Actinomycete for use in cannabinoid compound production, and production method using this same

Abstract

To provide: an actinomycete for more efficient production of cannabinoids; and a production method for cannabinoids using the actinomycete.SOLUTION: Provided is an actinomycete for cannabinoid compound production, engineered to incorporate a gene encoding a polyketide synthase and a gene encoding a cyclase.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present disclosure relates to actinomycetes used in the production of cannabinoid-like compounds and a method for producing cannabinoid-like compounds using the actinomycetes. [Background technology]

[0002] Cannabinoids are known as one of the chemical substances contained in cannabis (hemp). Cannabinoids derived from hemp include cannabidiol, tetrahydrocannabinol, cannabinol, cannabigerol, etc., and among them, cannabidiol in particular has come to be used for various purposes such as food and beverages, luxury goods, and cosmetics, as well as medical purposes. However, the production of such cannabinoids has remained dominated by the method of extraction from cultivated cannabis plants, and there have been problems with the production, such as the maintenance of fields and the complicated extraction process.

[0003] Patent Document 1 describes a method for producing synthetic cannabidiol by adding a base solution to a precursor compound of cannabidiol in a solvent-free state under atmospheric pressure and removing water and the solvent. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2021-130654 A Summary of the Invention [Problem to be solved by the invention]

[0005] Therefore, an objective of the present disclosure is to provide an actinomycete for more efficiently producing cannabinoids and a method for producing cannabinoids using the actinomycete. [Means for solving the problem]

[0006] In order to solve the above problems, the present inventors have found that it is possible to efficiently produce cannabinoid compounds by using actinomycetes into which a gene encoding an enzyme involved in the synthesis of a precursor of a cannabinoid compound has been introduced. Therefore, the first aspect of the present disclosure is to (1) An actinomycete for producing cannabinoid compounds, into which a gene encoding a polyketide synthase and a gene encoding a cyclase have been introduced.

[0007] A preferred embodiment of the present disclosure is (2) The actinomycete according to (1) above, wherein the polyketide synthase and the cyclase are enzymes capable of catalyzing the synthesis of olivetolic acid, which is a precursor of the cannabinoid compound.

[0008] A preferred embodiment of the present disclosure is (3) The polyketide synthase is an actinomycete according to (1) above, which is either (A1) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, (A2) a protein having polyketide synthase activity and an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence set forth in SEQ ID NO: 2, or (A3) a protein having polyketide synthase activity and an amino acid sequence that shows 60% or more identity to the amino acid sequence set forth in SEQ ID NO: 2.

[0009] A preferred embodiment of the present disclosure is (4) The actinomycete according to (1) above, wherein the cyclase is either (B1) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 3, (B2) a protein having cyclase activity and an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence set forth in SEQ ID NO: 3, or (A3) a protein having cyclase activity and an amino acid sequence that shows 60% or more identity to the amino acid sequence set forth in SEQ ID NO: 3.

[0010] In order to solve the above problems, the present inventors have further found that it is possible to efficiently produce cannabinoid compounds by using actinomycetes into which a gene encoding an enzyme involved in the synthesis of a precursor of a cannabinoid compound has been introduced. Therefore, the second aspect of the present disclosure is: (5) An actinomycete for producing cannabinoid compounds, into which a gene encoding an enzyme having the activity of synthesizing hexanoyl-CoA, which is used in the synthesis of olivetolic acid, a precursor of cannabinoid compounds, has been introduced.

[0011] A preferred embodiment of the present disclosure is (6) The enzyme having the activity of synthesizing hexanoyl-CoA is an actinomycete described in (5) above, which is either (C1) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1, (C2) a protein having an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence set forth in SEQ ID NO: 1, or (C3) a protein having an amino acid sequence that shows 60% or more identity to the amino acid sequence set forth in SEQ ID NO: 1.

[0012] A preferred embodiment of the present disclosure is (7) An actinomycete for producing cannabinoid compounds, into which a gene encoding geranyltransferase has been introduced.

[0013] In order to solve the above problems, the present inventors have further found that it is possible to produce cannabinoid compounds more efficiently by using actinomycetes into which a gene encoding an enzyme involved in geranyl group transfer to a precursor of a cannabinoid compound is introduced. Therefore, the third aspect of the present disclosure is: (8) The actinomycete according to (7) above, wherein the geranyltransferase is an enzyme capable of catalyzing the synthesis of cannabigerolic acid by geranyl transfer to olivetolic acid, which is a precursor of the cannabinoid compound.

[0014] A preferred embodiment of the present disclosure is (9) The actinomycete described in (7) above, wherein the geranyltransferase is either (D1) a protein consisting of the amino acid sequence set forth in SEQ ID NO:5, (D2) a protein having an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence set forth in SEQ ID NO:5, or (D3) a protein having an amino acid sequence that shows 60% or more identity to the amino acid sequence set forth in SEQ ID NO:5.

[0015] A preferred embodiment of the present disclosure is (10) The actinomycete according to (1) above, wherein the cannabinoid compound is at least one of cannabidiol, cannabidiolic acid, and cannabigerolic acid.

[0016] In order to solve the above problems, the present inventors have found that it is possible to efficiently produce cannabinoid compounds by using actinomycetes into which a gene encoding an enzyme involved in the synthesis of a precursor of a cannabinoid compound has been introduced. Therefore, the fourth aspect of the present disclosure is: (11) A method for producing a cannabinoid compound, comprising a step of culturing the actinomycete described in (1) above.

[0017] In order to solve the above problems, the present inventors have further found that it is possible to efficiently produce cannabinoid compounds by using actinomycetes into which a gene encoding an enzyme involved in the synthesis of a precursor of a cannabinoid compound has been introduced. Therefore, a fifth aspect of the present disclosure is: (12) A method for producing a cannabinoid compound, comprising a step of culturing the actinomycete described in (5) above.

[0018] In order to solve the above problems, the present inventors have further found that it is possible to efficiently produce cannabinoid compounds by using actinomycetes into which a gene encoding an enzyme involved in the synthesis of a precursor of a cannabinoid compound has been introduced. Therefore, a sixth aspect of the present disclosure is: (13) A method for producing a cannabinoid compound, comprising a step of culturing the actinomycete described in (7) above.

[0019] In order to solve the above problems, the present inventors have further found that it is possible to efficiently produce cannabinoid compounds by using actinomycetes into which a gene encoding an enzyme involved in the synthesis of a precursor of a cannabinoid compound has been introduced. Therefore, the seventh aspect of the present disclosure is: (14) A method for producing a cannabinoid compound, comprising the steps of: introducing into an actinomycete a gene encoding an enzyme having a hexanoyl-CoA synthesis activity; introducing into the actinomycete a gene encoding a polyketide synthase and a gene encoding a cyclase used to synthesize olivetolic acid from the hexanoyl-CoA; introducing into the actinomycete a gene encoding a geranyl transferase used to synthesize cannabigerolic acid from the olivetolic acid; and culturing the actinomycete into which the gene encoding the enzyme having a hexanoyl-CoA synthesis activity, the gene encoding the polyketide synthase, the gene encoding the cyclase, and the gene encoding the geranyl transferase have been introduced. Effect of the Invention

[0020] The present disclosure can provide an actinomycete for more efficiently producing cannabinoids and a method for producing cannabinoids using the actinomycete. [Brief description of the drawings]

[0021] [Figure 1] FIG. 1 is a diagram showing an example of the cannabidiol biosynthetic pathway. [Diagram 2] FIG. 2 shows the structure of the constructed expression vector, pKU592AT_aphII::CsTKS-CsOAC_GC. [Diagram 3] FIG. 3 shows a comparison of mass chromatograms between an olivetolic acid standard and an actinomycete into which a gene encoding CsTKS-CsOAC has been introduced. [Figure 4]FIG. 4 shows the structure of the constructed expression vector, pKU594AT_aac(3)IV::KSE_65520. [Diagram 5] FIG. 5 is a diagram showing the ability of SUKA25 / CsTKS-CsOAC / KSE_65520 to produce olivetolic acid. [Figure 6] FIG. 6 shows the structure of the constructed expression vector, pKU565tsr_2-cistron::gps-nphB_A232S_Y288A. [Figure 7] FIG. 7 is a diagram showing the ability of SUKA25 / CsTKS-CsOAC / KSE_65520 / gps-nphB_A232S_Y288A to produce cannabigerolic acid. [Figure 8] FIG. 8 is a diagram showing the production ability of cannabigerolic acid by SUKA25 / CsTKS-CsOAC / KSE_65520 / gps-Spru_nphB_A230S_Y286A. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] The actinomycetes used in the production of the cannabinoid compounds of the present disclosure and the embodiment for carrying out the method for producing the cannabinoid compounds using the actinomycetes will be described in detail below. However, the following embodiment is an example for explaining the present disclosure, and the present disclosure is not limited to only the embodiment. Note that, although the word "multiple" is used in the present disclosure, the term is not limited to a specific number (e.g., 2), and may include any number such as 3 or 4.

[0023] 1. Biosynthetic pathway of cannabinoids Cannabinoid compounds are one of the chemical substances contained in cannabis (cannabis), and the cannabinoid compounds in the present disclosure are not limited to extracts derived from cannabis. Such cannabinoid compounds include cannabinoids such as cannabidiol (CBD), tetrahydrocannabinol (THC), cannabinol (CBN), and cannabigerol (CBG), as well as precursors produced in the synthesis of these cannabinoids. Examples of such precursors include cannabidiolic acid (CBDA), cannabigerolic acid (CBGA), and olivetolic acid (OA). In the present disclosure, cannabidiol and its precursors (cannabidiolic acid or cannabigerolic acid) are particularly preferred examples of cannabinoid compounds from the viewpoint of their usefulness.

[0024] FIG. 1 shows an exemplary pathway for the biosynthesis of cannabidiol. The pathway shown in FIG. 1 includes the cannabinoid biosynthesis pathway for biosynthesizing cannabidiol from olivetolic acid, which is a precursor of cannabidiol, as well as the malonyl-CoA biosynthesis pathway used for the production of olivetolic acid. Malonyl-CoA is produced in vivo by acetyl-CoA carboxylase (ACC) catalyzing the carboxylation of acetyl-CoA. On the other hand, hexanoyl-CoA is produced by a catalytic reaction by a ligase (synthetic enzyme) with hexanoic acid derived from hexanoyl ACP, which is biosynthesized from acetyl-CoA in the fatty acid production pathway. For the sake of convenience, hexanoic acid has been described here as being produced by fatty acid synthesis, but it may of course be produced by fatty acid decomposition.

[0025] Here, such a synthetic enzyme can be suitably used as long as it is an endogenous enzyme in a living organism such as hemp (cannabis), bacteria, or eukaryotes, or an enzyme that can be introduced into a host. Examples of such synthetic enzymes include CsAAE1 and CsAAE3, which are synthetic enzymes used in the biosynthetic pathway in cannabis, ScFAA1, ScFAA2, ScFAA3, ScFAA4, ScFAT1, and ScFAT2, which are synthetic enzymes that can be introduced into the biosynthetic pathway in yeast, and KSE_65520 (SEQ ID NO: 1), which is a synthetic enzyme that can be introduced into the cannabinoid biosynthetic pathway in actinomycetes. Among these, KSE_65520 can be suitably used as a synthetic enzyme from the viewpoint of high production of olivetolic acid used in the biosynthesis of cannabidiol.

[0026] [SEQ ID NO:1] MTDRVPGTAPHALPNALPDGPFDATPDTAPDTTPAAASDGTPATTSATTSATTPAATPAAASDGRPGALPSLPDVLARRAVEQPDELAYAFLHNGEEVAETLTYRQLDARARAVAARLTALRLAGRSVLLLHPSGLGFVSDLLGCMYAGVAAAPVQVPSRARG LARLRAIADDAGTTVVLTTPEVRRDLLERFGALPELAGLTLHDPETLAAEAELTDPAAVAGWRPIGSEELALLQYTSGSTGTPKGVRVTHANFGANVDETDRLWPCRGDARVVNWLPLFHDMGMLFGVVLPLWAGIPSYLMAPDAFIRRPARWLEAVSRFGG THAAAPSFAYELCVRAVGEEGLPSGLDLSSWRVAANGAEPVRWQTVRAFTEALAPAGFRPEAMCPGYGLAENTLKATGSPEDRVPTVLWLSAEELRAGRAVRVPRSDAPGVLPAVGCGAVVGDSLVRIVDPVERTTRPEGRVGEIWVSGPPCVASGYHGRPEESE ETFRARRSDRTEQRSWLRTGDLGFLADGELFVTGRIKDVVIRQGRNFYPQDIELSAESADPGLHPNCAAAFSADDGTSERLVLVVEADGRTLRGGGELLRERIRRAVHENQRLEADEILLVRRGSLPKTSSGKVQRRETLRRYLDGEFGSAQAVAPRERVEAGR

[0027] More specifically, in the present disclosure, actinomycetes are used in the production of cannabinoid compounds. Therefore, by utilizing a gene involved in the production of a compound in which a medium-chain fatty acid-CoA such as hexanoyl-CoA is an intermediate in the biosynthesis of actinomycetes, it is expected that hexanoyl-CoA, which is the starting substrate in the production of olivetolic acid, can be produced more efficiently in actinomycetes. Therefore, by searching for secondary metabolites having a partial structure derived from hexanoic acid used in the production of hexanoyl-CoA from a large number of actinomycete strains and identifying the enzyme involved in the production of the secondary metabolite, it is possible to screen for synthetic enzymes that can be suitably used in the present disclosure.

[0028] The synthetic enzymes obtained by the screening include at least the following proteins (1) to (3): (1) A protein consisting of the amino acid sequence set forth in SEQ ID NO:1 (2) A protein having an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence set forth in SEQ ID NO: 1 and having an activity of synthesizing hexanoyl-CoA. (3) A protein having an amino acid sequence that shows 60% or more identity with the amino acid sequence set forth in SEQ ID NO:1 and has an activity of synthesizing hexanoyl-CoA. The identity of the amino acid sequence in (3) above is preferably 60% or more, more preferably 80% or more, even more preferably 90% or more, and particularly preferably 95% or more.

[0029] Next, the tetraketo intermediate shown in Figure 1 is generated by the catalyst of polyketide synthase using hexanoyl-CoA synthesized by the malonyl-CoA biosynthetic pathway as an initial substrate and malonyl-CoA as an elongation substrate. Furthermore, olivetolic acid is generated by catalyzing the aromatization of the tetraketo intermediate thus generated with the aid of a cyclization enzyme accompanied by dehydration.

[0030] Specifically, the polyketide synthase here catalyzes three condensation reactions of hexanoyl-CoA with malonyl-CoA to produce 3,5,7-trioxododecanoyl-CoA. Any polyketide synthase may be used as long as it can catalyze the condensation reaction of hexanoyl-CoA as described above, and from the viewpoint of high production of olivetolic acid, CsTKS (SEQ ID NO: 2) is an example.

[0031] [SEQ ID NO:2] MNHLRAEGPASVLAIGTANPENILLQDEFPDYYFRVTKSEHMTQLKEKFRKICDKSMIRKRNCFLNEEHLKQNPRLVEHEMQTLDARQDMLVVEVPKLGKDACAKAIKEWGQPKSKITHLIFTSASTTDMPGADYHCAKLLGLSPSVKRVMMYQLGCYGGGTVLRIAKDIAENNKGARVLAVCCDIMACLFR GPSESDLELLVGQAIFGDGAAAVIVGAEPDESVGERPIFELVSTGQTILPNSEGTIGGHIREAGLIFDLHKDVPMLISNNIEKCLIEAFTPIGISDWNSIFWITHPGGKAILDKVEEKLHLKSDKFVDSRHVLSEHGNMSSSTVLFVMDELRKRSLEEGKSTTGDGFEWGVLFGFGPGLTVERVVVRSVPIKY

[0032] The polyketide synthase includes at least the following proteins (1) to (3): (1) A protein consisting of the amino acid sequence set forth in SEQ ID NO:2 (2) A protein having an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence set forth in SEQ ID NO:2 and has polyketide synthase activity. (3) A protein having an amino acid sequence that shows 60% or more identity with the amino acid sequence set forth in SEQ ID NO:2 and has polyketide synthase activity. The identity of the amino acid sequence in (3) above is preferably 60% or more, more preferably 80% or more, even more preferably 90% or more, and particularly preferably 95% or more.

[0033] Specifically, the cyclase catalyzes the aromatization accompanied by dehydration of 3,5,7-trioxododecanoyl-CoA obtained by the condensation reaction to produce olivetolic acid. As described above, any cyclase can be used as long as it can catalyze the cyclization of 3,5,7-trioxododecanoyl-CoA, and an example of such a cyclase is CsOAC (SEQ ID NO: 3) from the viewpoint of high production of olivetolic acid.

[0034] [SEQ ID NO:3] MAVKHLIVLKFKDEITEAQKEEFFKTYVNLVNIIPAMKDVYWGKDVTQKNKEEGYTHIVEVTFESVETIQDYIIHPAHVGFGDVYRSFWEKLLIFDYTPRK

[0035] The cyclase includes at least the following proteins (1) to (3): (1) A protein consisting of the amino acid sequence set forth in SEQ ID NO:3 (2) A protein having a cyclase activity and an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence set forth in SEQ ID NO:3. (3) A protein having an amino acid sequence that shows 60% or more identity with the amino acid sequence set forth in SEQ ID NO:3 and has cyclase activity. The identity of the amino acid sequence in (3) above is preferably 60% or more, more preferably 80% or more, even more preferably 90% or more, and particularly preferably 95% or more.

[0036] Next, geranyltransferase catalyzes the geranylation of the C3 position of the produced olivetolic acid to produce cannabigerolic acid.

[0037] As such a geranyltransferase, any enzyme can be suitably used as long as it is an endogenous enzyme in living organisms such as cannabis (cannabis), bacteria, and eukaryotes, or an enzyme that can be introduced into a host. Examples of such geranyltransferases include CsPT4, a geranyltransferase used in the biosynthetic pathway in cannabis, and NphB, which can be introduced into the biosynthetic pathway in actinomycetes (reference 1: Kuzuyama, T. et al. Structural basis for the promiscuous biosynthetic prenylation of aromatic natural products. Nature 435, 983-987 (2005)), or an improved enzyme thereof (reference 2: Valliere, MA et al. A cell-free platfworm for the prenylation of natural products and application to cannabinoid production. Nat. Commun. 10, 565 (2019) with two amino acid substitutions (SEQ ID NO: 4) or reference 3: Lim, KJH et al. Structure-Guided Engineering of Prenyltransferase NphB for High-Yield and Regioselective Cannabinoid Production. ACS Catal. 12(8), 4628-4639). (2022)) is an example of a geranyltransferase. Among these, from the viewpoint of high production of cannabigerolic acid, an example is the two amino acid substitution mutant of NphB described in Reference 2 (SEQ ID NO: 4).

[0038] [SEQ ID NO:4] MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLSTFQDTLVEGGSVVVFSMASGRHSTELDFSISVPTSHGDPYATVVEKGLFPATGHPVDDLLADTQKHLPVSMFAIDGEVTGGFKKTYAFFPTDNMPGVAELSAIPSMPPAVAENAELFA RYGLDKVQMTSMDYKKRQVNLYFSELSAQTLEAESVLALVRELGLHVPNELGLKFCKRSFSVYPTLNWETGKIDRLCFSVISNDPTLVPSSDEGDIEKFHNYATKAPYAYVGEKRTLVYGLTLSPKEEYYKLGAAYHITDVQRGLLKAFDSLED

[0039] The two amino acid substitutions of NphB shown in SEQ ID NO: 4 have an amino acid sequence in which the 232nd alanine is replaced by serine and the 288th tyrosine is replaced by alanine. NphB is an enzyme involved in the biosynthesis of naphterpin, and actinomycetes capable of producing analogues of the compound are highly likely to have an endogenous enzyme exhibiting geranyl transfer activity similar to NphB. Thus, it is possible to search for NphB homologues by performing genome analysis on actinomycetes capable of producing analogues, and to identify enzymes exhibiting activity equal to or greater than that of NphB. Spru_NphB is an enzyme identified in this way. Furthermore, an enzyme (SEQ ID NO: 5) having an amino acid sequence in which the 230th alanine is replaced by serine and the 286th tyrosine is replaced by alanine is more preferable.

[0040] [SEQ ID NO:5] MSGAADVERVYAAMEEAAGLLGVTCAREKIYPLLTEFQDTLTDGVVVFSMASGRRSTELDFSISVPTSQGDPYATVVDKGLFPATGHPVDDLLADTQKHLPVSMFAIDGEVTGGFKKTYAFFPTDDMPGVAQLSAIPSMPASVAQNAELFAR YGLDKVQMTSMDYKKRQVNLYFSELSQQTLAPESVLALVRELGLHVPTELGLEFCKRSFSVYPTLNWDTGKIDRLCFSVISTDPTLVPSEDERDIEQFRDYGTKAPYAYVGEKRTLVYGLTLSPTEEYYKLGAAYHITDIQRRLLKAFDALED

[0041] The above geranyltransferase includes at least nucleic acids encoding the following proteins (1) to (6): (1) A protein consisting of the amino acid sequence set forth in SEQ ID NO:4 (2) A protein having geranyltransferase activity, which has an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence set forth in SEQ ID NO: 4. (3) A protein having an amino acid sequence that shows 60% or more identity with the amino acid sequence set forth in SEQ ID NO: 4 and has geranyltransferase activity. (4) A protein consisting of the amino acid sequence set forth in SEQ ID NO:5 (5) A protein having geranyltransferase activity, which has an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence set forth in SEQ ID NO:5. (6) A protein having geranyltransferase activity and an amino acid sequence showing 60% or more identity with the amino acid sequence set forth in SEQ ID NO:5. The identity of the amino acid sequences in (3) and (6) above is preferably 60% or more, more preferably 80% or more, even more preferably 90% or more, and particularly preferably 95% or more.

[0042] The generated cannabigerolic acid then acts as a substrate for cannabidiolic acid synthase, which catalyzes the generation of cannabidiolic acid. Cannabidiolic acid, the precursor of cannabidiol, is then decarboxylated by light or heat to generate cannabidiol.

[0043] In addition, in the present disclosure, as described above, the actinomycete used for producing the cannabinoid compound or the production method using the actinomycete is concerned, and the cannabinoid compound is not limited to the above-mentioned cannabidiol. The cannabinoid compound also includes cannabidiolic acid and cannabigerolic acid, which are precursors in the production of cannabidiol. In addition, although not specifically shown in FIG. 1, in addition to cannabidiol, tetrahydrocannabinol, cannabinol, cannabigerol, etc. are also produced in the biosynthetic pathway of cannabidiol. Therefore, the cannabinoid compound also includes tetrahydrocannabinol, cannabinol, and cannabigerol.

[0044] 2. Method for producing cannabinoid compounds For example, cannabidiol is biosynthesized as shown in FIG. 1, and actinomycetes into which genes encoding each enzyme are introduced and which are capable of expressing each enzyme activity are used to produce cannabinoid compounds such as cannabidiol. The actinomycetes are then cultured in a specific medium, and the target compound is extracted from the culture to produce the compound. Thus, the method for producing cannabinoid compounds in the present disclosure includes, as an example, (A) a step of selecting a host actinomycete, (B) a step of constructing an expression vector into which genes encoding each enzyme are introduced, (C) a step of introducing the expression vector into the host actinomycete, and (D) a step of culturing the actinomycete into which the expression vector is introduced in a specific medium. Note that the method for producing cannabinoid compounds in the present disclosure does not necessarily have to include all of the above steps. Each step will be described in detail below.

[0045] (A) Selection stage of host actinomycetes In the microbial production of plant-derived compounds, there are known examples of using yeast (Reference 4: Saccharomyces cerevisiae) as a host to introduce genes encoding required enzymes (Luo, X. et al. Complete biosynthesis of cannabinoids and their unnatural analogues in yeast, Nature, 567,123-126,(2019)). However, although yeast is suitable for the production of proteins such as enzymes, it is not necessarily an appropriate microorganism for the production of compounds that are secondary metabolites such as cannabinoid compounds due to the problem of the supply capacity of hexanoyl-CoA and geranyl diphosphate. On the other hand, in the present disclosure, actinomycetes are used as hosts in the production of secondary metabolites such as cannabinoid compounds. As is clear from the fact that actinomycetes produce various secondary metabolites, they have a higher substrate supply capacity than hosts such as yeast, and are therefore expected to produce cannabinoid compounds more efficiently through heterologous expression.

[0046] Examples of actinomycetes that can be used as hosts include actinomycetes belonging to any of the genera Corynebacterium, Mycobacterium, Rhodococcus, Propionibacterium, Streptomyces, Micrococcus, Frankia, and Bifidobacterium. Among these actinomycetes, from the viewpoint of production efficiency, actinomycetes belonging to the genus Streptomyces are preferred, and more preferably Streptomyces tuirus, Streptomyces avermitilis, Streptomyces arenae, Streptomyces fragilis, Streptomyces violaceoruber, Streptomyces collinus, Streptomyces flaveolus, Streptomyces misionennsis, Streptomyces coelicolor, Streptomyces griseus, Streptomyces maltophil ... griseus, Streptomyces heimi, Streptomyces hygroscopicus, Streptomyces albulus, and Streptomyces griseofuscus, more preferably Streptomyces avermitilis, particularly preferably Streptomyces avermitilis SUKA strain (Komatsu, M. et al.Genome-minimized Streptomyces host for the heterologous expression of secondary metabolism. Proc. Natl. Acad. Sci. 107,2646-2651(2010); Komatsu, M. et al.Engineered Streptomyces avermitilis host for heterologous expression of biosynthetic gene cluster for secondary metabolites. ACS Synth. Biol. 2,384-396(2013)) is used. Streptomyces avermitilis SUKA strains include SUKA17, SUKA22, SUKA24, SUKA25, SUKA34, and SUKA54, and any of these may be used. For example, SUKA17 is registered at the RIKEN BioResource Center under the deposit number "JCM18251". The actinomycetes exemplified above may be used alone or in combination of two or more kinds.

[0047] (B) constructing an expression vector into which a gene encoding each enzyme has been introduced Next, using an actinomycete consisting of one or a combination of two or more types selected from the above actinomycetes as a host, genes encoding each exogenous enzyme are introduced to heterologously produce cannabinoid compounds biosynthesized in cannabis.

[0048] The method of introducing nucleic acids such as genes encoding each enzyme into the host actinomycete can be appropriately selected depending on the host used, and as an example, a vector capable of being introduced into the host and expressing the incorporated nucleic acid is used. In the present disclosure, it is necessary to introduce genes encoding each enzyme, hexanoyl-CoA synthase, polyketide synthase, cyclase, and geranyl transferase, and these genes may be incorporated into one vector or into two or more different vectors. In addition, when multiple genes are incorporated into one vector, each gene may be expressed by a common promoter or each may be expressed by a separate promoter. In addition, vectors that can be used in the present disclosure are not only vectors already known, but also vectors in which regions related to transcription control, replication region, etc. of promoters, etc. are modified according to the purpose. Furthermore, the introduction method using a vector is one example of gene introduction, and other methods such as mutation and genome shuffling may be used.

[0049] In the present disclosure, as described above, an expression vector is incorporated into an actinomycete host. As expression vectors suitable for such actinomycete hosts, among the expression vectors described above, for example, circular plasmids such as pIJ101 and pSG5, linear plasmids such as SAP1 and SCP1, genome-integrated plasmids such as SLP1, pKU590, pKU591, pKU592, pKU593, pKU594 and pKU595, and derivatives thereof are used. Note that, although specific expression vectors are described below, these are merely examples and are not limited to the expression vectors.

[0050] [Construction of expression vector for hexanoyl-CoA synthase (e.g., KSE_65520)] As shown in Figure 1, in the synthesis of cannabinoid compounds, it is extremely important to efficiently produce hexanoyl-CoA, which is the starting substrate for the production of olivetolic acid. Therefore, from among the many actinomycetes exemplified above, secondary metabolites having a partial structure derived from hexanoic acid used for the production of hexanoyl-CoA are searched for, and enzymes involved in the production of the secondary metabolites are identified, thereby screening for a synthetic enzyme that can be suitably used in the present disclosure. In the present disclosure, as explained in Figure 1, KSE_65520 (SEQ ID NO: 1) is particularly suitably used among various synthetic enzymes.

[0051] The expression vector for the synthetic enzyme is first generated by amplifying the gene encoding the synthetic enzyme by PCR using the genomic DNA of the actinomycete from which the enzyme was screened as a template. The resulting fragment is inserted into the restriction enzyme site XbaI / HindIII of the pKU565 vector to generate a pKU565 vector into which the DNA fragment of KSE_65520 has been inserted. The pKU565 vector is then further digested with NheI and NsiI to obtain a fragment that is inserted into the restriction enzyme site NheI / NsiI of the pKU594 vector to generate a pKU594 vector into which the DNA fragment of KSE_65520 has been inserted.

[0052] [Construction of expression vectors for polyketide synthases and cyclases] As shown in FIG. 1, in the synthesis of cannabinoid compounds, it is extremely important to efficiently produce 3,5,7-trioxododecanoyl-CoA, which is an intermediate in the production of olivetolic acid. In the present disclosure, CsTKS (SEQ ID NO: 2) is particularly preferably used as a polyketide synthase that catalyzes a condensation reaction in the production of this 3,5,7-trioxododecanoyl-CoA. Also, as shown in FIG. 1, in the synthesis of cannabinoid compounds, high production of olivetolic acid is extremely important in the production of the final target cannabinoid compound. In the present disclosure, CsOAC (SEQ ID NO: 3) is particularly preferably used as a cyclase that catalyzes the aromatization of 3,5,7-trioxododecanoyl-CoA.

[0053] The construction of expression vectors for these polyketide synthases and cyclases begins, for example, with the synthesis of genes encoding amino acid sequences in which both enzymes are linked via a linker sequence, and the generation of a DNA fragment to be inserted into the expression vector. The generated fragment is inserted into the restriction enzyme site XbaI / HindIII of the pKU565 vector, generating a pKU565 vector into which the above DNA fragment has been inserted. The pKU565 vector is further digested with NheI and NsiI, and the resulting fragment is inserted into the restriction enzyme site NheI / NsiI of the pKU592 vector, generating a pKU592 vector into which DNA fragments of a polyketide synthase (e.g., CsTKS) and a cyclase (e.g., CsOAC) have been inserted.

[0054] In the above, a DNA fragment encoding amino acids in which a polyketide synthase and a cyclase are linked via a linker sequence is used. However, it is of course possible to synthesize DNA fragments encoding each enzyme separately and introduce them into a single expression vector, or each may be introduced into a different expression vector.

[0055] [Construction of expression vector for geranyltransferase] As shown in Figure 1, in the synthesis of cannabinoid compounds, it is extremely important to efficiently produce cannabigerolic acid, which is one of the cannabinoid compounds and an intermediate of cannabidiol. In the present disclosure, in the production of cannabigerolic acid, a two-amino acid substitution product of NphB (SEQ ID NO: 4) or a two-amino acid substitution product of Spru_NphB (SEQ ID NO: 5), which was discovered as an enzyme related to NphB, is particularly preferably used as a geranyl transferase.

[0056] Construction of an expression vector for geranyltransferase is, for example, initiated by synthesizing a gene encoding the amino acid sequence of the enzyme and generating a DNA fragment to be inserted into the expression vector. The generated fragment is inserted into the restriction enzyme sites XbaI / HindIII of the pKU565 vector to generate the pKU565 vector into which the DNA fragment has been inserted.

[0057] Although not specifically described above, the enzymes introduced into the expression vector are not limited to the enzymes exemplified above. Naturally, when the host actinomycete does not have the enzymes that catalyze the production of cannabinoid compounds, it is possible to introduce exogenous enzymes into the expression vector in the same manner as above.

[0058] The expression vector produced as described above can be directly introduced into the host actinomycete, but may be further subjected to a treatment such as demethylation.

[0059] (C) Introducing the expression vector into a host actinomycete. The expression vector produced as described above can be introduced into the host actinomycete using a known introduction method. Examples of such introduction methods include chemical methods, biological methods, fusion methods, and physical methods, and preferably biological methods or fusion methods, and more preferably conjugative transfer methods or polyethylene glycol (PEG) methods. By using such introduction methods, it is possible to introduce the expression vector into the host actinomycete.

[0060] (D) Cultivating the actinomycete into which the expression vector has been introduced in a specific medium. As described above, it is possible to produce cannabinoid compounds by culturing actinomycetes into which an expression vector has been introduced in a predetermined medium. For the culture, any medium, such as a liquid medium, a semi-liquid medium, or a solid medium, can be used as desired. In the present disclosure, among these media, a liquid medium is preferably used, and more preferably a GY medium or a GKdy medium is used. In addition to glucose as an energy source, the medium may appropriately contain a nitrogen source, an extract added as a growth promoter, a carbon source added for the purpose of supporting growth, a buffer for adjusting the pH to a range suitable for the growth of actinomycetes, water, nucleic acid, inorganic salt, etc.

[0061] When the actinomycete having the expression vector introduced into the medium is subcultured, it is cultured with shaking at a predetermined culture temperature for a predetermined culture period. The culture temperature is preferably 5°C to 45°C, more preferably 15°C to 40°C, and even more preferably 25°C to 35°C, and the culture period is preferably 1 hour to 60 days, more preferably 1 day to 30 days, and even more preferably 2 days to 10 days, and can be appropriately adjusted in consideration of production efficiency, etc. As described above, one example of the culture conditions is shaking culture, but it is not limited thereto and can be appropriately selected from culture conditions such as standing, aeration, and stirring depending on the strain.

[0062] In the above, actinomycetes are directly added to a liquid medium and cultured, but it is also possible to preculture them in advance. As in the above, any medium such as a liquid medium, a semi-liquid medium, or a solid medium can be used as the medium for such preculture, as desired. In the present disclosure, among these media, a liquid medium is preferably used, and a GSY medium is more preferably used. In addition to glucose as an energy source, the medium may appropriately contain a nitrogen source, an extract added as a growth promoter, a carbon source added for the purpose of supporting growth, a buffer for adjusting the pH to a range suitable for the growth of actinomycetes, water, nucleic acid, inorganic salt, etc.

[0063] When the actinomycete into which the expression vector has been introduced is inoculated into the above-mentioned medium, it is subjected to shaking culture as pre-culture at a predetermined culture temperature for a predetermined culture period. The culture temperature is preferably 5°C to 45°C, more preferably 15°C to 40°C, and even more preferably 25°C to 35°C, and the culture period is preferably 1 hour to 20 days, more preferably 12 hours to 10 days, and even more preferably 1 day to 5 days, and can be appropriately adjusted in consideration of production efficiency, etc. As described above, one example of the culture conditions is shaking culture, but it is not limited thereto and can be appropriately selected from culture conditions such as standing, aeration, and stirring depending on the strain. The main culture is performed by subculturing the culture solution obtained by the pre-culture in the above-mentioned liquid medium.

[0064] When the actinomycete into which the expression vector has been introduced is cultured as described above, the culture solution is separated into a medium supernatant fraction and a cell fraction by centrifugation, and ethyl acetate is added to the medium supernatant fraction or a 2:1 mixture of chloroform and methanol is added to the cell fraction for partition extraction. This makes it possible to extract the target cannabinoid compound. The cannabinoid compound can be recovered by separation and concentration using not only the above method but also known solid-phase extraction methods or other liquid-liquid extraction methods.

[0065] The cannabinoid compounds thus produced are finally extracted and used as active ingredients or additives in various applications such as medicines, luxury items, food and beverages, cosmetics, etc. Although the cannabinoid compounds are finally used in the extracted state in these applications, it is of course possible to use them as the culture solution itself or as a composition containing cannabinoid compounds.

[0066] As described above, according to the actinomycete or the production method using the actinomycete disclosed herein, it is possible to produce cannabinoid compounds more efficiently. EXAMPLES

[0067] The actinomycetes of the present disclosure or the production method using said actinomycetes will be explained in more detail below with reference to examples, but the invention according to the present disclosure is not limited thereto.

[0068] Example 1: Production of olivetolic acid in actinomycete hosts As described above, high production of olivetolic acid is an important factor in increasing the production efficiency of cannabinoid compounds. Here, we confirmed that olivetolic acid can be effectively produced by introducing genes encoding polyketide synthase and cyclase involved in the production of olivetolic acid into actinomycetes.

[0069] The polyketide synthase used was CsTKS having the amino acid sequence shown in SEQ ID NO:2, and the cyclization enzyme used was CsOAC shown in SEQ ID NO:3. The CsTKS-CsOAC amino acid sequence, in which the CsTKS and CsOAC enzymes are linked to each other via a linker sequence, was that described in Luo, X. et al. Complete biosynthesis of cannabinoids and their unnatural analogues in yeast. Nature 567, 123-126 (2019). Codon optimization was performed to introduce the gene encoding CsTKS-CsOAC into actinomycetes, and a gene (SEQ ID NO:6) was prepared with an XbaI recognition sequence at the 5' end and a HindIII recognition sequence at the 3' end.

[0070] [SEQ ID NO:6]

[0071] The gene encoding the CsTKS-CsOAC was obtained as pUC57-Kan::CsTKS-CsOAC_GC by a contract synthesis by a contract organization. pUC57-Kan::CsTKS-CsOAC_GC was digested with the restriction enzymes XbaI and HindIII, and inserted into the XbaI-HindIII site of the expression vector pKU565tsr_2-cistron (reference 5: Kim, JHet al. Distribution and functional analysis of the phosphopantetheinyl transferase superfamily in Actinomycetales microorganisms, Proc. Natl Acad. Sci.USA115, 6828-6833(2018)) to construct pKU565tsr_2-cistron::CsTKS-CsOAC_GC. pKU565tsr_2-cistron::CsTKS-CsOAC_GC is a vector in which the promoter of the SAV_2794 gene is located upstream of the XbaI recognition sequence (Amagai, K. et al. Identification of a gene cluster for telomestatin biosynthesis and heterologous expression using a specific promoter in a clean host. Sci. Rep. 7, 3382 (2017)). Next, pKU565tsr_2-cistron::CsTKS-CsOAC_GC was digested with NheI and NsiI and inserted into the NheI-NsiI site of pKU592AT_aphII (Reference 4) to construct pKU592AT_aphII::CsTKS-CsOAC_GC. Figure 2 shows the structure of the constructed expression vector, pKU592AT_aphII::CsTKS-CsOAC_GC. Thus, the expression vector is a genome integration vector having the TG1 integrase gene and the attP sequence of TG1 integrase.To obtain a vector that was not methylated, Escherichia coli GM2929 was transformed with pKU592AT_aphII::CsTKS-CsOAC_GC, and the vector was extracted from the culture medium of the transformant to obtain demethylated pKU592AT_aphII::CsTKS-CsOAC_GC.

[0072] Next, the actinomycete host was transformed by the protoplast-polyethylene glycol (PEG) method using the demethylated pKU592AT_aphII::CsTKS-CsOAC_GC. The actinomycete host was Streptomyces avermitilis SUKA25 strain. The SUKA25 strain is a strain in which the erythromycin resistance gene of Streptomyces avermitilis SUKA24 strain (Okubo, S. et al. Identification of functional cytochrome P450 and ferredoxin from Streptomyces sp. EAS-AB2608 by transcriptional analysis and their heterologous expression. Appl. Microbiol.Biotechnol.105,4177-4187 (2021)) was replaced with a hygromycin resistance gene. The SUKA25 strain was then transformed to construct SUKA25 / CsTKS-CsOAC. The transformants were confirmed to have the CsTKS-CsOAC gene by PCR using a forward primer for checking CsTKS-CsOAC (SEQ ID NO: 7) and a reverse primer for checking CsTKS-CsOAC (SEQ ID NO: 8).

[0073] [SEQ ID NO: 7] ATCTCCAACAACATCGAGAAGTG [SEQ ID NO:8] GTTCACGTACGTCTTGAAGAACTC

[0074] Next, SUKA25 / CsTKS-CsOAC was inoculated into 15 mL of GSY medium dispensed into a test tube and cultured at 27°C for 2 days with shaking (reciprocating) at 320 rpm. The components of the GSY medium used are as shown in Table 1. [Table 1]

[0075] The culture solution obtained by the above culture was subcultured in 450 μL (3% (v / v)) into 15 mL of GY medium dispensed into a 125 mL Erlenmeyer flask, and cultured at 27° C. for 7 days with shaking (rotary) at 180 rpm. The components of the GSY medium used are as shown in Table 2. [Table 2]

[0076] The obtained culture solution was separated into a medium supernatant fraction and a bacterial cell fraction by centrifugation, and an equal amount of ethyl acetate was added to the medium supernatant fraction for partition extraction. The ethyl acetate layer was dried to solidify, and the extract obtained was dissolved in methanol to prepare an analytical sample. The analytical sample was analyzed using a high-performance liquid chromatography / mass spectrometry device (ACQUITY UPLC / Synapt G2: manufactured by Waters) equipped with a column (ACQUITY UPLC BEH C18 Column (1.7 μm, 2.1 mm × 100 mm: manufactured by Waters), and the production of olivetolic acid was confirmed by comparing the retention time, ultraviolet absorption spectrum, and mass chromatogram with those of a purchased olivetolic acid standard (manufactured by A1 BioChem Labs, North Carolina, USA).

[0077] FIG. 3 shows a comparison of the mass chromatogram of a standard sample of olivetolic acid and the mass chromatogram of an analytical sample produced by an actinomycete into which a gene encoding CsTKS-CsOAC has been introduced. According to FIG. 3, it was confirmed that olivetolic acid can be produced similarly by an actinomycete into which a gene encoding CsTKS-CsOAC has been introduced. In other words, it was shown that the actinomycete is useful in the production of cannabinoid compounds using olivetolic acid as an intermediate.

[0078] <Example 2: Production of olivetolic acid using medium-chain fatty acid synthase> As described above, high production of olivetolic acid is an important factor in increasing the production efficiency of cannabinoid compounds. Here, we confirmed that olivetolic acid can be effectively produced by an actinomycete into which a gene encoding the enzyme KSE_65520 (SEQ ID NO: 1) was introduced as a synthetic enzyme used to synthesize hexanoyl-CoA, which is the starting substrate in the production of olivetolic acid.

[0079] First, the gene encoding KSE_65520 was amplified by PCR using the genomic DNA of Kitasatospora setae as a template and the forward primer of SEQ ID NO:9 and the reverse primer of SEQ ID NO:10.

[0080] [SEQ ID NO: 9] CGtctagaATGACCGATCGCGTTCCCGGCACCGCGCCG [SEQ ID NO: 10] GCGaagcttTCATCGTCCCGCCTCCACCCGTCGCGCGG

[0081] The resulting amplified fragment was digested with XbaI and HindIII and inserted into the XbaI-HindIII site of pKU565tsr_2-cistron to construct pKU565tsr_2-cistron::KSE_65520. Next, pKU565tsr_2-cistron::KSE_65520 was digested with NheI and NsiI and inserted into the NheI-NsiI site of pKU594AT_aac(3)IV to construct pKU594AT_aac(3)IV::KSE_65520. Here, FIG. 4 shows the structure of the constructed expression vector, pKU594AT_aac(3)IV::KSE_65520. Thus, this expression vector is a genome integration vector in which the TG1 integrase gene and the attP sequence of TG1 integrase of pKU592AT_aphII are replaced with the ΦBT1 integrase gene and the attP sequence of ΦBT1 integrase, respectively, and the kanamycin resistance gene (aphII gene) is replaced with the apramycin resistance gene (aac(3)IV gene).

[0082] pKU594AT_aac(3)IV::KSE_65520 was transformed into E. coli harboring pUB307, and then conjugatively transferred to SUKA25 / CsTKS-CsOAC to construct SUKA25 / CsTKS-CsOAC / KSE_65520.

[0083] The constructed SUKA25 / CsTKS-CsOAC / KSE_65520 was cultured with shaking in the same manner as in Example 1, and an analytical sample was prepared by partition extraction from the culture solution using ethyl acetate or the like. Then, the production of olivetolic acid was confirmed in the same manner as in Example 1.

[0084] FIG. 5 is a diagram showing the ability of SUKA25 / CsTKS-CsOAC / KSE_65520 to produce olivetolic acid. FIG. 5 shows the results of calculating the production amount from the LC / MS extraction chromatogram. SUKA25 / CsTKS-CsOAC / KSE_65520 ("with KSE_CoL" in FIG. 5) produced 908 μg / L of olivetolic acid, whereas SUKA25 / CsTKS-CsOAC (actinomycete obtained in Example 1: ("without KSE_CoL" in FIG. 5) using an endogenous synthetic enzyme without the gene encoding KSE_65520 was produced 52 μg / L. / L. It was confirmed that SUKA25 / CsTKS-CsOAC / KSE_65520 has 17 times the olivetolic acid production ability as compared with SUKA25 / CsTKS-CsOAC (the actinomycete obtained in Example 1). In other words, it was shown that olivetolic acid can be produced extremely efficiently by using the actinomycete into which the gene encoding KSE_65520 was introduced in the production of cannabinoid compounds using olivetolic acid as an intermediate.

[0085] <Example 3: Production of cannabigerolic acid using medium-chain fatty acid synthase> In Example 2, it was confirmed that olivetolic acid can be produced extremely efficiently by an actinomycete into which a gene encoding KSE_65520, a medium-chain fatty acid synthase, has been introduced. Here, olivetolic acid is converted to cannabigerolic acid by catalyzing geranylation with geranyl transferase CsPT4 in cannabis. On the other hand, although NphB is known as a geranyl transferase in actinomycetes, an enzyme that can catalyze the geranylation of olivetolic acid more efficiently and increase the efficiency of cannabigerolic acid production is required. Therefore, in Example 3, the production of cannabigerolic acid was attempted using a two-amino acid substitute (SEQ ID NO: 4) of NphB described in Reference 2.

[0086] The gene (SEQ ID NO: 11) encoding NphB_A232S_Y288A, which is a two amino acid substitution variant of NphB (SEQ ID NO: 4), was obtained as pUC57-Kan::nphB_A232S_Y288A by contract synthesis at a contract organization.

[0087] [SEQ ID NO: 11] tctagaTACAAGGAGCGGTCAGATGTCCGAAGCCGCTGATGTCGAGCGCGTGTACGCGGCCATGGAGGAAGCGGCTGGACTGCTGGGTGTGGCCTGCGCACGCGACAAGATCTATCCGCTGCTGAGCACGTTCCAGGACACGCTCGTCGAGGGCGGCAGCGTCGTCGTCTTCTCCATGGCGAGCGGGCGTCATTCCACGGAACTGGACTTCAGCATCTCGGTGCCGACCAGCCACGGCGACCCGTACGCCACCGTCGTGGAAAAGGGGCTGTTCCCGGCGACCGGCCACCCCGTGGACGACCTGCTCGCGGACACCCAGAAGCACCTTCCGGTCTCCATGTTCGCCATCGACGGCGAGGTCACCGGCGGCTTCAAGAAGACGTACGCCTTCTTCCCCACCGACAACATGCCCGGCGTCGCCGAGCTGAGCGCCATCCCCTCCATGCCGCCGGCCGTCGCCGAGAACGCGGAGCTGTTCGCCCGCTACGGTCTGGACAAGGTCCAGATGACGTCGATGGACTACAAGAAGCGGCAGGTCAACCTCTACTTCAGCGAGCTGAGCGCGCAGACCCTGGAGGCGGAATCCGTCCTCGCCCTGGTGCGCGAGCTGGGCCTGCACGTGCCGAACGAGCTGGGCCTGAAGTTCTGCAAGCGCTCCTTCTCGGTCTACCCCACCCTCAACTGGGAGACCGGCAAGATCGACCGGCTGTGTTTCAGCGTCATCTCCAACGACCCCACCCTGGTGCCGTCCTCGGACGAGGGCGACATCGAGAAGTTCCACAACTACGCGACCAAGGCGCCGTACGCGTACGTCGGCGAGAAGCGCACCCTCGTCTATGGGCTCACGCTGTCGCCCAAGGAGGAGTACTACAAGCTGGGCGCGGCCTACCACATCACCGATGTCCAGCGCGGACTGCTGAAGGCGTTCGACTCGCTGGAGGACTGAaagctt

[0088] pKU1021gps (GenBank ID: AB982124 (Reference 6: Yuuki Yamada et al. Terpene synthases are widely distributed in bacteria, Proc Natl Acad Sci USA, 112(3), 857-62(2015))) was digested with NheI and XbaI and inserted into the NheI-XbaI site of pKU565tsr_2-cistron to construct pKU565tsr_2-cistron::gps. pKU1021gps is a plasmid that contains the geranyl diphosphate synthase gene downstream of the rpsJ promoter (Yamada, Y. et al. Terpene synthases are widely distributed in bacteria. PNAS 112(3), 857-862(2014)). Next, pUC57-Kan::nphB_A232S_Y288A was digested with XbaI and HindIII and inserted into the XbaI-HindIII site of pKU565tsr_2-cistron::gps to construct pKU565tsr_2-cistron::gps-nphB_A232S_Y288A. Figure 6 shows the structure of the constructed expression vector, pKU565tsr_2-cistron::gps-nphB_A232S_Y288A. To obtain a plasmid that is not methylated, pKU565tsr_2-cistron::gps-nphB_A232S_Y288A was transformed into Escherichia coli GM2929. The plasmid was extracted from the culture medium of the transformant to obtain demethylated pKU565tsr_2-cistron::gps-nphB_A232S_Y288A.

[0089] pKU565tsr_2-cistron::gps-nphB_A232S_Y288A was transformed into E. coli harboring pUB307, and then conjugatively transferred to SUKA25 / CsTKS-CsOAC and SUKA25 / CsTKS-CsOAC / KSE_65520 to construct SUKA25 / CsTKS-CsOAC / gps-nphB_A232S_Y288A and SUKA25 / CsTKS-CsOAC / KSE_65520 / gps-nphB_A232S_Y288A.

[0090] The constructed SUKA25 / CsTKS-CsOAC / gps-nphB_A232S_Y288A and SUKA25 / CsTKS-CsOAC / KSE_65520 / gps-nphB_A232S_Y288A were cultured with shaking in the same manner as in Example 1, and an analytical sample was prepared by partition extraction from the culture solution using ethyl acetate or the like. Then, the production of cannabigerolic acid was confirmed in the same manner as in Example 1. However, at this time, the GKdy medium shown in Table 3 was used instead of the GY medium. [Table 3]

[0091] 7 is a diagram showing the cannabigerolic acid production ability of SUKA25 / CsTKS-CsOAC / KSE_65520 / gps-nphB_A232S_Y288A. According to FIG. 7, the result of calculating the production amount from the LC / MS extraction chromatogram is shown, and it was confirmed that cannabigerolic acid can be produced in both SUKA25 / CsTKS-CsOAC / gps-nphB_A232S_Y288A (without KSE_CoL) and SUKA25 / CsTKS-CsOAC / KSE_65520 / gps-nphB_A232S_Y288A (with KSE_CoL), and it was confirmed that the two amino acid substitution of NphB (SEQ ID NO: 4) is useful as a geranyl group transferase. Furthermore, the amount of cannabigerolic acid produced by SUKA25 / CsTKS-CsOAC / KSE_65520 / gps-nphB_A232S_Y288A was 21-fold higher than the amount of cannabigerol produced by SUKA25 / CsTKS-CsOAC / gps-nphB_A232S_Y288A, demonstrating that cannabigerolic acid can be produced extremely efficiently by using an actinomycete into which the gene encoding KSE_65520 as a medium-chain fatty acid synthase has been introduced.

[0092] <Example 4: Enhancement of cannabigerolic acid production using Spru_NphB> In Example 3, it was confirmed that cannabigerolic acid can be efficiently produced in actinomycetes by using a two amino acid substitution of NphB (SEQ ID NO: 4), but searching for a geranyl transferase with better productivity is extremely important for high production of cannabigerolic acid. Here, NphB is an enzyme involved in the biosynthesis of naphterpin, and actinomycetes capable of producing analogues of the compound are highly likely to have an endogenous enzyme that exhibits geranyl transfer activity similar to NphB. Therefore, NphB homologs were searched for by performing genome analysis on actinomycetes capable of producing analogues, and enzymes exhibiting activity equal to or greater than that of NphB were identified. As a result, Spru_NphB was identified. Furthermore, an enzyme having an amino acid sequence in which the 230th alanine is replaced by serine and the 286th tyrosine is replaced by alanine with respect to Spru_NphB (SEQ ID NO: 5: Spru_NphB_A230S_Y286A) was identified as a particularly preferred enzyme.

[0093] First, the gene (SEQ ID NO: 12) encoding Spru_NphB_A230S_Y286A shown in SEQ ID NO: 5 was obtained as pUC57-Kan::Spru_NphB_A230S_Y286A by contract synthesis by a contract organization.

[0094] [SEQ ID NO: 12] ATGTCCGGAGCCGCTGATGTCGAGCGCGTGTACGCGGCCATGGAGGAGGCGGCTGGACTGCTGGGCGTGACCTGCGCACGCGAGAAGATCTACCCGCTGCTGACCGAGTTCCAGGACACCCTCACCGACGGTGTCGTCGTCTTCTCCATGGCGAGCGGACGTCGTTCCACGGAGCTGGACTTCAGCATCTCGGTGCCGACCAGCCAGGGCGACCCGTACGCCACCGTGGTGGACAAGGGGCTGTTCCCGGCGACCGGTCACCCCGTGGACGATCTGCTGGCCGACACCCAGAAGCATCTGCCGGTGTCGATGTTCGCCATCGACGGCGAGGTCACCGGTGGCTTCAAGAAGACGTACGCGTTCTTCCCGACCGACGACATGCCCGGGGTCGCGCAGCTGAGCGCCATCCCCTCGATGCCGGCGTCCGTGGCTCAGAACGCGGAGCTGTTCGCCCGCTACGGGCTGGACAAGGTCCAGATGACGTCGATGGACTACAAGAAGCGCCAGGTCAACCTGTACTTCAGCGAGCTGAGCCAGCAGACGCTGGCGCCGGAGTCGGTGCTGGCTCTGGTGCGTGAGCTGGGTCTGCACGTGCCGACCGAGCTGGGGCTGGAGTTCTGCAAGCGCTCGTTCTCGGTCTACCCGACGCTCAACTGGGACACCGGCAAGATCGACCGGCTGTGCTTCTCCGTGATCTCCACCGACCCGACGCTGGTGCCGTCCGAGGACGAGCGCGACATCGAGCAGTTCCGTGACTACGGCACGAAGGCGCCGTACGCGTATGTCGGCGAGAAGCGCACCCTCGTGTACGGGCTGACGCTGTCGCCCACGGAGGAGTACTACAAGCTCGGCGCGGCCTACCACATCACCGATATCCAGCGGCGGCTGCTGAAGGCGTTCGACGCGCTGGAGGACTGA

[0095] Then, the Spru_NphB_A230S_Y286A expression plasmid pKU565tsr_2-cistron::gps-Spru_nphB_A230S_Y286A was constructed in the same manner as in the construction of the plasmid NphB_A232S_Y288A shown in Example 3. Further, by introducing it into actinomycetes in the same manner as in Example 3, SUKA25 / CsTKS-CsOAC / KSE_65520 / gps-Spru_nphB_A230S_Y286A was constructed. Thereafter, SUKA25 / CsTKS-CsOAC / KSE_65520 / gps-nphB_A232S_Y288A and SUKA25 / CsTKS-CsOAC / KSE_65520 / gps-Spru_nphB_A230S_Y286A were cultured in the same manner as described in Example 3.

[0096] 8 is a diagram showing the production ability of cannabigerolic acid of SUKA25 / CsTKS-CsOAC / KSE_65520 / gps-Spru_nphB_A230S_Y286A. According to FIG. 8, the result of calculating the production amount from the extraction chromatogram of LC / MS is shown, and the production amount of cannabigerolic acid of SUKA25 / CsTKS-CsOAC / KSE_65520 / gps-Spru_nphB_A230S_Y286A is two-fold higher than the production amount of cannabigerolic acid of SUKA25 / CsTKS-CsOAC / KSE_65520 / gps-nphB_A232S_Y288A, and it was shown that cannabigerolic acid can be produced very efficiently by using an actinomycete into which a gene encoding Spru_NphB shown in SEQ ID NO: 5 as a geranyl group transferase has been introduced.

Claims

1. An actinomycete for producing cannabinoid compounds, into which a gene encoding a polyketide synthase and a gene encoding a cyclase have been introduced.

2. The actinomycete according to claim 1 , wherein the polyketide synthase and the cyclase are enzymes capable of catalyzing the synthesis of olivetolic acid, which is a precursor of the cannabinoid compound.

3. The actinomycete according to claim 1, wherein the polyketide synthase is either (A1) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, (A2) a protein having polyketide synthase activity and an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence set forth in SEQ ID NO: 2, or (A3) a protein having polyketide synthase activity and an amino acid sequence that shows 60% or more identity to the amino acid sequence set forth in SEQ ID NO:

2.

4. The actinomycete according to claim 1, wherein the cyclase is either (B1) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 3, (B2) a protein having cyclase activity and an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence set forth in SEQ ID NO: 3, or (A3) a protein having cyclase activity and an amino acid sequence that shows 60% or more identity to the amino acid sequence set forth in SEQ ID NO:

3.

5. An actinomycete for producing cannabinoid compounds, into which a gene encoding an enzyme having activity of synthesizing hexanoyl-CoA, which is utilized in the synthesis of olivetolic acid, a precursor of cannabinoid compounds, has been introduced.

6. The actinomycete according to claim 5, wherein the enzyme having an activity of synthesizing hexanoyl-CoA is any one of: (C1) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1; (C2) a protein having an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence set forth in SEQ ID NO: 1; and (C3) a protein having an amino acid sequence showing 60% or more identity to the amino acid sequence set forth in SEQ ID NO:

1.

7. An actinomycete for producing cannabinoid compounds, into which a gene encoding geranyltransferase has been introduced.

8. The actinomycete according to claim 7 , wherein the geranyltransferase is an enzyme capable of catalyzing the synthesis of cannabigerolic acid by geranyl transfer to olivetolic acid, which is a precursor of the cannabinoid compound.

9. The actinomycete according to claim 7, wherein the geranyltransferase is either (D1) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 5, (D2) a protein having an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence set forth in SEQ ID NO: 5, or (D3) a protein having an amino acid sequence that shows 60% or more identity to the amino acid sequence set forth in SEQ ID NO:

5.

10. The actinomycete according to claim 1 , wherein the cannabinoid compound is at least one of cannabidiol, cannabidiolic acid, and cannabigerolic acid.

11. A method for producing a cannabinoid compound, comprising culturing the actinomycete of claim 1.

12. A method for producing a cannabinoid compound, comprising culturing the actinomycete of claim 5.

13. A method for producing a cannabinoid compound, comprising culturing the actinomycete of claim 7.

14. Introducing a gene encoding an enzyme having an activity of synthesizing hexanoyl-CoA into an actinomycete; introducing into the actinomycete a gene encoding a polyketide synthase and a gene encoding a cyclase used to synthesize olivetolic acid from the hexanoyl-CoA; introducing into said actinomycete a gene encoding a geranyltransferase used to synthesize cannabigerolic acid from said olivetolic acid; culturing the actinomycete into which a gene encoding an enzyme having an activity of synthesizing hexanoyl-CoA, a gene encoding a polyketide synthase, a gene encoding a cyclase, and a gene encoding a geranyl transferase have been introduced; A method for producing a cannabinoid compound, comprising:

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