RECOMBINANT POLYPEPTIDES HAVING BERBERINE BRIDGE ENZYME ACTIVITY USEFUL IN THE BIOSYNTHESIS OF CANNABINOIDS - Patent application

Abstract

The present application relates to recombinant polypeptides having berberine bridge enzyme (BBE) activity from a microorganism, such as Phytohabitans safuscus, Streptomyces sp. AJS327, Streptomyces bathobiensis, Actinomadura pelletieri, Streptomyces flaveolus, Streptomyces sp. Ru71, or Streptomyces sp. CNH287, and the use of these recombinant polypeptides in compositions and methods for the oxidative cyclization of prenylated compounds, such as CBGA, in carrying out the biosynthesis, including cell-free biosynthesis, of cyclized cannabinoid compounds, such as CBCA, THCA, and CBDA.

Description

[Technical field]

[0001] [CROSS REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 297,561, filed January 7, 2022, the entirety of which is incorporated herein by reference.

[0002] The present disclosure relates to recombinant polypeptides having berberine bridge enzyme (BBE) activity and the use of these polypeptides in compositions and methods for the biosynthesis of cannabinoids.

[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 "15041-003PV1.xml", created on January 4, 2023, and 445667 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 / 297,561 on January 7, 2022 with filename "15041-003PV1_SeqList_ST25.txt", created on December 14, 2021, and 445667 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 potential 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). Nearly all of the cannabinoids produced by the plant are derived from the precursors cannabigerolic acid (CBGA) or cannabigerovariic acid (CBGVA). CBGA and CBGVA are produced by enzymatic prenylation of the polyketides olivetolic acid (OA) or divalic acid (DA), respectively, with geranyl pyrophosphate (GPP) by the action of the membrane protein GPP:OA transferase (GOT). After the formation of CBGA (or CBGVA), the plant uses specific soluble berberine bridge enzymes (BBEs) such as THCA synthase (THCAS), CBDA synthase (CBDAS), or CBCA synthase (CBCAS) to cyclize the prenylated aromatic compound to the final downstream cannabinoids THCA, CBDA, or CBCA, respectively.

[0006] BBEs are a large family of FAD-dependent enzymes that catalyze a variety of oxidations, including oxidative cyclizations. However, currently, the only BBEs known to catalyze the oxidative cyclization of CBGA to the downstream cannabinoids THCA, CBDA, or CBCA are the THCAS, CBDAS, and CBCAS enzymes from Cannabis sativa. These synthases from C. sativa cannot be expressed in soluble form in Escherichia coli (E. coli) systems for purification and use in in vitro cannabinoid biosynthesis. Therefore, alternative recombinant polypeptides with BBE activity are needed. Summary of the Invention

[0007] The present disclosure relates generally to recombinant polypeptides having berberine bridge enzyme (BBE) activity from a bacterial host organism (not derived from C. sativa), such as Phytohabitans suffuscus, Streptomyces sp. AJS327, Streptomyces varsoviensis, Actinomadura pelletieri, Streptomyces flaveolus, Streptomyces sp. Ru71, or Streptomyces sp. CNH287, and the use of these recombinant polypeptides in compositions and methods for the oxidative cyclization of prenylated compounds, such as CBGA, in the biosynthesis of cyclized products, such as the cyclized cannabinoids CBCA, THCA, and / or CBDA. This Summary is intended to introduce the subject matter of the present disclosure, but is not exhaustive of 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.

[0008] In at least one embodiment, the disclosure also provides a method of producing a recombinant polypeptide having a BBE activity of the disclosure, the method comprising culturing a host cell comprising a polynucleotide or expression vector encoding the polypeptide, and isolating the polypeptide from the cultured host cell.

[0009] In at least one embodiment, the present disclosure also provides a compound of structural formula (I): [ka] (In the formula, R 1 is C 1 ~C 7 The method of producing a compound of structural formula (II): [ka] (In the formula, R 1 is C 1 ~C 7 In one embodiment, the method comprises contacting a compound of formula (I) (I is an alkyl group) with a recombinant polypeptide having BBE activity of the disclosure comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to a sequence selected from SEQ ID NOs: 8, 2, 4, 6, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, and 82.

[0010] In at least one embodiment of the method of making a compound of structural formula (I), (a) the compound of structural formula (I) is cannabichromenic acid (CBCA) and the compound of structural formula (II) is cannabigerolic acid (CBGA); (b) the compound of structural formula (I) is cannabichromevaric acid (CBCVA) and the compound of structural formula (II) is cannabigerovaric acid (CBGVA); or (c) the compound of structural formula (I) is cannabichromephorolic acid (CBCPA) and the compound of structural formula (II) is cannabigerophorolic acid (CBGPA).

[0011] In at least one embodiment of the method of producing a compound of structural formula (I), suitable reaction conditions include: (a) a cell-free solution; (b) a substrate compound of structural formula (II), 0.1 M buffer pH 8.0, and a recombinant polypeptide at 298 K for at least 1 hour; (c) a substrate compound of structural formula (II) at least about 0.6 g / L, at least about 1.2 g / L, 2 g / L, 6 g / L, 12 g / L, 18 g / L, 24 g / L, 30 g / L, or more; (d) a recombinant polypeptide concentration of about 0.1 g / L to about 5 g / L, or even lower; (e) a pH of about 4.0 to about 11.0, or about 5.0 to 10.0; and / or (f) a buffer solution of about 0.05 M Tris-Cl pH 8.0 to about 0.5 M Tris-Cl pH 8.0.

[0012] In at least one embodiment, the disclosure provides a composition comprising (a) a recombinant polypeptide having a BBE activity of the disclosure, and (b) one or more enzymes that generate a substrate for the recombinant polypeptide. In at least one embodiment of the composition, the recombinant polypeptide having BBE activity comprises an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to a sequence selected from SEQ ID NOs: 8, 2, 4, 6, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, and 82.

[0013] In at least one embodiment, a composition comprising a recombinant polypeptide having BBE activity and one or more enzymes that generate a substrate is present in a cell-free solution.

[0014] In at least one embodiment of the composition, the one or more enzymes that generate a substrate include an enzyme that converts a cannabinoid precursor compound (e.g., OA, DA, PA) to a prenylated aromatic compound (e.g., CBGA, CBGVA, CBGPA), and optionally the prenylated aromatic compound is selected from CBGA, CBGVA, CBGPA, and combinations thereof. In at least one embodiment, the composition includes an enzyme that is a prenyltransferase (e.g., NphB), and optionally the composition that includes a prenyltransferase further includes a substrate for the prenyltransferase (e.g., OA, DA, PA).

[0015] In at least one embodiment of the composition, the one or more enzymes include (a) a plurality of enzymes that convert isoprenol or prenol to geranyl pyrophosphate (GPP) and (b) enzymes that convert cannabinoid precursor compounds (e.g., OA, DA, PA) to prenylated aromatic compounds (e.g., CBGA, CBGVA, CBGPA).

[0016] In at least one embodiment of the composition, the one or more enzymes include (a) enzymes that convert isoprenol or prenol to geranyl pyrophosphate (GPP), (b) enzymes that convert cannabinoid precursors (e.g., OA, DA, PA) to prenylated aromatic compounds (e.g., CBGA, CBGVA, CBGPA), (c) enzymes that convert malonate and acetyl-CoA to malonyl-CoA, and / or (d) enzymes that convert ADP and / or AMP to ATP, and optionally, the enzymes that convert ADP and / or AMP to ATP also convert acetyl phosphate to acetate.

[0017] In at least one embodiment of the composition, the one or more enzymes include (i) acyl-activating enzyme 3 (AAE3), (ii) olivetol synthase (OLS), (iii) olivetolic acid cyclase (OAC), and / or (iv) prenyltransferase (NphB).

[0018] In at least one embodiment of the composition, the one or more enzymes comprise (i) acetylphosphate transferase (PTA), (ii) malonate decarboxylase alpha subunit (mdcA), (iii) acyl-activating enzyme 3 (AAE3), (iv) olivetol synthase (OLS), (v) olivetolic acid cyclase (OAC), (vi) prenyltransferase (NphB), (vii) hydroxyethylthiazole kinase (ThiM), (viii) isopentenyl kinase (IPK), (ix) isopentyl diphosphate isomerase (IDI), (x) diphosphomevalonate decarboxylase alpha subunit (MDCa), and / or (xi) geranyl PP synthase (GPPS) or farnesyl PP synthase mutant S82F (FPPSS82F).

[0019] In at least one embodiment, the disclosure provides a recombinant polypeptide having berberine bridge enzyme (BBE) activity comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:2 and having an amino acid residue difference at one or more positions selected from T325, F156, L238, L283, T325, and G340 compared to SEQ ID NO:2, optionally wherein the amino acid residue difference is selected from T325G, F156Y, L238S, L283S, and G340I.

[0020] In at least one embodiment, the recombinant polypeptide having BBE activity further comprises an amino acid residue difference at one or more positions selected from M101, A171, N267, L269, I271, V323, E370, A398, N400, H402, D404 and / or T438 compared to SEQ ID NO:2, optionally wherein the amino acid residue difference is selected from M101A, A171Y, N267V, L269M, I271H, V323Y, E370M, A398E, N400W, H402T, D404S and / or T438Y.

[0021] In at least one embodiment of a recombinant polypeptide having BBE activity, the polypeptide has a set of at least two amino acid residue differences selected from M101A / T438Y, M101A / L269M / I271H, A171Y / A398E, L269M / I271H, L269M / I271H / T438Y, I271H / T438Y, L283S / V323Y, E370M / T438Y, N400W / T438Y, N400W / H402T / D404S, N400W / H402T / D404S / T438Y, H402T / T438Y, and / or D404S / T438Y.

[0022] In at least one embodiment of a recombinant polypeptide having BBE activity, the polypeptide comprises an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to a sequence selected from the group consisting of SEQ ID NOs: 8, 2, 4, 6, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, and 82.

[0023] In at least one embodiment of a recombinant polypeptide having BBE activity, the BBE activity of the polypeptide is increased by at least 1.2-fold, at least 1.5-fold, at least 2-fold, at least 5-fold, or more, as compared to a polypeptide consisting of SEQ ID NO:2, and optionally, the BBE activity is measured as the conversion of CBGA to CBCA. In at least one embodiment, the BBE activity is measured in 2.5 mM OA, 5 mM GPP, 5 mM MgCl at pH 8.0 and 298 K. 2 It is measured as the conversion rate of the substrate cannabigerolic acid (CBGA) to the product THCA and / or CBCA under reaction conditions of 50 mM Tris.

[0024] In at least one embodiment of a recombinant polypeptide having BBE activity, the BBE activity of the polypeptide, as compared to a polypeptide consisting of SEQ ID NO:2, exhibits at least 1.2-fold, at least 1.5-fold, at least 2-fold, at least 5-fold, or more increased product selectivity, optionally where the product selectivity is measured as the conversion of the substrate CBGA to the product CBCA compared to the conversion of the substrate CBGA to the product THCA.

[0025] In at least one embodiment, the disclosure also provides a polynucleotide encoding a recombinant polypeptide having a BBE activity of the disclosure. In at least one embodiment, the polynucleotide encoding the polypeptide comprises a sequence having at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to a sequence selected from the group consisting of SEQ ID NOs: 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, and 81.

[0026] In at least one embodiment, the present disclosure also provides an expression vector comprising a polynucleotide encoding a recombinant polypeptide having a BBE activity of the present disclosure, and optionally, the expression vector comprises a regulatory sequence.

[0027] In at least one embodiment, the disclosure also provides a host cell comprising the polynucleotide, or an expression vector comprising the polynucleotide, where the polynucleotide encodes a recombinant polypeptide having a BBE activity of the disclosure.

[0028] 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]

[0029] [Figure 1] Schematic diagram of the steps, molecular inputs / outputs, and enzymes involved in the biosynthesis of various cannabinoid compounds related to the recombinant BBE polypeptide compositions and methods of the present disclosure, and their use for the biocatalytic production of cannabinoids. The general pathways illustrated are used in both cell-based (in vivo) and cell-free (in vitro) systems. [Diagram 2] FIG. 1 shows the chemical structures of exemplary cannabinoids THCA, THCVA, THCPA, CBCA, CBCVA, and CBCPA that can be biosynthesized using the recombinant BBE polypeptides of the present disclosure. [Diagram 3] Putative mechanistic diagram for FAD-dependent quinone methide (QM) formation of CBGA catalyzed by cannabinoid synthases from C. sativa, such as THCA synthase, CBDA synthase, or CBCA synthase. The bacterial BBE, Clz9, has been shown to catalyze a step in QM formation. The stereochemistry and / or isomerization of the resulting QM in the active site dictates the specific cyclization reaction that the highly reactive QM intermediate undergoes. Unlike the synthases from C. sativa, the active sites of Clz9 and other bacterial BBE homologs only allow for the formation of CBCA. [Figure 4] FIG. 1 shows a structural model of the Clz9 active site and the location of the mutations described in this disclosure. The blue sites, when mutated to the corresponding residues from THCAS in C. sativa, have a beneficial effect on enzyme activity. The L283 site (grey) can only be mutated in conjunction with the V323Y mutation to avoid loss of activity. [Diagram 5] FIG. 1 shows an alignment of the wild-type Clz9 amino acid sequence (SEQ ID NO: 2) together with four mutants Clz9M1, Clz9M2, Clz9M3 and Clz9Mut34. [Figure 6] FIG. 1 shows the results of cyclization reactions catalyzed by wild-type Clz9 and Clz9 mutants described in Example 1. [Figure 7] FIG. 1 shows representative BBE gene clusters from various bacterial source organisms that contain polyketide synthase (PKS) and / or prenyltransferase enzymes. [Figure 8]A: Standards of the substrate compound CBGA and the product compounds THCA and CBCA. B: Results of the cyclization reaction catalyzed by the BBE homologue of Clz9 from Phytohabitans safuscus (PsBBE) at pH 7.5, as described in Example 2. C: Results of the cyclization reaction catalyzed by the BBE homologue of Clz9 from Streptomyces bathobiensis (SvBBE) at pH 7.5, as described in Example 2. [Figure 8-1] D. Results of cyclization reactions catalyzed by the BBE homolog of Clz9 from Cannabis sativa THCAS at pH 7.5, as described in Example 2. E. Results of cyclization reactions catalyzed by the BBE homolog of Clz9 from Streptomyces sp. AJS327 (SAJBBE) at pH 7.5, as described in Example 2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context 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. The terms "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."

[0031] 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 dictates otherwise, as well as any other stated or intervening value within the stated range, are encompassed within the invention, unless the context 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.

[0032] 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").

[0033] 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, and other document was individually indicated to be incorporated by reference for all purposes.

[0034] 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.

[0035] definition "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. Cannabinoids referred to in this disclosure include, but are not limited to, exemplary naturally occurring and synthetic cannabinoid-producing compounds set forth in Table 1 below.

[0036] Table 1: Exemplary cannabinoid-producing compounds [Table 1] JPEG2025503632000005.jpg228159JPEG2025503632000006.jpg239159JPEG2025503632000007.jpg213159JPEG2025503632 000008.jpg226159JPEG2025503632000009.jpg210159JPEG2025503632000010.jpg216158JPEG2025503632000011.jpg80161

[0037] 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.

[0038] "Product" as used herein in the context of an enzyme-mediated process refers to a compound or molecule that results from the activity of an enzyme. In the context of the engineered polypeptides having BBE activity of the present disclosure, exemplary products include, but are not limited to, the cannabinoid compounds summarized in Table 1.

[0039] "Substrate" as used herein in the context of an enzyme-mediated process refers to a compound or molecule that is acted upon by an enzyme. In the context of the engineered polypeptides having BBE activity of the present disclosure, the substrates that are acted upon by the polypeptide may include a range of "cannabinoid" compounds, including, but not limited to, the exemplary cannabinoid compounds CBGA, CBGVA, and CBGPA, which vary in alkyl carbon chain length, as summarized in Table 1.

[0040] As used herein, "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, e.g., a polyketide substrate for a prenyltransferase that is prenylated to form a cannabinoid. Exemplary cannabinoid precursor compounds having a range of alkyl carbon lengths are provided in Table 2.

[0041] Table 2: Exemplary cannabinoid precursor substrate compounds [Table 2]

[0042] As used herein, "host cell" refers to a cell that can be functionally modified with a recombinant nucleic acid and that can function to express recombinant products, including polypeptides and compounds produced by the activity of the polypeptides.

[0043] 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.

[0044] "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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] "Coding sequence" refers to a nucleic acid segment (eg, a gene) that codes for the amino acid sequence of a protein.

[0049] 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.

[0050] "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.

[0051] "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., ASMPress, 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).

[0052] 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.

[0053] 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.

[0054] "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.

[0055] "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.

[0056] "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.

[0057] "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.

[0058] 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.

[0059] "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).

[0060] "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.

[0061] "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.

[0062] Recombinant Polypeptides Having BBE Activity The present disclosure provides a recombinant polypeptide having berberine bridge enzyme (BBE) activity and exhibiting high selectivity for the conversion of a prenylated aromatic cannabinoid substrate CBGA to a cyclized cannabinoid product CBCA compared to the production of the cannabinoid product THCA, as compared to the CBCA synthase from C. sativa. In particular, the recombinant polypeptide can convert a prenylated aromatic cannabinoid substrate compound such as CBGA, CBGVA or CBGPA to the corresponding cannabinoid producing compounds CBCA, CBCVA and CBCPA, respectively, with high selectivity compared to the alternative cyclized cannabinoid producing compounds THCA, THCVA and THCPA. Figure 2 shows various structures of these cyclized cannabinoid products. Without intending to be bound by mechanism, it is believed that the recombinant polypeptide having BBE activity of the present disclosure has an active site that favors the formation of a Z-shaped CBGA quinone methide intermediate, which leads to the selective formation of the CBCA product, as shown in Figure 3. This mechanistic interpretation of biocatalytic stereoselectivity is believed to be supported by the structure of the wild-type Clz9 active site shown in FIG. 4, and further by the site-directed mutagenesis results provided by the Examples disclosed herein.

[0063] In an exemplary embodiment, a recombinant polypeptide having BBE activity of the present disclosure is capable of converting the prenylated aromatic cannabinoid substrate cannabigerolic acid (CBGA) (compound (2)), as shown in Scheme 1, to the cyclized cannabinoid cannabichromenic acid (CBCA) (compound (1)), with increased selectivity over the production of the alternative cyclized cannabinoid THCA (compound (3)), as shown in Scheme 2. [ka]

[0064] The disclosed recombinant polypeptides with increased product selectivity and / or activity are derived from bacterial sources with very low sequence identity to the cyclized cannabinoid synthases CBCAS, THCAS and CBDAS from C. sativa. Unlike these synthase enzymes from C. sativa, which are membrane-bound and difficult to use in soluble cell-free systems, the disclosed bacterial homolog enzymes with BBE activity have been found to be well suited for expression in E. coli and use in soluble cell-free systems, as described elsewhere herein, including in the Examples. A series of exemplary recombinant polypeptides with BBE activity derived from bacterial sources and with the unexpected and surprising technical effect of increased CBCA selectivity are summarized in Table 3 below.

[0065] Table 3: Recombinant polypeptides with BBE activity from bacterial sources [Table 3] JPEG2025503632000015.jpg239161JPEG2025503632000016.jpg102161

[0066] As shown in Table 3, certain naturally occurring recombinant polypeptides with BBE activity from source organisms other than C. sativa can convert the prenylated aromatic cannabinoid CBGA (compound (2)) to the cyclized cannabinoid product CBCA (compound (1)) with higher selectivity than wild-type CBCAS from C. sativa. The recombinant polypeptide Clz9 (SEQ ID NO:2) from Streptomyces sp. CNH287 was further engineered by site-directed mutagenesis as described in the Examples to yield recombinantly engineered polypeptides with different amino acid residues compared to SEQ ID NO:2 and exhibiting BBE activity with various unexpected technical effects, including altered selectivity and / or activity compared to wild-type Clz9 in converting CBGA (compound (2)) to CBCA (compound (1)). These recombinantly engineered variants of Clz9 are summarized in Table 4 below.

[0067] Table 4: Recombinant mutants of Clz9 [Table 4] JPEG2025503632000018.jpg229161

[0068] In at least one embodiment, a recombinant polypeptide having BBE activity and altered product selectivity and / or increased activity has one or more residue differences compared to a reference recombinant polypeptide Clz9 of SEQ ID NO: 2. In at least one embodiment, the recombinant polypeptide has one or more residue differences at residue positions selected from M101, F156, A171, L238, N267, L269, I271, L283, V323, T325, G340, E370, A398, N400, H402, D404 and / or T438.

[0069] It should be understood that residue differences from SEQ ID NO:2 at residue positions associated with altered product selectivity and / or increased activity can be used in various combinations to form recombinant polypeptides having a combination of desired enzymatic properties, such as increased conversion, increased product yield, and / or improved substrate utilization. Exemplary combinations are described herein. For example, the disclosure provides recombinant polypeptides having BBE activity and altered product selectivity and / or increased activity, the polypeptide comprising an amino acid sequence having at least 80% identity to SEQ ID NO:2 and an amino acid residue difference at one or more positions selected from M101, F156, A171, L238, N267, L269, I271, L283, V323, T325, G340, E370, A398, N400, H402, D404, and / or T438, as compared to SEQ ID NO:2. In at least one embodiment, the amino acid residue differences are selected from M101A, F156Y, A171Y, L238S, N267V, L269M, I271H, L283S, V323Y, T325G, G340I, E370M, A398E, N400W, H402T, D404S and / or T438Y.

[0070] In at least one embodiment of a recombinant polypeptide having BBE activity and altered product selectivity and / or increased activity, it is contemplated that the polypeptide comprises an amino acid sequence having a specific set of amino acid residue differences relative to SEQ ID NO:2. In at least one embodiment, the set includes at least two amino acid residue differences selected from M101A / T438Y, M101A / L269M / I271H, A171Y / A398E, L269M / I271H, L269M / I271H / T438Y, I271H / T438Y, L283S / V323Y, E370M / T438Y, N400W / T438Y, N400W / H402T / D404S, N400W / H402T / D404S / T438Y, H402T / T438Y, and / or D404S / T438Y.

[0071] Based on the correlation of the functional information of the recombinant polypeptides provided herein with the sequence information presented in Table 3, Table 4 and the attached sequence listing, one of skill in the art will recognize that the present disclosure provides various recombinant polypeptides having BBE activity and having altered product selectivity and / or increased activity, the polypeptides being disclosed in any one of SEQ ID NOs: 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80 and 82 (SEQ ID NOs: The present invention can be recognized as including an amino acid sequence that includes one or more amino acid differences or a set of amino acid differences (relative to SEQ ID NO: 2) or that otherwise has at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to a sequence selected from the group consisting of SEQ ID NOs: 2, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, and 82.

[0072] Thus, in at least one embodiment, a recombinant polypeptide of the present disclosure having BBE activity and altered product selectivity and / or increased activity is disclosed in any one of SEQ ID NOs: 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, and 82 (as opposed to SEQ ID NO: 2). ) an amino acid sequence comprising one or more amino acid differences or a set of amino acid differences and may additionally have 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 11, 1 to 12, 1 to 14, 1 to 15, 1 to 16, 1 to 18, 1 to 20, 1 to 22, 1 to 24, 1 to 26, 1 to 30, 1 to 35, 1 to 40, 1 to 45, 1 to 50, 1 to 55, or 1 to 60 residue differences at other residue positions. In some embodiments, the number of differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35, 40, 45, 50, 55 or 60 residue differences at other residue positions.

[0073] In addition to the residue positions identified above, any of the engineered polypeptides having BBE activity and altered product selectivity and / or increased activity disclosed herein can further include other residue differences at other residue positions relative to the wild-type Clz9 polypeptide of SEQ ID NO:2. Residue differences at these other residue positions can provide additional variation in the amino acid sequence without adversely affecting the ability of the recombinant polypeptide to carry out a desired biocatalytic conversion (e.g., conversion of compound (2) to compound (1)). In some embodiments, the recombinant polypeptide can additionally have 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40 residue differences at other amino acid residue positions compared to SEQ ID NO:2. In some embodiments, the number of differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35, and 40 residue differences at other residue positions. The residue differences at these other positions can include conservative or non-conservative changes. In some embodiments, the residue differences can include conservative and non-conservative substitutions compared to the wild-type Clz9 polypeptide of SEQ ID NO:2.

[0074] In some embodiments, recombinant polypeptides of the present disclosure may be in the form of a fusion polypeptide in which the engineered polypeptide is fused to other polypeptides, such as, by way of example and not limitation, an antibody tag (e.g., a myc epitope), a purification sequence (e.g., a His tag for binding to metals), and a cellular localization signal (e.g., a secretion signal), although the recombinant polypeptides described herein may thus be used with or without fusion to other polypeptides. It is also contemplated that the recombinant polypeptides described herein are not limited to genetically encoded amino acids. In addition to genetically encoded amino acids, the polypeptides described herein may be composed in whole or in part of naturally occurring and / or synthetic non-encoded amino acids.

[0075] In another aspect, the disclosure provides a polynucleotide encoding a recombinant polypeptide having BBE activity and altered product selectivity and / or increased activity as described herein. The polynucleotide can be operably linked to one or more heterologous regulatory sequences that control gene expression to generate a recombinant polynucleotide capable of expressing the polypeptide. An expression construct containing a heterologous polynucleotide encoding a recombinant polypeptide can be introduced into a suitable host cell to express the corresponding polypeptide. Since the codons corresponding to the various amino acids are known, the availability of a protein sequence provides a description of all polynucleotides capable of encoding the subject. Due to the degeneracy of the genetic code, where the same amino acid is coded for by alternative or synonymous codons, a very large number of nucleic acids can be generated, all of which will code for the improved transaminase enzymes disclosed herein. Thus, once a particular amino acid sequence is identified, one of skill in the art can generate any number of different nucleic acids by simply modifying the sequence of one or more codons in a manner that does not change the amino acid sequence of the protein. In this regard, the present disclosure specifically contemplates every possible variation of polynucleotides that could be made by selecting combinations based on possible codon choices, and all such variations are considered to be specifically disclosed for any polypeptide disclosed herein, including the amino acid sequences presented in Tables 3 and 4, and the Sequence Listing.

[0076] Codons can be selected to be compatible with the host cell in which the protein is produced. For example, preferred codons used in bacteria are used for expression of genes in bacteria, preferred codons used in yeast are used for expression in yeast, and preferred codons used in mammals are used for expression in mammalian cells. It is contemplated that not all codons need to be replaced to optimize the codon usage of a recombinant polypeptide, since native sequences contain preferred codons and preferred codon usage may not be required for every amino acid residue. As a result, a codon-optimized polynucleotide encoding a recombinant polypeptide may contain preferred codons at more than about 40%, 50%, 60%, 70%, 80%, or 90% of the codon positions in the full-length coding region.

[0077] In at least one embodiment, a polynucleotide encoding a recombinant polypeptide having BBE activity and altered product selectivity and / or increased activity comprises an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the wild-type Clz9 sequence of SEQ ID NO: 2. In some embodiments, the polynucleotide encodes a recombinant polypeptide comprising an amino acid sequence having percent identity to SEQ ID NO: 2, as described above, and having one or more amino acid residue differences, e.g., at residue positions selected from M101, F156, A171, L238, N267, L269, I271, L283, V323, T325, G340, E370, A398, N400, H402, D404, and / or T438, compared to SEQ ID NO: 2 set forth elsewhere herein (e.g., in Table 4). In at least one embodiment, the polynucleotide sequence comprises a sequence having at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to a sequence selected from the group consisting of SEQ ID NOs: 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, and 81.

[0078] The present disclosure provides an expression vector comprising a polynucleotide encoding a recombinant polypeptide having BBE activity and altered product selectivity and / or enhanced activity, and one or more expression control regions, such as a promoter, terminator, origin of replication, depending on the type of host into which it is to be introduced. The various nucleic acids and control sequences described above can be joined together to generate a recombinant expression vector that contains one or more convenient restriction sites, allowing for the insertion or substitution of a nucleic acid sequence encoding a recombinant polypeptide at such site. Alternatively, the polynucleotide sequence of the present disclosure can be expressed by inserting the nucleic acid sequence or a nucleic acid construct containing this sequence into a suitable vector for expression. In making an expression vector, a coding sequence is placed in the vector such that the coding sequence is operably linked to a suitable control sequence for expression. The recombinant expression vector can be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can result in expression of a polynucleotide sequence. The choice of vector will typically depend on the compatibility of the vector with the host cell into which it is to be introduced. The vector can be a linear or closed circular plasmid.

[0079] The expression vector may be an autonomously replicating vector, i.e. a vector that exists as an extrachromosomal entity and whose replication is independent of chromosomal replication, e.g. a plasmid, an extrachromosomal element, a minichromosome or an artificial chromosome. The vector may contain any means to ensure self-replication. Alternatively, the vector may be a vector that, when introduced into a host cell, is integrated into the genome and replicated together with the chromosome(s) into which it is integrated. Furthermore, a single vector or plasmid, or two or more vectors or plasmids, or transposons, may be used that together contain the total DNA to be introduced into the genome of the host cell. In at least one embodiment, the expression vector further comprises one or more selection markers that allow easy selection of transformed cells.

[0080] The present disclosure also provides a host cell comprising a polynucleotide or expression vector encoding a recombinant polypeptide of the present disclosure, the polynucleotide being operably linked to one or more control sequences for expression of a polypeptide having BBE activity in the host cell. Host cells used for expression of a polypeptide encoded by an expression vector of the present invention are known in the art and include, but are not limited to, bacterial cells such as E. coli, or fungal cells such as Saccharomyces cerevisiae or Pichiapastoris, insect cells such as Drosophila S2 and Spodoptera Sf9, animal cells such as CHO, COS, BHK, 293, and plant cells. Appropriate culture media and growth conditions for the above host cells are known in the art. In at least one embodiment, the present disclosure provides a method for producing cannabinoids comprising (a) culturing a recombinant host cell of the present disclosure in a suitable medium and (b) recovering the produced cannabinoids.

[0081] The BBE activity of the recombinant polypeptides of the present disclosure can result in the oxidative cyclization of prenylated aromatic compounds, such as CBGA, to form cyclic compounds, such as CBCA, which is a key enzymatic step in the biosynthesis of cannabinoids and many other compounds of interest. Thus, it is contemplated that the recombinant polypeptides having BBE activity of the present disclosure can be incorporated into methods and compositions useful in a variety of in vitro cell-free systems or in vivo recombinant host cell systems for the biosynthesis of compounds requiring such an oxidative cyclization step.

[0082] In at least one embodiment, the present disclosure contemplates the use of recombinant polypeptides having BBE activity in in vitro cell-free systems that require a biosynthetic oxidative cyclization step to produce the compound. Indeed, the recombinant polypeptides of the present disclosure (e.g., the polypeptides of Tables 3 and 4) are expressed in soluble form in E. coli and can be directly integrated into known cell-free biosynthetic systems and processes. For example, they can be used in methods and compositions for the cell-free biosynthesis of cannabinoid compounds, such as those 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, and WO 2020 / 028722, which are incorporated herein by reference. Moreover, recombinant polypeptides having BBE activity of the present disclosure can be used to perform known cell-free cannabinoid biosynthesis methods, which may result in more efficient and selective conversion of CBGA, CBGVA and / or CBGPA to the cyclized cannabinoid products CBCA, CBCVA and CBCPA, respectively.

[0083] FIG. 1 shows a schematic diagram of the molecular inputs / outputs and enzymes involved in an exemplary system for the biosynthesis of cannabinoid compounds. On the right side of the scheme in FIG. 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 biosynthesis enzymes. Alternatively, butyryl-CoA is converted to divaleric acid (DA) or the precursor octanoyl-CoA is converted to sphaerophorolicacid (PA). The cannabinoid precursors OA, DA or PA can act as cannabinoid precursor substrate compounds or as "polyketide inputs" for prenyltransferases, respectively. The left side of this scheme in Figure 1 shows the terpene biosynthetic pathway that converts a glucose input molecule to geranyl pyrophosphate (GPP), which is the co-substrate that prenyltransferases use to convert the cannabinoid precursor compounds OA, DA or PA to the corresponding cannabinoid-producing compounds CBGA, CBGVA or CBGPA. These exemplary cannabinoid-producing compounds differ only in the length of the alkyl carbon chain, as indicated by the general structure shown in Figure 1.

[0084] As shown in the bottom of the scheme in FIG. 1, these cannabinoid products (e.g., CBGA, CBGVA, or CBGPA) are themselves precursor substrate compounds for the final step of oxidative cyclization. In the case of C. sativa, this oxidative cyclization is carried out by cannabinoid synthase enzymes (e.g., CBDAS, THCAS, or CBCAS) to produce the cyclized cannabinoid compounds THCA, CBDA, CBCA, and other structural analogs. These plant-derived synthase enzymes are membrane-bound and have proven difficult to incorporate into cell-free soluble biosynthetic systems. Thus, the present disclosure contemplates that recombinant polypeptides with BBE activity expressed in soluble form in E. coli can be used in place of various synthases from C. sativa to carry out this final step of oxidative cyclization in such cell-free biosynthetic systems.

[0085] Thus, in at least one embodiment, the disclosure provides a composition of enzymes useful in a cell-free biosynthetic system comprising a recombinant polypeptide having a BBE activity of the disclosure (e.g., a polypeptide of Table 3 or Table 4) and one or more enzymes capable of producing a substrate desired to undergo oxidative cyclization catalyzed by the recombinant polypeptide. In at least one embodiment, the composition can further comprise a compound that is a substrate for the one or more enzymes that produce a substrate for oxidative cyclization catalyzed by the recombinant polypeptide having BBE activity.

[0086] Depending on the final biosynthetic product(s) desired from the system, one or more of a wide variety of enzymes may be incorporated into the composition. In at least one embodiment, the composition comprises a prenyltransferase that converts cannabinoid precursor compounds (e.g., OA, DA, PA) and co-substrates, such as GPP, to prenylated aromatic compounds (e.g., CBGA, CBGVA, CBGPA). It is therefore contemplated that a composition useful for the biosynthetic production of cyclized cannabinoids (e.g., CBCA) may comprise a recombinant polypeptide of the present disclosure having BBE activity, a prenyltransferase (e.g., NphB), and a substrate for the prenyltransferase (e.g., OA, DA, PA, and GPP).

[0087] Those skilled in the art will recognize that this simple cell-free biosynthetic composition may be further expanded to include additional enzymes capable of producing cannabinoid precursor compounds and / or GPP substrates. Thus, in at least one embodiment of the composition, in addition to the recombinant polypeptide having BBE activity, the one or more enzymes of the biosynthetic system may include one or more of: (a) enzymes that convert isoprenol or prenol to geranyl pyrophosphate (GPP); (b) enzymes that convert cannabinoid precursors (e.g., OA, DA, PA) to prenylated aromatic compounds (e.g., CBGA, CBGVA, CBGPA); (c) enzymes that convert malonate and acetyl-CoA to malonyl-CoA; and / or (d) enzymes that convert ADP and / or AMP to ATP, and optionally, the enzymes that convert ADP and / or AMP to ATP also convert acetyl phosphate to acetate. Table 5 provides a list of exemplary enzymes that can be used in a cell-free biosynthetic system incorporating a recombinant prenyltransferase polypeptide of the present disclosure.

[0088] Table 5: Exemplary enzymes useful for cell-free biosynthesis [Table 5]

[0089] As described herein, recombinant polypeptides having BBE activity of the present disclosure, altered product selectivity and / or increased activity can be incorporated into any biosynthetic process requiring a BBE-catalyzed biocatalytic step. Thus, in at least one embodiment, the recombinant polypeptide (e.g., the exemplary polypeptides of Tables 3 and 4) has structural formula (I): [ka] (In the formula, R 1 is C 1 ~C 7 The biosynthetic method can be used to produce cannabinoid compounds of structural formula (II) (wherein the alkyl is an alkyl group). The biosynthetic method can be used to produce cannabinoid compounds of structural formula (II) (wherein the alkyl is an alkyl group). The biosynthetic method can be used to produce cannabinoid compounds of structural formula (II) (wherein the alkyl is an alkyl group). [ka] (In the formula, R 1 is C 1 ~C 7 The method includes contacting the compound of formula (I) with a compound of formula (I) (I),

[0090] Exemplary cyclized cannabinoid compounds of structural formula (I) (CBCA, CBCVA, CBCPA) that can be prepared in stereoselective excess using recombinant polypeptides of the present disclosure are shown in FIG. 2. FIG. 3 shows a proposed mechanism of the biocatalytic reaction carried out by recombinant polypeptides of the present disclosure (e.g., polypeptides of Table 3 or Table 4) resulting in cyclized cannabinoid products CBCA, CBCVA, CBCPA. The cannabinoid compound substrate cannabigerolic acid (CBGA) can be converted in stereoselective excess to the cyclized cannabinoid compound product CBCA, rather than the alternative cyclization product THCA. Similarly, the cannabinoid compound substrate cannabigerolic acid (CBGVA) can be converted in stereoselective excess to the cyclized cannabinoid compound product CBCVA, rather than the alternative cyclization product THCVA. It is therefore contemplated that the recombinant polypeptides of the present disclosure will exhibit BBE activity towards other cannabinoid compounds that are structural analogs of the prenylated aromatic cannabinoids CBGA, CBGVA and CBGPA, including but not limited to the exemplary cannabinoid compounds listed in Table 1.

[0091] Thus, in at least one embodiment of the method, the compound of structural formula (I) is cannabichromenic acid (CBCA) and the compound of structural formula (II) is cannabigerolic acid (CBGA), or the compound of structural formula (I) is cannabichromevaric acid (CBCVA) and the compound of structural formula (II) is cannabigerovaric acid (CBGVA), or the compound of structural formula (I) is cannabichromephorolic acid (CBCPA) and the compound of structural formula (II) is cannabigerophorolic acid (CBGPA).

[0092] The present disclosure contemplates a range of suitable reaction conditions that may be used in the present methods, including, but not limited to, ranges of pH, temperature, buffer, solvent system, substrate loading, polypeptide loading, co-substrate or cofactor loading, atmosphere, and reaction time. The present disclosure also contemplates that a method comprising the biocatalytic conversion of a substrate compound of structural formula (II) to a product compound of structural formula (I) using a recombinant polypeptide having BBE activity of the present disclosure may further comprise additional chemical or biocatalytic steps performed on the product compound, work-up, extraction, isolation, purification, and / or crystallization of the product compound, each of which may be performed under a variety of conditions.

[0093] Further suitable reaction conditions for the biocatalytic conversion of a substrate compound of structural formula (II) to a product compound of structural formula (I) using a recombinant polypeptide having BBE activity as described herein can be readily optimized by routine experimentation, including, but not limited to, contacting the recombinant polypeptide with the substrate under experimental reaction conditions of concentration, pH, temperature, and solvent conditions, and detecting the production of the desired compound of structural formula (I), for example, using the methods described in the Examples presented herein.

[0094] Generally, the biosynthetic reaction involving the conversion of a cannabinoid compound of formula (II) to a cannabinoid compound of formula (I) by a recombinant polypeptide catalyst can be carried out according to reaction conditions for cell-free biosynthesis of cannabinoids known in the art (see, for example, Valliere et al. 2020, or WO2020028722) or as described herein. In some embodiments of the method, suitable reaction conditions may include a temperature range of about 293 K to about 318 K. In one embodiment, suitable reaction conditions include a temperature of about 310 K.

[0095] It is also contemplated that the improved thermostability of the recombinant polypeptides having BBE activity of the present disclosure may allow for a range of substrate loadings in the reaction. Thus, in some embodiments of the method for producing a cannabinoid compound of structural formula (I), suitable reaction conditions may include a cannabinoid substrate loading of at least about 0.6 g / L, at least about 1.2 g / L, 2 g / L, 6 g / L, 12 g / L, 18 g / L, 24 g / L, 30 g / L, or more. Specifically, when the cannabinoid substrate is selected from CBGA, CBGVA, and CBGPA, the substrate loading may be at least about 0.6 g / L, at least about 1.2 g / L, 2 g / L, 6 g / L, 12 g / L, 18 g / L, 24 g / L, 30 g / L, or more.

[0096] The recombinant polypeptides having BBE activity of the present disclosure may allow the reaction to be carried out at a higher biocatalytic conversion rate. Thus, in some embodiments, it is contemplated that the recombinant polypeptide-catalyzed conversion of a cannabinoid compound of formula (II) to a cannabinoid compound of formula (I) can be carried out at lower concentrations of recombinant polypeptides having BBE activity. Thus, in at least one embodiment of the present method, suitable reaction conditions include recombinant polypeptide concentrations of about 0.1 g / L to about 5 g / L, or even lower.

[0097] As noted elsewhere herein, suitable pH and buffer conditions for the biosynthesis of cannabinoids are known in the art and may be used with the recombinant polypeptides having BBE activity of the present disclosure. Thus, in at least one embodiment of the method for producing a cannabinoid compound of structural formula (I) using engineered polypeptides of the present disclosure, suitable reaction conditions may include (a) a pH of about 5.0 to about 11.0, or about 4.0 to 10.0, and / or a buffer solution of about 0.05 M Tris-Cl pH 8.0 to about 0.5 M Tris-Cl pH 8.0. In at least one embodiment, suitable reaction conditions for producing the cannabinoid compound CBCA include cannabigerolic acid (CBGA), 0.1 M buffer pH 8.0, and the recombinant polypeptide at 310 K for at least 1 hour. Identical or very similar conditions are contemplated for the biosynthetic production of CBCVA or CBCPA. Suitable reaction conditions for the various recombinant polypeptides of the present disclosure can be readily determined using routine techniques for optimizing biocatalytic reaction conditions well known to those of skill in the art.

[0098] In at least one embodiment, a recombinant polypeptide having BBE activity of the present disclosure can be further engineered for use in a biosynthetic reaction for the production of a cyclized cannabinoid compound or a composition comprising a cyclized cannabinoid compound. It is contemplated that the cannabinoid compounds produced may include, but are not limited to, the cannabinoid compounds in Table 1. Thus, in at least one embodiment, the biosynthetic reaction can be carried out to produce cannabichromenic acid (CBCA), cannabichromene (CBC), cannabichromenic acid isomer B (CBCA-B), cannabidiolic acid (CBDA), cannabidiol (CBD), Δ 9 -Tetrahydrocannabinolic acid (Δ 9 -THCA), Δ 9 -Tetrahydrocannabinol (Δ 9 -THC), Δ 8 -Tetrahydrocannabinolic acid (Δ 8 -THCA), Δ 8 -Tetrahydrocannabinol (Δ 8-THC), cannabinolic acid (CBNA), cannabinol (CBN), cannabidivarinic acid (CBDVA), cannabidivarin (CBDV), Δ 9 -Tetrahydrocannabivarinic acid (Δ 9 -THCVA), Δ 9 -Tetrahydrocannabivarin (Δ 9 -THCV), cannabidibutolic acid (CBDBA), cannabidibutol (CBDB), Δ 9 -Tetrahydrocannabutolic acid (Δ 9 -THCBA), Δ 9 -Tetrahydrocannabutol (Δ 9 -THCB), cannabidiphorolic acid (CBDPA), cannabidiphorol (CBDP), Δ 9 -Tetrahydrocannabifolic acid (Δ 9 -THCPA), Δ 9 -Tetrahydrocannabiphorol (Δ 9 cannabinoid compound selected from the group consisting of cannabidiol-THCP, cannabichromevaric acid (CBCVA), cannabichromevarin (CBCV), cannabigerovaric acid (CBGVA), cannabigerovarin (CBGV), cannabicyclol acid (CBLA), cannabicyclol (CBL), cannabiersoic acid (CBEA), cannabiersoin (CBE), cannabicitranic acid (CBTA), cannabicitran (CBT), and any combination thereof.

[0099] 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 polypeptide of the present disclosure having BBE activity 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. In some embodiments, it is contemplated that 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.

[0100] 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 enzymes. The host cells can then be lysed and the lysate containing one or more enzymes (including recombinant polypeptides having BBE activity) can be combined with an appropriate buffer and substrates (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.

[0101] In at least one embodiment of the method of producing cannabinoids, a heterologous nucleic acid encoding a recombinant polypeptide having BBE activity and altered product selectivity and / or increased activity (e.g., an exemplary recombinant polypeptide of Table 3 or Table 4) can be introduced into a recombinant host cell. The recombinant host cell can then be used to produce the polypeptide or can be incorporated into a biocatalytic process that utilizes the BBE activity of the recombinant polypeptide expressed by the host cell to catalyze oxidative cyclization of a substrate, such as cyclization of CBGA to produce CBCA. In at least one embodiment, the recombinant host cell can further comprise a pathway of enzymes capable of producing a cannabinoid substrate (e.g., CBGA) in addition to a recombinant polypeptide having BBE activity of the present disclosure. 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 cyclized cannabinoids (e.g., CBCA) in terms of titer, yield and production rate due to improved conversion rate and / or product selectivity of the expressed BBE activity.

[0102] Thus, in at least one embodiment, the disclosure provides a method for producing a cannabinoid derivative, the method comprising: (a) culturing a recombinant host cell of the disclosure in a suitable medium; and (b) recovering the produced cannabinoid derivative. In at least one embodiment, the method for producing a cannabinoid derivative further comprises contacting a cell-free extract of the culture containing the produced cannabinoid with a biocatalytic or chemical reagent capable of converting the cannabinoid to a cannabinoid derivative. In at least one embodiment, the biocatalytic reagent is an enzyme capable of converting the produced cannabinoid to a different cannabinoid or cannabinoid derivative compound. In at least one embodiment, the chemical reagent can chemically modify the produced cannabinoid to produce a different cannabinoid or cannabinoid derivative compound. In at least one embodiment of the method for producing a cannabinoid, the method may further comprise contacting a cell-free extract of the culture containing the produced cannabinoid with a biocatalytic or chemical reagent.

[0103] It is contemplated that the cannabinoids or cannabinoid derivatives produced using the methods of the present disclosure may be produced and / or recovered from the reaction in salt form. In at least one embodiment, the salt of the recovered cannabinoid or cannabinoid derivative is a pharmaceutically acceptable salt. Such pharmaceutically acceptable salts retain the biological effectiveness and properties of the free base compound. EXAMPLES

[0104] 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.

[0105] Example 1: Preparation and characterization of Clz9 wild-type BBE and engineered Clz9 mutants This example describes the preparation of a recombinant polypeptide with BBE activity, the wild-type Clz9 of SEQ ID NO: 2, and various site-directed mutants of this parent wild-type polypeptide. This example further describes screening of the site-directed mutants for activity in converting the cannabinoid substrate CBGA to the cyclized cannabinoid product CBCA, relative to the recombinant wild-type polypeptide of SEQ ID NO: 2.

[0106] Materials and Methods A. Wild-type Clz9 with or without MBP cloned into pET28a A codon-optimized gene of SEQ ID NO:1 encoding Clz9 from Streptomyces sp. CNH287 was synthesized and cloned into the pET28a expression vector as follows: The amino acid sequence of Clz9 BBE from Streptomyces sp. CNH287 was provided by Twist DNA. This sequence was codon-optimized by Twist DNA, with the addition of 20 bp complementary to the NdeI site 20 bp upstream and the XhoI site 20 bp downstream, and cloned into the pET28a expression vector via Gibson. The resulting cloned Clz9 construct is 6×HIS tagged for expression in E. coli. For cloning of Clz9 fused to MBP, the pET28a vector with MBP cloned between NdeI and XhoI was used as the recipient vector, and Clz9 was cloned downstream of MBP as described above for the pET28a construct.

[0107] B. Expression of MBP-Clz9 Expression of recombinant Clz9 in E. coli having the amino acid sequence of SEQ ID NO:2 was performed as follows: The cloned gene in the pET28a expression vector or the pET28a-MBP 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 mg / mL) and grown at 37°C and 250 rpm. After 12 hours, the overnight culture was inoculated into 1 L LB+kanamycin (50 mg / mL). OD 600 At 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.

[0108] C. Site-directed mutagenesis of Clz9 Mutant genes encoding Clz9 variants were obtained by site-directed mutagenesis using mutagenic primers containing the desired mutations. The wild-type Clz9 gene of SEQ ID NO: 1 was used as a template to introduce mutations by polymerase chain reaction (PCR) using mutagenic primers. Mutations were confirmed by Sanger sequencing. After confirmation, recombinant Clz9 variants were expressed in E. coli.

[0109] The cloned genes in the pET28a expression vector were transformed into BL21-Gold(DE3) competent cells using standard chemical transformation methods. A single colony was used to inoculate 4 mL LB+kanamycin (50 mg / mL) and grown at 37°C and 250 rpm. After 12 hours, the overnight culture was inoculated into 1 LLB+kanamycin (50 mg / mL). OD 600 At 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.

[0110] D. BBE Activity Assay To assess BBE activity, 98 μL of Clz9 mutant polypeptide sample at 2 mg / mL concentration in 25 mM Tris pH 7.5 was mixed with 2 μL of 5 mM CBGA. BBE reactions (100 μL final) were allowed to proceed for 4 hours at 32°C and then quenched by adding 900 μL of methanol. Protein precipitates were removed by centrifugation (16000 g for 3 min) and CBGA, THCA and CBCA were analyzed by HPLC. Relative activities correspond to the activity of wild-type or mutant Clz9 in the cell-free conversion of CBGA to CBCA at 32°C for 4 hours under the assay conditions described above. Under these conditions, wild-type Clz9 converts approximately 50% of the input CBGA to CBCA. Relative rates are indicated as follows: +=less than 25% conversion in 4 hours; ++=more than 50% conversion in 4 hours; +++=more than 75% conversion in 4 hours; ++++=100% conversion in 4 hours; -=no conversion.

[0111] result Table 4 provides an overview of the specific site-directed mutations found in the 34 recombinant mutants of the Clz9 polypeptide of SEQ ID NO:2 that were prepared and screened for activity. Figure 5 shows an alignment of the four mutants (Clz9M1, Clz9M2, Clz9M3 and Clz9Mut34) and the wild-type Clz9 sequence of SEQ ID NO:2. Figure 6 shows raw HPLC data from the screening assay showing the relative production of CBCA (and depletion of the substrate CBGA) by the 12 Clz9 mutants. Table 4 summarizes the relative activity of the 34 Clz9 mutants compared to the activity of the parent wild-type of SEQ ID NO:2 under the same assay conditions.

[0112] Example 2: Identification of bacterial homologues of Clz9 and preparation of additional recombinant polypeptides with BBE activity This example describes the preparation and characterization of a series of recombinant polypeptides with BBE activity from bacterial sources that were identified based on their homology to wild-type Clz9 of SEQ ID NO:2.

[0113] Materials and Methods A. Homology search using wild-type Clz9 The sequence of wild-type Clz9 in SEQ ID NO:2, which exhibits BBE activity in the conversion of CBGA to CBCA, was used to search for bacterial homologs that clustered with either putative PKS gene clusters and / or prenyltransferases (NphB homologs). To identify clusters, the nucleotide sequence of NphB or Clz9 (SEQ ID NO:1) was used as a template for a BlastN search. Resulting positive hits with 25%-90% identity were further analyzed by submitting genomic regions 40 kb upstream and 40 kb downstream (80 kb in total) to 2ndfind (see biosyn.nih.go.jp / 2ndfind / ). The resulting annotated genes and clusters were then analyzed for the presence of BBE in addition to prenyltransferases, polyketide synthases, or both. Representative BBE gene clusters from various bacterial source organisms containing polyketide synthase (PKS) and / or prenyltransferase enzymes are shown in Figure 7. Putative bacterial homologs of Clz9 with BBE activity were identified in Streptomyces flaveolus (SflBBE), Streptomyces Ru71 (SR71BBE), Phytohabitans safuscus (PsBBE), Streptomyces AJS327 (SAJBBE), Streptomyces bathobiensis (SvBBE), and Actinomadura pelletieri (ApBBE). An overview of these bacterial homologs is presented in Table 3 and the attached sequence listing.

[0114] Cloning and expression of a bacterial homologue of B.Clz9 Genes encoding the identified bacterial homolog polypeptides of SEQ ID NOs: 4, 6, 8, 10, 12 and 14 were constructed, cloned and expressed in E. coli fused to MBP as described above for Clz9. Expression of the bacterial homologs or Clz9 transformed into E. coli BL21Gold(DE3) was increased to OD 100 by addition of 0.4 mM isopropyl β-d-1-thiogalactopyranoside (IPTG). 600The yeast was induced at approximately 0.6 and grown for an additional 16 hours at 18° C. and 250 rpm. After 16 hours, protein purification was performed using standard Ni-NTA methods.

[0115] D. BBE Activity Assay The bacterial homolog polypeptides of SEQ ID NOs: 4, 6, 8, 10, 12 and 14 were evaluated for BBE activity in the conversion of CBGA to CBCA as described in Example 1.

[0116] result Table 3 shows a summary of the recombinant polypeptides of bacterial homologues of Clz9 that were screened and their BBE activity compared to Clz9.

[0117] Figures 8B, 8C and 8E show HPLC results showing the products of cyclization reactions catalyzed at pH 7.5 by BBE homologs from Phytohabitans safuscus (PsBBE) (Figure 8B), Streptomyces bathobiensis (SvBBE) (Figure 8C) and Streptomyces sp. AJS327 (SAJBBE) (Figure 8E) performed as described in Example 2. These active bacterial BBE homologs of Clz9 produce only CBCA at all pH values ​​tested. In contrast, THCAS from C. sativa (Figure 8D) produces mainly CBCA with some THCA at pH 7.5 and mainly THCA with some CBCA at pH 5.5. Standard profiles of the cannabinoid substrate CBGA and the cannabinoid products THCA and CBCA are shown in Figure 8A.

[0118] 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.

[0119] Additional embodiments of the invention are set forth in the accompanying claims.

[0120] 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.

[0121] Drawing translation Figure 1 Glucose TerpeneBiosynthesis geranylpyrophosphate prenyltransferase enzyme FattyAcid Biosynthesis Hexanoyl-CoA + 3 Malonyl-CoA (or Butyryl-CoAor Octanoyl-CoA) Polyketide Chalcone Biosynthesis Olivetolic acid divarinicacid sphaerophorolicacid sphaerophoric acid cannabigerolic acid cannabigerovarinic acid cannabigerophorolic acid cannabinoids Figure 3 rotatable bond cannabigerolic acid cannabigerovarinic acid cannabigerophorolic acid THCA / CBDA Synthase CBCA Synthase or Clz9 or homolog FAD initiated quinone methide (QM) formation Figure 6 Response Time (min) Blank Figure 7 Streptomyces sp. Phytohabitans suffuscus Streptomyces varsoviensis Actinomadura pelletieri Actinomadura pelletieri Streptomyces flaveolus PP-binding PP-binding Figure 8A RESPONSE TIME (min) STANDARDS Standard substances Figure 8B RESPONSE TIME (min) Figure 8C RESPONSE TIME (min) Figure 8D RESPONSE TIME (min) Figure 8E RESPONSE TIME (min)

Claims

1. Structural formula (I): 【Chemistry 1】 (In the formula, R 1 is C 1 ~C 7 1. A method for preparing a compound of structural formula (II): 【Chemistry 2】 (In the formula, R 1 is C 1 ~C 7 8, 2, 4, 6, 10, 12, and 14.

2. The suitable reaction conditions are (a) cell-free solution, (b) a substrate compound of formula (II), 0.1 M buffer, pH 8.0, and the recombinant polypeptide at 298 K for at least 1 hour; (c) a loading of the substrate compound of structural formula (II) of at least about 0.6 g / L, at least about 1.2 g / L, 2 g / L, 6 g / L, 12 g / L, 18 g / L, 24 g / L, 30 g / L, or more; and / or (d) the recombinant polypeptide concentration is about 0.1 g / L to about 5 g / L, or even lower; 2. The method of claim 1, comprising:

3. (a) the compound of structural formula (I) is cannabichromenic acid (CBCA) and the compound of structural formula (II) is cannabigerolic acid (CBGA); (b) the compound of formula (I) is cannabichromevaric acid (CBCVA) and the compound of formula (II) is cannabigerovaric acid (CBGVA), or (c) The method of claim 1 or 2, wherein the compound of structural formula (I) is cannabichromephoric acid (CBCPA) and the compound of structural formula (II) is cannabigerophoric acid (CBGPA).

4. A composition comprising: (a) a recombinant polypeptide having BBE activity, wherein the recombinant polypeptide comprises an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to a sequence selected from SEQ ID NOs: 8, 2, 4, 6, 10, 12, and 14; and (b) one or more enzymes that produce a substrate for the recombinant polypeptide.

5. the one or more enzymes (a) an enzyme that produces a prenylated aromatic compound, optionally wherein the prenylated aromatic compound is selected from CBGA, CBGVA, CBGPA, and combinations thereof; (b) an enzyme that is a prenyltransferase, optionally the prenyltransferase is NphB; (c) an enzyme that converts isoprenol or prenol to geranyl pyrophosphate (GPP); (d) an enzyme that converts malonate and acetyl-CoA to malonyl-CoA; (e) an enzyme that converts ADP and / or AMP to ATP, and optionally, the enzyme that converts ADP and / or AMP to ATP also converts acetyl phosphate to acetate; (f) acyl-activating enzyme 3 (AAE3), olivetol synthase (OLS), olivetolic acid cyclase (OAC), and prenyltransferase (NphB), and / or 5. The composition of claim 4, comprising: (g) acetylphosphate transferase (PTA), malonate decarboxylase alpha subunit (mdcA), acyl-activating enzyme 3 (AAE3), olivetol synthase (OLS), olivetolic acid cyclase (OAC), prenyltransferase (NphB), hydroxyethylthiazole kinase (ThiM), isopentenyl kinase (IPK), isopentyl diphosphate isomerase (IDI), diphosphomevalonate decarboxylase alpha subunit (MDCa), and geranyl PP synthase (GPPS) or farnesyl PP synthase mutant S82F (FPPSS82F).

6. 5. The composition of claim 4, further comprising geranyl pyrophosphate (GPP) and a cannabinoid precursor substrate selected from olivetolic acid (OA), divaleric acid (DA) and sphaerophoric acid (PA).

7. The composition of claim 6, which is in a cell-free solution.

8. A recombinant polypeptide having berberine bridge enzyme (BBE) activity, comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 2, and having an amino acid residue difference at one or more positions selected from T325, F156, L238, L283, T325 and G340 compared to SEQ ID NO: 2, optionally wherein the amino acid residue difference is selected from T325G, F156Y, L238S, L283S and G340I.

9. 9. The polypeptide of claim 8, wherein the polypeptide further comprises an amino acid residue difference at one or more positions selected from M101, A171, N267, L269, 1271, V323, E370, A398, N400, H402, D404 and / or T438 compared to SEQ ID NO: 2, and optionally the amino acid residue difference is selected from M101A, A171Y, N267V, L269M, 1271H, V323Y, E370M, A398E, N400W, H402T, D404S and / or T438Y.

10. 10. The polypeptide of claim 9, wherein the polypeptide has a set of at least two amino acid residue differences selected from M101A / T438Y, M101A / L269M / I271H, A171Y / A398E, L269M / I271H, L269M / I271H / T438Y, I271H / T438Y, L283S / V323Y, E370M / T438Y, N400W / T438Y, N400W / H402T / D404S, N400W / H402T / D404S / T438Y, H402T / T438Y, and / or D404S / T438Y.

11. 9. The polypeptide of claim 8, wherein the polypeptide comprises an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to a sequence selected from the group consisting of SEQ ID NOs: 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, and 82.

12. 9. The polypeptide of claim 8, wherein the BBE activity of the polypeptide is increased by at least 1.2-fold, at least 1.5-fold, at least 2-fold, at least 5-fold, or more compared to the polypeptide consisting of SEQ ID NO:

2.

13. 13. The polypeptide of claim 12, wherein the BBE activity is measured as a conversion rate of the substrate cannabigerolic acid (CBGA) to the product THCA and / or CBCA under reaction conditions of 0.05 mM CBGA and 50 mM Tris at pH 5.5 to 9.5 and 298 K.

14. A polynucleotide encoding the polypeptide of claim 8.

15. 15. The polynucleotide of claim 14, wherein the polynucleotide sequence comprises a sequence having at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to a sequence selected from the group consisting of SEQ ID NOs: 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, and 81.

16. An expression vector comprising the polynucleotide of claim 14.

17. 17. The expression vector of claim 16, comprising a regulatory sequence.

18. 17. A host cell comprising the polynucleotide of claim 14 or the expression vector of claim 16.

19. 19. A method for producing the polypeptide of claim 8, comprising culturing the host cell of claim 18 and isolating the polypeptide from the cell.

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