Microorganisms of the genus Corynebacterium that produce L-glutamic acid and a method for producing L-glutamic acid using the same

Abstract

This invention relates to a Corynebacterium microorganism that produces L-glutamic acid and a method for producing L-glutamic acid using the same. More specifically, it relates to a myo-inositol facilitator IolT2 mutant, polynucleotides and transformants involved in the L-glutamic acid biosynthesis pathway, and a method for producing L-glutamic acid using the same. In the myo-inositol facilitator IolT2 mutant according to the present invention, the activity of the protein is altered by the substitution of one or more amino acids in the amino acid sequence constituting myo-inositol facilitator IolT2, and recombinant microorganisms containing this mutant can efficiently produce L-glutamic acid.

Description

Technical Field

[0001] The present invention relates to a Corynebacterium microorganism that produces L-glutamic acid and a method for producing L-glutamic acid using the same, and specifically, to a myo-inositol facilitator IolT2 variant, a polynucleotide, and a transformant involved in the biosynthetic pathway of L-glutamic acid, and a method for producing L-glutamic acid using the same.

Background Art

[0002] L-glutamic acid is a typical amino acid produced by microbial fermentation. Monosodium L-glutamate (MSG) enhances the preference for foods such as meat, fish, chicken, vegetables, sauces, soups, and seasonings by balancing the overall taste of food, and can enhance the taste of low-salt foods with up to 30% salt reduction, and is widely used as a seasoning for household and processed food production.

[0003] A brief look at the fermentation pathway of L-glutamic acid reveals that glucose is primarily metabolized via the glycolytic pathway, but some is also metabolized via the pentose phosphate pathway into two molecules of pyruvic acid. One of these molecules fixes CO2 to become oxaloacetic acid, while the other combines with acetyl-CoA to form citric acid. Furthermore, oxaloacetic acid and citric acid enter the citric acid cycle (TCA cycle) to become alpha-ketoglutaric acid. Here, the oxidative metabolic pathway that oxidizes alpha-ketoglutaric acid to succinic acid is absent, and because isocitrate dehydrogenase and glutamate dehydrogenase are closely involved, the reductive amino acid conversion reaction of alpha-ketoglutaric acid proceeds efficiently to produce L-glutamic acid.

[0004] L-glutamic acid can be produced using wild-type strains obtained in nature or mutant strains modified to enhance their glutamic acid production capacity. Recently, in order to improve the efficiency of L-glutamic acid production, genetic engineering techniques have been applied to microorganisms such as Escherichia coli and Corynebacterium, which are widely used in the production of useful substances such as amino acids and nucleic acids. This has led to the development of diverse recombinant or mutant strains with superior L-glutamic acid production capabilities and methods for producing L-glutamic acid using these strains. In particular, there have been attempts to increase L-glutamic acid production by inducing mutations in genes such as enzymes, transcription factors, and transport proteins involved in the L-glutamic acid biosynthesis pathway, or in promoters that regulate their expression. However, since there are dozens to hundreds of types of proteins, including enzymes, transcription factors, and transport proteins, that are directly or indirectly related to L-glutamic acid production, much research is still needed to determine whether changes in the activity of such proteins increase L-glutamic acid production capacity. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] U.S. Registered Patent No. 6,852,516 [Patent Document 2] U.S. Registered Patent No. 6,962,805 [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] The present invention aims to provide a novel myo-inositol facilitator IolT2 variant.

[0007] Furthermore, the present invention aims to provide a polynucleotide for encrypting (coding) the aforementioned variants.

[0008] Furthermore, the present invention aims to provide a transformant comprising the aforementioned mutant or polynucleotide.

[0009] Furthermore, the present invention aims to provide a method for producing L-glutamic acid using the aforementioned transformant. [Means for solving the problem]

[0010] One aspect of the present invention provides a myo-inositol facilitator IolT2 variant comprising the amino acid sequence of SEQ ID NO: 2, wherein the 424th glutamic acid in the amino acid sequence of SEQ ID NO: 4 is replaced with lysine.

[0011] The "myo-inositol facilitator IolT2" used in this invention is a protein involved in sugar transport as a myo-inositol transporter, and may be a polypeptide or protein that has myo-inositol facilitator IolT2 activity, encoded by the iolT2 or Cgl3058 gene.

[0012] The nucleic acid and protein sequence information of the myo-inositol facilitator IolT2 can be obtained through known sequence databases (e.g., GenBank, UniProt).

[0013] According to one specific example of the present invention, the myo-inositol facilitator IolT2 consists of the amino acid sequence of SEQ ID NO: 4 and may be encrypted by the nucleotide sequence of SEQ ID NO: 3.

[0014] The amino acid sequence of the myo-inositol facilitator IolT2 according to the present invention, or the nucleotide sequence that encodes it, may include a nucleotide sequence or amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% homology or identity compared to the nucleotide sequence of SEQ ID NO: 3. Here, "homology" or "identity" means the percentage of agreement between two sequences when the reference nucleotide sequence or amino acid sequence and any other nucleotide sequence or amino acid sequence are aligned and analyzed to maximize correspondence.

[0015] According to one specific example of the present invention, the myo-inositol facilitator IolT2 may be derived from wild-type Corynebacterium glutamicum.

[0016] In this invention, "mutant" refers to a variant in which one or more amino acids in the amino acid sequence undergo conservative substitution and / or modification at the N-terminus, C-terminus, and / or internally due to a mutation in the base sequence of the gene encoding the protein, resulting in a sequence different from the amino acid sequence before the mutation, but maintaining the functions or properties. Here, "conservative substitution" means replacing one amino acid with another amino acid that has similar structural and / or chemical properties, and has little or no effect on the activity of the protein or polypeptide. "Modification" refers to amino acid substitution, insertion, deletion, etc. The aforementioned amino acids were selected from alanine (Ala, A), isoleucine (Ile, I), valine (Val, V), leucine (Leu, L), methionine (Met, M), asparagine (Asn, N), cysteine ​​(Cys, C), glutamine (Gln, Q), serine (Ser, S), threonine (Thr, T), phenylalanine (Phe, F), tryptophan (Trp, W), tyrosine (Tyr, Y), aspartic acid (Asp, D), glutamic acid (Glu, E), arginine (Arg, R), histidine (His, H), lysine (Lys, K), glycine (Gly, G), and proline (Pro, P).

[0017] Furthermore, mutants may include those in which one or more parts, such as the N-terminal leader sequence or transmembrane domain, are removed, or in which a portion of the N- and / or C-terminus of a mature protein is removed.

[0018] Such mutants may exhibit increased (enhanced), unchanged, or decreased (weakened) capabilities compared to the pre-mutation protein. Here, "increased or enhanced" includes cases where the activity of the protein itself increases compared to the pre-mutation protein, cases where the overall level of protein activity within the cell is higher than that of the wild-type strain or strains expressing the pre-mutation protein due to increased expression or translation of the gene that codes for the protein, and combinations thereof. "Decreased or weakened" includes cases where the activity of the protein itself decreases compared to the pre-mutation protein, cases where the overall level of protein activity within the cell is lower than that of the wild-type strain or strains expressing the pre-mutation protein due to inhibited expression or translation of the gene that codes for the protein, and combinations thereof. In this invention, mutants can be mixed with mutant types, deformities, mutant polypeptides, mutated proteins, mutations, etc.

[0019] The myo-inositol facilitator IolT2 variant according to the present invention may contain an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% homology or identity with the amino acid sequence of SEQ ID NO: 2, except for the mutation site (amino acid residue 424).

[0020] Another aspect of the present invention provides a polynucleotide that encodes the myo-inositol facilitator IolT2 variant.

[0021] The term "polynucleotide" as used in this invention refers to a polymer of nucleotides in which nucleotide units (monomers) are covalently linked together in a long chain, and is a DNA or RNA chain of a certain length or longer. More specifically, it refers to a polynucleotide fragment that encodes the aforementioned variant.

[0022] According to one specific example of the present invention, the polynucleotide may include a base sequence that encodes the amino acid sequence of SEQ ID NO: 2.

[0023] More specifically, the polynucleotide can include the nucleotide sequence of SEQ ID NO: 1 in which the 1270th base g in the nucleotide sequence of SEQ ID NO: 3 encoding myo-inositol facilitator IolT2 is substituted with a.

[0024] Another aspect of the present invention provides a vector comprising a polynucleotide encoding the myo-inositol facilitator IolT2 mutant.

[0025] Also, another aspect of the present invention provides a transformant comprising the myo-inositol facilitator IolT2 mutant or polynucleotide.

[0026] As used in the present invention, the "vector" means all types of nucleic acid sequence carrier structures used as means for transferring and expressing a target gene in a host cell. Unless otherwise specified, the vector can mean one in which the carried nucleic acid sequence is inserted into the host cell genome and expressed and / or expressed independently. Such a vector includes essential regulatory elements operably linked so that the gene insert is expressed, and "operably linked" means that the target gene and its regulatory sequences are linked in a manner that enables genetic expression by being functionally bound to each other. The "regulatory element" includes a promoter for transcription, any operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence for regulating the termination of transcription and translation.

[0027] The vectors used in this invention are not particularly limited as long as they are replicable within host cells, and any vector known in the art can be used. Examples of such vectors include plasmids, cosmids, viruses, and bacteriophages in their native or recombinant state. For example, phage vectors or cosmid vectors include pWE15, M13, λMBL3, λMBL4, λIXII, λASHII, λAPII, λt10, λt11, Charon4A, Charon21A, etc., and plasmid vectors include, but are not limited to, the pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, and pET series.

[0028] The aforementioned vectors can typically be constructed as vectors for cloning or vectors for expression. For expression, standard vectors used in the art to express foreign genes or proteins in plants, animals, or microorganisms can be used, and can be constructed by a variety of methods known in the art.

[0029] The "recombinant vector" used in this invention may, after being transformed into a suitable host cell, be able to replicate independently of the host cell's genome or be stitched into the genome itself. In this case, the "suitable host cell" is one in which the vector can replicate and may include an origin of replication, which is a specific base sequence from which replication is initiated. For example, if the vector used is an expression vector and the host is a prokaryotic cell, it typically includes a strong promoter that can advance transcription (e.g., pLλ promoter, CMV promoter, trp promoter, lac promoter, tac promoter, T7 promoter), a ribosome binding site for initiating sequencing, and a transcription / sequence termination sequence. If the host is a eukaryotic cell, the origins of replication that act in eukaryotic cells and are included in the vector include, but are not limited to, the f1 origin of replication, SV40 origin of replication, pMB1 origin of replication, adeno origin of replication, AAV origin of replication, and BBV origin of replication. Furthermore, promoters derived from the genome of mammalian cells (e.g., metallothione promoter) or promoters derived from mammalian viruses (e.g., late adenovirus promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, HSV tk promoter) may be used, and generally have a polyadenylated sequence as the transcription termination sequence.

[0030] The recombinant vector may contain a selection marker, which is used to select transformants (host cells) transformed by the vector. Since only cells expressing the selection marker can survive in a culture medium treated with the selection marker, the transformed cells can be selected. Typical examples of the selection marker include, but are not limited to, kanamycin, streptomycin, and chloramphenicol.

[0031] A transformant can be created by inserting a recombinant vector into a host cell, and the transformant may be obtained by introducing the recombinant vector into a suitable host cell. The host cell is a cell that can stably and continuously clone or express the expression vector, and any host cell known in the art can be used.

[0032] When transforming prokaryotic cells to produce recombinant microorganisms, the host cells may include, but are not limited to, a variety of Enterobacteria and bacterial strains such as E. coli DH5α, E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, and E. coli XL1-Blue; Bacillus species such as Bacillus subtilis and Bacillus thuringensis; Corynebacterium species such as Corynebacterium glutamicum and Corynebacterium stachyonis; Salmonella tiphimurium, Serratia marcescens, and Pseudomonas species.

[0033] When transforming eukaryotic cells to produce recombinant microorganisms, the host cells may include, but are not limited to, yeast (e.g., Saccharomyces cerevisiae), insect cells, plant cells, and animal cells, such as Sp2 / 0, CHO K1, CHO DG44, PER.C6, W138, BHK, COS7, 293, HepG2, Huh7, 3T3, RIN, and MDCK cell lines.

[0034] In this invention, "transformation" refers to the phenomenon of artificially inducing genetic changes by introducing external DNA into a host cell, while "transformat" refers to a host cell into which external DNA has been introduced and which maintains stable expression of the target gene.

[0035] The transformation can be performed by selecting a suitable vector introduction technique for the host cell to express the target gene or a recombinant vector containing it within the host cell. For example, vector introduction may be performed by electroporation, heat shock, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cation liposome method, lithium acetate-DMSO method, or a combination thereof, but is not limited to these. The transformed gene can be expressed within the host cell, and is not limited to those located inside or outside the host cell's chromosome.

[0036] The transformants include cells that have been transfected, transformed, or infected with the recombinant vector according to the present invention in vivo or in vitro, and can be used as synonymous with recombinant host cells, recombinant cells, or recombinant microorganisms.

[0037] The gene inserted into the recombinant vector of the present invention may be introduced into a host cell, such as a microorganism of the genus Corynebacterium, by homologous recombination cross-recombination.

[0038] According to one specific example of the present invention, the transformed organism may be a microorganism of the genus Corynebacterium.

[0039] The aforementioned Corynebacterium species include Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium callunae, Corynebacterium suranareeae, Corynebacterium lubricantis, Corynebacterium doosanense, Corynebacterium efficiens, and Corynebacterium uterechii. Corynebacterium uterequi), Corynebacterium stationis, Corynebacterium pacaense, Corynebacterium singulare, Corynebacterium humireducens, Corynebacterium marinum, Corynebacterium halotolerans, Corynebacterium spheniscorum, Corynebacterium freiburgense, Corynebacterium striatum (Corynebacterium Corynebacterium striatum), Corynebacterium canis, Corynebacterium ammoniagenes, Corynebacterium renale, Corynebacterium portisoliThis may also be, but is not limited to, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium caspium, Corynebacterium testudinoris, Corynebacterium pseudopelargi, or Corynebacterium flavescens.

[0040] The transformant in the present invention may be, but is not limited to, a strain containing the myo-inositol facilitator IolT2 mutant or a polynucleotide encoding it, or a vector containing the same, a strain expressing the myo-inositol facilitator IolT2 mutant or polynucleotide, or a strain having activity against the myo-inositol facilitator IolT2 mutant.

[0041] The transformants in this invention may include other protein variants or gene mutations in addition to the myo-inositol facilitator IolT2 variant.

[0042] According to one specific example of the present invention, the transformant may have the ability to produce L-glutamic acid.

[0043] The transformant may naturally possess the ability to produce L-glutamate, or it may have been artificially given the ability to produce L-glutamate.

[0044] According to one specific example of the present invention, the transformant may have altered activity of the myo-inositol facilitator IolT2, resulting in improved L-glutamic acid production capacity.

[0045] In this invention, "improved production capacity" means an increase in L-glutamic acid production compared to the parent strain. The parent strain refers to the wild type or mutant strain that is the target of mutation, and includes strains that are directly targeted for mutation or transformed with recombinant vectors, etc. In this invention, the parent strain may be a wild-type Corynebacterium strain or a Corynebacterium strain that has mutated from the wild type.

[0046] The transformant according to the present invention exhibits increased L-glutamic acid production capacity compared to the parent strain, due to the introduction of the myo-inositol facilitator IolT2 mutant, which alters the activity of the myo-inositol facilitator IolT2. More specifically, the transformants may, but are not limited to, those whose L-glutamic acid production is increased by at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to the parent strain, or by 1.1 times, 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 5.5 times, 6 times, 6.5 times, 7 times, 7.5 times, 8 times, 8.5 times, 9 times, 9.5 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, or 100 times. As an example, the transformant containing the myo-inositol facilitator IolT2 mutant may have a L-glutamic acid production that is 5% or more, specifically 5-100% (preferably 10-80%) higher than that of the parent strain.

[0047] Another aspect of the present invention provides a method for producing L-glutamic acid, comprising the step of culturing the transformants in a culture medium.

[0048] The culture may be carried out using appropriate culture media and conditions known in the art, or a typical technician can easily adjust and use the culture media and conditions. Specifically, the culture media may be, but is not limited to, a liquid culture. The culture method may include, but is not limited to, batch culture, continuous culture, fed-batch culture, or a combination thereof.

[0049] According to one specific example of the present invention, the culture medium must meet the requirements of a specific bacterial strain in an appropriate manner and can be modified as appropriate by an ordinary technician. Culture media for Corynebacterium strains can be found in, but are not limited to, known literature (Manual of Methods for General Bacteriology. American Society for Bacteriology. Washington DC, USA, 1981).

[0050] According to one specific example of the present invention, the culture medium may contain a variety of carbon sources, nitrogen sources, and trace element components. Usable carbon sources include sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch, and cellulose; oils and fats such as soybean oil, sunflower oil, castor oil, and coconut oil; fatty acids such as palmitic acid, stearic acid, and linoleic acid; alcohols such as glycerol and ethanol; and organic acids such as acetic acid. These substances may be used individually or in mixtures, but are not limited thereto. Usable nitrogen sources may include peptone, yeast extract, meat juice, malt extract, corn maceration, soybean meal, and urea, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate. Nitrogen sources may also be used individually or in mixtures, but are not limited thereto. Usable phosphorus sources may include, but are not limited thereto, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or corresponding sodium-containing salts. Furthermore, the culture medium may, but is not limited to, contain metal salts such as magnesium sulfate or iron sulfate necessary for growth. Other essential growth substances such as amino acids and vitamins may also be included. In addition, suitable precursors can be used in the culture medium. The medium or individual components may, but are not limited to, be added to the culture medium in a batch or continuous manner in a manner appropriate to the culture process.

[0051] According to one specific example of the present invention, the pH of the microbial culture medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid to the culture medium in an appropriate manner during cultivation. Furthermore, the formation of bubbles can be suppressed during cultivation using an antifoaming agent such as fatty acid polyglycol ester. Additionally, oxygen or an oxygen-containing gas (e.g., air) can be injected into the culture medium to maintain an aerobic state. The temperature of the culture medium is typically 20-45°C, for example, 25-40°C. The cultivation period can be continued until the useful substance is obtained in the desired yield, for example, 10-160 hours.

[0052] The method for producing L-glutamic acid according to the present invention may further include the step of recovering L-glutamic acid from the transformant or the culture medium in which the transformant was cultured.

[0053] According to one specific example of the present invention, the step of recovering L-glutamic acid from the cultured transformant or the culture medium in which the transformant was cultured can be performed by collecting or recovering the L-glutamic acid produced from the culture medium using a suitable method known in the art, depending on the culture method. For example, methods such as centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, fractional dissolution (e.g., ammonium sulfate precipitation), and chromatography (e.g., ion exchange, affinity, hydrophobicity, and size exclusion) can be used, but are not limited thereto.

[0054] According to one specific example of the present invention, the step of recovering L-glutamic acid can be performed by removing biomass by slow centrifugation of the culture medium and separating the resulting supernatant by ion exchange chromatography.

[0055] According to one specific example of the present invention, the step of recovering L-glutamic acid may include a step of purifying L-glutamic acid. [Effects of the Invention]

[0056] In the myo-inositol facilitator IolT2 mutant according to the present invention, the activity of the protein is altered by the substitution of one or more amino acids in the amino acid sequence constituting the myo-inositol facilitator IolT2, and recombinant microorganisms containing this mutant can efficiently produce L-glutamic acid. [Brief explanation of the drawing]

[0057] [Figure 1] This is the structure of a pK19msb plasmid according to one embodiment of the present invention. [Modes for carrying out the invention]

[0058] The present invention will be described in more detail below. However, such description is provided only as an example for the purpose of understanding the present invention, and the scope of the present invention is not limited by such exemplary description.

[0059] Example 1. Creation of a bacterial strain expressing the myo-inositol facilitator IolT2 mutant. To investigate the effect of a mutant of myo-inositol facilitator IolT2 (SEQ ID NO: 2), in which glutamic acid (E) at position 424 of the amino acid sequence (SEQ ID NO: 4) is replaced with lysine (K), on L-glutamic acid production, a vector expressing the myo-inositol facilitator IolT2 mutant and a bacterial strain into which the vector was introduced were constructed.

[0060] 1-1. Vector construction for the expression of the myo-inositol facilitator IolT2 mutant. Using genomic DNA from wild-type Corynebacterium glutamicum ATCC13869 as a template, PCR was performed using primer pairs 1 and 2 and primer pairs 3 and 4. Subsequently, overlapping PCR was performed using primer pairs 1 and 4 on the two PCR products, respectively, to ligate them into a single fragment. The PCR fragment and the pK19msb plasmid (SEQ ID NO: 5) were treated with restriction enzyme smaI (NEB), and cloned using T4 ligase. The constructed plasmid was named pK_iolT2 (E424K).

[0061] For PCR, a pfu premix (bioneer) was used. After denaturation at 95°C for 5 minutes, the mixture was incubated at 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute, repeating this 30 times, followed by a 5-minute reaction at 72°C.

[0062] The primer sequences used to construct the plasmid are shown in Table 1 below.

[0063] [Table 1]

[0064] 1-2. Creation of mutant strains into which the myo-inositol facilitator IolT2 mutant has been introduced. Corynebacterium glutamicum U3 (KCCM13218P) was used as the parent strain to introduce the myo-inositol facilitator IolT2 mutant. For the transformation of the U3 strain, a modified electrocompetent cell production method based on van der Rest's method was used.

[0065] First, the U3 strain was cultured in 10 ml of 2YT medium (containing 16 g / l tryptone, 10 g / l yeast extract, and 5 g / l sodium chloride) supplemented with 2% glucose to prepare a seed culture. Subsequently, 100 ml of 2YT medium (without glucose) was mixed with 1 mg / ml of isonicotinic acid hydrazine and 2.5% glycine. After that, OD (Oral Dissociation) was performed. 610 After inoculating the seed culture solution to achieve a value of 0.3, incubate at 18°C ​​and 180 rpm for 12-16 hours to obtain an OD (Oxygen Demand). 610 The values ​​were adjusted to 1.2-1.4. After leaving the culture medium on ice for 30 minutes, it was centrifuged at 4°C and 4000 rpm for 15 minutes. Then, the supernatant was discarded, and the precipitated U3 strain was washed four times with 10% glycerol solution. Finally, competent cells were prepared by resuspending them in 0.5 ml of 10% glycerol solution. Electroporation was performed using a Bio-Rad electroporator. After adding the prepared competent cells and the fabricated pK_iolT2(E424K) vector to an electroporation cuvette (0.2 mm), an electric shock was applied under conditions of 2.5 kV, 200 Ω, and 12.5 μF. Immediately after the electric shock, 1 ml of regenerated medium (containing Brain Heart infusion 18.5 g / l and sorbitol 0.5 M) was added, and the medium was heat-treated at 46°C for 6 minutes. After cooling to room temperature, the mixture was transferred to a 15 ml capped tube and incubated at 30°C for 2 hours. The mixture was then streaked onto a selection medium (containing 5 g / l tryptone, 5 g / l NaCl, 2.5 g / l yeast extract, 18.5 g / l Brain Heart infusion powder, 15 g / l agar, 91 g / l sorbitol, and 20 μg / l kanamycin). Colonies were incubated at 30°C for 72 hours to induce secondary recombination in BHI medium. -2 ~10 -3The sample was diluted to [specific concentration] and spread onto antibiotic-free 2YT agar plates (containing 10% sucrose). Strains that showed no kanamycin resistance and could grow in a medium containing 10% sucrose were selected and named iolT2(E424K).

[0066] Experimental Example 1. Evaluation of L-glutamate production capacity of a bacterial strain expressing the myo-inositol facilitator IolT2 mutant. We compared the L-glutamate production capacity of the parent strain U3 and the mutant strain iolT2(E424K), which was introduced with the myo-inositol facilitator IolT2 mutant.

[0067] Each bacterial strain (parent strain or mutant strain) was inoculated at a volume of 1% into a 100 mL flask containing 10 mL of the L-glutamic acid production medium shown in Table 2 below, and cultured with shaking at 30°C and 200 rpm for 48 hours. After the culture period, the concentration of L-glutamic acid in the medium was measured using HPLC (Agilent), and the results are shown in Table 3 below.

[0068] [Table 2]

[0069] [Table 3]

[0070] As shown in Table 3 above, mutant strains into which the myo-inositol facilitator IolT2 mutant was introduced showed an approximately 40.76% increase in L-glutamate production compared to the parent strain, due to the substitution of glutamate at position 424 with lysine. These results suggest that the introduction of point mutations in the myo-inositol facilitator IolT2 has an effective effect on L-glutamate productivity.

[0071] The present invention has been described above, focusing on its preferred embodiments. Those with ordinary skill in the art to which the present invention pertains will understand that the present invention can be realized in modified forms without departing from its essential characteristics. Therefore, the disclosed embodiments should be considered in an explanatory rather than restrictive manner. The scope of the present invention is shown in the claims, not in the above description, and all differences within an equivalent scope should be interpreted as being included in the present invention.

[0072] [Accession Number] Depository name: Korea Center for Microbial Conservation (KCCM) Accession number: KCCM13218P Date of acceptance: 20220629

Claims

1. The myo-inositol facilitator IolT2 mutant consists of the amino acid sequence of SEQ ID NO: 2, in which the 424th glutamic acid in the amino acid sequence of SEQ ID NO: 4 is replaced with lysine.

2. A polynucleotide encoding the variant described in claim 1.

3. A transformant comprising the mutant described in claim 1 or the polynucleotide described in claim 2.

4. The transformant according to claim 3, wherein the transformant is a microorganism of the genus Corynebacterium.

5. The transformant according to claim 3, wherein the transformant has the ability to produce L-glutamic acid.

6. A method for producing L-glutamic acid, comprising the step of culturing the transformant described in claim 3 in a culture medium.

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