Microorganism having ability to produce l-theanine, and method for producing l-theanine by using same

Abstract

The present invention relates to a microorganism having the ability to produce L-theanine, and a method for producing L-theanine by using same. Specifically, provided are: a microorganism including a recombinant vector into which a gene expressing foreign γ-glutamyl methylamide synthetase (GMAS) and dihydrofolate reductase (DHFR) is introduced; and a method for producing L-theanine with high efficiency by using same.

Description

Microorganism capable of producing L-theanine and method for producing L-theanine using the same

[0001] The present invention relates to a microorganism having the ability to produce L-theanine with the DHFR gene introduced, and a method for producing L-theanine using the same.

[0002] L-theanine is a natural amino acid found in tea plants that is a major component of umami flavor and can exhibit various physiological effects. It is known to have positive functions, such as relaxation, inhibition of excitement, improved concentration, and enhanced learning ability. Consequently, it is recognized for its value as a food and beverage additive, and commercial development is underway.

[0003] A common method for producing L-theanine is extraction by drying tea leaves. However, this method was not suitable for mass production because the yield of L-theanine is only about 1.0 to 1.5% of the dried tea leaves. Consequently, a synthetic method for L-theanine emerged, accounting for 70% of the global market. However, the aforementioned synthetic method has aspects that do not meet consumer needs, as D-type theanine (D-theanine) remains due to the synthetic reaction and it is not a natural method.

[0004] Meanwhile, there is an enzymatic method for producing L-theanine using a conversion enzyme. This method uses high-purity purified L-glutamine and ethylamine as raw materials and produces L-theanine through the reaction of a conversion enzyme, such as L-glutaminase, e.g., γ-glutamyl transpeptidase. However, the aforementioned enzymatic method has limitations in that the manufacturing cost is very high because it requires the excessive use of expensive enzymes and utilizes high-quality ethylamine and L-glutamine as raw materials. Furthermore, glutaminase exhibits optimal activity in a strongly alkaline environment, but a large amount of reaction byproducts derived from L-glutamine, which is unstable in such environments, are generated. Consequently, complex processes and facilities are required to recover and purify L-theanine from these byproducts, which limits the reduction of manufacturing costs.

[0005] In addition, technology utilizing microorganisms to produce L-theanine has recently been reported in Japan, China, and other countries. Specifically, a method has been disclosed in which a signal peptide of gamma-glutamyl transpeptidase (GGT) derived from Bacillus subtilis activates the secretory expression of GGT in a Corynebacterium glutamicum system and increases the conversion rate of L-theanine through an extracellular enzymatic conversion reaction using improved enzyme activity (CN 106085938 A).

[0006] In addition, a method for producing L-theanine with high efficiency using Escherichia coli and Corynebacterium, which introduce an enzyme derived from the genus Pseudomonas that synthesizes ethylamine using acetaldehyde and alanine as substrates, thereby eliminating the need to supply ethylamine from an external source, and enhancing the activity of gamma-glutamyl methylamide synthetase and glutaminase, has been disclosed (WO 2018 / 190398 A1).

[0007] A method for producing L-theanine without the addition of ethylamine from an external source has been disclosed (WO 2022 / 014196 A1) by introducing alanine decarboxylase derived from the genus Bacillus that converts alanine into ethylamine, gamma-glutamyl methylamide synthase and glutaminase derived from the genus Pseudomonas, and using E. coli or the like with enhanced activity thereof.

[0008] As such, the method of producing L-theanine using microorganisms is a natural and eco-friendly process that meets consumer needs and enables high-efficiency production, thus requiring technological development in this area.

[0009] In the production of recombinant microorganisms into which foreign genes have been introduced, antibiotic resistance genes are most commonly utilized as selection markers to identify only microorganisms containing vectors that include foreign genes. However, numerous studies have been published reporting that microorganisms containing such antibiotic resistance genes and products produced therefrom may pose potential risks to the health and safety of the environment, humans, and animals.

[0010] The present invention aims to provide a microorganism of the genus Corynebacterium that can effectively select a target recombinant microorganism even when an antibiotic resistance gene is not introduced as a selection marker, has a low dropout rate of the recombinant vector, and has further enhanced L-theanine production capacity, as well as a method for producing L-theanine using the same.

[0011] To solve the above-mentioned problem, the present invention provides a microorganism of the genus Corynebacterium capable of producing L-theanine, comprising a recombinant vector expressing gamma-glutamyl methylamide synthetase (γ-glutamyl methylamide synthetase, GMAS) and dihydrofolate reductase (DHFR).

[0012] In the above microorganism, the recombinant vector may not contain an antibiotic resistance gene.

[0013] In the above microorganism, the antibiotic resistance gene may be an ampicillin resistance gene, a kanamycin resistance gene, an erythromycin resistance gene, a chloramphenicol resistance gene, a spectinomycin resistance gene, a streptomycin resistance gene, a thiamphenicol resistance gene, a trimethoprim resistance gene, or a tetracycline resistance gene.

[0014] In the above microorganism, the gene encoding the gamma-glutamyl methylamide synthase may be composed of the nucleotide sequence of SEQ ID NO. 2.

[0015] In the above microorganism, the gene encoding the dehydrofolic acid reductase may be composed of the nucleotide sequence of SEQ ID NO. 3.

[0016] In the above microorganism, the recombinant vector may have a cat promoter introduced into the upstream region of the gene encoding dihydrofolic acid reductase.

[0017] In the above microorganism, the microorganism may be Corynebacterium glutamicum.

[0018] In the above microorganism, the microorganism may be Corynebacterium glutamicum BTC-THA-4D1 (KCCM13542P).

[0019] In addition, the present invention provides a method for producing L-theanine comprising the step of culturing the microorganism in a culture medium.

[0020] The above production method may further include a step of recovering L-theanine from the cultured microorganism or culture medium.

[0021] In the above production method, the medium may contain ethylamine.

[0022] In the above production method, the microorganism may be cultured in the presence of trimethoprim.

[0023] According to the microorganism of the present invention and the method for producing L-theanine using the same, L-theanine can be produced by a natural / eco-friendly method using a microorganism transformed to have the ability to produce L-theanine.

[0024] In addition, according to the microorganism and production method of the present invention, L-glutamic acid, which serves as a reaction raw material, is biosynthesized from glucose, and L-theanine is produced from the synthesized L-glutamic acid using a microorganism into which the γ-glutamyl methylamide synthetase (GMAS) gene has been introduced. Accordingly, since expensive enzymes and raw materials for L-glutamic acid production are not used or the amount of external supply of L-glutamic acid can be reduced, the manufacturing cost of L-theanine can be lowered.

[0025] In addition, the microorganism of the present invention can grow normally even when the antibiotic trimethoprim is present at a certain concentration, as dihydrofolate reductase (DHFR) is overexpressed. Accordingly, since the expression of GMAS can be stably maintained without the introduction of antibiotic resistance genes, L-theanine can be produced without the potential risk caused by antibiotic resistance genes.

[0026] In addition, according to the microorganism and production method of the present invention, since GMAS expressed in the recombinant microorganism is used for the production of L-theanine, L-theanine can be produced with high efficiency without process problems associated with the use of glutaminase, which requires strong basic conditions.

[0027] Figure 1 is a figure showing each step in the pathway for synthesizing tetrahydrofolate, an active form of folic acid, from starting materials, and the types of enzymes involved therein.

[0028] Figure 2 is a schematic diagram illustrating the structure of the GMAS gene and the DHFR gene within the GMAS-DHFR-pXMJ19-cat-HindIII vector.

[0029] Figure 3 is a schematic diagram showing the process of constructing the GMAS-DHFR-pXMJ19-β vector by removing the cat gene, an antibiotic resistance gene, from the GMAS-DHFR-pXMJ19-cat-HindIII vector, focusing on the structures of the GMAS gene and the DHFR gene.

[0030] Figure 4 is a schematic map of the GMAS-DHFR-pXMJ19-β vector introduced into the BTC-THA-4D1 strain.

[0031] Figure 5 is a graph comparing the growth of wild-type ATCC 14067 strain (WT) and GMAS-DHFR-pXMJ19-β vector-introduced strain (BTC-THA-4D1) according to trimethoprim concentration.

[0032] Figure 6 is a graph comparing the growth of BTC-THA-4 strain and BTC-THA-4D1 strain according to culture time.

[0033] The present invention will be described in more detail. Details not described in this specification are omitted because they can be sufficiently recognized and inferred by those skilled in the art or a similar field of the present invention. Each description and embodiment disclosed in this application may be applied to other descriptions and embodiments. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the scope of this application should not be considered limited by the specific descriptions provided below.

[0034] A person skilled in the art can recognize or identify a number of equivalents to the specific embodiments of the present application described herein by using only ordinary experiments. Furthermore, such equivalents are intended to be included in the present application.

[0035] In this application, the term "microorganism (or strain)" includes both wild-type microorganisms and microorganisms that have undergone natural or artificial genetic modification. These microorganisms are those in which specific mechanisms are weakened or strengthened due to causes such as the insertion of external genes or the weakening of the activity of endogenous genes, and may include genetic modification for the production of a desired polypeptide, protein, or product.

[0036] In this application, the term "weakening" is a concept that encompasses both reduced activity and lack of activity compared to intrinsic activity. The term "weakening" may be used interchangeably with terms such as inactivation, deficiency, down-regulation, decrease, reduce, and attenuation.

[0037] In addition, in this application, the term "enhancement" means an increase relative to intrinsic activity. The enhancement may be used interchangeably with terms such as activation, up-regulation, overexpression, and increase.

[0038] Furthermore, in this application, the term "intrinsic activity" refers to the activity of a specific polypeptide originally possessed by the parent strain or the non-modified microorganism prior to the change in traits caused by genetic variation due to natural or artificial factors. This term may be used interchangeably with "pre-modification activity."

[0039] In addition, the term "non-mutated microorganism" in this application does not exclude strains containing mutations that may naturally occur in microorganisms, and may refer to wild-type strains or natural-type strains themselves, or strains prior to changes in traits caused by genetic mutations due to natural or artificial factors.

[0040] In addition, in this application, the term "having the ability to produce L-theanine" refers to a microorganism that naturally possesses the ability to produce L-theanine, or a microorganism to which the ability to produce L-theanine has been conferred upon a parent strain that lacks the ability to produce L-theanine.

[0041] In addition, in this application, the term "transformation" means introducing a vector containing a gene (polynucleotide) encoding a target protein into a host cell so that the protein encoded by said gene can be expressed within the host cell.

[0042] Additionally, the gene comprises DNA and / or RNA encoding a target protein. The gene may be introduced in any form, provided that it can be introduced into a host cell and expressed. For example, the gene may be introduced into a host cell in the form of an expression cassette, which is a gene structure containing all elements necessary for self-expression. The expression cassette may typically include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the gene. The expression cassette may be in the form of a self-replicating expression vector, but is not limited thereto.

[0043] Furthermore, in this application, the term "introduction" refers to the manifestation of activity of a specific protein as a result of a gene that was not originally possessed by the microorganism being expressed within the microorganism, or the manifestation of increased or enhanced activity compared to the intrinsic activity or pre-modification activity of the said protein.

[0044] Additionally, in this application, the term "operably connected" means that a promoter sequence and a gene sequence are functionally connected to initiate and mediate the transcription of a gene encoding the target protein of the present invention.

[0045] Additionally, in this application, the terms "homology" or "identity" refer to the degree of similarity between two given amino acid sequences or base sequences and may be expressed as a percentage. The terms homology and identity may often be used interchangeably.

[0046] Whether any two polynucleotide or polypeptide sequences have homology, similarity, or identity can be determined using known computer algorithms. Examples include, but are not limited to, the "FASTA" program, the Needleman program of the EMBOSS package, and the Needleman-Wunsch algorithm.

[0047] Furthermore, in this application, the term "culture" refers to growing the microorganisms of the genus Corynebacterium of the present invention under appropriately controlled environmental conditions. The culture process of this application may be carried out according to suitable media and culture conditions known in the art. Such a culture process can be easily adjusted and used by those skilled in the art depending on the selected strain. Specifically, the culture may be batch, continuous, and / or fed-batch, but is not limited thereto.

[0048] Furthermore, in this application, the term "medium" refers to a substance mixed with nutrients as the main component required to culture the microorganisms of the genus Corynebacterium of the present invention, and supplies nutrients and growth factors, including water, which is indispensable for survival and growth. The medium and other culture conditions may be used without special limitations as long as they are media used for the culture of ordinary microorganisms; however, the microorganisms of the genus Corynebacterium 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, phosphorus, inorganic salts, amino acids, and / or vitamins.

[0049] The microorganism of the present invention is a microorganism of the genus Corynebacterium capable of producing L-theanine, and is characterized by comprising a recombinant vector capable of expressing GMAS and DHFR by introducing a gene encoding gamma-glutamyl methylamide synthetase (GMAS) (hereinafter also referred to as the 'gene of GMAS') and a gene encoding dihydrofolate reductase (DHFR) (hereinafter also referred to as the 'gene of DHFR').

[0050] The above gamma-glutamyl methylamide synthetase (GMAS) synthesizes L-theanine from L-glutamic acid and ethylamine.

[0051] In one embodiment of the present invention, the GMAS gene may be derived from Methylovorus mays, a salt-tolerant microorganism, preferably from a wild-type GMAS gene derived from Methylovorus mays No. 9 (Japanese DNA Database Bank (DDBJ); accession number: AB333782), and more preferably from a GMAS gene obtained by undergoing a step of codon optimization of the base sequence of a wild-type GMAS gene derived from Methylovorus mays No. 9 (hereinafter also referred to as the 'codon-optimized GMAS gene').

[0052] The above codon optimization step can be performed by a method that can be sufficiently recognized and inferred by a person skilled in the technical field of the present invention or a similar field. For example, it may be performed by introducing a wild-type GMAS gene derived from Methylroborus mace No. 9 into each of about 5 to 1,000 colonies of Corynebacterium glutamicum ATCC 14067 and transforming them, and then selecting colonies with high L-theanine formation activity and high L-theanine production, but is not limited thereto.

[0053] The above codon-optimized GMAS gene may be composed of the nucleotide sequence of SEQ ID NO. 2, or may be composed of a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% or more homology or identity with the nucleotide sequence of SEQ ID NO. 2. Additionally, if the nucleotide sequence codes for a protein exhibiting efficacy corresponding to the above codon-optimized GMAS, variants having a sequence in which some sequences are deleted, modified, substituted, conservatively substituted, or added may also be included in the gene of the above codon-optimized GMAS. For example, this includes cases of sequence additions that do not alter the function of the above codon-optimized GMAS, naturally occurring mutations, silent mutations thereof, or conservatively substituted.

[0054] Tetrahydrofolic acid is a cofactor for the synthesis of amino acids and nucleic acids in microorganisms and is synthesized from dihydrofolic acid by dihydrofolate reductase (DHFR). Trimethoprim, an antibiotic, is a competitive inhibitor of DHFR and inhibits the growth of microorganisms by interfering with the synthesis of tetrahydrofolic acid (Fig. 1).

[0055] The microorganism of the present invention, comprising a recombinant vector into which the gene for DHFR has been introduced, has DHFR overexpressed and can synthesize sufficient tetrahydrofolate necessary for normal growth even in the presence of a certain concentration of trimethoprim.

[0056] In one embodiment of the present invention, the recombinant vector may not contain an antibiotic resistance gene. As described above, since the microorganism of the present invention can grow even in the presence of a certain concentration of trimethoprim, it is possible to select a transformed strain into which the recombinant vector has been introduced without introducing an antibiotic resistance gene into the recombinant vector, and to stably maintain the expression of GMAS within the strain.

[0057] The above antibiotic resistance genes may be, but are not limited to, ampicillin resistance genes, kanamycin resistance genes, erythromycin resistance genes, chloramphenicol resistance genes, spectinomycin resistance genes, streptomycin resistance genes, thiamphenicol resistance genes, trimethoprim resistance genes, or tetracycline resistance genes.

[0058] In one embodiment of the present invention, the gene of the DHFR may be derived from a microorganism of the genus Corynebacterium, preferably from Corynebacterium glutamicum, and more preferably from Corynebacterium glutamicum ATCC 14067.

[0059] The gene of the DHFR above may be composed of the nucleotide sequence of SEQ ID NO. 3, or may be composed of a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% or more homology or identity with the nucleotide sequence of SEQ ID NO. 3. In addition, if the nucleotide sequence codes for a protein exhibiting efficacy corresponding to the DHFR above, a variant having a sequence in which some sequence is deleted, modified, substituted, conservatively substituted, or added may also be included in the gene of the DHFR above. For example, this includes cases of sequence additions that do not alter the function of the DHFR above, naturally occurring mutations, silent mutations thereof, or conservatively substituted.

[0060] In one embodiment of the present invention, the DHFR gene may have a cat promoter introduced in an upstream region. That is, a cat promoter may be operably linked to an upstream region of the nucleotide sequence encoding DHFR. The cat promoter may be derived from the pXMJ19 vector and, preferably, may be composed of the nucleotide sequence of SEQ ID NO. 4.

[0061] The gene of DHFR, in which a cat promoter is operably connected to the upstream region above, may consist of the nucleotide sequence of SEQ ID NO. 5, but is not limited thereto.

[0062] The above-mentioned microorganisms of the genus Corynebacterium refer to all microorganisms belonging to the genus Corynebacterium. For example, Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, It may be Corynebacterium imitans, Corynebacterium testudinoris, or Corynebacterium flavescens, preferably Corynebacterium glutamicum, and more preferably Corynebacterium glutamicum BTC-THA-4D1 (KCCM13542P).

[0063] Meanwhile, since Brevibacterium flavum is currently classified as Corynebacterium glutamicum, the Brevibacterium flavum strain is also included in the scope of the present invention.

[0064] The vector for preparing the above-mentioned recombinant vector is not particularly limited, and any vector known in the art may be used. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages in their natural or recombinant state. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A may be used as phage vectors or cosmid vectors, and pBR-based, pUC-based, pBluescriptII-based, pGEM-based, pTZ-based, pCL-based, and pET-based vectors may be used as plasmid vectors. Specifically, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC, pXMJ19 vectors may be used.

[0065] Any method for transduction of a vector into a host cell that can be sufficiently recognized and inferred by a person skilled in the technical field of the present invention or a similar field is possible. Examples include, but are not limited to, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and lithium acetate-DMSO method.

[0066] In addition, the present invention provides a composition for producing L-theanine comprising the microorganism of the present invention described above.

[0067] The above composition for L-theanine production comprises a microorganism capable of producing L-theanine and may further include, without limitation, additional components necessary for L-theanine production. The additional components necessary for L-theanine production may include, for example, any suitable excipient or component of a culture medium commonly used in compositions for fermentation. Examples of excipients include, but are not limited to, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, or isotonic agents.

[0068] In addition, the present invention provides a method for producing L-theanine comprising the step of culturing the microorganism of the present invention described above in a culture medium.

[0069] The microorganism of the present invention is a microorganism of the genus Corynebacterium that can biosynthesize L-glutamic acid from glucose, and a GMAS expressed from a recombinant vector synthesizes L-theanine from the L-glutamic acid and ethylamine added to the medium.

[0070] The cultivation of microorganisms may include a seed culture and a main culture, and the media used for the seed culture and the main culture may be the same or different. Any media and other culture conditions used for culturing the microorganisms of the present application may be used without special limitations as long as they are media used for culturing microorganisms of the genus Corynebacterium. Specifically, the microorganisms of the present application may be cultured in a conventional medium containing a suitable carbon source, nitrogen source, inorganic salts, trace growth factors, amino acids, antibiotics, and any vitamins, etc., under aerobic or anaerobic conditions while controlling the temperature, pH, etc.

[0071] The above carbon sources may include, but are not limited to, carbohydrates such as glucose, fructose, sucrose, maltose, etc.; alcohols such as sugar alcohols, glycerol, ethanol, etc.; fatty acids such as palmitic acid, stearic acid, linoleic acid; organic acids such as pyruvate, lactic acid, acetic acid, citric acid; and amino acids such as glutamic acid, methionine, lysine, etc. Additionally, natural organic nutrient sources such as starch hydrolysate, molasses, blackstrap molasses, rice bran, cassava, sugarcane residue, and corn steep liquid may be used. Furthermore, carbohydrates such as sterilized pre-treated molasses (i.e., molasses converted into reducing sugars) may be used, and other appropriate amounts of carbon sources may be used in various ways without limitation. These carbon sources may be used individually or in combination of two or more types.

[0072] The above nitrogen sources may include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, urea, etc.; and organic nitrogen sources such as amino acids such as glutamic acid, methionine, glutamine, etc., peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquid, casein hydrolysate, soy protein hydrolysate, fish or its decomposition products, defatted soybean cake or its decomposition products, etc. These nitrogen sources may be used alone or in combination of two or more, but are not limited thereto.

[0073] Potassium phosphate, potassium chloride, magnesium sulfate, iron sulfate, zinc sulfate, copper sulfate, manganese sulfate, etc., may be used as the above inorganic salts, and these may be used individually or in combination of two or more types.

[0074] The above-mentioned trace growth factors may include thiamine hydrochloride, biotin, pyridoxine, calcium pantothenate, etc., and these may be used individually or in combination of two or more. As antibiotics, trimethoprim, etc. may be used.

[0075] In addition, the above medium may contain vitamins and / or suitable precursors, etc. The above medium or precursors may be added to the culture in a batch or continuous manner, but are not limited thereto.

[0076] In the present application, the pH of the culture may be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc., to the culture in an appropriate manner during the culture of microorganisms. Additionally, during the culture, the formation of bubbles may be suppressed by using an antifoaming agent such as a fatty acid polyglycol ester. Furthermore, to maintain an aerobic state of the culture, oxygen or an oxygen-containing gas may be injected into the culture, or nitrogen, hydrogen, or carbon dioxide gas may be injected without gas injection to maintain an anaerobic and microaerobic state.

[0077] In one embodiment of the present invention, the temperature of the culture may be 25 to 40°C, more specifically 28 to 37°C, but is not limited thereto. The culture period may continue until a desired amount of useful substance is obtained, specifically 1 to 200 hours, but is not limited thereto.

[0078] Since an excess amount of biotin in the culture medium can inhibit microorganisms from producing L-glutamic acid, it may be preferable for the microorganisms to be cultured under conditions where biotin is absent or limited. For example, biotin may be included in the culture medium in a range of 0.01 to 1000 μg / L, preferably 0.1 to 100 μg / L, and more preferably 0.1 to 10 μg / L.

[0079] In addition, since the presence of surfactant components such as fatty acid esters in the culture medium can promote the production of L-glutamic acid by microorganisms, it may be desirable for the microorganisms to be cultured under conditions where fatty acid esters are present. For example, as a surfactant component, Tween ® 60 (CAS No. 9005-67-8) can be added to the culture medium.

[0080] In one embodiment of the present invention, the production method comprises measuring the OD during culture using a spectrophotometer (Mecasys Optizen 2120UV). 600 A feeding solution of glucose, ethylamine, and Tween 60 may be administered at a level of 30 to 35 by measuring the value. The feeding solution is not particularly limited as long as it is suitable for use by a person skilled in the art, and for example, a solution containing glucose, ethylamine, and fatty acid esters may be used.

[0081] The above production method allows for the termination of cultivation when the residual sugar concentration becomes 0% and the pH of the medium increases. At the time of cultivation termination and at periodic intervals during the cultivation process, the culture medium is aliquoted to check the residual sugar concentration and OD. 600 The values, and the concentrations of L-theanine, L-glutamic acid, and ethylamine can be confirmed through HPLC analysis.

[0082] The concentration analysis of L-theanine, L-glutamic acid, and ethylamine can be performed using a derivatization method utilizing o-phthalaldehyde (OPA). After derivatizing L-theanine, L-glutamic acid, and ethylamine, analysis can be performed using HPLC (Agilent 1260 Infinity 2), and a UV detector (DAD) wavelength of 338 nm can be used. Specifically, a solution containing 10 mM Na2HPO4 and 10 mM Na2B4O4 can be used as mobile phase A, and a solution containing acetic acid (ACN), methanol (MeOH), and distilled water in a ratio of 45:45:10 can be used as mobile phase B. Analysis can be performed for 30 minutes to 2 hours with a column temperature of 20°C and a flow rate of 1.0 mL / min, with a gradient applied over time, but is not limited thereto.

[0083] Residual sugar concentration analysis can be performed using a spectrophotometer (Mecasys Optizen 2120UV) after inducing an enzyme reaction using Megazyme's GOPOD assay kit.

[0084] In one embodiment of the present invention, the production method may further include a step of recovering L-theanine from the cultured microorganism or culture medium. Specifically, after the step of culturing the microorganism in the medium, the method may include a step of recovering L-theanine from one or more substances selected from the microorganism, the culture medium, the culture product, the supernatant of the culture product, the extract of the culture product, and the lysate of the microorganism.

[0085] Depending on the method of culturing microorganisms, such as batch, continuous, or fed-batch culture methods, the target substance L-theanine can be recovered from the culture medium using a suitable method known in the relevant art. Recovery may be achieved by methods such as precipitation, centrifugation, filtration, chromatography, and crystallization, and the recovery step may include, but is not limited to, a purification process.

[0086] In one embodiment of the present invention, the microorganism of the present invention may be cultured in the presence of trimethoprim. As explained above, the recombinant vector included in the microorganism of the present invention can express DHFR in excess even without containing antibiotic resistance genes, and thus can grow normally in the presence of trimethoprim at a certain concentration. The concentration of trimethoprim may be less than 30 μg / ml, preferably 10 to 20 μg / ml, more preferably 15 to 20 μg / ml, and most preferably 15 μg / ml.

[0087] The present invention will be explained in more detail below through examples. However, the following examples are merely illustrative of the present invention, and the scope of the present invention is not limited by the examples.

[0088] Example 1. Transformation of wild-type microorganisms and codon optimization of GMAS genes

[0089] The microbial strain used for transformation and codon optimization was Corynebacterium glutamicum ATCC 14067.

[0090] In order for the microbial strain to produce GMAS, a wild-type GMAS gene (AB333782, SEQ ID NO. 1) derived from Methylovorus mays No. 9 was transfected into the said microbial strain in the form of a vector. Codon optimization was performed on the said transformed microbial strain to form 10 colonies. Each colony was named BTC-THA-1 to BTC-THA-10.

[0091] While culturing strains BTC-THA-1 to BTC-THA-10 in a flask, the concentration (g / L) of L-theanine produced was measured after adding ethylamine to the flask. The concentrations of L-theanine for each strain are shown in Table 1 below.

[0092]

[0093] It was confirmed that L-theanine was produced in BTC-THA-2, 4, 5, and 8 to 10, with relatively high concentrations of L-theanine produced in BTC-THA-4 and 9. In the other strains, D-theanine was produced, L-theanine was not produced, or L-theanine was produced in very small amounts, so it appears that L-theanine was not detected. A codon-optimized GMAS gene (Sequence No. 2) was obtained from the above BTC-THA-4.

[0094] Example 2. Construction of a recombinant vector for the introduction of codon-optimized GMAS and DHFR genes

[0095] A DHFR expression vector containing the codon-optimized GMAS obtained in Example 1 and the promoter portion of the cat gene derived from the pXMJ19 vector was constructed as follows.

[0096] The codon-optimized GMAS obtained in Example 1 was treated with HindIII / XbaI, and then inserted into the pXMJ19-cat-HindIII vector having a replication starter pBL1 treated with the same restriction enzyme using T4 DNA ligase to construct the GMAS-pXMJ19-cat-HindIII vector.

[0097] PCR was performed using the pXMJ19 vector as a template and a primer pair consisting of the nucleotide sequences of SEQ ID NOs. 6 and 7 to obtain a fragment of the cat promoter (CP), and PCR was performed using the gDNA (genomic DNA) of Corynebacterium glutamicum ATCC 14067 as a template and a primer pair consisting of the nucleotide sequences of SEQ ID NOs. 8 and 9 to obtain a fragment of the open reading frame (DHFR) of the DHFR gene. Pfu DNA polymerase was used as the polymerase, and PCR was performed by repeating the following cycles 25 times: pre-denaturation at 94°C for 1 minute, denaturation at 94°C for 20 seconds, annealing at 61°C for 30 seconds, and extension at 72°C for 30 seconds.

[0098] The two fragments (CP and DHFR) obtained above were linked together by performing overlap-extension PCR using them as templates. Specifically, the upstream and downstream were linked by performing overlap PCR without primers using a mixture of the two fragments as a template, and the CP-DHFR fragment was obtained by performing purification PCR after adding a primer pair consisting of the nucleotide sequences of SEQ ID NOs. 6 and 9. Pfu DNA polymerase was used as the polymerase, and for the overlap PCR, denaturation at 94°C for 20 seconds, annealing at 52°C for 30 seconds, and extension at 72°C for 50 seconds were repeated 5 times, and for the purification PCR, denaturation at 94°C for 20 seconds, annealing at 62°C for 30 seconds, and extension at 72°C for 50 seconds were repeated 25 times.

[0099] After treating the CP-DHFR fragment obtained above with XbaI / EcoRI, the GMAS-pXMJ19-cat-HindIII vector was constructed by inserting it into the GMAS-pXMJ19-cat-HindIII vector treated with the same restriction enzyme using T4 DNA ligase (Fig. 2).

[0100] The GMAS-DHFR-pXMJ19-cat-HindIII vector constructed above was treated with HindIII to remove the cat gene (antibiotic resistance gene), and then the vector was reconnected by treating with T4 DNA ligase to construct the GMAS-DHFR-pXMJ19-β vector (Figs. 3 and 4).

[0101] The nucleotide sequences of the primers used in this example are shown in Table 2 below.

[0102]

[0103] Example 3. Production of Corynebacterium microorganisms into which foreign GMAS and DHFR genes were introduced

[0104] (1) Prior to full-scale production of strains introduced with the GMAS-DHFR-pXMJ19-β vector, the optimal trimethoprim concentration for selecting transformed strains was determined. Wild-type ATCC 14067 strains and GMAS-DHFR-pXMJ19-β vector-introduced strains were cultured overnight in BHI (3.7g brain heart infusion powder) medium supplemented with trimethoprim (DMSO solution) at concentrations of 0, 10, 15, 20, and 30 μg / mL, and then each cell concentration was measured at a wavelength of 600 nm. As a result, the growth of the wild-type ATCC 14067 strain was significantly inhibited at a trimethoprim concentration of 10 μg / mL and completely inhibited at a trimethoprim concentration of 15 μg / mL (Fig. 5). Therefore, the trimethoprim concentration for the selection of strains introduced with the GMAS-DHFR-pXMJ19-β vector was determined to be 15 μg / ml.

[0105] (2) The GMAS-DHFR-pXMJ19-β vector obtained in Example 2 was introduced into the Corynebacterium glutamicum ATCC 14067 strain and cultured at 30°C for 3 hours, then applied to LB medium containing 15 μg / ml of trimethoprim. The culture was continued for about 2 days until colonies formed in the medium, and the resulting strain was named BTC-THA-4D1. The BTC-THA-4D1 strain was named Corynebacterium glutamicum BTC-THA-4D1 and was deposited with the Korean Culture Collection Center (KCCM) under accession number KCCM13542P on January 21, 2025, under the Treaty of Budapest.

[0106] Example 4. Confirmation of L-theanine production ability of Corynebacterium microorganisms introduced with foreign GMAS and DHFR genes

[0107] The L-theanine production ability of the strain (BTC-THA-4D1) prepared in Example 3 above was confirmed using the BTC-THA-4 strain as a control.

[0108] One loop of a strain cultured statically at 30°C for 3 days on LB agar medium was taken and inoculated into a seed culture flask containing 220 ml of seed culture medium, and then cultured with shaking at 30°C and 150 rpm for 15 hours.

[0109] The main culture medium was prepared by sterilizing it in an autoclave at 121°C for 15 minutes; however, glucose and divalent cations, which can cause browning reactions with other components, and potassium phosphate, which can cause precipitation reactions, were sterilized separately. The inoculum of a sterilized 5L jar fermenter containing 2.2L of the main culture medium (based on a working volume) was flame-sterilized, and 220mL of seed culture was inoculated. Culture was then carried out under conditions of 900rpm, an aeration rate of 1vvm, and 32°C. The pH was maintained at 7.0 using 9% ammonia solution. During culture, cell density at a wavelength of 600nm was measured using a spectrophotometer (Mecasys Optizen 2120UV). At an absorbance level of 30, the temperature was shifted to 30°C while simultaneously initiating the feeding of ethylamine, which acts directly as a substrate for L-theanine conversion.

[0110] During cultivation, the culture medium is sampled at regular intervals to determine the cell density (OD). 600 ), residual sugar concentration, L-theanine concentration, and plasmid drop rate were analyzed (Table 3 and Figure 6).

[0111] For the analysis of L-theanine concentration, L-theanine standard from Sigma-Aldrich was diluted to an appropriate concentration and derivatized with o-phthalaldehyde (OPA), and an HPLC (Agilent 1260 Infinity 2) with a UV detector (DAD) at a wavelength of 338 nm was used.

[0112] The method for analyzing the plasmid dropout rate is as follows. During culture, the culture medium was sampled at regular intervals to perform serial dilution. LB agar medium without the antibiotic trimethoprim was used as the control group, and LB agar medium with trimethoprim was used as the experimental group. 100 µl of each diluted sample was plated onto the experimental and control groups, and after static incubation at 30°C for 3 days, the number of colonies in each medium was counted, and the plasmid dropout rate was calculated according to Equation 1 below.

[0113] [Equation 1]

[0114] Plasmid dropout rate (%) = {(Control group CFU / mL - Experimental group CFU / mL) / Control group CFU / mL} * 100

[0115] <Seed Culture Medium>

[0116] - LB 25 g / L

[0117] <Main Culture Medium>

[0118] - Carbon source: Glucose 110 g / L

[0119] - Nitrogen source: Ammonium sulfate 30 g / L, corn soaking liquid 5.21 g / L, ethylamine 110 g / L

[0120] - Inorganic salts: Potassium monophosphate 7.57 g / L, magnesium sulfate 0.4 g / L, ferrous sulfate 10 mg / L, manganese sulfate 10 mg / L

[0121] - Trace growth factors: Thiamine hydrochloride 200 µg / L, Biotin 6 µg / L

[0122] - Amino acids: Glutamic acid 11.4g / L

[0123]

[0124] As shown in Table 3 above, it can be confirmed that the BTC-THA-4D1 strain according to the present invention has superior L-theanine production ability compared to the control strain BTC-THA-4, a significantly lower plasmid drop rate, and can further improve L-theanine productivity by shortening the fermentation time.

[0125] [Consignment Number]

[0126] Depository: Korean Culture Collection of Microorganisms

[0127] Trustee Number: KCCM13542P

[0128] Date of Deposit: 20250121

[0129] [Correction pursuant to Rule 91 06.02.2026]

Claims

1. As a microorganism of the genus Corynebacterium capable of producing L-theanine, Microorganism comprising a recombinant vector expressing gamma-glutamyl methylamide synthetase (GMAS) and dihydrofolate reductase (DHFR).

2. In Claim 1, The above recombinant vector is a microorganism that does not contain an antibiotic resistance gene.

3. In Claim 2, The above antibiotic resistance gene is a microorganism that is an ampicillin resistance gene, a kanamycin resistance gene, an erythromycin resistance gene, a chloramphenicol resistance gene, a spectinomycin resistance gene, a streptomycin resistance gene, a thiamphenicol resistance gene, a trimethoprim resistance gene, or a tetracycline resistance gene.

4. In Claim 1, The gene encoding the above gamma-glutamyl methylamide synthase is a microorganism composed of the nucleotide sequence of SEQ ID NO.

2.

5. In Claim 1, The gene encoding the above-mentioned dehydrofolic acid reductase is a microorganism composed of the nucleotide sequence of SEQ ID NO.

3.

6. In Claim 1, The above recombinant vector is a microorganism in which a cat promoter is introduced into the upstream region of a gene encoding dihydrofolate reductase.

7. In Claim 1, The above microorganism is a microorganism that is Corynebacterium glutamicum.

8. In Claim 7, The above microorganism is a microorganism that is Corynebacterium glutamicum BTC-THA-4D1 (KCCM13542P).

9. A method for producing L-theanine, comprising the step of culturing the microorganism of any one of claims 1 to 8 in a culture medium.

10. In Claim 9, A method for producing L-theanine, further comprising the step of recovering L-theanine from the cultured microorganism or culture medium.

11. In Claim 9, The above medium is a method for producing L-theanine containing ethylamine.

12. In Claim 9, A method for producing L-theanine in which the above-mentioned microorganism is cultured in the presence of trimethoprim.

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