Corynebacterium mutant microorganism capable of producing L-glutamic acid and method for producing L-glutamic acid using the same

Abstract

The present invention relates to a Corynebacterium mutant microorganism that produces L-glutamic acid and a method for producing L-glutamic acid using the same, and more specifically to a novel mutant, polynucleotide, and transformant of pantoate-beta-alanine ligase involved in the L-glutamic acid biosynthetic pathway, and a method for producing L-glutamic acid using the same. The pantoate-beta-alanine ligase mutant of the present invention has altered enzymatic activity due to the substitution of one or more amino acids in the amino acid sequence constituting pantoate-beta-alanine ligase, and a recombinant microorganism containing the same can efficiently produce L-glutamic acid.

Description

[Technical Field]

[0001] The present invention relates to a mutant Corynebacterium microorganism that produces L-glutamic acid and a method for producing L-glutamic acid using the same. More specifically, the present invention relates to a novel mutant, polynucleotide, and transformant of pantoate-beta-alanine ligase involved in the biosynthetic pathway of L-glutamic acid, and a method for producing L-glutamic acid using the same. [Background technology]

[0002] L-glutamic acid is a typical amino acid produced by microbial fermentation. Monosodium L-glutamate (MSG) balances the overall taste of food, increasing the palatability of meat, fish, chicken, vegetables, sauces, soups, and seasonings. It can also enhance the taste of low-salt foods with up to 30% less salt, making it widely used as a seasoning for home use and processed food production.

[0003] A brief look at the fermentation pathway of L-glutamate shows that glucose primarily passes through the glycolytic pathway, but some of it is metabolized via the pentose phosphate pathway to two molecules of pyruvic acid. One of these molecules fixes CO2 to become oxaloacetic acid, and the other molecule combines with acetyl CoA to become citric acid. Furthermore, oxaloacetic acid and citric acid enter the citric acid cycle to become α-ketoglutaric acid. Here, the oxidative metabolic pathway for oxidizing alpha-ketoglutaric acid to succinic acid is lacking, and in addition, isocitrate dehydrogenase and glutamate dehydrogenase are closely involved, so 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 under natural conditions or mutant strains modified to enhance glutamic acid production. Recently, to improve L-glutamic acid production efficiency, genetic engineering has been applied to microorganisms such as Escherichia coli and Corynebacterium, which are commonly used to produce useful substances such as amino acids and nucleic acids, to develop a variety of recombinant or mutant strains with excellent L-glutamic acid production capabilities and L-glutamic acid production methods using these strains. In particular, attempts have been made to increase L-glutamic acid production by targeting genes for enzymes, transcription factors, transport proteins, and other proteins involved in the L-glutamic acid biosynthesis pathway or by inducing mutations in the promoters that regulate their expression. However, because there are dozens of proteins, including enzymes, transcription factors, and transport proteins, directly or indirectly involved in L-glutamic acid production, much research is needed to determine whether altering the activity of these proteins can increase L-glutamic acid production. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] U.S. Patent No. 6,852,516 [Patent Document 2] U.S. Patent No. 6,962,805 Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide a novel pantoate-beta-alanine ligase mutant.

[0007] Another object of the present invention is to provide a polynucleotide encoding the mutant.

[0008] A further object of the present invention is to provide a transformant comprising the mutant or polynucleotide.

[0009] Another object of the present invention is to provide a method for producing L-glutamic acid using the transformant. [Means for solving the problem]

[0010] One aspect of the present invention provides a pantoate-beta-alanine ligase mutant consisting of the amino acid sequence of SEQ ID NO: 2, in which the proline at position 174 in the amino acid sequence of SEQ ID NO: 4 has been substituted with serine.

[0011] The "pantoate-beta-alanine ligase" used in the present invention catalyzes the condensation of pantoate with beta-alanine to form pantothenate and is involved in the amino acid biosynthesis pathway. The pantoate-beta-alanine ligase may be a gene encoding the pantoate-beta-alanine ligase or a sequence substantially identical thereto. Here, "substantial identity" means that when the respective gene sequences, i.e., base sequences or nucleotide sequences, are aligned and analyzed with any other nucleotide sequence for maximum correspondence, the any other nucleotide sequence has 70% or more, 80% or more, 90% or more, or 98% or more sequence identity with the respective nucleotide sequence.

[0012] The pantoate-beta-alanine ligase of the present invention is encoded by the ncgl2595 gene and comprises the amino acid sequence of SEQ ID NO:4.

[0013] According to one embodiment of the present invention, the amino acid sequence of SEQ ID NO: 4 may be derived from a wild-type microorganism of the genus Corynebacterium.

[0014] More specifically, the Corynebacterium microorganism may be Corynebacterium glutamicum.

[0015] As used herein, a "variant" refers to a polypeptide in which one or more amino acids are conservatively substituted, deleted, modified, or added at the N-terminus, C-terminus, and / or internally in the amino acid sequence of a specific gene, resulting in a difference from the amino acid sequence of the variant before the mutation, but maintaining its functions or properties. Here, a "conservative substitution" refers to the substitution of 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. The amino acid is selected from alanine (Ala), isoleucine (Ile), valine (Val), leucine (Leu), methionine (Met), asparagine (Asn), cysteine ​​(Cys), glutamine (Gln), serine (Ser), threonine (Thr), phenylalanine (Phe), tryptophan (Trp), tyrosine (Tyr), aspartic acid (Asp), glutamic acid (Glu), arginine (Arg), histidine (His), lysine (Lys), glycine (Gly), and proline (Pro).

[0016] Variants also include those in which one or more portions have been removed, such as the N-terminal leader sequence or transmembrane domain, or portions have been removed from the N- and / or C-terminus of the mature protein.

[0017] The ability of such mutants may be increased (strengthened), unchanged, or decreased (weakened) compared to the protein before mutation. Here, "increased or enhanced" includes cases where the activity of the protein itself is increased compared to the protein before mutation, cases where the overall level of enzymatic activity in cells is higher than that of a wild-type strain or a strain expressing the protein before mutation due to increased expression or translation of the gene encoding the protein, or a combination thereof. Furthermore, "decreased or weakened" includes cases where the activity of the protein itself is decreased compared to the protein before mutation, cases where the overall level of enzymatic activity in cells is lower than that of a wild-type strain or a strain expressing the protein before mutation due to inhibited expression or translation of the gene encoding the protein, or a combination thereof. In the present invention, "mutant" may be interchangeably referred to as "mutated type," "transformed," "mutated polypeptide," "mutated protein," "mutation," etc.

[0018] The mutant of the present invention is a pantoate-beta-alanine ligase in which the proline at the 174th amino acid in the amino acid sequence of SEQ ID NO: 4 is replaced with serine, and may consist of the amino acid sequence of SEQ ID NO: 2.

[0019] Another aspect of the present invention provides polynucleotides encoding the pantoate-beta-alanine ligase variants.

[0020] The term "polynucleotide" as used herein refers to a nucleotide polymer in which nucleotide units are linked in a long chain by covalent bonds, and is a DNA or RNA chain of a certain length or more, and more specifically, refers to a polynucleotide fragment encoding the above variant.

[0021] The polynucleotide may comprise a base sequence encoding the amino acid sequence of SEQ ID NO:2.

[0022] According to one embodiment of the present invention, the polynucleotide may comprise the base sequence shown in SEQ ID NO:1.

[0023] Another aspect of the present invention provides a vector comprising a polynucleotide encoding the pantoate-beta-alanine ligase mutant.

[0024] Another aspect of the present invention provides a transformant comprising the pantoate-beta-alanine ligase mutant or polynucleotide.

[0025] As used herein, the term "vector" refers to any type of nucleic acid sequence delivery structure used to transfer and express a gene of interest in a host cell. Unless otherwise specified, the term "vector" can refer to a vector in which the nucleic acid sequence carried therein is inserted into the host cell genome and expressed, and / or a vector in which the nucleic acid sequence is expressed independently. Such vectors contain essential regulatory elements operably linked to allow expression of the gene insert. "Operably linked" means that the gene of interest and its regulatory sequences are functionally linked to each other to enable gene expression. "Regulatory elements" include a promoter for transcription, an optional operator sequence for transcription regulation, a sequence encoding a suitable mRNA ribosomal binding site, and sequences for regulating the termination of transcription and translation.

[0026] The vector used in the present invention is not particularly limited as long as it is replicable in host cells, and any vector known in the art can be used. Examples of such vectors include naturally occurring or recombinant plasmids, cosmids, viruses, and bacteriophages. Examples of phage or cosmid vectors include pWE15, M13, λMBL3, λMBL4, λIXII, λASHII, λAPII, λt10, λt11, Charon4A, and Charon21A. Examples of plasmid vectors include, but are not limited to, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, and pET.

[0027] The vector can typically be constructed as a vector for cloning or a vector for expression. Expression vectors can be those commonly used in the art to express foreign genes or proteins in plants, animals, or microorganisms, and can be constructed by various methods known in the art.

[0028] The "recombinant vector" used in the present invention may be replicable independently of the genome of a suitable host cell after transformation, or may be integrated into the genome itself. In this regard, the "suitable host cell" refers to a cell in which the vector is replicable and may contain a replication origin, which is a specific base sequence from which replication is initiated. For example, when the vector used is an expression vector and a prokaryotic cell is used as the host, it typically contains a strong promoter capable of driving transcription (e.g., pLλ promoter, CMV promoter, trp promoter, lac promoter, tac promoter, T7 promoter), a ribosome binding site for the initiation of transcription, and a transcription / transcription termination sequence. When a eukaryotic cell is used as the host, replication origins operable in eukaryotic cells and contained in the vector include, but are not limited to, the f1 origin, SV40 origin, pMB1 origin, adenovirus origin, AAV origin, and BBV origin. Alternatively, promoters derived from the genome of mammalian cells (e.g., metallothionine promoter) or promoters derived from mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, HSV tk promoter) may be used, and generally have a polyadenylation sequence as a transcription termination sequence.

[0029] The recombinant vector may contain a selection marker, which is used to select transformants (host cells) transformed with the vector, and only cells expressing the selection marker can survive in a medium treated with the selection marker, allowing for the selection of transformed cells. Representative examples of the selection marker include, but are not limited to, kanamycin, streptomycin, and chloramphenicol.

[0030] A transformant can be produced 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.

[0031] When transforming prokaryotic cells to produce recombinant microorganisms, host cells that can be used include, but are not limited to, Escherichia coli strains such as E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli X1776, E. coli W3110, and E. coli XL1-Blue; Bacillus strains such as Bacillus subtilis and Bacillus thuringiensis; Corynebacterium strains; and various Enterobacteriaceae strains such as Salmonella typhimurium, Serratia marcescens, and Pseudomonas species.

[0032] When transforming eukaryotic cells to produce recombinant microorganisms, host cells may be used, including, but 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.

[0033] As used herein, "transformation" refers to the phenomenon of artificially causing genetic changes by introducing exogenous DNA into a host cell, and "transformant" refers to a host cell into which exogenous DNA has been introduced and which stably maintains expression of a target gene.

[0034] For the transformation, a suitable vector introduction technique can be selected depending on the host cell, allowing the target gene or a recombinant vector containing the gene to be expressed in the host cell. For example, vector introduction may be performed by, but is not limited to, electroporation, heat shock, calcium phosphate (CaPO) precipitation, calcium chloride (CaCl) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, lithium acetate-DMSO method, or a combination thereof. The transformed gene may be inserted into the host cell's chromosome or located extrachromosomally, as long as it can be expressed in the host cell.

[0035] The transformant includes cells transfected, transformed, or infected with a recombinant vector according to the present invention in vivo or in vitro, and may be used interchangeably with recombinant host cells, recombinant cells, or recombinant microorganisms.

[0036] According to one embodiment of the present invention, the transformant may be a microorganism of the genus Corynebacterium.

[0037] More specifically, examples of the Corynebacterium microorganism include Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium callunae, Corynebacterium suranareeae, Corynebacterium lubricantis, Corynebacterium doosanense, Corynebacterium efficiens, and Corynebacterium uteri. Corynebacterium uterequi, Corynebacterium stationis, Corynebacterium pacaense, Corynebacterium singulare, Corynebacterium humireducens, Corynebacterium marinum, Corynebacterium halotolerans, Corynebacterium spheniscorum, Corynebacterium freiburgense, Corynebacterium striatum striatum, Corynebacterium canis, Corynebacterium ammoniagenes, Corynebacterium renale, Corynebacterium portisoriiExamples of suitable bacteria include, but are not limited to, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium caspium, Corynebacterium testudinoris, Corynebacterium pseudopelargi, or Corynebacterium flavescens.

[0038] The transformant of the present invention may be, but is not limited to, a strain containing the above-mentioned pantoate-beta-alanine ligase mutant or a polynucleotide encoding it, or a strain containing a vector containing it, a strain expressing the pantoate-beta-alanine ligase mutant or polynucleotide, or a strain having activity against the pantoate-beta-alanine ligase mutant.

[0039] According to one embodiment of the present invention, the transformant may have the ability to produce L-glutamic acid.

[0040] The transformant may naturally have the ability to produce L-glutamic acid, or may be one to which the ability to produce L-glutamic acid has been artificially imparted.

[0041] According to one embodiment of the present invention, the transformant may have an altered pantoate-beta-alanine ligase activity, thereby improving L-glutamic acid production ability.

[0042] As used herein, "improved production ability" means increased L-glutamic acid productivity compared to the parent strain. The parent strain refers to a wild-type or mutant strain that can be mutated, and includes strains that can be directly mutated or transformed with a recombinant vector. In the present invention, the parent strain may be a wild-type Corynebacterium microorganism or a Corynebacterium microorganism mutated from the wild type.

[0043] The transformant of the present invention exhibits increased L-glutamic acid productivity compared to the parent strain due to altered pantoate-beta-alanine ligase activity caused by the introduction of the pantoate-beta-alanine ligase mutant. More specifically, the transformant may exhibit at least a 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% increase in L-glutamic acid production compared to the parent strain, or a 1.1-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, or 10-fold increase in L-glutamic acid production compared to the parent strain, but is not limited thereto. For example, a transformant containing the pantoate-beta-alanine ligase mutant may have an increased production of L-glutamic acid of 5% or more, specifically 5 to 50% (preferably 7 to 30%), compared to the parent strain.

[0044] Another aspect of the present invention provides a method for producing L-glutamic acid, comprising the steps of culturing the transformant in a medium and recovering L-glutamic acid from the transformant or from the medium in which the transformant has been cultured.

[0045] The culture may be performed using an appropriate medium and culture conditions known in the art, and those skilled in the art can easily adjust the medium and culture conditions. Specifically, the medium may be, but is not limited to, a liquid medium. The culture method may include, but is not limited to, batch culture, continuous culture, fed-batch culture, or a combination thereof.

[0046] According to one embodiment of the present invention, the medium should be appropriately adapted to meet the requirements of a specific strain and can be modified by a person skilled in the art. Culture media for Corynebacterium microorganisms can be found in known literature (Manual of Methods for General Bacteriology, American Society for Bacteriology, Washington DC, USA, 1981), but are not limited thereto.

[0047] According to one embodiment of the present invention, the medium can 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 can be used individually or in mixtures, but are not limited to these. Usable nitrogen sources include peptone, yeast extract, broth, malt extract, corn steep liquor, soybean meal, and urea, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate. Nitrogen sources can also be used individually or in mixtures, but are not limited to these. Usable phosphorus sources include, but are not limited to, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts. The culture medium may also contain, but is not limited to, metal salts necessary for growth, such as magnesium sulfate or iron sulfate. Other essential growth substances, such as amino acids and vitamins, may also be included. Furthermore, appropriate precursors can be used for the culture medium. The medium or individual components may be added to the culture solution in a suitable manner during the culture process, such as batchwise or continuous, but not limited to these.

[0048] According to one embodiment of the present invention, the pH of the culture medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid to the microbial culture medium in an appropriate manner during cultivation. Furthermore, foam formation can be suppressed during cultivation using an antifoaming agent such as a 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 in the culture medium. The temperature of the culture medium can generally be 20°C to 45°C, for example, 25°C to 40°C. The cultivation period can be continued until a desired amount of useful substance is produced, and can be, for example, 10 to 160 hours.

[0049] According to one embodiment of the present invention, the step of recovering L-glutamic acid from the cultured transformant or the medium in which the transformant has been cultured can be carried out by using a suitable method known in the art depending on the culture method, such as, but not limited to, centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, differential dissolution (e.g., ammonium sulfate precipitation), and chromatography (e.g., ion exchange, affinity, hydrophobic, and size exclusion).

[0050] According to one embodiment of the present invention, the step of recovering L-glutamic acid can be carried out by centrifuging the culture medium at low speed to remove the biomass, and separating the resulting supernatant by ion exchange chromatography.

[0051] According to one embodiment of the present invention, the step of recovering L-glutamic acid can include a step of purifying L-glutamic acid. [Effects of the Invention]

[0052] The pantoate-beta-alanine ligase mutant of the present invention has altered enzymatic activity due to the substitution of one or more amino acids in the amino acid sequence constituting the pantoate-beta-alanine ligase, and a recombinant microorganism containing the mutant can efficiently produce L-glutamic acid. [Brief explanation of the drawings]

[0053] [Figure 1] FIG. 1 shows the structure of plasmid pK19msb according to one embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0054] The present invention will be described in more detail below. However, such description is merely provided as an example for understanding the present invention, and the scope of the present invention is not limited by such exemplary description.

[0055] Example 1. Construction of vectors for expression of pantoate-beta-alanine ligase mutants A vector was constructed that expresses a mutant in which the proline (P) at position 174 in the amino acid sequence of pantoate-beta-alanine ligase (SEQ ID NO: 4) was substituted with serine (S).

[0056] Using wild-type Corynebacterium glutamicum ATCC13869 gDNA as a template, PCR was performed using the primer pair Primer 1 and Primer 2 and the primer pair Primer 3 and Primer 4, respectively. Subsequently, overlapping PCR was performed again using the mixture of the two PCR products as a template and Primer 1 and Primer 4 to obtain a fragment. Takara PrimeSTAR Max DNA polymerase was used as the polymerase. PCR amplification conditions were denaturation at 95°C for 5 minutes, followed by 30 cycles of 95°C for 30 seconds, 58°C for 30 seconds, and 72°C for 1 minute 30 seconds, followed by 72°C for 5 minutes. The pK19msb vector was digested with smaI and ligated with the PCR product (fragment). The resulting plasmid was designated pK_panC(P174S).

[0057] The primer sequences used to construct the vectors are shown in Table 1 below.

[0058] [Table 1]

[0059] Example 2. Construction of mutant strains incorporating pantoate-beta-alanine ligase mutants Corynebacterium glutamicum U3 (KCCM13218P) was used as the parent strain for introducing the pantoate-beta-alanine ligase mutant, and a modified electrocompetent cell preparation method based on the method of van der Rest et al. was used to transform the U3 strain.

[0060] First, a seed culture of the U3 strain was prepared by primary cultivation in 100 ml of 2YT medium (containing 16 g / L tryptone, 10 g / L yeast extract, and 5 g / L sodium chloride) supplemented with 2% glucose. Subsequently, 100 ml of glucose-free 2YT medium was supplemented with 1 mg / ml isonicotinic acid hydrazine and 2.5% glycine. The seed culture was inoculated to an OD610 value of 0.3 and then cultured at 18°C ​​and 180 rpm for 12–16 hours until an OD610 value of 1.2–1.4 was reached. The culture was then placed on ice for 30 minutes and centrifuged at 4°C and 4000 rpm for 15 minutes. The supernatant was discarded, and the precipitated U3 strain was washed four times with 10% glycerol solution. Finally, it was resuspended in 0.5 ml of 10% glycerol solution to prepare competent cells. Electroporation was performed using a Bio-Rad electroporator. The prepared competent cells and pK_panC(P174S) vector were added to an electroporation cuvette (0.2 mm), and an electric shock was applied at 2.5 kV, 200 Ω, and 12.5 μF. Immediately after the electric shock, 1 ml of regeneration medium (containing Brain Heart infusion 18.5 g / L and sorbitol 0.5 M) was added, and the cells were heated at 46°C for 6 minutes. After cooling at room temperature, the mixture was transferred to a 15 ml cap tube and cultured at 30°C for 2 hours, and then smeared on a selective medium (containing tryptone 5 g / L, NaCl 5 g / L, yeast extract 2.5 g / L, Brain Heart infusion powder 18.5 g / L, agar 15 g / L, sorbitol 91 g / L, and kanamycin 20 μg / L). The colonies formed after 72 hours of culture at 30°C were cultured in BHI medium until stationary phase to induce secondary recombination, and then 10 -5 ~10 -7 The resulting mixture was diluted to 100°C and smeared on antibiotic-free 2YT plates (containing 10% sucrose). A strain that lacked kanamycin resistance and could grow on the medium containing 10% sucrose was selected and named PAN1.

[0061] Experimental Example 1: Evaluation of L-glutamic acid production ability of mutant strains into which pantoate-beta-alanine ligase mutants were introduced The L-glutamic acid production ability of the parent strain U3 was compared with that of the mutant strain PAN1 into which the pantoate-beta-alanine ligase mutant had been introduced.

[0062] Each strain (parent strain or mutant strain) was inoculated at 1% volume into a 100 mL flask containing 10 mL of the 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 was completed, the concentration of L-glutamic acid in the medium was measured using HPLC (Agilent), and the results are shown in Table 3 below.

[0063] [Table 2]

[0064] [Table 3]

[0065] As shown in Table 3, the mutant strains into which the pantoate-beta-alanine ligase mutant was introduced showed an increase in L-glutamic acid production by about 13% compared to the parent strain. These results suggest that the pantoate-beta-alanine ligase mutation increases the flow of carbon sources in the glutamic acid production pathway, thereby increasing L-glutamic acid production.

[0066] The present invention has been described above with a focus on its preferred embodiments. Those skilled in the art will understand that the present invention can be realized in modified forms without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a restrictive perspective. The scope of the present invention is defined by the claims, not the above description, and all variations within the scope of the claims should be construed as being within the scope of the present invention.

[0067] [Accession number] Depository institution: Korea Center for Microorganisms (KCCM) Accession number: KCCM13218P Date of acceptance: 20220629

Claims

1. A pantoate-beta-alanine ligase mutant consisting of the amino acid sequence of SEQ ID NO: 2, in which the 174th proline in the amino acid sequence of SEQ ID NO: 4 is replaced with serine.

2. A polynucleotide encoding the variant of claim 1.

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

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

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

6. Cultivating the transformant according to claim 3 in a medium; and recovering L-glutamic acid from the transformant or from the medium in which the transformant has been cultured.

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