Microorganism having l-theanine production ability and method for producing l-theanine using same

Abstract

The present invention relates to a microorganism having the ability to produce L-theanine and a method for producing L-theanine using same. Specifically, the present invention provides: a recombinant microorganism in which a foreign glutamyl methylamide synthetase (GMAS) gene is directly inserted into the genome of the microorganism; and a method for producing L-theanine with high efficiency 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 capable of producing L-theanine with an inserted GMAS gene 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 γ-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] Furthermore, although the initial expression of transgenic microorganisms into which plasmid vectors containing foreign genes have been introduced is stable, problems such as changes in the plasmid copy number, mutations, or an increased plasmid shedding rate may occur upon prolonged fermentation or exposure to various external environments, which leads to a decrease in L-theanine production capacity.

[0011] Accordingly, the present invention aims to provide a microorganism of the genus Corynebacterium with enhanced L-theanine production capacity and a method for producing L-theanine using the same, which fundamentally prevents problems such as deletion of recombinant vectors, changes in copy number, and mutations, and prevents environmental harm caused by antibiotic resistance genes.

[0012] To solve the above-mentioned problem, the present invention provides a microorganism of the genus Corynebacterium capable of producing L-theanine, wherein a gene encoding gamma-glutamyl methylamide synthetase (γ-glutamyl methylamide synthetase, GMAS) is directly inserted into the genome of said microorganism of the genus Corynebacterium.

[0013] In the above microorganism, the gene encoding the gamma-glutamyl methylamide synthase may be directly inserted into two or more sites of the genome of the genus Corynebacterium.

[0014] In the above microorganism, the gene encoding the gamma-glutamyl methylamide synthase may be directly inserted into three sites at different locations in the genome of the genus Corynebacterium.

[0015] In the above microorganism, the gene encoding the gamma-glutamyl methylamide synthase may be composed of the nucleotide sequence of SEQ ID NO. 2.

[0016] In the above microorganism, the microorganism may not contain a recombinant vector into which an exogenous antibiotic resistance gene has been introduced.

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

[0018] In the above microorganism, the microorganism may be Corynebacterium glutamicum BTC-THA-18 (KCCM13543P).

[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] 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.

[0023] 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 L-glutamic acid using a microorganism in which the gamma-glutamyl methylamide synthetase (GMAS) gene is directly introduced into the genome. 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 production cost of L-theanine can be lowered.

[0024] In addition, the microorganism of the present invention has the gamma-glutamyl methylamide synthase (GMAS) gene directly introduced into its genome, so it can stably maintain the expression of GMAS without including a recombinant vector containing an antibiotic resistance gene, and thus can produce L-theanine without the potential risk caused by the antibiotic resistance gene.

[0025] 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.

[0026] FIG. 1 is a schematic diagram showing a helper vector and a donor vector for the transformation of microorganisms according to the present invention.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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."

[0033] 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.

[0034] 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.

[0035] Furthermore, in this application, the term "transformation" refers to introducing a vector containing a gene (polynucleotide) encoding a target protein into a host cell, or directly inserting the gene encoding the target protein into the genome of a host cell so that the protein encoded by said gene can be expressed within the host cell.

[0036] 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. The promoter may be, for example, the promoter disclosed in Korean Registered Patent Publication No. 10-2632294, but is not limited thereto.

[0037] 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.

[0038] Furthermore, in this application, the term "operably linked" means that a gene sequence is functionally linked to a promoter sequence that initiates and mediates the transcription of a gene encoding the target protein of the present invention. For example, a gene encoding GMAS, which is the target protein of the present invention, may be functionally linked to a promoter sequence disclosed in Korean Registered Patent Publication No. 10-2632294 so that the transcription of the gene may be initiated and mediated.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] Additionally, in this application, the term "transposase" refers to an enzyme that binds to the end of a transposon and catalyzes its movement to another part of the genome by a cleavage and paste mechanism or a replicative translocation mechanism; that is, an enzyme that catalyzes translocation.

[0044] Additionally, in this application, the term "translocation" may refer to a complex genetic rearrangement process including, but is not limited to, the movement of a DNA sequence from one location to another, and insertion between, for example, a genome and a DNA construct such as a plasmid, vacmid, cosmid, and viral vector.

[0045] Additionally, in this application, the term "transposon" refers to transposable elements capable of moving or transposing from one location to another on the genome, such as Tn5, Tn7, and Tn10, but is not limited thereto.

[0046] Additionally, in this application, the term "genome" may mean the genetic material of an organism, for example, a chromosome.

[0047] The microorganism of the present invention is a microorganism of the genus Corynebacterium capable of producing L-theanine, characterized in that a gene encoding gamma-glutamyl methylamide synthetase (GMAS) (hereinafter also referred to as the "gene of GMAS") is directly inserted into the genome of the said microorganism of the genus Corynebacterium. Accordingly, the deletion, copy number change, and mutation of a recombinant vector containing a foreign gene are fundamentally blocked, thereby significantly improving the L-theanine production capacity.

[0048] The above gamma-glutamyl methylamide synthetase (GMAS) synthesizes L-theanine from L-glutamic acid and ethylamine.

[0049] 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').

[0050] 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.

[0051] 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.

[0052] The method of directly inserting the above GMAS gene into the genome of a microorganism of the genus Corynebacterium may use the CRISPR CAS system, but is not limited thereto; preferably, the Tn7-like type IF CRISPR CAS system may be used, and more preferably, the gene delivery vector system within the genome of Corynebacterium glutamicum disclosed in Korean Patent Publication No. 10-2024-0055211 may be used.

[0053] For detailed information regarding the above-mentioned vector system for gene delivery within the genome of Corynebacterium glutamicum, reference may be made to the contents described in Korean Published Patent Application No. 10-2024-0055211, and the contents described in Korean Published Patent Application No. 10-2024-0055211 may be applied in the same way to the present invention.

[0054] The above-mentioned vector system for gene delivery within the genome of Corynebacterium glutamicum may include a first vector and a second vector, wherein the first vector includes crRNA, TniQ-Cascade, and transposase, and the second vector may include a transposase recognition site and a foreign gene.

[0055] The first vector may consist of a CRISPR system containing crRNA, TniQ-Cas876 (TniQ-cascade), and a Tn7-like transposon system including the transposase TnsABC, but is not limited thereto. In the present invention, the first vector may refer to the same vector as pCoryne_Integrate_Helper or helper vector.

[0056] The second vector above may consist of a right end (RE) and a left end (LE), which are recognition sites for the transposon enzyme, and a foreign gene cassette to be inserted at a location designated by crRNA, but is not limited thereto. In the present invention, the second vector may refer to the same vector as pCoryne_Integrate_Donor or the donor vector.

[0057] The transposase recognition site may include one or more of RE (Right End) or LE (Left End), but is not limited thereto. Additionally, the second vector may further include one or more of BsaI restriction enzyme sites or red fluorescent protein, but is not limited thereto.

[0058] That is, the second vector above may be configured to allow convenient cloning of a foreign gene cassette desired by the user by inserting two Golden Gate enzyme (BsaI restriction enzyme) sites between the sequences of RE and LE, which are recognition sites of the Tn6677 transposon enzyme, but is not limited thereto.

[0059] The first vector above may include crRNA, TRc promoter, TniQ, Cas8, Cas7, Cas6, TnsA, TnsB, and TnsC in order, but is not limited thereto.

[0060] The second vector may include RE, a foreign gene, and LE in that order, but is not limited thereto. Additionally, the second vector may have a red fluorescent protein (RFP) located at the foreign gene cassette insertion site, so that when the target foreign gene cassette is inserted during cloning, the red fluorescent protein disappears, making it possible to select a Red / White colony in which the colony containing the desired plasmid turns white, but is not limited thereto.

[0061] In one embodiment of the present invention, the gene delivery vector system within the Corynebacterium glutamicum genome can be operated by the principle of using two types of vectors to attach TniQ-cascade to a target location via crRNA, forming a transposase TnsABC transposon and moving it toward TniQ-cascade, and then inserting the transposon at a location specified by crRNA at a certain distance behind the target DNA location.

[0062] More specifically, when the completed first vector, pCoryne_Integrate_Helper, is expressed within Corynebacterium glutamicum, Cas876 and TniQ are expressed to form a complex (TniQ-Cascade), which recognizes PAM and attaches to a designated location on the genome. Subsequently, TnsC, a non-sequence-specific DNA-binding protein and one of the transposases, binds to TniQ. The transposon is recognized and bound by the transposases TnsA and TnsB to the RE and LE sequences present in the second vector, pCoryne_Integrate_Donor, forming a paired-end complex. The formed transposon is cleaved from the pCoryne_Integrate_Donor vector and, via TnsC, is gathered at a target site on the DNA recognized by TniQ-Cascade. A transposon is inserted at a fixed distance (47 to 52 bp) behind the TniQ-Cascade bound to DNA, characterized by target site duplication (TSD) in which 5 bp of the target site is replicated. Through this process, Tn7-like transposon-based gene insertion can occur.

[0063] In one embodiment of the present invention, the productivity of L-theanine can be increased in proportion to the number of copies of the gene encoding gamma-glutamyl methylamide synthase introduced into the genome of a microorganism of the genus Corynebacterium. Accordingly, in the microorganism of the present invention, it is preferable that the gene encoding gamma-glutamyl methylamide synthase is directly inserted into two or more sites in the genome of the microorganism of the genus Corynebacterium, and it is even more preferable that the gene encoding gamma-glutamyl methylamide synthase is directly inserted into three or more sites in the genome of the microorganism of the genus Corynebacterium.

[0064] In addition, in one embodiment of the present invention, the microorganism of the present invention may have a gene encoding the gamma-glutamyl methylamide synthase directly inserted into three sites at different locations in the genome of the genus Corynebacterium, and preferably, one copy each may be directly inserted into the idsA, inter2, and inter20 locations in the genome of the genus Corynebacterium.

[0065] The above idsA, inter2, and inter20 targets may be designated by crRNA included in the helper vector. Specifically, the target location idsA may be designated by crRNA consisting of the nucleotide sequence of SEQ ID NO. 5, the target location inter2 may be designated by crRNA consisting of the nucleotide sequence of SEQ ID NO. 6, and the target location inter20 may be designated by crRNA consisting of the nucleotide sequence of SEQ ID NO. 7.

[0066] In one embodiment of the present invention, the microorganism of the present invention may have a gene encoding gamma-glutamyl methylamide synthase directly inserted into the genome of Corynebacterium glutamicum ATCC 14067, preferably a gene encoding gamma-glutamyl methylamide synthase directly inserted into two or more sites of the genome of Corynebacterium glutamicum ATCC 14067, and more preferably a gene encoding gamma-glutamyl methylamide synthase directly inserted into three sites at different locations of the genome of Corynebacterium glutamicum ATCC 14067.

[0067] In addition, in one embodiment of the present invention, the microorganism of the present invention may have a gene encoding gamma-glutamyl methylamide synthase directly inserted into one or more sites selected from idsA, composed of the nucleotide sequence of SEQ ID NO. 5, inter2, composed of the nucleotide sequence of SEQ ID NO. 6, and inter20, composed of the nucleotide sequence of SEQ ID NO. 7, present in the genome of Corynebacterium glutamicum ATCC 14067; preferably, the microorganism may have a gene encoding gamma-glutamyl methylamide synthase directly inserted into two or more sites selected from idsA, composed of the nucleotide sequence of SEQ ID NO. 5, inter2, composed of the nucleotide sequence of SEQ ID NO. 6, and inter20, composed of the nucleotide sequence of SEQ ID NO. 7, present in the genome of Corynebacterium glutamicum ATCC 14067; and more preferably, the microorganism comprising the nucleotide sequence of SEQ ID NO. 5 present in the genome of Corynebacterium glutamicum ATCC 14067 A gene encoding gamma-glutamyl methylamide synthase may be directly inserted into the sites of idsA, inter2 consisting of the nucleotide sequence of SEQ ID NO. 6, and inter20 consisting of the nucleotide sequence of SEQ ID NO. 7.

[0068] A microorganism in which one copy of the gene encoding gamma-glutamyl methylamide synthase is inserted at the idsA position of the genome of the above-mentioned microorganism of the genus Corynebacterium may contain the nucleotide sequence of SEQ ID NO. 14.

[0069] In addition, a microorganism having one copy of a gene encoding gamma-glutamyl methylamide synthase inserted at the inter2 position of the genome of the above-mentioned microorganism of the genus Corynebacterium may contain the nucleotide sequence of SEQ ID NO. 15.

[0070] In addition, a microorganism having one copy of a gene encoding gamma-glutamyl methylamide synthase inserted at the inter20 position of the genome of the above-mentioned microorganism of the genus Corynebacterium may contain the nucleotide sequence of SEQ ID NO. 16.

[0071] In one embodiment of the present invention, the microorganism of the present invention may not include a recombinant vector into which an exogenous antibiotic resistance gene has been introduced. Accordingly, it is desirable to be able to produce L-theanine without the potential risk associated with the exposure of the antibiotic resistance gene to the external environment.

[0072] 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-18 (KCCM13543P).

[0073] Meanwhile, since Brevibacterium flavum is currently classified as Corynebacterium glutamicum, the Brevibacterium flavum strain is also included in the scope of the present invention.

[0074] 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.

[0075] 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.

[0076] In addition, the present invention provides a composition for producing L-theanine comprising the microorganism of the present invention described above.

[0077] 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.

[0078] 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.

[0079] The microorganism of the present invention is a microorganism of the genus Corynebacterium that can biosynthesize L-glutamic acid from glucose, and a GMAS directly inserted into the genome synthesizes L-theanine from the L-glutamic acid and ethylamine added to the medium.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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 types.

[0085] 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.

[0086] 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.

[0087] In one embodiment of the present invention, the temperature of the culture may be 25 to 40°C, more specifically 28°C 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.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] Residual sugar concentration analysis can be performed using a spectrophotometer (Mecasys Optizen 2120UV) after inducing an enzymatic reaction using Megazyme's GOPOD assay kit.

[0094] 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.

[0095] 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.

[0096] 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.

[0097] Preparation Example 1. Transformation of wild-type microorganisms and codon optimization of GMAS genes

[0098] The microbial strain used for transformation and codon optimization was Corynebacterium glutamicum ATCC 14067.

[0099] 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.

[0100] 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.

[0101]

[0102] 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.

[0103] Preparation Example 2. Construction of a Tn7-like type IF crispr CAS vector-based recombinant vector for codon-optimized GMAS gene insertion

[0104] A recombinant vector for Corynebacterium glutamicum strains was designed based on a Tn7-like type IF crispr CAS vector and composed of two types of plasmids. Specifically, the first plasmid (pCoryne_Integrate_Helper, hereinafter 'helper vector') (Sequence No. 3) was prepared to include sequences encoding TniQ-Cascade (TniQ, Cas8, Cas7, Cas6), CRISPR RNA (crRNA), and transposases (TnsA, TnsB, TnsC). Additionally, the second plasmid (pCoryne_Integrate_Donor, hereinafter 'donor vector') (Sequence No. 4) was prepared to include the Right end (RE) and Left end (LE) sequences, which are recognition sites for the foreign gene cassette and transposon enzyme to be inserted at a specific location on the genome designated by crRNA (Fig. 1).

[0105] Next, idsA (Geranylgeranyl pyrophosphate synthase), intergenic region 2 (inter2), and intergenic region 20 (inter20) present in the genome of Corynebacterium glutamicum were selected as targets for codon-optimized GMAS gene insertion, and each target sequence is shown in Table 2 below.

[0106]

[0107] A helper vector containing three different crRNAs was constructed by inserting CRISPR RNA into a helper vector using Golden Gate assembly to guide the TniQ-cascade to the target location based on each crRNA sequence of 32 bp length to target each of the above locations.

[0108] The codon-optimized GMAS gene obtained in Preparation Example 1 was subjected to a polymerase chain reaction using primers attached to a BsaI restriction enzyme site, and the resulting gene fragment was inserted between the RE and LE sequences inside the donor vector using Golden Gate Assembly to construct a donor vector containing the codon-optimized GMAS gene.

[0109] It was confirmed that antibiotic resistance genes were not introduced into the above-described helper vector and donor vector.

[0110] Example 1. Production of Corynebacterium microorganisms into which foreign GMAS genes were introduced

[0111] The helper vector and donor vector prepared in Preparation Example 2 above were inserted into a wild-type strain of Corynebacterium glutamicum ATCC 14067 to produce a strain capable of Tn7-like transposon-based gene insertion.

[0112] Specifically, helper vectors containing each crRNA consisting of the nucleotide sequences of SEQ ID NOs. 5–7 were introduced into a Corynebacterium glutamicum strain containing a donor vector to produce a strain in which a total of 3 copies of the foreign GMAS gene were introduced, with 1 copy each at the idsA, inter2, and inter20 positions of the genome, and the strain produced accordingly was named BTC-THA-18. The above-mentioned BTC-THA-18 strain was named Corynebacterium glutamicum BTC-THA-18 and was deposited with the Korean Culture Collection Center (KCCM) on January 21, 2025, under the Treaty of Budapest, with accession number KCCM13543P.

[0113] In addition, helper vectors containing crRNAs composed of the nucleotide sequences of SEQ ID NOs. 5 and 6 were introduced into a Corynebacterium glutamicum strain containing a donor vector to produce a strain in which a total of 2 copies of the foreign GMAS gene were introduced, with 1 copy each at the idsA and inter2 positions, and the strain produced accordingly was named BTC-THA-19.

[0114] In addition, a helper vector containing a crRNA consisting of the nucleotide sequence of SEQ ID NO. 6 was introduced into a Corynebacterium glutamicum strain containing a donor vector to produce a strain in which one copy of the foreign GMAS gene was introduced at the inter2 position, and the strain produced accordingly was named BTC-THA-20.

[0115] At this time, to determine whether a gene was inserted, colonies with the above phenotype were primarily selected using the characteristic that the color of the colony appears white when the foreign GMAS gene is inserted at the idsA site. For the selected colonies, fragments were obtained by amplifying the target gene through polymerase chain reaction using primer pairs consisting of the nucleotide sequences of SEQ ID NOs. 8 and 9, SEQ ID NOs. 10 and 11, and SEQ ID NOs. 12 and 13. The fragments were separated by size using electrophoresis to confirm whether insertion occurred, and the fragments confirmed to have gene insertion were submitted for DNA sequencing analysis as samples to finally confirm that the foreign gene was inserted into the targeted region.

[0116] The nucleotide sequences of the primers used in this example are shown in Table 3 below.

[0117]

[0118] Example 2. Confirmation of L-theanine production ability of Corynebacterium microorganisms introduced with foreign GMAS gene

[0119] The L-theanine production ability of the BTC-THA-18, BTC-THA-19, and BTC-THA-20 strains produced in Example 1 above was confirmed.

[0120] 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.

[0121] 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.

[0122] During cultivation, the culture medium is sampled at regular intervals to determine the cell density (OD). 600 ), residual sugar concentration and L-theanine concentration were analyzed (Table 4).

[0123] 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.

[0124] <Seed Culture Medium>

[0125] - LB 25 g / L

[0126] <Main Culture Medium>

[0127] - Carbon source: Glucose 110 g / L

[0128] - Nitrogen source: Ammonium sulfate 30 g / L, corn soaking liquid 5.21 g / L, ethylamine 110 g / L

[0129] - Inorganic salts: Potassium monophosphate 7.57 g / L, magnesium sulfate 0.4 g / L, ferrous sulfate 10 mg / L, manganese sulfate 10 mg / L

[0130] - Trace growth factors: Thiamine hydrochloride 200 µg / L, Biotin 6 µg / L

[0131] - Amino acids: Glutamic acid 11.4g / L

[0132]

[0133] As shown in Table 4 above, the BTC-THA-18, BTC-THA-19, and BTC-THA-20 strains according to the present invention exhibited excellent L-theanine production capabilities. In particular, the BTC-THA-18 strain, into which a total of three copies of the foreign GMAS gene—one copy each at the idsA, inter2, and inter20 positions of the Corynebacterium glutamicum ATCC 14067 strain genome—were introduced, showed the highest L-theanine concentration, confirming that the productivity of L-theanine can be further improved compared to the BTC-THA-19 and BTC-THA-20 strains.

[0134] [Consignment Number]

[0135] Depository Name: Korean Culture Collection of Microorganisms

[0136] Trustee Number: KCCM13543P

[0137] Date of Deposit: 20250121

[0138]

Claims

1. As a microorganism of the genus Corynebacterium capable of producing L-theanine, A microorganism in which a gene encoding gamma-glutamyl methylamide synthetase (GMAS) is directly inserted into the genome of the said genus Corynebacterium.

2. In Claim 1, A microorganism in which the gene encoding the above-mentioned gamma-glutamyl methylamide synthase is directly inserted into two or more sites of the genome of the above-mentioned microorganism of the genus Corynebacterium.

3. In Claim 1, A microorganism in which the gene encoding the above-mentioned gamma-glutamyl methylamide synthase is directly inserted at three different sites in the genome of the above-mentioned microorganism of the genus Corynebacterium.

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 above microorganism is a microorganism that does not contain a recombinant vector into which an exogenous antibiotic resistance gene has been introduced.

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

7. In Claim 6, The above microorganism is a microorganism that is Corynebacterium glutamicum BTC-THA-18 (KCCM13543P).

8. A method for producing L-theanine comprising the step of culturing the microorganism of any one of claims 1 to 7 in a culture medium.

9. In Claim 8, A method for producing L-theanine, further comprising the step of recovering L-theanine from the cultured microorganism or culture medium.

10. In Claim 8, The above medium is a method for producing L-theanine containing ethylamine.

Images