Transformed recombinant microorganism with ability to produce cannabigerolic acid and method for producing cannabigerolic acid using same

Abstract

The present invention relates to a transformed recombinant microorganism having the ability to produce cannabigerolic acid, and a method for producing cannabigerolic acid using same. More specifically, the present invention relates to a recombinant microorganism capable of sustainably producing cannabigerolic acid with high efficiency even without cultivation of Cannabis sativa L., by employing a modularization vector for optimal expression of a key enzyme gene involved in cannabigerolic acid production.

Description

Transformed recombinant microorganism having the ability to produce cannabizerol acid and a method for producing cannabizerol acid using the same

[0001] The present invention relates to a transformed recombinant microorganism having the ability to produce cannabizerol acid and a method for producing cannabizerol acid using the same. More specifically, the invention relates to a recombinant microorganism capable of producing cannabizerol acid sustainably and with excellent efficiency without hemp cultivation by using a modular vector for the optimal expression of a key enzyme gene involved in cannabizerol acid production.

[0002] Cannabis sativa L. is an annual plant belonging to the hemp family that has been widely cultivated in tropical and temperate regions, primarily in Central Asia, for 12,000 years. In Korea, it has also been used as a raw material for hemp clothing since ancient times. Cannabis is a substance obtained from the leaves and flowers of this plant, containing over 400 types of chemical substances. Most of these are cannabinoids, terpenes, and phenolic compounds, among which there are about 90 types of cannabinoids, which are important natural medicinal components, and many of these components are found only in cannabis.

[0003] Among the cannabinoids found in marijuana, delta-9 tetrahydrocannabinol (THC) is the substance with the strongest psychoactive effects. Therefore, marijuana containing higher levels of THC can be considered to cause greater harm to the human body, as just a few hundred micrograms (µg) of THC can induce hallucinogenic symptoms. Additionally, cannabigerol (CBG) is a non-psychoactive cannabinoid that can be applied in various ways in the medical field. CBG is known to interact with CB1 and CB2 receptors in the endocannabinoid system to increase dopamine levels, thereby regulating sleep, mood, and appetite; it also interferes with the absorption of GABA (GABA receptors) in the brain and blocks serotonin receptors. Furthermore, it is known to be effective for inflammatory bowel disease, neuronal degeneration, appetite stimulation, and bladder dysfunction. Next, cannabigerolic acid (CBGA) is known as a precursor to tetrahydrocannabinol acid (THCA), cannabidiol acid (CBDA), cannabicromenic acid (CBCA), and cannabigerorol (CBG), and is known to promote early apoptosis as a natural inhibitor. Additionally, cannabigerolic acid exhibits antibacterial, anti-inflammatory, and pain-relieving effects, and has been reported to work in conjunction with the body's cannabinoid system to have a positive effect on mood and appetite.

[0004] Accordingly, the inventors conducted research using recombinant microorganisms to produce cannabizerolic acid, a type of cannabinoid, without cannabis. As a result, they selected a combination of promoters and terminators for the optimal expression of key genes involved in the modules using a geranylpyrophosphate (GPP) module and a CBGA module, manufactured a modular vector containing the same, and confirmed that cannabizerolic acid can be stably produced within recombinant microorganisms using this, thereby completing the present invention.

[0005] The present invention aims to provide a modular vector for the production of cannabiserolic acid and a recombinant microorganism for the production of cannabiserolic acid transformed with said recombinant vector.

[0006] The technical problems of the present invention are not limited to those mentioned above, and other unmentioned technical problems will be clearly understood by those skilled in the art from the description below.

[0007] According to an embodiment of the present invention, a modular vector for producing cannabigerolic acid (CBGA) is provided, comprising one or more transcription units including a promoter-target protein-terminator, wherein the gene encoding the target protein is one or more selected from the group consisting of a gene encoding idi represented by the nucleotide sequence of SEQ ID NO. 1; a gene encoding ERG20-WW represented by the nucleotide sequence of SEQ ID NO. 2; and a gene encoding NphB represented by the nucleotide sequence of SEQ ID NO. 3.

[0008] According to an embodiment of the present invention, a modular vector for producing cannabigerolic acid (CBGA) is provided, comprising one or more transcription units including a promoter-target protein-terminator, wherein the gene encoding the target protein is one or more selected from the group consisting of a gene encoding idi represented by the nucleotide sequence of SEQ ID NO. 11; a gene encoding idsA represented by the nucleotide sequence of SEQ ID NO. 12; and a gene encoding NphB represented by the nucleotide sequence of SEQ ID NO. 13.

[0009] According to an embodiment of the present invention, a modular vector for producing cannabigerolic acid (CBGA) is provided, comprising one or more transcription units including a promoter-target protein-terminator, wherein the gene encoding the target protein is one or more selected from the group consisting of a gene encoding idi represented by the nucleotide sequence of SEQ ID NO. 21; a gene encoding idsA represented by the nucleotide sequence of SEQ ID NO. 22; and a gene encoding NphB represented by the nucleotide sequence of SEQ ID NO. 23.

[0010] According to another embodiment of the present invention, a recombinant microorganism for producing cannabiserolic acid, transformed with the vector, is provided.

[0011] According to another embodiment of the present invention, a method for producing cannabiserolic acid is provided, comprising the steps of: culturing a recombinant microorganism transformed with the vector; and reacting the recombinant microorganism culture obtained above with olivetolic acid (OLA).

[0012] The modular vector according to the present invention is designed to optimize the expression of three enzyme genes for the production of cannabizerolic acid, so that cannabizerolic acid can be produced continuously and stably using recombinant microorganisms without direct cannabis cultivation, and can be usefully utilized in the pharmaceutical field where cannabizerolic acid and various cannabinoids synthesized therefrom are used.

[0013] A brief description of each drawing is provided to help to better understand the drawings cited in the detailed description of the invention.

[0014] Figure 1 is a schematic diagram showing the cannabigelol acid production pathway in Corynebacterium glutamicum.

[0015] Figure 2 is a diagram showing the configuration of the GPP module.

[0016] Figure 3 is a diagram showing the CBGA module configuration.

[0017] Figure 4 is a schematic diagram showing the pTU and pSPU construction strategy for diversifying the expression of core genes.

[0018] Figure 5 is a schematic diagram showing the number of pTU and pSPU combinations.

[0019] Figure 6 is a figure showing a list of constructed SPU plasmids.

[0020] Figure 7 shows a list of cannabiserolic acid-producing strains using SPU plasmid.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a skilled expert in the art to which the present invention pertains. In general, the nomenclature used herein is well known and commonly used in the art. Furthermore, in describing embodiments of the present invention, detailed descriptions of related known components or functions are omitted if it is determined that such detailed descriptions would hinder understanding of the embodiments of the present invention. Additionally, while embodiments of the present invention will be described below, the technical concept of the present invention is not limited or restricted thereto and can be modified and implemented in various ways by those skilled in the art.

[0022] In this specification, when a part is described as including a certain component, it means that, unless specifically stated otherwise, it does not exclude other components but may include additional components. In this specification, the term "and / or" includes a combination of a plurality of related items or any one of a plurality of related items.

[0023]

[0024] According to an embodiment of the present invention, a modular vector for producing cannabigerolic acid (CBGA) is provided, comprising one or more transcription units including a promoter-target protein-terminator, wherein the gene encoding the target protein is one or more selected from the group consisting of a gene encoding idi represented by the nucleotide sequence of SEQ ID NO. 1; a gene encoding ERG20-WW represented by the nucleotide sequence of SEQ ID NO. 2; and a gene encoding NphB represented by the nucleotide sequence of SEQ ID NO. 3.

[0025] According to an embodiment of the present invention, a modular vector for the production of cannabigerolic acid (CBGA) is provided, comprising one or more transcription units including a promoter-target protein-terminator, wherein the gene encoding the target protein is one or more selected from the group consisting of a gene encoding idi represented by the nucleotide sequence of SEQ ID NO. 11; a gene encoding idsA represented by the nucleotide sequence of SEQ ID NO. 12; and a gene encoding NphB represented by the nucleotide sequence of SEQ ID NO. 13.

[0026] According to an embodiment of the present invention, a modular vector for the production of cannabigerolic acid (CBGA) is provided, comprising one or more transcription units including a promoter-target protein-terminator, wherein the gene encoding the target protein is one or more selected from the group consisting of a gene encoding idi represented by the nucleotide sequence of SEQ ID NO. 21; a gene encoding idsA represented by the nucleotide sequence of SEQ ID NO. 22; and a gene encoding NphB represented by the nucleotide sequence of SEQ ID NO. 23.

[0027] In the present invention, a ‘transcription unit (TU)’ means containing a gene encoding a target protein between a promoter and a terminator for the expression of a target protein. One or more of the transcription units may be included in the vector, and accordingly, the number of genes expressed in the vector may be controlled.

[0028] In the present invention, the term 'gene' should be considered in the broadest sense and refers to a molecule encoding a target protein. The gene of the present invention is not limited to a nucleic acid molecule encoding a specific amino acid sequence (polypeptide) described above, but is interpreted to include a nucleic acid molecule encoding a polypeptide having an amino acid sequence that exhibits substantial identity with respect to the specific amino acid sequence as described above. The substantial identity refers to an amino acid sequence that exhibits at least 60% homology, more preferably at least 80% homology, and most preferably at least 90% homology when the amino acid sequence encoded by the gene of the present invention is aligned with any other sequence to the greatest extent possible and the aligned sequence is analyzed using an algorithm commonly used in the art. Furthermore, the polypeptide having the identity includes, for example, a polypeptide of an amino acid sequence in which one or more amino acids are lost, substituted, inserted, and / or added.

[0029] In the present invention, 'promoter' refers to a nucleic acid that is functionally linked to a nucleic acid to be transcribed and regulates the transcription of said nucleic acid. A promoter module refers to a form in which IIS-type restriction enzyme sites are added to both ends of the promoter to enable Golden Gate assembly. Furthermore, promoter activity refers to the amount of RNA formed by the promoter within a specific time, i.e., the transcription rate. The promoter may be classified into high (H), intermediate (I), and low (L) promoters according to the expression intensity. For example, a high-expression promoter is a promoter with an RFP / Abs600 value of 4,000 or higher, an intermediate-expression promoter is a promoter between 1,000 and 4,000, and a low-expression promoter is a promoter with a value of 1,000 or lower, but is not limited thereto, and may be selected to regulate the expression level of a gene encoding a target protein. Additionally, the promoter of the present invention may be a sigma A or sigma B-based promoter, but is not limited thereto, and may be selected to regulate the expression level of a gene encoding a target protein. In one embodiment of the present invention, a promoter represented by a nucleotide sequence selected from the group consisting of SEQ ID NOs 4 to 6 was used. In another embodiment of the present invention, a promoter represented by a nucleotide sequence selected from the group consisting of SEQ ID NOs 14 to 16 was used. In another embodiment of the present invention, a promoter represented by a nucleotide sequence selected from the group consisting of SEQ ID NOs 24 to 26 was used.

[0030] In the present invention, "terminator" refers to a nucleic acid that is functionally connected to the end of a nucleic acid to be transcribed and capable of stopping the transcription of said nucleic acid. A terminator module refers to a form in which IIS-type restriction enzyme sites are added to both ends of said terminator to enable Golden Gate assembly. In one embodiment of the present invention, a terminator represented by a nucleotide sequence selected from the group consisting of SEQ ID NOs 7 to 9 was used. In another embodiment of the present invention, a terminator represented by a nucleotide sequence selected from the group consisting of SEQ ID NOs 17 to 19 was used. In another embodiment of the present invention, a terminator represented by a nucleotide sequence selected from the group consisting of SEQ ID NOs 27 to 29 was used. The above terminator was previously designed by the inventors, and specifically, the relevant details are disclosed in Korean Registered Patent Publication No. 10-2632294, which is incorporated into the present invention in its entirety.

[0031] In the present invention, 'vector' refers to a gene construct containing a nucleotide sequence of a gene operably linked to a suitable regulatory sequence so as to express a target gene within a suitable host. In the present invention, 'modular vector' refers to a vector that can be constructed to include two or more target genes to be artificially expressed, thereby enabling the simultaneous expression of two or more target proteins upon transformation. A vector according to the present invention may include a pCg1 replication origin or a ribosome binding sequence derived from Corynebacterium glutamicum. More specifically, a vector according to the present invention may include a mutated pCg1 replication origin derived from Corynebacterium glutamicum having a relatively high copy number, and said mutated pCg1 replication origin may include a mutated BsaI restriction enzyme recognition sequence that reduces the number of restriction enzymes without affecting the function of the replication origin itself. A cloning vector of Corynebacterium glutamicum containing a pCg1 replication origin derived from the above-mentioned mutated Corynebacterium glutamicum was previously designed by the inventors, specifically disclosed in Korean Patent Publication No. 10-2024-0104304, which is incorporated into the present invention in its entirety.

[0032] In addition, the vector according to the present invention may include ColE1, the replication initiation site of Escherichia coli, an antibiotic resistance gene, or one or more, specifically two, Type IS restriction enzyme sites. If the vector includes the Type IS restriction enzyme sites, modular cloning through Golden Gate Assembly is possible, and if it includes commonly used antibiotic resistance genes, such as ampicillin resistance genes, chloramphenicol resistance genes, penicillin resistance genes, streptomycin resistance genes, etc., the transformation status can be confirmed through this.

[0033] In the present invention, “ribosomal binding site” may refer to an RNA sequence found in mRNA to which a ribosome binds to initiate translation. A typical RBS sequence is located about 6 nucleotides upstream of the start codon in mRNA. Also referred to as a Shine-Dalgarno sequence, it refers to an A / G-rich polynucleotide sequence located 30 bases or less upstream of the translation start codon.

[0034] All genes within the vectors disclosed herein are operably linked to one another, and "operably linked" means that a nucleic acid expression regulatory sequence and a nucleic acid sequence encoding a target protein are functionally linked to perform a general function. For example, a promoter and a nucleic acid sequence encoding a protein or RNA may be operably linked to influence the expression of the coding sequence. This does not necessarily require a direct linkage in a chemical sense. Operatory linkage with the recombinant vector can be prepared using gene recombination techniques well known in the art, and site-specific DNA cleavage and linkage can be performed using enzymes, etc., generally known in the art.

[0035] In the present invention, 'goldengate assembly' is one of the molecular cloning methods capable of assembling various DNA fragments using Type IS restriction enzymes and T4 ligae. Type IS restriction enzymes, like general restriction enzymes, play a role in recognizing and cutting specific sites. Examples of such types include BsaI, AarI, Eco31I, Esp3I, Bpil, MnII, BsmI, Alw26I, MboII, BseGI, etc., but any known Type IS restriction enzyme is included in the present invention without being limited thereto.

[0036] In one embodiment of the present invention, the vector according to the present invention may include a GPP module and a CGBA module for the production of cannabizerolic acid, the GPP module may include idi and GPPS as key genes for producing GPP (geranyl pyrophosphate), which is a biosynthetic precursor of cannabizerolic acid, and the CGBA module may include prenyltransferase to produce cannabizerolic acid through GPP and OLA.

[0037] According to a specific embodiment of the present invention, in order to express genes encoding enzymes for producing GPP and CBGA in a modular vector with optimal efficiency, a promoter and a terminator were selected for each gene to derive an optimal combination of transcription units. More specifically, in the case of idi, a core gene of the GPP module, it may be derived from Corynebacterium glutamicum and formed a transcription unit together with promoter Ib, represented by the nucleotide sequence of SEQ ID NO. 4, which is a promoter based on medium strength sigma B, and terminator 5 (T5), represented by the nucleotide sequence of SEQ ID NO. 7. More specifically, in the case of ERG20_WW (N96W / 127W) derived from Saccharomyces cerevisiae, a type of GPPS, which is another core gene of the GPP module, it formed a transcription unit with promoter Ib, represented by the nucleotide sequence of SEQ ID NO. 5, which is a promoter based on intermediate strength sigma B, and terminator 6 (T6), represented by the nucleotide sequence of SEQ ID NO. 8. More specifically, in the case of NphB-M23 (Y288A, G286S(M23)), derived from Streptomyces, which is a core gene of the CBGA module, it formed a transcription unit with promoter Ib, represented by the nucleotide sequence of SEQ ID NO. 6, which is a promoter based on intermediate strength sigma B, and terminator 4 (T4), represented by the nucleotide sequence of SEQ ID NO. 9. A modular vector according to a specific embodiment of the present invention is characterized by including all three of the aforementioned transcription units.

[0038] In the modular vector according to the present invention, the modular vector containing all three transcription units described above can be represented by the nucleotide sequence of SEQ ID NO. 10, and in one embodiment of the present invention, it was named pBbECk-Ib-idi-T5-Ib-ERG20-WW-T6-Ib-NphB-T4.

[0039] According to another specific embodiment of the present invention, in order to express genes encoding enzymes for producing GPP and CBGA in a modular vector with optimal efficiency, a promoter and a terminator were selected for each gene to derive an optimal combination of transcription units. More specifically, in the case of idi, a core gene of the GPP module, it may be derived from Corynebacterium glutamicum and formed a transcription unit with promoter Ia, represented by the nucleotide sequence of SEQ ID NO. 14, which is a promoter based on medium strength sigma A, and terminator 5 (T5), represented by the nucleotide sequence of SEQ ID NO. 17. More specifically, in the case of idsA derived from Corynebacterium glutamicum, which is a type of GPPS, another core gene of the GPP module, it formed a transcription unit together with promoter Ib, represented by the nucleotide sequence of SEQ ID NO. 15, which is a promoter based on medium-strength sigma B, and terminator 6 (T6), represented by the nucleotide sequence of SEQ ID NO. 18. More specifically, in the case of NphB-M23 (Y288A, G286S(M23)), derived from Streptomyces, which is a core gene of the CBGA module, it formed a transcription unit together with promoter H, represented by the nucleotide sequence of SEQ ID NO. 16, which is a promoter based on strong-strength sigma A, and terminator 4 (T4), represented by the nucleotide sequence of SEQ ID NO. 19. The modularization vector according to a specific embodiment of the present invention is characterized by including all three transcription units described above.

[0040] In the modular vector according to the present invention, the modular vector containing all three transcription units described above can be represented by the nucleotide sequence of SEQ ID NO. 20, and in one embodiment of the present invention, it was named pBbECk-Ia-idi-T5-Ib-idsA-T6-H-NphB-T4.

[0041] According to another specific embodiment of the present invention, in order to express genes encoding enzymes for producing GPP and CBGA in a modular vector with optimal efficiency, a promoter and a terminator were selected for each gene to derive an optimal combination of transcription units. More specifically, in the case of idi, a core gene of the GPP module, it may be derived from Corynebacterium glutamicum and formed a transcription unit with promoter Ib, represented by the nucleotide sequence of SEQ ID NO. 24, which is a promoter based on medium strength sigma B, and terminator 5 (T5), represented by the nucleotide sequence of SEQ ID NO. 27. More specifically, in the case of idsA derived from Corynebacterium glutamicum, which is a type of GPPS, another core gene of the GPP module, it formed a transcription unit together with promoter Ib, represented by the nucleotide sequence of SEQ ID NO. 25, which is a promoter based on medium strength sigma B, and terminator 6 (T6), represented by the nucleotide sequence of SEQ ID NO. 28. More specifically, in the case of NphB-M23 (Y288A, G286S(M23)), derived from Streptomyces, which is a core gene of the CBGA module, it formed a transcription unit together with promoter H, represented by the nucleotide sequence of SEQ ID NO. 26, which is a promoter based on strong strength sigma A, and terminator 4 (T4), represented by the nucleotide sequence of SEQ ID NO. 29. The modularization vector according to a specific embodiment of the present invention is characterized by including all three transcription units described above.

[0042] In the modular vector according to the present invention, the modular vector containing all three transcription units described above can be represented by the nucleotide sequence of SEQ ID NO. 30, and in one embodiment of the present invention, it was named pBbECk-Ib-idi-T5-Ib-idsA-T6-H-NphB-T4.

[0043] When the modular vector of the present invention is introduced into a microorganism, cannabiserolic acid can be produced in high yield through a simple process consisting of cultivation and extraction of the microorganism without a complex manufacturing process.

[0044]

[0045] According to another embodiment of the present invention, a recombinant microorganism for producing cannabiserolic acid, transformed with the vector, is provided.

[0046] In the present invention, 'recombinant microorganism' includes all microorganisms that have undergone artificial genetic modification, and may be microorganisms in which a specific mechanism is weakened or strengthened due to causes such as the insertion of an external gene to express an external protein or the enhancement or weakening of the activity of an endogenous gene, and may be microorganisms that include genetic modification for the production of a desired protein or product.

[0047] In the present invention, the microorganism may be Corynebacterium glutamicum.

[0048] The recombinant microorganism according to the present invention may be transformed together with a vector capable of expressing genes constituting the IUP module, in addition to the modularization vector containing the GPP and CBGA modules described above. This is intended to increase the GPP pool, which is one of the important precursors of CBGA biosynthesis, and may utilize a plasmid containing CK (Choline kinase) and IPK (Isopentenyl phosphate kinase), which are key genes for producing GPP from Isoprenol. More specifically, the CK may be derived from Saccharomyces cerevisiae, and the IPK may be derived from Arabidopsis thaliana, but is not limited thereto. More specifically, the vector capable of expressing genes constituting the IUP module may be, for example, pBbEBc-I-ScCK-IPK, but is not limited thereto as long as it can induce the expression of the CK and IPK.

[0049] In the present invention, the method for introducing a recombinant vector into a cell may utilize methods known in the art, preferably a transformation method. Here, "transformation" means introducing DNA into a host so that the DNA becomes replicable as an extrachromosomal factor or through the completion of chromosomal integration. Transformation includes any method of introducing nucleic acid molecules into an organism, cell, tissue, or organ, and can be performed by selecting a standard technique suitable for the host cell as known in the art. Such methods may include, but are not limited to, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE (diethylaminoethyl)-dextran method, cationic liposome method, and lithium acetate-DMSO method.

[0050]

[0051] According to another embodiment of the present invention, a method for producing cannabizerolic acid is provided, comprising the step of culturing a recombinant microorganism transformed with the vector.

[0052] In the above production method, olivetolic acid may be provided during the microbial culture step, and the olivetolic acid may be provided directly or indirectly, for example, by directly supplying olivetolic acid into the culture medium or by providing it through a recombinant microorganism that produces olivetolic acid, but is not limited thereto.

[0053] In the present invention, the step of culturing the recombinant microorganism may be performed using a commonly known culture method. Preferably, the recombinant microorganism of the present invention may be cultured under aerobic conditions while controlling the temperature, pH, etc., in a conventional medium containing a suitable carbon source, nitrogen source, amino acids, vitamins, etc., but is not limited thereto. In a specific embodiment of the present invention, the medium may further include an inorganic compound, and may further include amino acids, vitamins, and suitable precursors, etc. These media or precursors may be added to the culture in a batch or continuous manner.

[0054] In the present invention, the separation and recovery of the target substance from the culture medium may be performed using known methods suitable for the physical and chemical properties of the protein, such as distillation, electrodialysis, pervaporation, chromatography, solvent extraction, reaction extraction, HPLC, etc., and may be performed in combination, but is not limited thereto.

[0055] In the following, examples and experimental examples are presented to further explain the present invention in more detail, but the present invention is not limited thereto.

[0056]

[0057] Example 1. Design of a metabolic pathway for CBGA production

[0058] The production pathway of cannabigerolic acid (CBGA) within Corynebacterium glutamicum is shown in Fig. 1. Specifically, for the production of CBGA within a recombinant microorganism, the inventors intended to construct a recombinant plasmid comprising a GPP module for producing GPP, a precursor of cannabigerolic acid, and a CBGA module for producing cannabigerolic acid through said GPP and OLA.

[0059]

[0060] 1-1. Design of Key Genes Constituting the GPP Module

[0061] As shown in Figure 2, the key enzymes constituting the GPP module are idi and GPPS (GPP Synthase). The inventors selected three enzymes derived from different species as candidates for GPPS, and specific information is shown in Table 1. idi was derived from Corynebacterium glutamicum.

[0062]

[0063] 1-2. Design of Core Genes Constituting the CBGA Module

[0064] As shown in Figure 3, a prenyl group transfer enzyme is required to produce CBGA through GPP and OLA. The inventors selected NphB derived from Streptomyces as the prenyl group transfer enzyme, and specific information is shown in Table 2.

[0065]

[0066] Example 2. Design of a Key Gene Expression Vector for CBGA Production

[0067] For the expression of the core gene for CBGA production selected in Example 1, a pTU vector previously designed by the inventors (Korean Patent Registration No. 10-2604399) was used. Specifically, TU refers to a Transcription Unit containing a promoter, a gene, and a terminator. In addition, a recombinant vector expressing multiple genes was prepared by constructing a pSPU containing multiple pTUs based on the above pTU. The above pSPU was also designed based on a vector previously designed by the inventors (Korean Patent Publication No. 10-2024-0104304), which is a high-replication vector for Corynebacterium glutamicum that incorporates a pCg1 replication origin with a high copy number and, in particular, includes a mutated BsaI restriction enzyme recognition sequence, thereby enabling efficient expression of the target gene. A schematic diagram for constructing the above pTU and pSPU vectors is shown in Fig. 4.

[0068] More specifically, to determine the optimal expression conditions for each enzyme gene selected in Example 1, various combinations of pTU expression vectors with different expression levels for each enzyme gene were designed by corresponding synthetic promoters and terminators of varying strengths. Specific information is shown in Table 3. Here, H represents a strong promoter and I represents an intermediate promoter; in the case of the I promoter, it was selected from two types, Sigma A (Ia) and Sigma B (Ib), depending on the mechanism of action. For the H promoter, a Sigma A-based promoter was used.

[0069]

[0070] Therefore, in the GPP module, a total of 12 pTUs were designed, consisting of 3 for idi and 9 for 3 types of GPPS, and in the CBGA module, a total of 14 pTUs were designed by designing 2 pTUs for NphB-M23.

[0071] Next, using the above pTUs, we intended to construct pSPUs including a combination of CBGA modules and GPP modules, and designed a total of 54 (3x9x2) pSPUs by combining 3 pTUs for the expression of idi, 9 pTUs for the expression of GPPS, and 2 pTUs for the expression of NphB-M23. A schematic diagram illustrating the above process is shown in Fig. 5.

[0072] During the process of manufacturing the aforementioned 54 pSPU recombinant vectors, it was confirmed that the expression efficiency tended to be low in combination with specific promoters for each gene. Specifically, for the GPPS gene, results showed low cloning efficiency by Golden Gate Assembly when the promoter was based on Sigma A; therefore, for the genes belonging to GPPS—ERG20_WW, iGPPS2, and idsA—only Ib, a promoter based on Sigma B, was used to design 18 (3x3x2) pSPUs. The list of the 18 finally constructed SPU plasmids is shown in Fig. 6 (named PLASMID SAMPLE 27 to PLASMID SAMPLE 44).

[0073]

[0074] Example 3. Construction of a recombinant strain for CBGA production

[0075] Recombinant strains for CBGA production were prepared by transforming wild-type Corynebacterium glutamicum strains with 18 SPU plasmids prepared in Example 2. At this time, in order to increase the GPP pool, which is one of the important precursors for CBGA biosynthesis, recombinant strains were prepared by expressing the recombinant plasmid pBbEBc-I-ScCK-IPK together with a total of 18 pSPUs composed of the aforementioned combination of GPP and CBGA modules, in order to co-express genes constituting the IUP module capable of producing GPP from Isoprenol. The list of finally constructed recombinant strains is shown in Figure 7. Meanwhile, in the case of recombinant strains transformed with plasmid PLASMID SAMPLE 39, no colonies were obtained, so a total of 17 recombinant strains (named HKCBGA_05 to HKCBGA_21) were constructed.

[0076]

[0077] Example 4. Evaluation of CBGA Production Capacity and Selection of Optimized Strains

[0078] The CBGA production capacity was evaluated using 17 recombinant strains prepared in Example 3 above (corresponding to HKCBGA_05 to HKCBGA_21 and ID-05 to ID21). More specifically, the culture was performed by adding 2% glucose to CGXII, a minimal medium for culturing Corynebacterium glutamicum, and the final culture volume was 600 µl using a 96-deep-well plate. The temperature conditions were set to 30°C and 1200 rpm for culture; 1 mM olivetolic acid (OLA) and 50 mM isoprenol were added 8 hours after the start of culture, and an additional 1% glucose was added at 12 hours. After culturing for a final 96 hours, the culture medium was collected, and the results of confirming the CBGA concentration are shown in Table 4. In addition, for the HKCBGA_12 strain, the culture medium was obtained after culturing for 72 hours using the same method, and the results of checking the CGBA concentration are shown in Table 5.

[0079]

[0080]

[0081] As shown in Table 4, the HKCBGA_10 strain produced cannabizerolic acid at a concentration of about 4.5 mg / L, which showed significantly superior production capacity compared to other recombinant strains. The HKCBGA_10 strain was transformed by the PLASMID SAMPLE 32 plasmid (pBbECk-Ib-idi-T5-Ib-ERG20-WW-T6-Ib-NphB-T4), which is characterized by containing a combination of three transcription units of Ib-idi-T5, Ib-ERG20-WW-T6, and Ib-NphB-T4 as a modular vector, and the entire sequence being represented by SEQ ID NO. 10. In addition, as shown in Table 4, the HKCBGA_13 strain produced cannabizerolic acid at a concentration of about 3.9 mg / L, which showed significantly superior production capacity compared to other recombinant strains. The HKCBGA_13 strain was transformed by the PLASMID SAMPLE 35 plasmid (pBbECk-Ib-idi-T5-Ib-idsA-T6-H-NphB-T4), which is characterized by containing a combination of three transcription units of Ib-idi-T5, Ib-idsA-T6, and H-NphB-T4 as a modular vector, and the entire sequence being represented by SEQ ID NO. 30.

[0082] In addition, as shown in Table 5, the HKCBGA_12 strain produced cannabizerolic acid at a concentration of about 4.4 mg / L, which showed significantly superior production capacity compared to other recombinant strains. The HKCBGA_12 strain was transformed by the PLASMID SAMPLE 34 plasmid (pBbECk-Ia-idi-T5-Ib-idsA-T6-H-NphB-T4), which is characterized by containing a combination of three transcription units of Ia-idi-T5, Ib-idsA-T6, and H-NphB-T4 as a modular vector, and the entire sequence being represented by SEQ ID NO. 20.

[0083] In summary, it was confirmed that cannabizerol acid can be produced with excellent efficiency in a microbial culture medium by selecting a combination of promoters and terminators that enable optimal expression of three genes for the production of cannabizerol acid, and by preparing a recombinant microorganism transformed with a vector containing this combination.

[0084]

[0085] Foregoing, specific parts of the content of the present invention have been described in detail. It will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the present invention. Accordingly, the actual scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A vector comprising one or more transcription units containing a promoter-gene-terminator encoding a target protein, A modular vector for the production of cannabigerolic acid (CBGA), wherein the gene encoding the above-mentioned target protein is one or more selected from the group consisting of the gene encoding idi represented by the nucleotide sequence of SEQ ID NO. 1; the gene encoding ERG20-WW represented by the nucleotide sequence of SEQ ID NO. 2; and the gene encoding NphB represented by the nucleotide sequence of SEQ ID NO.

3.

2. In Paragraph 1, A modular vector for the production of cannabizerolic acid, wherein the above transcription unit comprises a promoter Ib represented by the nucleotide sequence of SEQ ID NO. 4, a gene encoding idi represented by the nucleotide sequence of SEQ ID NO. 1, and a terminator 5 (T5) represented by the nucleotide sequence of SEQ ID NO.

7.

3. In Paragraph 1, A modular vector for the production of cannabizerolic acid, wherein the above transcription unit comprises a promoter Ib represented by the nucleotide sequence of SEQ ID NO. 5, a gene encoding ERG20-WW represented by the nucleotide sequence of SEQ ID NO. 2, and a terminator 6 (T6) represented by the nucleotide sequence of SEQ ID NO.

8.

4. In Paragraph 1, A modular vector for the production of cannabizerolic acid, wherein the above transcription unit comprises a promoter Ib represented by the nucleotide sequence of SEQ ID NO. 6, a gene encoding NphB represented by the nucleotide sequence of SEQ ID NO. 3, and a terminator 4 (T4) represented by the nucleotide sequence of SEQ ID NO.

9.

5. In Paragraph 1, The above vector is a modular vector for the production of cannabizelolic acid, comprising a pCg1 replication origin or ribosome binding sequence derived from Corynebacterium glutamicum.

6. In Paragraph 1, The above vector is a modular vector for the production of cannabizelolic acid, represented by the nucleotide sequence of SEQ ID NO.

10.

7. A vector comprising one or more transcription units containing a promoter-gene-terminator encoding a target protein, A modular vector for the production of cannabigerolic acid (CBGA), wherein the gene encoding the above-mentioned target protein is one or more selected from the group consisting of the gene encoding idi represented by the nucleotide sequence of SEQ ID NO. 11; the gene encoding idsA represented by the nucleotide sequence of SEQ ID NO. 12; and the gene encoding NphB represented by the nucleotide sequence of SEQ ID NO.

13.

8. In Paragraph 7, A modular vector for the production of cannabizerolic acid, wherein the above transcription unit comprises a promoter Ia represented by the nucleotide sequence of SEQ ID NO. 14, a gene encoding idi represented by the nucleotide sequence of SEQ ID NO. 11, and a terminator 5 (T5) represented by the nucleotide sequence of SEQ ID NO.

17.

9. In Paragraph 7, A modular vector for the production of cannabizerolic acid, wherein the above transcription unit comprises a promoter Ib represented by the nucleotide sequence of SEQ ID NO. 15, a gene encoding idsA represented by the nucleotide sequence of SEQ ID NO. 12, and a terminator 6 (T6) represented by the nucleotide sequence of SEQ ID NO.

18.

10. In Paragraph 7, A modular vector for the production of cannabizerolic acid, wherein the above transcription unit comprises a promoter H represented by the nucleotide sequence of SEQ ID NO. 16, a gene encoding NphB represented by the nucleotide sequence of SEQ ID NO. 13, and a terminator 4 (T4) represented by the nucleotide sequence of SEQ ID NO.

19.

11. In Paragraph 7, The above vector is a modular vector for cannabigellic acid production comprising a pCg1 replication origin or ribosome binding sequence derived from Corynebacterium glutamicum.

12. In Paragraph 7, The above vector is a modular vector for the production of cannabizelolic acid, represented by the nucleotide sequence of SEQ ID NO.

20.

13. A vector comprising one or more transcription units including a promoter-gene-terminator encoding a target protein, A modular vector for the production of cannabigerolic acid (CBGA), wherein the gene encoding the above-mentioned target protein is one or more selected from the group consisting of the gene encoding idi represented by the nucleotide sequence of SEQ ID NO. 21; the gene encoding idsA represented by the nucleotide sequence of SEQ ID NO. 22; and the gene encoding NphB represented by the nucleotide sequence of SEQ ID NO.

23.

14. In Paragraph 13, A modular vector for the production of cannabizerolic acid, wherein the above transcription unit comprises a promoter Ib represented by the nucleotide sequence of SEQ ID NO. 24, a gene encoding idi represented by the nucleotide sequence of SEQ ID NO. 21, and a terminator 5 (T5) represented by the nucleotide sequence of SEQ ID NO.

27.

15. In Paragraph 13, A modular vector for the production of cannabizerolic acid, wherein the above transcription unit comprises a promoter Ib represented by the nucleotide sequence of SEQ ID NO. 25, a gene encoding idsA represented by the nucleotide sequence of SEQ ID NO. 22, and a terminator 6 (T6) represented by the nucleotide sequence of SEQ ID NO.

28.

16. In Paragraph 13, A modular vector for the production of cannabizerolic acid, wherein the above transcription unit comprises a promoter H represented by the nucleotide sequence of SEQ ID NO. 26, a gene encoding NphB represented by the nucleotide sequence of SEQ ID NO. 23, and a terminator 4 (T4) represented by the nucleotide sequence of SEQ ID NO.

29.

17. In Paragraph 13, The above vector is a modular vector for the production of cannabizelolic acid, comprising a pCg1 replication origin or ribosome binding sequence derived from Corynebacterium glutamicum.

18. In Paragraph 13, The above vector is a modular vector for the production of cannabizelolic acid, represented by the nucleotide sequence of SEQ ID NO.

30.

19. A recombinant microorganism for producing cannabizelol acid, transformed with a vector of any one of claims 1 to 18.

20. In Paragraph 19, The above microorganism is a recombinant microorganism for producing cannabiserolic acid, which is Corynebacterium glutamicum.

21. A method for producing cannabizerolic acid, comprising the step of culturing a recombinant microorganism transformed with a vector of any one of claims 1 to 18.

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