Transformed recombinant microorganism capable of producing cannabidiolic acid, and method for producing cannabidiolic acid by using same

Abstract

The present invention relates to a transformed recombinant microorganism capable of producing cannabidiolic acid, and a method for producing cannabidiolic acid by using same. More specifically, the present invention relates to a recombinant microorganism capable of synthesizing cannabidiolic acid from cannabigerolic acid without hemp by using a recombinant vector containing a codon-optimized gene that encodes a cannabidiolic acid synthase.

Description

Transformed recombinant microorganism having cannabidiolic acid production ability and method for producing cannabidiolic acid using the same

[0001] The present invention relates to a transformed recombinant microorganism having the ability to produce cannabidiolic acid and a method for producing cannabidiolic acid using the same, and more specifically, to a recombinant microorganism capable of synthesizing cannabidiolic acid from cannabigenolic acid without cannabis by using a recombinant vector containing a gene encoding a codon-optimized cannabidiolic acid synthase.

[0002] Hemp (Cannabis sativa L.) is an annual plant of the hemp family that has been widely cultivated in tropical and temperate regions, primarily in Central Asia, for over 12,000 years. In Korea, it has also been used as a raw material for hemp clothing since ancient times. Cannabis, a substance obtained from the leaves and flowers of this plant, contains over 400 chemical compounds, mostly cannabinoids, terpenes, and phenolic compounds. Of these, around 90 are medically important natural cannabinoids, many of which are unique to cannabis.

[0003] Among the cannabinoids found in cannabis, the one with the strongest psychoactive effect is delta-nine tetrahydrocannabinol (THC). Therefore, the higher the THC content in cannabis, the greater the potential harm to the human body. Even a few hundred micrograms (㎍) of THC can cause hallucinations. Furthermore, cannabidiol (CBD), a cannabinoid found in cannabis, is known to affect the brain by inhibiting the breakdown of brain chemicals that affect pain, mood, and mental function, although its exact mechanism of action remains unknown. CBD is non-narcotic and is used as a medication to treat conditions such as pain, intractable epilepsy, and multiple sclerosis.

[0004] Accordingly, the inventors of the present invention conducted research using recombinant microorganisms to produce cannabidiol without cannabis, and as a result, by codon-optimizing the CBDAS gene derived from Cannabis sativa, producing a recombinant vector containing the same, and using the same to produce a recombinant microorganism that produces a transformed cannabidiolic acid synthase, confirmed that cannabigenoic acid can be synthesized into cannabidiolic acid in a microbial culture, and completed the present invention.

[0005] [Prior Art Literature]

[0006] [Patent Document]

[0007] (Patent Document 1) KR 102277523 B1

[0008] The purpose of the present invention is to provide a recombinant vector for producing cannabidiolic acid synthase and a recombinant microorganism for producing cannabidiolic acid transformed with the recombinant vector.

[0009] The technical problems of the present invention are not limited to the technical problems mentioned above, and other technical problems not mentioned will be clearly understood by those skilled in the art from the description below.

[0010] According to an embodiment of the present invention, a recombinant vector for producing cannabidiolic acid synthase is provided, which comprises a gene encoding codon-optimized cannabidiolic acid synthase (CBDAS) represented by the base sequence of SEQ ID NO: 1.

[0011] Additionally, the recombinant vector may comprise an α-factor for extracellular secretion within the recombinant strain.

[0012] Additionally, the recombinant vector may be for expression in a Pichia pastoris strain.

[0013] According to another embodiment of the present invention, a recombinant microorganism for producing cannabidiolic acid (CBDA) transformed with the recombinant vector is provided.

[0014] According to another embodiment of the present invention, a method for producing cannabidiolic acid (CBDA) using the recombinant microorganism is provided.

[0015] The recombinant microorganism according to the present invention can continuously synthesize cannabigenoic acid into cannabidiolic acid without cannabis by secreting cannabidiolic acid synthase outside the cell, and thus can be usefully utilized in the pharmaceutical field where cannabidiolic acid and cannabidiol are used.

[0016] In order to more fully understand the drawings cited in the detailed description of the present invention, a brief description of each drawing is provided.

[0017] Figure 1 is a schematic diagram showing the synthesis process of cannabidiol (CBD).

[0018] Figure 2 is a vector map of a recombinant vector containing a codon-optimized CBDAS gene according to the present invention.

[0019] Figure 3 shows the SDS-PAGE results of the culture supernatant of the recombinant microorganism according to the present invention before optimization.

[0020] Figure 4 shows the SDS-PAGE results of the culture supernatant of the recombinant microorganism according to the present invention after optimization.

[0021] According to an embodiment of the present invention, a recombinant vector for producing cannabidiolic acid synthase is provided, which comprises a gene encoding codon-optimized cannabidiolic acid synthase (CBDAS) represented by the base sequence of SEQ ID NO: 1.

[0022] Additionally, the recombinant vector may comprise an α-factor for extracellular secretion within the recombinant strain.

[0023] Additionally, the recombinant vector may be for expression in a Pichia pastoris strain.

[0024] According to another embodiment of the present invention, a recombinant microorganism for producing cannabidiolic acid (CBDA) transformed with the recombinant vector is provided.

[0025] According to another embodiment of the present invention, a method for producing cannabidiolic acid (CBDA) using the recombinant microorganism is provided.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. In general, the nomenclature used herein is well known and commonly used in the art. In addition, when describing embodiments of the present invention, if a detailed description of a related known structure or function is judged to hinder the understanding of the embodiments of the present invention, a detailed description thereof will be omitted. In addition, although embodiments of the present invention will be described below, the technical idea 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] When a part in this specification is said to include a certain component, this does not exclude other components, unless otherwise specifically stated, but rather means that other components may be included. In this specification, the term "and / or" includes a combination of multiple related items or any one of multiple related items.

[0028] According to an embodiment of the present invention, a recombinant vector for producing cannabidiolic acid synthase is provided, which comprises a gene encoding cannabidiolic acid synthase (CBDAS) that is codon optimized and represented by the base sequence of SEQ ID NO: 1.

[0029] In the present invention, the term "gene" should be considered in the broadest sense and can encode a protein of interest. Furthermore, two or more genes may be operably linked. 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 to the specific amino acid sequence 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, a polypeptide having the above identity includes, for example, a polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, and / or added.

[0030] In the present invention, the term 'vector' means a genetic construct containing a base sequence of a gene operably linked to a suitable regulatory sequence so as to enable expression of a target gene in a suitable host, wherein the regulatory sequence may include a promoter capable of initiating transcription, an arbitrary operator sequence for regulating such transcription, and a sequence for regulating the termination of transcription and translation.

[0031] In the present invention, "operably linked" refers to a functional connection between a nucleic acid expression regulatory sequence and a nucleic acid sequence encoding a target protein, such that the nucleic acid sequence is 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 affect the expression of the coding sequence. The operably linked sequence with a recombinant vector can be produced using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation can be performed using enzymes generally known in the art.

[0032] The gene encoding cannabidiolic acid synthase (CBDAS) included in the recombinant vector of the present invention has sequence number 1, which is codon optimized for expression in a Pichia pastoris strain based on the base sequence of the CBDAS gene derived from Cannabis sativa.

[0033] In the present invention, 'codon optimization' means modifying the target nucleic acid sequence to reflect the codon preference of the selected host cell so that the target nucleic acid sequence is properly expressed and further expressed at a higher level in the host cell transformed with the target base sequence (nucleic acid sequence). The 'codon preference' refers to the nucleic acid sequence frequently used to code each amino acid depending on the organism. If the most frequently used codon among the codons of a given host microorganism is used, the possibility of translation generally increases, and thus the expression level of the desired sequence can also increase.

[0034] In the present invention, "expression of a target gene" may mean expressing the target gene to produce a protein encoded by the target gene. In the present invention, the method for expressing the target gene is a method of culturing a transformant host cell transformed with a vector containing the target gene to express the protein encoded by the target gene, thereby producing the final product of the biosynthetic pathway in which the protein is involved.

[0035] The vector of the present invention is not particularly limited as long as it is replicable in a cell, and any vector known in the art can be used, for example, a plasmid, a cosmid, a phage particle, a viral vector, and preferably a plasmid.

[0036] The recombinant vector according to the present invention may include an α-factor for extracellular secretion of the target substance (cannabidiolic acid synthase) within the recombinant strain, and more specifically, may be represented by a vector map according to FIG. 2.

[0037] When the recombinant vector of the present invention is introduced into a microorganism, cannabidiolic acid synthase can be produced at a high yield through a simple process consisting of culturing and extracting the microorganism without a complicated manufacturing process, thereby synthesizing cannabidiolic acid from cannabigenolic acid.

[0038] According to another embodiment of the present invention, a recombinant microorganism for producing cannabidiolic acid (CBDA) transformed with the recombinant vector is provided.

[0039] In the present invention, a 'recombinant microorganism' includes all microorganisms that have undergone artificial genetic modification, and may be a microorganism that has a specific mechanism weakened or strengthened due to a cause such as the insertion of an external gene to express an external protein or the strengthening or weakening of the activity of an endogenous gene, and may be a microorganism that includes genetic modification for the production of a desired protein or product.

[0040] In the present invention, the microorganism may be selected from the group consisting of Escherichia coli, bacteria, yeast and mold, and preferably may be a Pichia pastoris strain.

[0041] In the present invention, the method for introducing a recombinant vector into a cell can be a method known in the art, and is preferably a transformation method. Here, 'transformation' means introducing DNA into a host so that the DNA becomes replicable as an extrachromosomal element or by chromosomal integration. Transformation includes any method for introducing a nucleic acid molecule into an organism, cell, tissue, or organ, and can be performed by selecting an appropriate standard technique depending on 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.

[0042] When the recombinant microorganism according to the present invention is cultured under optimized conditions, cannabidiolic acid synthase is secreted into the culture medium, and cannabidiolic acid can be synthesized from cannabigenolic acid using this enzyme.

[0043] According to another embodiment of the present invention, a method for producing cannabidiolic acid (CBDA) is provided, comprising: (S1) a step of culturing a recombinant microorganism transformed with the recombinant vector; and (S2) a step of reacting the recombinant microorganism culture solution obtained in (S1) with cannabigenolic acid (CBGA).

[0044] In the present invention, the step of culturing the recombinant microorganism can be performed using a commonly known culturing method. Preferably, the recombinant microorganism of the present invention may be cultured in a conventional medium containing an appropriate carbon source, nitrogen source, amino acid, vitamin, etc., under aerobic conditions while controlling temperature, pH, 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 appropriate precursors. These media or precursors may be added to the culture in a batch or continuous manner.

[0045] In the present invention, the separation and recovery of the target substance from the culture solution can be accomplished using a known method suitable for the physical and chemical properties of the protein, for example, distillation, electrodialysis, pervaporation, chromatography, solvent extraction, reaction extraction, HPLC, etc., and a combination of these can be used, but is not limited thereto.

[0046] Specifically, in order to induce the expression of cannabidiolic acid synthase (CBDAS) during the cultivation of the recombinant microorganism in the above step (S1), methanol may be included at a concentration of 0.6 to 1.5%.

[0047] In addition, in order to optimize the expression of cannabidiolic acid synthase (CBDAS), the culture conditions of the recombinant microorganism in the step (S1) may be, but are not limited to, pH 4.5 to 5.5 and a temperature of 25 to 29 °C. In addition, in order to stably express cannabidiolic acid synthase (CBDAS), it is preferable that the culture in the step (S1) be performed for at least 48 hours, preferably 72 hours, and more preferably 96 hours.

[0048] Hereinafter, examples and experimental examples are presented to explain the present invention more specifically, but the present invention is not limited thereto.

[0049]

[0050] Example 1. Preparation of a recombinant vector for CBDAS production

[0051] As shown in Fig. 1, cannabigenoic acid (CBGA) reacts with cannabidiolic acid synthase (CBDAS) to produce cannabidiolic acid (CBDA). The inventors of the present invention utilized a Pichia pastoris strain, which has a wide optimal growth pH range of pH 3 to 7, as a recombinant strain, noting that cannabidiolic acid synthase (CBDAS) is active at pH 5. Furthermore, codon optimization of the CBDAS gene derived from Cannabis sativa was performed to produce CBDAS within the strain.

[0052] More specifically, in order to enable extracellular secretion of CBDAS using the a-factor, the codon-optimized CBDAS gene (SEQ ID NO: 1) derived from Cannabis sativa was inserted into an expression plasmid for P. pastoris containing the a-factor (pPICZα, Invitrogen, V19520) to construct a recombinant vector as shown in Fig. 2.

[0053]

[0054] Example 2. Production of recombinant strains through transformation

[0055] The recombinant vector prepared in Example 1 was transformed into Pichia pastoris to produce a recombinant strain in which CBDAS was integrated into the Pichia pastoris genome.

[0056] More specifically, 80 μl of the Pichia pastoris strain culture was mixed with 5–10 μg of the recombinant vector pPICZα DNA prepared in Example 1 (in 5–10 μl sterile water) and transferred to an ice-cold (0°C) 0.2 cm electroporation cuvette. The cuvette was incubated with the cells on ice for 5 min. The cells were pulsed under conditions of 1500 V, 25 μF, and 200 Ω, and immediately 1 ml of ice-cold 1 M sorbitol was added to the cuvette, and the contents of the cuvette were transferred to a sterile 15 ml tube. The tube was incubated at 30°C (220 rpm) for 1 h. Add 1 ml of YPD medium to each tube, spread 200 μl onto separately labeled YPDS plates containing the appropriate concentration of Zeocin™, and incubate the plates at 30°C for 2–3 days until colonies formed. Finally, 10 colonies were selected and purified on fresh YPD or YPDS plates containing the appropriate concentration of Zeocin™ to prepare recombinant strains.

[0057]

[0058] Example 3. Confirmation of CBDAS production in recombinant strains

[0059] The recombinant strain prepared in Example 2 was cultured using previously known culture methods and conditions (EasySelect™ Pichia Expression Kit, Invitrogen), and the presence of a CBDAS band in the culture supernatant was confirmed through SDS-PAGE. That is, a system was used in which the expression of the target gene is induced by methanol (used as an inducer) in Pichia pastoris using BMMY medium. The specific culture conditions are as shown in Table 1, and the results are shown in Fig. 3.

[0060]

[0061] As shown in Fig. 3, no CBDAS band was observed in any lane when previously known culture methods and conditions were used.

[0062] Therefore, to increase CBDAS expression, the culture conditions were optimized as shown in Table 2. Specifically, the same BMMY culture medium was used, but the pH was lowered from 6 to 5, and the culture temperature was lowered by 2°C to 28°C. In addition, to increase the amount of CBDAS secreted outside the cells, the overall amount of cultured strain was increased, the inoculum amount was increased from 1 to 4 based on OD600, and the culture period was extended by 24 hours, and the culture solution was harvested at 96 hours. Finally, to increase the expression amount, the amount of methanol, an inducer, was doubled from the previous amount to 1.0% of the culture solution, and the culture was performed. The results are shown in Fig. 4.

[0063]

[0064] As shown in Fig. 4, a CBDAS band at 74 kDa was confirmed in all lanes, confirming that CBDAS was normally excreted from cells in the recombinant strain according to the present invention.

[0065]

[0066] Example 4. Confirmation of CBDA production through reaction with recombinant strains.

[0067] CBGA was produced by adding 100 mM sodium citrate buffer (pH 5.0), 5 mM MgCl2, and 5 mM KCl to the recombinant Pichia pastoria supernatant obtained in Example 3, and then reacting. LC / MS analysis confirmed that CBDA was produced by confirming a peak at the CBDA retention time. Therefore, when using the recombinant strain of the present invention, CBDA can be produced without cannabis.

[0068] In summary, by codon-optimizing the CBDAS gene derived from Cannabis sativa, producing a recombinant vector containing the same, and using the same to produce a recombinant microorganism producing cannabidiolic acid synthase, it was confirmed that cannabigenoic acid can be synthesized into cannabidiolic acid in a microbial culture medium.

[0069] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A recombinant vector for producing cannabidiolic acid synthase, comprising a gene encoding cannabidiolic acid synthase (CBDAS), which is codon-optimized and represented by the base sequence of sequence number 1.

2. In paragraph 1, A recombinant vector comprising an α-factor for extracellular secretion within a recombinant strain.

3. In paragraph 1, The above recombinant vector is a recombinant vector for expression in a Pichia pastoris strain.

4. A recombinant microorganism for producing cannabidiolic acid (CBDA), transformed with a recombinant vector according to any one of claims 1 to 3. 5.(S1) A step of culturing a recombinant microorganism transformed with a recombinant vector of any one of claims 1 to 3; and (S2) A method for producing cannabidiolic acid (CBDA), comprising a step of reacting the recombinant microbial culture obtained in (S1) with cannabigenolic acid (CBGA).

6. In paragraph 5, A production method comprising culturing a recombinant microorganism in the above step (S1) by including methanol at a concentration of 0.6 to 1.5%.

7. In paragraph 5, A production method wherein the culture conditions of the recombinant microorganism in the above step (S1) are pH 4.5 to 5.5 and a temperature of 25 to 29 °C.

8. In paragraph 5, A production method, wherein the above production method additionally includes a step of recovering cannabidiolic acid from the recombinant microbial culture solution reacted in step (S2).

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