, the recovered salt of cyclolavandulyl-substituted cannabinoids, flavonoids, alkaloids, or other aromatic compounds is a pharmaceutically acceptable salt.Such pharmaceutically acceptable salts retain the biological effectiveness and properties of the free base compound. EXAMPLES

[0115] Various features and embodiments of the present disclosure are described in the following representative examples, which are intended to be illustrative and not limiting. Those skilled in the art will readily appreciate that the specific examples are merely illustrative of the invention as more fully described in the appended claims. It should be understood that all embodiments and features described in this application are interchangeable and combinable with all embodiments contained therein.

[0116] Example 1: In vitro biosynthesis of the cyclolavanduryl-substituted cannabinoid CBCLA This example shows the preparation of a cyclolavandulyl substituted cannabinoid compound, cannabicyclolavolic acid (CBCLA), via a cell-free biosynthetic reaction using recombinantly produced cyclolavandulyl diphosphate synthase CLDS from Streptomyces sp. CL190 and recombinant prenyltransferase NphB.

[0117] Materials and Methods A. Cloning and Expression of CLDS The gene encoding CLDS (SEQ ID NO:2) from Streptomyces sp. CL190, GenBank accession AB872045.1, was codon-optimized for expression in Escherichia coli and synthesized by TwistDNA with an N-terminal 6x-HIS tag to obtain a cloned gene in the pET28a expression vector. The expressed HIS-tagged CLDS protein corresponds to SEQ ID NO:4. The cloned gene in the pET28a expression vector was transformed into BL21-Gold(DE3) competent cells using standard chemical transformation methods. A single colony was used to inoculate 4 mL LB + kanamycin (50 μg / mL) and grown at 37°C and 250 rpm. After 12 hours, the overnight culture was used to inoculate 1 L LB + kanamycin (50 μg / mL). At an OD600 of approximately 0.6, the cultures were induced by adding 0.4 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) and grown at 18°C ​​and 250 rpm. After 12 h, protein purification was performed using standard Ni-NTA methods.

[0118] B. Cell-free biosynthesis of cannabinoid derivatives using CLDS The production of CBCLA using a cell-free biosynthetic reaction was carried out using a similar modified procedure as described in Non-Patent Document 3 and Non-Patent Document 4, where the prenyltransferase substrate cyclolavandulyl pyrophosphate (CLPP) was first synthesized from DMAPP. After CLPP was synthesized, olivetolic acid (OA) and the prenyltransferase NphBM31s were added to the solution to form the cyclolavandulyl derivative of OA. NphBM31s (SEQ ID NO: 6) was prepared by cloning and expressing in the pET28 vector as described above for CLDS or as described in Patent Document 5. Briefly, 10 mM DMAPP, 5 mM MgCl2, 100 mM Tris-HCl (pH=8), and 4 mg / mL CLDS (SEQ ID NO: 4) were combined in a 2.25 mL reaction solution and incubated at 28° C. for 48 hours. Then, 5 mM olivetolic acid and 1.26 mg / mL NphBM31s were added to the reaction mixture and incubated at 28° C. for an additional 48 hours.

[0119] C. HPLC Analysis of Biosynthetic Reaction Products Analysis of the reaction mixture was performed as follows: 50 μL of the reaction mixture was added to 1 mL MeOH. The resulting solution was vortexed, centrifuged at 17200 g for 5 min, and transferred to HPLC vials for analysis. Samples were analyzed using an UltiMate 3000 HPLC equipped with a 100 x 4.6 mm 3 μm Syncronis C8 column and a mobile phase consisting of HO (0.1% TFA) and ACN (0.1% TFA).

[0120] result As shown by the HPLC chromatogram shown in Figure 3, the cyclolavandulyl cannabinoid compound CBCLA was produced in a cell-free reaction at a titer of about 0.5 g / L. HPLC analysis confirmed that this compound was distinct from CBGA and that only a single specific product was produced. Figure 4 shows exemplary LC-MS plots of the in vitro biosynthesis of the prenylated cannabinoids CBGVA, CBGA and CBGPA compared to the LC-MS plots of the cyclolavandulyl cannabinoid compounds CBCLVA and CBCLA.

[0121] Example 2: Biosynthesis of cyclolavandulyl-substituted aromatic compounds using various prenyltransferases This example demonstrates the in vitro cyclolavandulylation of various aromatic compounds in a cell-free biosynthetic reaction using recombinantly produced cyclolavandulyl diphosphate synthase CLDS (SEQ ID NO: 4) and various enzymes with prenyltransferase activity.

[0122] Materials and Methods A. Preparation of prenyltransferase The following prenyltransferase-encoding genes were codon-optimized, synthesized, and cloned into the pET28a vector for protein expression and isolation / purification as described in Example 1.

[0123] NphB (Accession: 1ZB6_A) (SEQ ID NO: 8) AbPT (Accession: KPI30840.1) (SEQ ID NO: 9) SkPT (Accession: Q2L6E3.1) (SEQ ID NO: 10) SvPT (Accession: WP_078899560.1) (SEQ ID NO: 11) Sr1310PT (Accession: WP_057602682.1) (SEQ ID NO: 12) NapT8 (Accession: ABS50461.1) (SEQ ID NO: 13) NapT9 (Accession: ABS50490.1) (SEQ ID NO: 14) Mcl23PT (Accession: AGH68908.1) (SEQ ID NO: 15) CnqP3PT (Accession: WP_047018069.1) (SEQ ID NO: 16) AtaWTPT (Accession: AMB20850.1) (SEQ ID NO: 17) MzPT1 (Accession: WP_135330628.1) (SEQ ID NO: 18) MzPT2 (Accession: WP_135330626.1) (SEQ ID NO: 19)

[0124] B. Cell-free biosynthetic reactions using CLPP and various aromatic substrates Cyclolabanulyl diphosphate synthase CLDS was prepared as described in Example 1 and used for the synthesis of CLPP.

[0125] Stocks of various aromatic compounds used as substrates for cyclolavandurylation activity screening were obtained from the following commercial sources: Resveratrol (Fisher Scientific) 200 mM DMSO stock Luteolin (Fisher Scientific) 200 mM DMSO stock Naringenin (Fisher Scientific) 200 mM DMSO stock Tryptamine (Fisher Scientific) 200 mM DMSO stock Tryptophan (Fisher Scientific) 40 mM aqueous stock Olivetolic acid (Toronto Research Chemicals) 200 mM DMSO stock Divalinic acid (Toronto Research Chemicals) 200 mM DMSO stock Pholoic acid (prepared in-house) 200 mM DMSO stock Olivetol (Fisher Scientific) 200 mM DMSO stock

[0126] The cell-free biosynthesis screening was performed as follows: 10 mM DMAPP, 5 mM MgCl2, 100 mM Tris-HCl (pH=8), and 4 mg / mL CLDS (SEQ ID NO:4) were combined in a 6.80 mL reaction solution and incubated at 28°C for 48 hours. The mixture was dispensed into a 96-well plate (80 μL per well). The various prenyltransferases described above (18 μL, final concentration approximately 1 g / L) were screened with the various substrates (2 μL of stock solution, variable concentrations as described above), added to the wells, and incubated at 28°C for another 48 hours.

[0127] C. Analysis of reaction products Analysis of the reaction mixture was performed as follows: 50 μL of the reaction mixture was added to 1 mL MeOH. The resulting solution was vortexed, centrifuged at 17200 g for 5 min, and transferred to HPLC vials for analysis. HRMS-QTOF analysis of the cell-free reaction was performed by Agilent Technologies 6545 Accurate Mass QTOF LC / MS equipped with a reversed-phase column (Agilent Infinity LabPoroshell 120 ECC18, 2.7 μm, 3.0 × 50 mm) with positive ESI in 1% MeCN-H2O (0.1% FA) for 2 min, followed by a linear gradient of 1%–99% MeCN-H2O (0.1% FA) for 14 min at a flow rate of 0.4 mL / min.

[0128] Mass spectrometry (MS) of the chromatograms was performed using MassHunter Qualitative Analysis 10.0. The protonated forms [H + Targeted metabolomics using the exact m / z of ] demonstrated the presence of cyclolavandulylation products. The results are shown in Table 7.

[0129] [Table 7]

[0130] result As shown by the results summarized in Table 7, the following prenyltransferases produced cyclolavandurylated aromatic compounds when combined with CLDS and DMAPP from Streptomyces sp. CL190 in cell-free biosynthetic reactions: NphBm31s (SEQ ID NO: 6), NphB (SEQ ID NO: 8), AbPT (SEQ ID NO: 9), NapT9 (SEQ ID NO: 14), AtaWTPT (SEQ ID NO: 17), and MzPT1 (SEQ ID NO: 18). HPLC plots of five exemplary cell-free biosynthetic reactions with and without prenyltransferases are shown in Figures 5A-5E. Reactions with added prenyltransferases produced HPLC peaks that were further analyzed by MS and found to match the cyclolavandurylated aromatic compound products shown in the plots. Although some of the prenyltransferases tested did not exhibit detectable cyclolavandurylating activity toward the aromatic compounds tested, it is contemplated that these enzymes may be engineered (e.g., using directed evolution methods) and / or reaction conditions optimized to provide such activity.

[0131] Although the foregoing disclosure of the present invention has been described in some detail by way of examples and illustrations for purposes of clarity and understanding, the present disclosure, including the examples, explanations, and embodiments described herein, are intended to be illustrative and exemplary and should not be construed as limiting the present disclosure. It will be apparent to those skilled in the art that various modifications or variations to the examples, explanations, and embodiments described herein are possible and are intended to be within the spirit and scope of the present disclosure and the appended claims. Moreover, those skilled in the art will recognize numerous methods and procedures equivalent to those described herein. Such equivalents are understood to be within the scope of the present disclosure and are covered by the appended claims.

[0132] Additional embodiments of the invention are set forth in the accompanying claims.

[0133] The disclosures of all publications, patent applications, patents, or other documents mentioned in this specification are expressly incorporated herein by reference in their entirety for all purposes as if each such publication, patent, patent application, or other document was individually and specifically indicated to be incorporated herein by reference in its entirety for all purposes and set forth in the specification. In case of conflict, the present specification, including designated language, will control.

[0134] Drawing translation Figure 1 Glucose Terpene Biosynthesis isoprenol geranyl pyrophosphate ALTERNATE GPP ROUTE GOT or NphB GOT or NphB mutant prenyltransferase enzyme Fatty Acid Biosynthesis Hexanoyl CoA + 3 Malonyl-CoA (or Butyryl-CoA or Octanoylt-CoA) Polyketide chalcone biosynthesis Olivetolic acid Divarinic acid Sphaerophorolic acid POLYKETIDE INPUTS cannabigerolic acid cannabigerovarinic cannabigerophorolic acid cannabinoids Figure 3 TIME (min) Figure 5A Resveratrol cyclolavandulylation Cyclolavandulyl-resveratrol Figure 5B Luteolin cyclolavandulylation Cyclolavandulyl-luteolin Figure 5C Naringenin cyclolavandulylation Cyclolavandulyl-naringenin Figure 5D typtophan cyclolavandulylation Cyclolavandulyl-typtophan Cyclolavandulyl-tryptophan Figure 5E Olivetolic acid cyclolavandulylation Cyclolavandulyl-olivetolic acid

Claims

1. Compounds with structural formula (Ia) or (Ib): 【Chemistry 1】 (In the formula, R 1 teeth, 【Chemistry 2】 (In the formula, R 2 is -H or -OH, R 3 is -H or -COOH, R 4 The group is -H, -OH, linear or branched C1-C10 alkyl, linear or branched C1-C10 alkylamide, linear or branched C1-C10 alkylamine, linear or branched C1-C10 alkylalkylene, linear or branched C1-C10 alkylalkoxy, or C1-C10 alkylaryl, where any of the C1-C10 groups is optionally -OH, -OCH 3 (Selected from, or substituted with halogen).

2. R 1 but, 【Transformation 3】 (In the formula, R 2 is -H or -OH, R 3 is -H or -COOH, R 4 is a linear or branched C1-C10 alkyl, linear or branched C1-C10 alkyl amide, linear or branched C1-C10 alkyl amine, linear or branched C1-C10 alkyl alkylene, linear or branched C1-C10 alkyl alkoxy, or C1-C10 alkyl aryl, where any of the C1-C10 groups is optionally substituted with -OH, -OCH 3 , or halogen), and is a compound according to claim 1.

3. (a) R 2 is -OH, and R 3 is -COOH, and / or, (b) R 4 CH 3 CH 2 CH 3 , (CH 2 ) 2 CH 3 , (CH 2 ) 3 CH 3 , (CH 2 ) 4 CH 3 , (CH 2 ) 5 CH 3 , and (CH 2 ) 6 CH 3 Selected from, optionally, R 4 (CH 2 ) 2 CH 3 , (CH 2 ) 4 CH 3 , and (CH 2 ) 6 CH 3 A compound according to claim 2, selected from the following.

4. The aforementioned compound has structural formulas (IIa), (IIb), (IIc), or (IId): 【Chemistry 4】 (In the formula, R 3 is -H or -COOH, R 4 The C1-C10 alkyl group is a linear or branched C1-C10 alkylamide, a linear or branched C1-C10 alkylamine, a linear or branched C1-C10 alkylalkylene, a linear or branched C1-C10 alkylalkoxy, or a C1-C10 alkylaryl group, where any of the C1-C10 groups can be -OH, -OCH 3 , or substituted with halogen, The compound according to claim 2, wherein R4 is optionally selected from CH3, CH2CH3, (CH2)2CH3, (CH2)3CH3, (CH2)4CH3, (CH2)5CH3, (CH2)6CH3, and (CH2)7CH3, and optionally R4 is selected from (CH2)2CH3, (CH2)4CH3, and (CH2)6CH3).

5. The compound is compound (2b), (2d), (2a), (2c), (2e), (2f), (2g), (2h), (2i), (2j), (2k), (2l), ( 2m), (2n), (2o), (2p), (2q), (2r), (2s), (2t), (2u), (2v), (2w), (2x), (2y), (2z), (2a a), (2bb), (2cc), (2dd), (2ee), (2ff), (2gg), (2hh), (2ii), (2jj), (2kk), (2ll), (2 mm), (2nn), (2oo), (2pp), (2qq), (2rr), (2ss), (2tt), (2uu), (2vv), (2ww), and (2xx): 【Transformation 5】 【change】 【change】 【change】 【change】 A compound according to claim 4, selected from the above.

6. R 1 but, 【Transformation 6】 【change】 A compound according to claim 1, selected from the following.

7. A method for producing cyclolavanduryl-substituted aromatic compounds, (a) Under appropriate reaction conditions, prenyltransferase and cyclolavandurylpyrophosphate of compound (1) 【Transformation 7】 and the compounds of structural formulas (III), (IV), (V), and (VI) 【Transformation 8】 (In the formula, R 2 is -H or -OH, R 3 is -H or -COOH, R 4 The group is -H, -OH, linear or branched C1-C10 alkyl, linear or branched C1-C10 alkylamide, linear or branched C1-C10 alkylamine, linear or branched C1-C10 alkylalkylene, linear or branched C1-C10 alkylalkoxy, or C1-C10 alkylaryl, where any of the C1-C10 groups is optionally -OH, -OCH 3 Contacting an aromatic compound selected from (or substituted with a halogen), (b) Recovering the cyclolavandulyl-substituted aromatic compound from the reaction mixture, Methods that include...

8. Prenyltransferase and cyclolavandurylpyrophosphate of compound (1) 【Chemistry 9】 And the compounds of structural formulas (III), (IV), (V), and (VI): 【Chemistry 10】 (In the formula, R 2 is -H or -OH, R 3 is -H or -COOH, R 4 The group is -H, -OH, linear or branched C1-C10 alkyl, linear or branched C1-C10 alkylamide, linear or branched C1-C10 alkylamine, linear or branched C1-C10 alkylalkylene, linear or branched C1-C10 alkylalkoxy, or C1-C10 alkylaryl, where any of the C1-C10 groups is optionally -OH, -OCH 3 A composition comprising an aromatic compound selected from (or substituted with a halogen).

9. A method for producing cyclolavanduryl-substituted aromatic compounds, (a) Under appropriate reaction conditions, cyclolavandulyl diphosphate synthase (CLDS), dimethylallyl pyrophosphate, prenyltransferase, and the compounds of structural formulas (III), (IV), (V), and (VI). 【Chemistry 11】 (In the formula, R 2 is -H or -OH, R 3 is -H or -COOH, R 4 The group is -H, -OH, linear or branched C1-C10 alkyl, linear or branched C1-C10 alkylamide, linear or branched C1-C10 alkylamine, linear or branched C1-C10 alkylalkylene, linear or branched C1-C10 alkylalkoxy, or C1-C10 alkylaryl, where any of the C1-C10 groups is optionally -OH, -OCH 3 Contacting an aromatic compound selected from (or substituted with a halogen), (b) Recovering the cyclolavandulyl-substituted aromatic compound from the reaction mixture, Methods that include...

10. The cyclolavanduryl-substituted aromatic compounds are compounds (4a), (4b), (5a), (5b), (6a), (6b), (7a), (7b), (8a), and (8b): 【Chemistry 12】 The method according to claim 9, selected from the following.

11. The cyclolavanduryl-substituted aromatic compound is defined by structural formula (IIa), (IIb), (IIc), or (IId): 【Chemistry 13】 (In the formula, R 3 is -H or -COOH, R 4 The C1-C10 alkyl group is a linear or branched C1-C10 alkylamide, a linear or branched C1-C10 alkylamine, a linear or branched C1-C10 alkylalkylene, a linear or branched C1-C10 alkylalkoxy, or a C1-C10 alkylaryl group, where any of the C1-C10 groups can be -OH, -OCH 3 , or substituted with halogen, The method according to claim 7 or 9, wherein R4 is optionally selected from CH3, CH2CH3, (CH2)2CH3, (CH2)3CH3, (CH2)4CH3, (CH2)5CH3, (CH2)6CH3, and (CH2)7CH3, and optionally R4 is selected from (CH2)2CH3, (CH2)4CH3, and (CH2)6CH3).

12. The recovered cyclolavanduryl-substituted compound is a cyclolavanduryl-substituted cannabinoid, and optionally, the cyclolavanduryl-substituted cannabinoid is compound (2a), (2b), (2c), (2d), (2e), (2f), (2g), (2h), (2i), (2j), (2k), (2l), (2m), (2n), (2o), (2p), (2q), (2r), (2s), (2t), (2u), (2v), (2w), (2x), (2y), (2z), (2aa The method according to claim 11, further comprising the step of decarboxylating the recovered cyclolavanduryl-substituted compound, selected from (2bb), (2cc), (2dd), (2ee), (2ff), (2gg), (2hh), (2ii), (2jj), (2kk), (2ll), (2mm), (2nn), (2oo), (2pp), (2qq), (2rr), (2ss), (2tt), (2uu), (2vv), (2ww), and (2xx, and / or optionally).

13. Cyclolavanduryl diphosphate synthase (CLDS), dimethylallyl pyrophosphate, prenyltransferase, and compounds of structural formulas (III), (IV), (V), and (VI). 【Chemistry 14】 (In the formula, R 2 is -H or -OH, R 3 is -H or -COOH, R 4 The group is -H, -OH, linear or branched C1-C10 alkyl, linear or branched C1-C10 alkylamide, linear or branched C1-C10 alkylamine, linear or branched C1-C10 alkylalkylene, linear or branched C1-C10 alkylalkoxy, or C1-C10 alkylaryl, where any of the C1-C10 groups is optionally -OH, -OCH 3 A composition comprising an aromatic compound selected from (or substituted with a halogen).

14. The method according to claim 9, wherein the cyclolavandulyl diphosphate synthase (CLDS) is a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or 4, or a variant of SEQ ID NO: 2 or 4, and optionally the variant of SEQ ID NO: 2 or 4 comprises an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 2 or 4.

15. The composition according to claim 13, wherein the cyclolavandulyl diphosphate synthase (CLDS) is a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or 4, or a variant of SEQ ID NO: 2 or 4, and optionally the variant of SEQ ID NO: 2 or 4 comprises an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 2 or 4.

16. The aforementioned prenyltransferase (i) A variant of NphB containing an amino acid sequence that has at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity with NphB (SEQ ID NO: 8), (ii) A variant of NphBM31s having an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to NphBM31s (SEQ ID NO: 6), (iii) Prenyltransferase comprising any one of the amino acid sequences or SEQ ID NOs. 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19, and (iv) A mutant prenyltransferase having an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity with any one of sequence numbers 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19, The method according to claim 7 or 9, selected from the above.

17. The prenyltransferase, (i) A variant of NphB containing an amino acid sequence that has at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity with NphB (SEQ ID NO: 8), (ii) A variant of NphBM31s having an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to NphBM31s (SEQ ID NO: 6), (iii) Prenyltransferase comprising any one of the amino acid sequences or SEQ ID NOs. 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19, and (iv) A mutant prenyltransferase having an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity with any one of sequence numbers 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19, A composition according to claim 8 or 13, selected from the above.

18. The aforementioned aromatic compound is the compound of structural formula (III): 【Chemistry 15】 (In the formula, R 2 is -H or -OH, R 3 is -H or -COOH, R 4 The C1-C10 alkyl group is a linear or branched C1-C10 alkylamide, a linear or branched C1-C10 alkylamine, a linear or branched C1-C10 alkylalkylene, a linear or branched C1-C10 alkylalkoxy, or a C1-C10 alkylaryl group, where any of the C1-C10 groups can be -OH, -OCH 3 , or replaced with halogen, Optionally, (a) R2 is -OH, R3 is -COOH, and / or (b) R4 is selected from CH3, CH2 CH3, (CH2)2 CH3, (CH2)3 CH3, (CH2)4 CH3, (CH2)5 CH3, (CH2)6 CH3, and (CH2)7 CH3, and optionally R4 is selected from (CH2)2 CH3, (CH2)4 CH3, and (CH2)6 CH3, (c) The aromatic compound is a cannabinoid precursor compound selected from divalic acid (DA), olivetolic acid (OA), and hololic acid (PA), and / or (d) The aromatic compound is compound (3a), (3b), (3c), (3d), (3e), (3f), (3g), and (3h): 【Chemistry 16】 The method according to claim 7 or 9, wherein the method is selected from any one of the following.

19. The aromatic compound is a compound of structural formula (III): 【Chemistry 17】 (In the formula, R2 is either -H or -OH. R3 is either -H or -COOH. R4 is a linear or branched C1-C10 alkyl, linear or branched C1-C10 alkylamide, linear or branched C1-C10 alkylamine, linear or branched C1-C10 alkylalkylene, linear or branched C1-C10 alkylalkoxy, or C1-C10 alkylaryl, where any one of the C1-C10 groups is optionally substituted with -OH, -OCH3, or a halogen. Optionally, (a) R2 is -OH, R3 is -COOH, and / or (b) R4 is selected from CH3, CH2 CH3, (CH2)2 CH3, (CH2)3 CH3, (CH2)4 CH3, (CH2)5 CH3, (CH2)6 CH3, and (CH2)7 CH3, and optionally R4 is selected from (CH2)2 CH3, (CH2)4 CH3, and (CH2)6 CH3, (c) The aromatic compound is a cannabinoid precursor compound selected from divalic acid (DA), olivetolic acid (OA), and hololic acid (PA), and / or (d) The aromatic compound is compound (3a), (3b), (3c), (3d), (3e), (3f), (3g), and (3h): [Chemistry 18] The composition according to claim 8 or 13, which is selected from any one of the following.

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