Transformed recombinant microorganism capable of producing cannabigerolic acid and method for producing cannabigerolic acid using same

Abstract

The present invention relates to a transformed recombinant microorganism capable of producing cannabigerolic acid and a method for producing cannabigerolic acid using same. More specifically, the present invention relates to a recombinant microorganism in which two types of modular vectors, such as an IUP / OLA modular vector and a CBGA / GPP modular vector, are used for optimal expression of key enzyme genes involved in cannabigerolic acid production, and which can thus sustainably produce cannabigerolic acid with excellent efficiency without the cultivation of Cannabis sativa L.

Description

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

[0001] The present invention relates to a transformed recombinant microorganism having the ability to produce cannabiserolic acid and a method for producing cannabiserolic acid using the same. More specifically, the invention relates to a recombinant microorganism capable of producing cannabiserolic acid sustainably and with excellent efficiency without hemp cultivation by using two types of modular vectors, such as the IUP / OLA modular vector and the CBGA / GPP modular vector, together for the optimal expression of a key enzyme gene involved in the production of cannabiserolic acid.

[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; it contains over 400 types of chemical compounds, most of which are cannabinoids, terpenes, and phenolic compounds. Among these, there are about 90 types of cannabinoids, which are important natural medicinal components, many of which 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 cannabigerol (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 cannabiserolic acid, a type of cannabinoid, without cannabis. As a result, they identified the isopentenol utilization pathway (IUP) module, the olivetolic acid (OLA) module, the geranylpyrophosphate (GPP) module, and the CBGA module, which are complex metabolic pathways involved in the synthesis of cannabiserolic acid. After selecting combinations of promoters and terminators for the optimal expression of key genes involved in the said modules, they manufactured two types of modular vectors containing these modules. They confirmed that using these, cannabiserolic acid can be stably produced within a single recombinant microorganism without the need for an external supply of OLA, thereby completing the present invention.

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

[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 set of modular vectors for the production of cannabigerolic acid (CBGA) comprises one or more transcription units including a promoter-target protein-terminator, wherein the first modular vector comprises a gene encoding OLS represented by the nucleotide sequence of SEQ ID NO. 1, a gene encoding OAC represented by the nucleotide sequence of SEQ ID NO. 2, a gene encoding LvaE represented by the nucleotide sequence of SEQ ID NO. 3, a gene encoding accBC represented by the nucleotide sequence of SEQ ID NO. 4, and a gene encoding accD1 represented by the nucleotide sequence of SEQ ID NO. 5, and the second modular vector comprises a gene encoding one or more target proteins selected from the group consisting of a gene encoding idi represented by the nucleotide sequence of SEQ ID NO. 8, a gene encoding idsA represented by the nucleotide sequence of SEQ ID NO. 9, and a gene encoding NphB represented by the nucleotide sequence of SEQ ID NO. 10. A set of modular vectors is provided.

[0008] According to another embodiment of the present invention, a recombinant microorganism for producing cannabiserolic acid, transformed with the set of modular vectors, is provided.

[0009] According to another embodiment of the present invention, a method for producing cannabiserolic acid is provided, comprising the step of culturing a recombinant microorganism transformed with the set of modular vectors.

[0010] The recombinant strain according to the present invention was transformed into two types of IUP / OLA module and CBGA / GPP module vectors to enable the production of cannabiserolic acid within the strain without the supply of external OLA, and was designed to optimize the expression of genes related to said modules. As such, cannabiserolic acid can be produced continuously and stably using recombinant microorganisms without direct cannabis cultivation, and cannabiserolic acid and various cannabinoids synthesized therefrom can be usefully utilized in the pharmaceutical field.

[0011] A brief description of each drawing is provided to help to better understand the drawings cited in the detailed description of the invention.

[0012] Figure 1 is a schematic diagram showing the cannabinoid acid production pathway in Corynebacterium glutamicum.

[0013] Figure 2 is a diagram showing the configuration of the OLA module.

[0014] Figure 3 is a diagram showing the configuration of the IUP module.

[0015] Figure 4 is a diagram showing the configuration of the GPP module.

[0016] Figure 5 is a diagram showing the CBGA module configuration.

[0017] Figure 6 is a schematic diagram showing the pTU and pSPU construction strategy for diversifying the expression of core genes.

[0018] Figure 7 is a schematic diagram showing the number of pTU and pSPU combinations in the OLA module.

[0019] Figure 8 is a figure showing a list of SPU plasmids related to the constructed OLA module.

[0020] Figure 9 is a schematic diagram showing the number of pTU and pSPU combinations in a CBGA / GPP module.

[0021] Figure 10 is a figure showing a list of SPU plasmids related to the constructed CBGA / GPP module.

[0022] Figure 11 shows the results of verifying the olibetolic acid biosynthesis efficiency using four types of SPU plasmids related to the constructed OLA module.

[0023] Figure 12 shows a list of SPU plasmids related to the constructed IUP / OLA module.

[0024] Figure 13 shows a list of cannabiserolic acid-producing strains using two types of SPU plasmids together.

[0025] Figure 14 shows the CBGA production capacity of the HKCBGA_28 strain.

[0026] 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.

[0027] 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.

[0028]

[0029] According to an embodiment of the present invention, a set of modular vectors comprising one or more transcription units including a promoter-target protein-gene-terminator, wherein the first modular vector comprises a gene encoding one or more target proteins selected from the group consisting of a gene encoding OLS represented by the nucleotide sequence of SEQ ID NO. 1, a gene encoding OAC represented by the nucleotide sequence of SEQ ID NO. 2, a gene encoding LvaE represented by the nucleotide sequence of SEQ ID NO. 3, a gene encoding accBC represented by the nucleotide sequence of SEQ ID NO. 4, and a gene encoding accD1 represented by the nucleotide sequence of SEQ ID NO. 5, and the second modular vector comprises a gene encoding one or more target proteins selected from the group consisting of a gene encoding idi represented by the nucleotide sequence of SEQ ID NO. 8, a gene encoding idsA represented by the nucleotide sequence of SEQ ID NO. 9, and a gene encoding NphB represented by the nucleotide sequence of SEQ ID NO. 10, for the production of cannabigerolic acid (CBGA). A set of modular vectors is provided.

[0030] 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.

[0031] 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.

[0032] In the present invention, 'promoter' collectively 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 above promoters can be classified into high (H), intermediate (I), and low (L) promoters according to the intensity of expression. For example, when the RFP / Abs600 value is set to 1 when the control lac I repressor and trc promoter are induced with 0h and 1mM IPTG, a high-expression promoter is classified when the RFP / Abs600 value is 10 or higher, an intermediate-expression promoter is classified when it is 5 or higher but less than 10, and a low-expression promoter is classified when it is less than 5. Although they can be grouped according to the intensity of the promoter as described above, they are not limited thereto and can be selected to control the expression level of a gene encoding a target protein. In addition, the promoter of the present invention may be a promoter based on sigma A or sigma B, but is not limited thereto, and may be selected to control 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 11 to 14 and 19 to 21 was used.

[0033] 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. 15 to 18 and 22 to 24 was used. The said terminator was previously designed by the inventors, and specifically, related details are disclosed in Korean Registered Patent Publication No. 10-2632294, which is incorporated into the present invention in its entirety.

[0034] 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.

[0035] In addition, the vector according to the present invention may include ColE1, the replication origin of Escherichia coli, an antibiotic resistance gene, and one or more, specifically two, Type IS restriction enzyme sites. If the vector includes the Type IS restriction enzyme sites, modular cloning via 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, and streptomycin resistance genes, the presence of transformation can be confirmed through this. The present invention comprises two types of modular vectors, each having a different replication origin and thus having the characteristic of enabling simultaneous expression within the recombinant microorganism.

[0036] 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.

[0037] 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.

[0038] In the present invention, 'goldengate assembly' is one of the molecular cloning methods that can assemble various DNA fragments using Type IS restriction enzymes and T4 ligae. Type IS restriction enzymes, like general restriction enzymes, recognize and cut specific sites. Examples of these types include BsaI, AarI, Eco31I, Esp3I, Bpil, MnII, BsmI, Alw26I, MboII, BseGI, etc., but are not limited to any known Type IS restriction enzymes.

[0039] In one embodiment of the present invention, the vector according to the present invention may include an OLA module, an IUP module, a GPP module, or a CBGA module for the production of cannabinoid acid, and may include two or more modules in a single vector. Specifically, the OLA module may include OLS, OAC, AAS, and AAC as key genes for producing olivetolic acid (OLA), which is a major precursor of CBGA biosynthesis and the most important intermediate in cannabinoid biosynthesis. The IUP module may include CK and IPK as key genes for producing GPP (geranyl pyrophosphate), a biosynthetic precursor of cannabinoid acid from isopronol. The GPP module may include idi and GPPS as key genes for producing GPP (geranyl pyrophosphate). The above CBGA module may include prenyltransferase to produce cannabinoid acid through GPP and OLA.

[0040] According to a specific embodiment of the present invention, two types of modular vectors were prepared. For the first modular vector, an optimal combination of transcription units was derived by selecting a promoter and a terminator for each gene to express a gene encoding an enzyme for producing OLA at optimal efficiency, and for the second modular vector, an optimal combination of transcription units was derived by selecting a promoter and a terminator for each gene to express a gene encoding an enzyme for producing GPP and CBGA at optimal efficiency. Additionally, the first modular vector may further include a gene involved in the IUP module to further increase the production efficiency of GPP. More specifically, OLS, a core gene of the OLA module, can be included in the vector in the form of an operon as a single gene together with OAC, another core gene, and can form a transcription unit with promoter Ib, represented by the nucleotide sequence of SEQ ID NO. 11, which is a promoter based on medium strength sigma B, and terminator 1 (T1), represented by the nucleotide sequence of SEQ ID NO. 15. More specifically, in the case of AAS, a core gene of the OLA module, LvaE can be used and can form a transcription unit with promoter H, represented by the nucleotide sequence of SEQ ID NO. 12, which is a promoter based on strong strength sigma A, and terminator 2 (T2), represented by the nucleotide sequence of SEQ ID NO. 16. More specifically, AAC, the core gene of the OLA module, can be included in the vector as a single gene with accBC and accD1, and can form a transcription unit together with promoter Ib, represented by the nucleotide sequence of SEQ ID NO. 13, which is a promoter based on intermediate strength sigma B, and terminator 3 (T3), represented by the nucleotide sequence of SEQ ID NO. 17.More specifically, in the case of idi derived from Corynebacterium glutamicum, a core gene of the GPP module, it can form a transcription unit with promoter Ia, represented by the nucleotide sequence of SEQ ID NO. 19, which is a promoter based on medium strength sigma A, and terminator 5 (T5), represented by the nucleotide sequence of SEQ ID NO. 22. More specifically, in the case of idsA derived from Corynebacterium glutamicum, a type of GPPS, another core gene of the GPP module, it can form a transcription unit with promoter Ib, represented by the nucleotide sequence of SEQ ID NO. 20, which is a promoter based on medium strength sigma B, and terminator 6 (T6), represented by the nucleotide sequence of SEQ ID NO. 23. More specifically, in the case of NphB-M23 (Y288A, G286S(M23), mixed with NphB), which is the core gene of the CBGA module derived from Streptomyces, it can form a transcription unit together with promoter H, represented by the nucleotide sequence of SEQ ID NO. 21, which is a promoter based on strong sigma A, and terminator 4 (T4), represented by the nucleotide sequence of SEQ ID NO. 24. A modular vector according to a specific embodiment of the present invention may include all three transcription units, including the genes of OLS-OAC, LvaE, and accBC-accD1 described above, as a first vector, and may include all three transcription units, including the genes of idi, idsA, and NphB-M23 described above, as a second vector. In addition, the first vector may further include another transcription unit comprising CK (Choline kinase) and IPK (Isopentenyl phosphate kinase) to increase the production of GPP. 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, CK and IPK can form a transcription unit together with promoter H, represented by the nucleotide sequence of SEQ ID NO. 14, which is a strong sigma A-based promoter, and rrnB-T1 terminator of E. coli origin represented by the nucleotide sequence of SEQ ID NO. 18.

[0041] The first modularization vector according to the present invention can be represented by the nucleotide sequence of SEQ ID NO. 25, and in one embodiment of the present invention, it was named pBbEBc-Ib-OLS-OAC-T1-H-PpLvaE-T2-Ib-accBC-accD1-T3-H-ScCK-IPK-rrnB-T1. The second modularization vector according to the present invention can be represented by the nucleotide sequence of SEQ ID NO. 26, and in one embodiment of the present invention, it was named pBbECk-Ia-idi-T5-Ib-idsA-T6-H-NphB-T4.

[0042] When two types of the modular vectors of the present invention are introduced together into microorganisms, cannabiserolic acid can be produced in high yield with only a simple process consisting of culture and extraction of said microorganisms, without complex manufacturing processes.

[0043]

[0044] According to another embodiment of the present invention, a recombinant microorganism for producing cannabiserolic acid, transformed with the set of modular vectors, is provided.

[0045] 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.

[0046] In the present invention, the microorganism may be Corynebacterium glutamicum.

[0047] The recombinant microorganism according to the present invention can be transformed at once by the two modular vectors described above, and accordingly, it can produce its own OLA from hexanoic acid within the microorganism without the supply of external OLA, and biosynthesis of CBGA is possible therefrom.

[0048] 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.

[0049]

[0050] According to another embodiment of the present invention, a method for producing cannabiserolic acid is provided, comprising the step of culturing a recombinant microorganism transformed with the set of modular vectors.

[0051] 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.

[0052] 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.

[0053] 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.

[0054]

[0055] Example 1. Design of a metabolic pathway for CBGA production

[0056] The production pathway of cannabigerolic acid (CBGA) within Corynebacterium glutamicum is shown in Fig. 1. Specifically, the inventors intended to construct a recombinant plasmid comprising an OLA module for producing OLA from hexanoic acid, an IUP or GPP module for producing GPP, which is a precursor of cannabigerolic acid, and a CBGA module for producing cannabigerolic acid through the GPP and OLA, in order to ultimately produce CBGA through self-production of OLA from hexanoic acid without the supply of external OLA within the recombinant microorganism.

[0057]

[0058] 1-1. Design of Key Genes Constituting the OLA Module

[0059] As shown in Figure 2, the core enzymes constituting the OLA module are AAC, MCS, AAE1, LvaE, OLS, OAC, etc. OLA is synthesized using hexanoyl-CoA and three molecules of malonyl-CoA; however, supplying a sufficient amount of malonyl-CoA for this purpose is not easy, and since the process of metabolizing hexanoic acid (hexanoate) to produce hexanoly-CoA is not an intrinsic metabolic process of the wild type of Corynebacterium glutamicum, it was determined that optimizing this module is important and will act as a rate-limiting step. Accordingly, the inventors designed the process by dividing it into two strategies: one that increases the pool of malonyl-CoA, and another that synthesizes olivetolic acid (OLA) from hexanoic acid via hexanoyl-CoA. Specific information is shown in Table 1.

[0060]

[0061] 1-2. Design of Core Genes Constituting the IUP Module

[0062] As shown in Figure 3, IPP and DMAPP are required to synthesize GPP, and the inventors intended to utilize the Isopentenol utilization pathway (IUP), which is known to allow for more economical use of coenzymes compared to the Methylertyritol 4-phosphate (MEP) pathway inherently possessed by Corynebacterium glutamicum. The inventors selected a source derived from Saccharomyces cerevisiae for the choline kinase (CK) and a source derived from Arabidopsis thaliana for the IPK, and specific information is shown in Table 2.

[0063]

[0064] 1-3. Design of Key Genes Constituting the GPP Module

[0065] As shown in Fig. 4, the core 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 3. idi was derived from Corynebacterium glutamicum.

[0066]

[0067] 1-4. Design of Core Genes Constituting the CBGA Module

[0068] As shown in Figure 5, 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 4.

[0069]

[0070] Example 2. Design of a Key Gene Expression Vector for CBGA Production

[0071] 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. 6.

[0072]

[0073] 2-1. OLA Module Vector Design

[0074] To determine the optimal expression conditions for each enzyme gene applied to the OLA module selected in Example 1, various combinations of pTU expression vectors with different expression levels of each enzyme gene were designed by corresponding synthetic promoters and terminators of varying strengths. Specific information is shown in Table 5. Here, H represents a strong promoter and I represents an intermediate promoter; for the I promoter, two types were selected based on the mechanism of action, namely Sigma A (Ia) and Sigma B (Ib), and Ib was used. For the H promoter, a Sigma A-based promoter was used.

[0075]

[0076] More specifically, four core genes were selected for the OLA module: OLS, OAC, AAS, and AAC. Among these, OLS and OAC were treated as a single gene and expressed in the form of an operon under the same promoter (two types). For AAS, AAE and LvaE derived from different species were used, along with two types of promoters, while for AAC, accBC-accD1 was used with two types of promoters. Therefore, a total of eight pTUs were designed in the OLA module: two for OLS-OAC, four for the two types of AAS, and two for AAC.

[0077] Next, we intended to construct pSPUs using the above pTUs, and designed a total of 16 (2x4x2) pSPUs by combining 2 pTUs for the expression of OLS-OAC, 4 pTUs for the expression of AAS, and 2 pTUs for the expression of AAC. A schematic diagram illustrating the above process is shown in Fig. 7.

[0078] During the process of manufacturing the aforementioned 16 pSPU recombinant vectors, it was confirmed that cloning did not occur in combination with specific promoters for each gene. Specifically, for the OLS-OAC and AAC genes (accBC-accD1), cases of non-cloning occurred depending on the subsequent gene when combined with the H promoter (a strong sigma A-dependent promoter); this was determined to be caused by the toxicity to the host strain depending on the characteristics of the expressed genes. Therefore, the actual pSPUs were constructed using pTUs corresponding to one promoter of Ib for OLS-OAC, two promoters of H and Ib for AAS (LvaE, AAE1), and one promoter of Ib for AAC, and the list of the four finally constructed SPU plasmids is shown in Fig. 8 (named PLASMID SAMPLE-23 to PLASMID SAMPLE-26).

[0079]

[0080] 2-2. CBGA / GPP Module Vector Design

[0081] To determine the optimal expression conditions for each enzyme gene applied to the CBGA / GPP module 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 6.

[0082]

[0083] 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.

[0084] 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. 9.

[0085] During the process of manufacturing the aforementioned 54 pSPU recombinant vectors, it was confirmed that cloning did not occur in combination with specific promoters for each gene; this was determined to be caused by the toxicity to the host strain depending on the characteristics of the expressed genes. Specifically, in the case of the GPPS gene, it was confirmed that cloning by Golden Gate Assembly did not occur 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. 10 (named PLASMID SAMPLE-27 to PLASMID SAMPLE-44).

[0086]

[0087] 2-3. IUP / OLA Module Vector Design

[0088] To compare the OLA production of recombinant strains transformed with the OLA module vector prepared in Example 2-1 above, the olivetolic acid production ability was confirmed after transforming each of the four vectors (PLASMID SAMPLE 23 to 26) described above into Corynebacterium glutamicum strains. Specifically, wild-type Corynebacterium glutamicum strains were transformed using PLASMID SAMPLE 23 to 26, respectively, and the transformed strains were named CG 01 to 04, respectively. The results are shown in Fig. 11.

[0089] As shown in Fig. 11, two pSPUs (PLASMID SAMPLE 24, PLASMID SAMPLE 25) were selected by confirming that CG-02 showed superior production among CG-01 and CG-02, and CG-03 showed superior production among CG-03 and CG-04.

[0090] Next, to further increase the production efficiency of GPP, an IUP / OLA module vector was constructed that allows the OLA module and the IUP module to be expressed together within a single plasmid by adding genes encoding CK and IPK, which belong to the IUP module in microorganisms, to the selected pSPU combination. CK, which belongs to the IUP module, is derived from Saccharomyces cerevisiae (S cCK), and IPK is derived from Arabidopsis thaliana; the two genes can form transcriptional units with promoter H, represented by the nucleotide sequence of SEQ ID NO. 14, which is a promoter based on strong sigma A, and rrnB T1 terminator derived from E. coli, represented by the nucleotide sequence of SEQ ID NO. 18. The list of the two finally constructed SPU plasmids (named PLASMID SAMPLE-45 to PLASMID SAMPLE-46) is shown in Fig. 12.

[0091]

[0092] Example 3. Construction of a recombinant strain for CBGA production

[0093] Thirty-six recombinant strains for CBGA production were prepared by transforming wild-type Corynebacterium glutamicum strains together with 18 SPU plasmids constructed during the preparation of the CBGA / GPP module vector in Example 2 and 2 SPU plasmids constructed during the preparation of the IUP / OLA module vector. Each plasmid can be simultaneously expressed within the strain using a different replication origin. Specifically, the vector for CBGA / GPP module expression has a variant pCG1 replication origin, while the vector for IUP / OLA module expression has a pBL1 replication origin. The two vectors differ in copy number; pCG1 belongs to the high copy number category, resulting in a large number of vectors within a single strain, whereas pBL1 corresponds to the middle copy number category, resulting in a relatively smaller number of vectors replicated per strain compared to pCG1. A specific list of recombinant strains is shown in Figure 13.

[0094]

[0095] Example 4. Evaluation of CBGA Production Capacity and Selection of Optimized Strains

[0096] CBGA production capacity was evaluated using 36 recombinant strains (corresponding to HKCBGA_22 to HKCBGA_48 and ID-22 to ID48) prepared in Example 3 above. More specifically, 2% glucose was added to CGXII, a minimal medium for culturing Corynebacterium glutamicum, and the antibiotics Cm and Km were added at 7.5 ulg / L and 25 ulg / L, respectively. The culture volume was set to 600 ul, and the OD600 was set to 0.6. The temperature conditions were set to 30°C and 1200 rpm. 0.82 ulM of Biotin, 1 mM of Hexanoate, and 50 mM of Isoprenol were added 8 hours after the start of culture, and 1% glucose was additionally added at 12 hours. It was cultured for a final 144 hours, and the biosynthesis of CBGA was evaluated without the supply of external OLA.

[0097] As a result, CBGA was not detected in most strains, and it was confirmed that the CBGA peak was detected only in the HKCBGA_28 strain transformed with two types of plasmids (PLASMID SAMPLE 34 (pBbECk-Ia-idi-T5-Ib-idsA-T6-H-NphB-T4) and PLASMID SAMPLE 45 (pBbEBc-Ib-OLS-OAC-T1-H-PpLvaE-T2-Ib-accBC-accD1-T3-H-ScCK-IPK-rrnB-T1)). The results are shown in Figure 14.

[0098] As shown in Figure 14, it was confirmed that only the HKCBGA_28 strain biosynthesized CBGA by exhibiting a peak at a retention time of 3.34 minutes, similar to the CBGA standard peak. It was determined that the reason CBGA was not detected in most strains was because the amount of OLA produced by Corynebacterium glutamicum itself was small.

[0099]

[0100] In summary, it was confirmed that cannabiserolic acid can be produced with excellent efficiency in a recombinant microbial culture medium by preparing IUP / OLA module and CBGA / GPP module vectors for the production of cannabiserolic acid within a recombinant strain without the supply of external OLA, and by selecting combinations of promoters and terminators that enable optimal expression of enzyme genes.

[0101]

[0102] 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 set of modular vectors comprising one or more transcription units containing a promoter-gene-terminator encoding a target protein, The first modularization vector comprises one or more genes encoding a target protein selected from the group consisting of a gene encoding OLS represented by the nucleotide sequence of SEQ ID NO. 1, a gene encoding OAC represented by the nucleotide sequence of SEQ ID NO. 2, a gene encoding LvaE represented by the nucleotide sequence of SEQ ID NO. 3, a gene encoding accBC represented by the nucleotide sequence of SEQ ID NO. 4, and a gene encoding accD1 represented by the nucleotide sequence of SEQ ID NO.

5. A set of modular vectors for the production of cannabigerolic acid (CBGA), wherein the second modular vector comprises a gene encoding one or more target proteins selected from the group consisting of a gene encoding idi represented by the nucleotide sequence of SEQ ID NO. 8, a gene encoding idsA represented by the nucleotide sequence of SEQ ID NO. 9, and a gene encoding NphB represented by the nucleotide sequence of SEQ ID NO.

10.

2. In Paragraph 1, A set of modular vectors for the production of cannabiserolic acid, wherein the first modular vector further comprises a gene encoding CK represented by the nucleotide sequence of SEQ ID NO. 6 or a gene encoding IPK represented by the nucleotide sequence of SEQ ID NO.

7.

3. In Paragraph 1, A set of modular vectors for the production of cannabiserolic acid, wherein the first modular vector comprises a promoter Ib represented by the nucleotide sequence of SEQ ID NO. 11, a gene encoding OLS represented by the nucleotide sequence of SEQ ID NO. 1, a gene encoding OAC represented by the nucleotide sequence of SEQ ID NO. 2, and a transcription unit comprising terminator 1 (T1) represented by the nucleotide sequence of SEQ ID NO.

15.

4. In Paragraph 1, A set of modular vectors for the production of cannabiserolic acid, wherein the first modular vector comprises a promoter H represented by the nucleotide sequence of SEQ ID NO. 12, a gene encoding LvaE represented by the nucleotide sequence of SEQ ID NO. 3, and a transcription unit comprising a terminator 2 (T2) represented by the nucleotide sequence of SEQ ID NO.

16.

5. In Paragraph 1, A set of modular vectors for the production of cannabiserolic acid, wherein the first modular vector comprises a transcription unit comprising a promoter Ib represented by the nucleotide sequence of SEQ ID NO. 13, a gene encoding accBC represented by the nucleotide sequence of SEQ ID NO. 4, a gene encoding accD1 represented by the nucleotide sequence of SEQ ID NO. 5, and a terminator 3 (T3) represented by the nucleotide sequence of SEQ ID NO.

17.

6. In Paragraph 1, A set of modular vectors for the production of cannabiserolic acid, wherein the second modular vector comprises a transcription unit comprising a promoter Ia represented by the nucleotide sequence of SEQ ID NO. 19, a gene encoding idi represented by the nucleotide sequence of SEQ ID NO. 8, and a terminator 5 (T5) represented by the nucleotide sequence of SEQ ID NO.

22.

7. In Paragraph 1, A set of modular vectors for the production of cannabiserolic acid, wherein the second modular vector comprises a promoter Ib represented by the nucleotide sequence of SEQ ID NO. 20, a gene encoding idsA represented by the nucleotide sequence of SEQ ID NO. 9, and a transcription unit comprising a terminator 6 (T6) represented by the nucleotide sequence of SEQ ID NO.

23.

8. In Paragraph 1, A set of modular vectors for the production of cannabiserolic acid, wherein the second modular vector comprises a promoter H represented by the nucleotide sequence of SEQ ID NO. 21, a gene encoding NphB represented by the nucleotide sequence of SEQ ID NO. 10, and a transcription unit comprising a terminator 4 (T4) represented by the nucleotide sequence of SEQ ID NO.

24.

9. In Paragraph 1, A set of modular vectors for the production of cannabiserolic acid, wherein the above modular vector comprises a pCG1 replication origin or ribosome binding sequence derived from Corynebacterium glutamicum.

10. In Paragraph 1, A set of modular vectors for the production of cannabiserolic acid, wherein the first modular vector is represented by the nucleotide sequence of SEQ ID NO.

25.

11. In Paragraph 1, A set of modular vectors for the production of cannabiserolic acid, wherein the second modular vector is represented by the nucleotide sequence of SEQ ID NO.

26.

12. A recombinant microorganism for producing cannabiserolic acid, transformed with a set of modular vectors according to any one of claims 1 to 11.

13. In Paragraph 12, The above microorganism is a recombinant microorganism for producing cannabiserolic acid, which is Corynebacterium glutamicum.

14. A method for producing cannabiserolic acid, comprising the step of culturing a recombinant microorganism transformed with a set of modular vectors according to any one of claims 1 to 11.

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