Mutant strain with improved L-glutamic acid production ability and method for producing L-glutamic acid using the same

Abstract

The present invention relates to a mutant strain with improved L-glutamic acid production ability and a method for producing L-glutamic acid using the same. In one embodiment of the present invention, the mutant strain has excellent L-glutamic acid production ability due to reduced or inactivated citramalate synthase activity, thereby reducing the production of the by-product citramalate. Additionally, a strain in which the YggB protein has been mutated enhances glutamic acid excretion, thereby improving the L-glutamic acid production yield. Therefore, the use of the mutant strain allows for more efficient L-glutamic acid production.

Description

[Technical field]

[0001] The present invention relates to a mutant strain having improved L-glutamic acid production ability and a method for producing L-glutamic acid using the same, more specifically, to a Corynebacterium sp. mutant strain having reduced L-glutamic acid production ability and reduced production of the by-product citramalate by weakening or inactivating citramalate synthase activity, and a method for producing L-glutamic acid using the same. [Background technology]

[0002] L-glutamic acid is a representative amino acid produced by microbial fermentation, and monosodium L-glutamate (MSG) is widely used as a seasoning for home and processed food production to balance and harmonize the overall flavor of food and beverages, increase the preference for foods such as meat, fish, chicken, vegetables, sauces, soups, and condiments, and enhance the taste of low-salt foods with up to 30% less salt.

[0003] L-glutamic acid is usually produced by fermentation using strains of the genus Brevibacterium or Corynebacterium and their mutants. In order to increase the amount of L-glutamic acid produced by microbial culture, methods have been used to increase the expression of genes in the biosynthetic pathway of L-glutamic acid by increasing the copy number of the relevant gene or modifying the promoter of the gene to regulate the activity of enzymes in the biosynthetic pathway. Specifically, the amount of L-glutamic acid produced has been increased by amplifying specific genes such as the pyc gene and fasR (U.S. Pat. No. 6,852,516) or by manipulating the promoter sites of the gdh, gltA, icd, pdh, and argG genes (U.S. Pat. No. 6,962,805).

[0004] Citramalate synthase (cimA) is an enzyme that synthesizes citramalate using acetyl-CoA and pyruvate as substrates. The cimA gene does not exist in Corynebacterium glutamicum, and it is known that the leuA gene, which is highly homologous to the cimA gene in Corynebacterium glutamicum, plays the role of citramalate synthase.

[0005] In the fermentation pathway of L-glutamic acid, acetyl-CoA is used as a substrate. Therefore, the inventors of the present application attempted to further improve the productivity of L-glutamic acid by conserving the substrate acetyl-CoA through weakening or inactivating the activity of citramalate synthase.

[0006] On the other hand, the yggB gene of coryneform bacteria is a homologue of the yggB gene of Escherichia coli (FEMS Microbiol Lett. 2003, 218(2), p305-309, Mol Microbiol. 2004, 54(2), p420-438), and has been reported to be a type of mechanosensitive channel (EMBO J. 1999, 18(7):1730-7). As a method for imparting L-glutamic acid producing ability to coryneform bacteria, there have been no reports specifically on the effect of the mutation of the present invention on L-glutamic acid production.

[0007] Therefore, the inventors of the present application have prepared a mutant strain in which the activity of citramalate synthase has been weakened or inactivated, and further prepared a mutant strain by inducing a mutation in the YggB protein involved in the excretion of glutamic acid, and have confirmed that the mutant strain has excellent L-glutamic acid producing ability, thereby completing the present invention. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Korean Patent No. 10-1138289 Summary of the Invention [Problem to be solved by the invention]

[0009] An object of the present invention is to provide a mutant strain of Corynebacterium sp. in which citramalate synthase activity is weakened or inactivated, thereby improving L-glutamic acid productivity.

[0010] Another object of the present invention is to (a) culturing a Corynebacterium genus mutant strain in a medium; (b) recovering L-glutamic acid from the cultured mutant strain or the culture medium. [Means for solving the problem]

[0011] One aspect of the present invention provides a mutant strain of Corynebacterium sp. in which citramalate synthase activity is weakened or inactivated, thereby improving L-glutamic acid productivity.

[0012] In the present invention, the term "citramalate synthase" refers to an enzyme that catalyzes the process of converting acetyl-CoA and pyruvate into citramalate and coenzyme A.

[0013] In the present invention, a strain with "improved productivity" means one that has increased L-glutamic acid productivity compared to the parent strain.

[0014] In the present invention, the term "mother strain" refers to a wild-type or mutant strain to be mutated, including a strain that is directly mutated or transformed with a recombinant vector, etc. In the present invention, the mother strain may be a wild-type Corynebacterium glutamicum strain or a strain mutated from the wild-type. Preferably, the mother strain may be the Corynebacterium glutamicum KCTC 11558BP strain.

[0015] According to one embodiment of the present invention, the mutant strain of Corynebacterium sp. having improved L-glutamic acid production ability may have a weakened or inactivated citramalate synthase activity compared to the parent strain.

[0016] In the present invention, "weakened activity" refers to the case where the level of citramalate synthase enzyme activity in cells is reduced compared to that of the parent strain, such as a wild-type strain or an untransformed strain. The weakening concept includes a case where the activity of the enzyme itself is reduced compared to the activity of the enzyme that the microorganism originally has due to a mutation in the gene encoding the enzyme, and a case where the expression level of the enzyme is reduced due to inhibition of transcription or translation of the gene encoding the enzyme, and the overall enzyme activity in cells is lower compared to that of a wild-type strain or an untransformed strain. In addition, in the present invention, "inactivated" refers to a state where the expression of the gene encoding citramalate synthase is not expressed at all compared to a wild-type strain or an untransformed strain, or where the expression is inactive even if it is expressed.

[0017] According to one embodiment of the present invention, the attenuation or inactivation of the citramalate synthase enzyme activity can be achieved by application of various methods known in the art. For example, a method of replacing a gene encoding the enzyme on a chromosome with a gene mutated to reduce the activity of the enzyme, including when the activity of the enzyme is completely eliminated; a method of introducing a mutation into an expression regulatory sequence (e.g., a promoter) of a gene encoding the enzyme on a chromosome; a method of replacing an expression regulatory sequence of a gene encoding the enzyme with a sequence that has weak or no activity; a method of deleting all or part of a gene encoding the enzyme on a chromosome; a method of introducing an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to a transcript of the gene on the chromosome and inhibits translation of the mRNA into an enzyme; a method of artificially adding a sequence complementary to the SD sequence to the front of the SD sequence of the gene encoding the enzyme to form a secondary structure to make it impossible for ribosomes to attach, and a reverse transcription engineering (RTE) method of adding a promoter to the 3' end of the ORF (open reading frame) of the relevant sequence so that it is reverse transcribed, and combinations thereof can also be used, but are not limited to these.

[0018] According to one embodiment of the present invention, the citramalate synthase may be encoded by the leuA (2-isopropylmalate synthase) gene.

[0019] In the present invention, the leuA (2-isopropylmalate synthase) gene is highly homologous to the cimA (citramalate synthase) gene, which is known to play the role of citramalate synthase in Corynebacterium sp. According to one embodiment of the present invention, the leuA gene has the nucleotide sequence of SEQ ID NO: 14.

[0020] According to one embodiment of the present invention, the activity of citramalate synthase is attenuated or inactivated by reducing the expression of the leuA gene encoding citramalate synthase, thereby attenuating or inactivating the activity of citramalate synthase.

[0021] In the present invention, "reduced expression" means that the expression level of a gene is reduced from the original expression level, and includes the case where expression is completely inhibited and no expression occurs. When it is desired to reduce expression in a microorganism, the expression can be reduced by replacing the initiation codon of gene translation, or the expression level of an existing gene can be reduced by genetic engineering. The method of modifying the sequence to regulate expression may be to induce a mutation in the expression regulatory sequence by deletion, insertion, non-conservative or conservative substitution, or a combination of these, in the nucleic acid sequence of the expression regulatory sequence, or to replace the promoter with a weaker promoter. The expression regulatory sequence includes, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating the termination of transcription and translation. When a gene whose expression is to be reduced exists in a microorganism to be mutated, the expression of the gene can be reduced, for example, by replacing a native promoter that drives the expression of the gene with a weak promoter or by introducing a mutation. Furthermore, the expression of the gene can be reduced by deleting all or a part of the existing gene.

[0022] According to one embodiment of the present invention, the expression of the leuA gene can be reduced by introducing mutations into the nucleic acid sequence, such as deletions, insertions, non-conservative or conservative substitutions, or a combination thereof, or by replacing it with a weaker promoter.

[0023] According to one embodiment of the present invention, the mutant Corynebacterium strain lacking the leuA gene produces less citramalate as a by-product than the parent strain, and conserves acetyl-CoA, thereby increasing the production of L-glutamic acid.

[0024] According to one embodiment of the present invention, the Corynebacterium mutant strain may be additionally mutated by replacing one or more leucines selected from the group consisting of leucine, the 93rd amino acid, and leucine, the 109th amino acid, in the amino acid sequence of the YggB protein represented by SEQ ID NO: 15, with alanine or valine.

[0025] In the present invention, the YggB gene refers to a gene encoding a mechanosensitive channel. The YggB gene corresponds to a complementary sequence of the sequence of 1,336,092 to 1,337,693 in the genome sequence registered in the NCBI database under Genbank Accession No. NC_003450, and is also referred to as NCgl1221. The YggB protein encoded by the YggB gene is registered under Genbank Accession No. NP_600492. According to one embodiment of the present invention, the YggB protein encoded by the YggB gene consists of the amino acid sequence of SEQ ID NO: 15.

[0026] According to one specific example, a mutation in which one or more leucines selected from the group consisting of leucine at the 93rd amino acid and leucine at the 109th amino acid in the amino acid sequence of the YggB protein represented by SEQ ID NO: 15 are substituted with alanine or valine removes the methyl group involved in the inside of the pore of the YggB protein capable of exporting L-glutamic acid, thereby increasing the diameter of the pore, thereby improving the L-glutamic acid production ability of a strain having the mutation.

[0027] Specifically, the mutant strain may contain the mutant YggB protein by mutation, or may be transformed with a vector containing a polynucleotide encoding the mutant YggB protein.

[0028] In the present invention, a "vector" refers to any medium for cloning and / or transferring a base to a host cell. A vector may be a replican to which other DNA fragments can be attached and cause replication of the attached fragment. A "replicon" refers to any genetic unit (e.g., a plasmid, a phage, a cosmid, a chromosome, a virus) that functions as an independent unit of DNA replication in vivo, i.e., that can replicate under its own control. In the present invention, the vector is not particularly limited as long as it can replicate in a host, and any vector known in the art can be used. The vector used to construct the recombinant vector may be in a natural state or a recombinant plasmid, cosmid, virus, or bacteriophage. For example, pWE15, M13, λEMBL3, λEMBL4, λFIXII, λDASHII, λZAPII, λgt10, λgt11, Charon4A, Charon21A, etc. can be used as phage vectors or cosmid vectors, and pDZ vectors, pBR series, pUC series, pBluescriptII series, pGEM series, pTZ series, pCL series, pET series, etc. Can be used vectors are not particularly limited, and known expression vectors can be used, but are not limited thereto.

[0029] In the present invention, "transformation" refers to the introduction of a gene into a host cell so that it can be expressed in the host cell, and the transformed gene can be contained regardless of whether it is inserted into the host cell's chromosome or located extrachromosomally, so long as it can be expressed in the host cell.

[0030] According to one embodiment of the present invention, a mutant Corynebacterium strain in which the leuA gene has been deleted and the YggB protein has been mutated can produce L-glutamic acid more efficiently than the parent strain, and the culture time for producing L-glutamic acid can be shortened.

[0031] According to one embodiment of the present invention, the Corynebacterium strain may be Corynebacterium glutamicum.

[0032] According to one embodiment of the present invention, the Corynebacterium strain is Corynebacterium ammoniagenes, Corynebacterium acetoacidophilum, Corynebacterium acetoglutamicum, Corynebacterium alkanolyticum, Corynebacterium callunae, Corynebacterium lilium, Corynebacterium melassecola, Corynebacterium thermoaminogenes, Corynebacterium efficiens, Corynebacterium efficiens, or Corynebacterium herculis, but is not limited thereto.

[0033] According to one embodiment of the present invention, the Corynebacterium sp. strain is most preferably Corynebacterium glutamicum KCTC 11558BP.

[0034] Another aspect of the present invention is (a) culturing a Corynebacterium genus mutant strain in a medium; and (b) recovering L-glutamic acid from the cultured mutant strain or the culture medium.

[0035] According to one embodiment of the present invention, the culture may be performed using a suitable medium and culture conditions known in the art, and a person skilled in the art can easily adjust and use the medium and culture conditions. Specifically, the medium may be a liquid medium, but is not limited thereto. The culture method may include, but is not limited to, for example, batch culture, continuous culture, fed-batch culture, or a combination thereof.

[0036] According to one embodiment of the present invention, the medium should be appropriately modified to meet the requirements of a specific strain by a skilled artisan. For the culture medium of Corynebacterium sp. strains, a known reference (Manual of Methods for General Bacteriology. American Society for Bacteriology. Washington DC, USA, 1981) may be referred to, but is not limited thereto.

[0037] According to one embodiment of the present invention, the medium may contain various carbon sources, nitrogen sources, and trace elemental components. Usable carbon sources include sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch, cellulose, oils and fats such as soybean oil, sunflower oil, castor oil, coconut oil, etc., fatty acids such as palmitic acid, stearic acid, linoleic acid, alcohols such as glycerol, ethanol, and organic acids such as acetic acid. These substances may be used individually or as mixtures, but are not limited to these. Usable nitrogen sources may 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 may also be used individually or as mixtures, but are not limited to these. Usable phosphorus sources may 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 metal salts necessary for growth, such as, but not limited to, magnesium sulfate or iron sulfate. Other essential growth substances, such as amino acids and vitamins, may also be included. Precursors suitable for the culture medium may also be used. The medium or individual components may be added to the culture solution during the culture process in a suitable manner, such as, but not limited to, in a batch or continuous manner.

[0038] According to one embodiment of the present invention, during the cultivation, a compound such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid can be added to the microorganism culture solution in an appropriate manner to adjust the pH of the culture solution. Also, during the cultivation, an antifoaming agent such as fatty acid polyglycol ester can be used to suppress the generation of bubbles. Additionally, oxygen or an oxygen-containing gas (e.g., air) can be injected into the culture solution to maintain the aerobic state of the culture solution. The temperature of the culture solution may usually be 20°C to 45°C, for example, 25°C to 40°C. The cultivation period may continue until a useful substance is obtained in a desired production amount, for example, 10 to 160 hours.

[0039] According to one embodiment of the present invention, the step of recovering L-glutamic acid from the cultured mutant strain and the culture medium can be carried out by 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), chromatography (e.g., ion exchange, affinity, hydrophobic, and size exclusion), etc.

[0040] According to one embodiment of the present invention, the step of recovering glutamic acid can be carried out by subjecting the culture to low speed centrifugation to remove the biomass, and separating the resulting supernatant by ion exchange chromatography.

[0041] According to one embodiment of the present invention, the step of recovering L-glutamic acid may include a step of purifying L-glutamic acid. Effect of the Invention

[0042] In one embodiment of the present invention, the mutant strain has excellent L-glutamic acid production ability due to the weakened or inactivated activity of citramalate synthase, thereby reducing the production of the by-product citramalate. In addition, a strain in which the YggB protein is mutated enhances the excretion of glutamic acid, thereby improving the production yield of L-glutamic acid. Therefore, by using the mutant strain, L-glutamic acid can be produced more effectively. [Brief description of the drawings]

[0043] [Figure 1] 1 shows a synthetic route for citramalate. [Diagram 2] 1 shows the pKLA vector prepared for disrupting the leuA gene in the Corynebacterium glutamicum KCTC 11558BP strain. [Diagram 3] The positions of primers 1 and 4 in the deleted portion of the leuA gene are shown. [Figure 4] A shows the structure of the YggB protein, B shows the pore structure of the YggB protein in which the 93rd leucine in the amino acid sequence of the YggB protein has been replaced with alanine, and C shows the pore structure of the YggB protein in which the 109th leucine in the amino acid sequence of the YggB protein has been replaced with alanine. [Diagram 5] 1 shows the pKY93 vector constructed to replace the 93rd amino acid in the amino acid sequence of the YggB protein in the I10 strain with another amino acid. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0044] One or more specific examples will be described in more detail below with reference to examples. However, these examples are merely for illustrative purposes and are not intended to limit the scope of the present invention.

[0045] Example 1. Analysis of intracellular metabolites in the fermentation solution of a bacterial strain Corynebacterium glutamicum KCTC 11558BP is a mutant strain obtained by treating the I6 strain with nitrosoguanidine (NTG), a chemical that causes DNA mutations. Since it has molasses resistance, it can produce L-glutamic acid at a high yield by using molasses, which is a low-cost raw material, as the main carbon source (Korean Patent Publication No. 10-2011-0034718).

[0046] The amounts of metabolites and L-glutamic acid in the cells of Corynebacterium glutamicum I6 (hereinafter, I6) and Corynebacterium glutamicum KCTC 11558BP (hereinafter, KCTC 11558BP), which have glutamic acid producing ability, were analyzed.

[0047] KCTC 11558BP and I6 were inoculated into 25 ml of CM liquid medium (glucose 1%, polypeptone 1%, yeast extract 0.5%, beef extract 0.5%, NaCl 0.25%, urea 0.2%, 50% NaOH 100 l, pH 6.8, 1 L standard) and cultured at 30°C with shaking at 200 rpm. When the cell growth reached the exponential phase during the culture, the cells were separated from the culture medium by rapid vacuum filtration (Durapore HV, 0.45 m; Millipore, Billerica, MA). The filter with the cells adsorbed was washed twice with 10 ml of cold water and then immersed in methanol containing 5 M morpholine ethanesulfonic acid and 5 M methionine sulfone for 10 min to stop the reaction. The resulting extract was then mixed with an equal volume of chloroform and 0.4 volumes of water, and the aqueous phase was applied to a spin column to remove protein contaminants. The filtered extract was analyzed for relative citramalate content by capillary electrophoresis mass spectrometry, and the results are shown in Table 1 below.

[0048] In addition, high performance liquid chromatography (HPLC; Agilent, USA) using a C18 column (Apollo C18, 5 μm, 150 mm × 4.6 mm) was performed to analyze the production amount of L-glutamic acid. The results are shown in Table 1 below. The analysis buffer, which has a composition of KH2PO4 8.85 g / L, tetrabutyl ammonium hydroxide solution 1.652 g / L, acetonitrile 10 ml / L, and sodium azide 12.5 mg / L and has a pH of 3.15 (adjusted with phosphoric acid), was used after filtering with a 0.45 μm membrane filter (HA).

[0049] As shown in Table 1, it was confirmed that the amount of citramalate produced by KCTC 11558BP was increased by about 3 times compared to the control I6.

[0050] [Table 1]

[0051] Example 2. Preparation of a mutant strain of Corynebacterium glutamicum lacking the leuA gene FIG. 1 shows the synthesis pathway of citramalate. Citramalate is produced by the cimA (citramalate synthase) gene in Escherichia coli, but the cimA gene does not exist in Corynebacterium glutamicum. However, it is known that the leuA gene, which is highly homologous to the cimA gene, plays the role of citramalate synthase in Corynebacterium glutamicum. Therefore, we attempted to further improve the productivity of L-glutamic acid by disrupting leuA (2-isopropylmalate synthase) to reduce the by-product citramalate and conserve acetyl-CoA, a substrate used in the production of L-glutamic acid. SEQ ID NO: 14 shows the nucleotide sequence of the leuA gene.

[0052] Example 2-1. Construction of leuA gene disruption vector Corynebacterium glutamicum KCTC 11558BP, which has L-glutamic acid producing ability, was used to disrupt the leuA gene (NCgl0245) in Corynebacterium glutamicum.

[0053] Chromosomal DNA of Corynebacterium glutamicum KCTC 11558BP strain was isolated and used as a template for PCR using the following primer sets 1 and 2 and primer sets 3 and 4, respectively, at 95°C for 30 seconds, 57°C for 30 seconds, and 72°C for 60 seconds, repeated for 30 cycles. The resulting PCR products (H1 and H2 in Figure 2) were purified.

[0054] The purified PCR products were further used as templates for crossover polymerase chain reaction (PCR) and amplified once more with primer set 1, 4.

[0055] The 1600 bp crossover PCR product for deleting the leuA gene was purified, digested with XbaI and SalI restriction enzymes (Takara, Japan), and then cloned into pK19mobSacB vector (Gene, 145:69-73, 1994) digested with the same restriction enzymes to prepare a vector for disrupting the leuA gene, which was named pKLA. Figure 2 shows the pKLA vector prepared for disrupting the leuA gene in the Corynebacterium glutamicum KCTC 11558BP strain.

[0056] [Table 2]

[0057] Example 2-2. Transformation of Corynebacterium glutamicum strain and preparation of leuA deletion mutant strain Using the method of van der Rest (Appl. Microbiol. Biotechnol., 52, 541-545, 1999), the vector pKLA produced in Example 2-1 was introduced by electric shock into Corynebacterium glutamicum KCTC 11558BP strain (hereinafter referred to as KCTC 11558BP) which had been made transformable by the electrocompetent cell production method.

[0058] Specifically, the KCTC 11558BP strain was primarily cultured in 100 ml of 2YT medium (tryptone 16 g / l, yeast extract 10 g / l, sodium chloride 5 g / l) supplemented with 2% glucose, and the same medium was supplemented with 1 mg / ml of isonicotinic acid hydrazine and 2.5% glycine, except for glucose. Then, the OD 610After inoculating the seed culture to an OD value of 0.3, the culture was incubated at 18°C ​​and 180 rpm for 12 to 16 hours. 610 The pH was adjusted so that the pH value was 1.2 to 1.4. After leaving it on ice for 30 minutes, it was centrifuged at 4000 rpm at 4℃ for 15 minutes. The supernatant was then discarded, and the precipitated KCTC 11558BP strain was washed four times with 10% glycerol solution, and finally resuspended in 0.5 ml of 10% glycerol solution. Electroporation was performed using a Bio-Rad electroporator. The competent cells prepared by the above method were placed in an electroporation cuvette (0.2 mm), and the pKLA vector prepared in Example 2-1 was added, followed by electric shock at 2.5 kV, 200 Ω, and 12.5 μF. Immediately after the electric shock, 1 ml of regeneration medium (Brain Heart infusion 18.5 g / l, sorbitol 0.5 M) was added, and the mixture was heat-treated at 46℃ for 6 minutes. After cooling at room temperature, the tubes were transferred to 15 ml cap tubes and cultured at 30°C for 2 hours, and then smeared on selective medium (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, kanamycin 20 μg / l). The colonies generated after 72 hours of culture at 30°C were cultured in BHI medium until stationary phase to induce secondary recombination, and then cultured for 10 hours. -5 ~10 -7 The resulting solution was diluted to 100 μl and smeared on an antibiotic-free plate (containing 10% sucrose) to select strains that had no kanamycin resistance and grew in a medium containing 10% sucrose. The selected strains were identified as Corynebacterium glutamicum strains lacking the leuA gene using primer set 1 and 4 shown in Table 2, and the strain lacking the leuA gene was named "I10." Figure 3 shows the positions of primers 1 and 4 in the portion where the leuA gene was deleted.

[0059] Example 2-3. Confirmation of auxotrophy of leuA-deficient strain (I10) The leuA-deficient strain (hereinafter, I10) prepared in Example 2-2 is a leucine auxotrophic strain because the leuA gene is deleted. In order to confirm this, the strain was smeared on a minimal medium and a leucine-supplemented medium prepared with the compositions shown in Table 3 below, and cultured at 30° C. for 24 hours to measure the degree of growth. The results are shown in Table 4.

[0060] [Table 3]

[0061] As shown in Table 4, in minimal medium without leucine, the growth of the I10 strain was rapidly reduced compared to the parent strain, Corynebacterium glutamicum KCTC 11558BP strain, and growth was restored by adding leucine, confirming that the I10 strain is deficient in the leuA gene.

[0062] [Table 4]

[0063] Example 3. Confirmation of L-glutamic acid and citramalate production by Corynebacterium glutamicum strain (I10) lacking the leuA gene Using the active plate medium prepared with the contents shown in Table 5 below, a leuA-deficient strain (hereinafter, I10) and Corynebacterium glutamicum KCTC 11558BP (hereinafter, KCTC 11558BP) were cultured at 30°C for 24 hours. Then, 10 ml of L-glutamic acid flask medium prepared with the contents shown in Table 6 below was placed in a 100 ml flask, and the bacteria cultured above were inoculated using a loop and cultured at 30°C, 200 rpm for 48 hours. The culture solution was used to analyze the amount of L-glutamic acid produced and intracellular metabolites. The amount of citramalate relative to the amount of L-glutamic acid produced was measured in the same manner as in Example 1.

[0064] Table 7 shows the amount of L-glutamic acid produced and the relative amount of citramalate in I10 and KCTC 11558BP. As shown in Table 7, it was confirmed that the amount of L-glutamic acid produced in I10 strain was increased and the amount of citramalate was significantly decreased.

[0065] [Table 5]

[0066] [Table 6]

[0067] [Table 7]

[0068] Example 4. Preparation of YggB mutant strain Analysis of metabolites in the leuA-deficient strain (I10) confirmed that a large amount of L-glutamic acid had accumulated inside the cells, and we attempted to enhance the excretion of L-glutamic acid.

[0069] YggB is a mechanosensitive channel homologue and is known to play an important role in the efflux of L-glutamate in Corynebacterium glutamicum. SEQ ID NO: 15 shows the amino acid sequence of YggB protein. Figure 4A shows the structure of YggB protein. The protein structure of YggB was analyzed to search for mutations that can enhance efflux.

[0070] FIG. 4B shows the pore structure of the YggB protein in which the 93rd leucine in the amino acid sequence of the YggB protein has been replaced with alanine, and FIG. 4C shows the pore structure of the YggB protein in which the 109th leucine in the amino acid sequence of the YggB protein has been replaced with alanine.

[0071] Example 4-1. Construction of a strain with a substitution at the 93rd amino acid in the amino acid sequence of the YggB protein As shown in Figure 4B, in order to expand the L-glutamate transport pathway, we presumed that if the 93rd leucine in the amino acid sequence of the YggB protein was replaced with alanine, the methyl loop involved in the narrow inner part of the pore would be removed and the diameter of the pore would increase, so we created a mutant strain.

[0072] The chromosomal DNA of the I10 strain prepared in Example 2-2 was isolated, and PCR was performed using the chromosomal DNA as a template with primers 5 and 6 set and primers 7 and 8 set under the conditions of 95°C for 30 seconds, 57°C for 30 seconds, and 72°C for 60 seconds, with 30 cycles repeated, and the PCR product obtained was purified. The purified PCR product was used as a template and amplified by crossover PCR with primers 5 and 8. The amplified product was confirmed for mutation by base sequence analysis, and then cleaved with XbaI and SalI restriction enzymes (Takara, Japan). The pK19mobSacB vector was cleaved with the same restriction enzyme and ligated with the amplified crossover PCR product to prepare a vector, which was named pKY93. The primer sequences used are shown in Table 8. Figure 5 shows the pKY93 vector for replacing the 93rd amino acid in the amino acid sequence of the YggB protein in the I10 strain.

[0073] [Table 8]

[0074] The pKY93 vector was introduced into the I10 strain in the same manner as in Example 2-2, and a transformed strain was selected and named I10-93. In the I10-93 mutant strain, the 93rd amino acid, leucine, of the YggB protein is replaced with alanine.

[0075] Example 4-2. Construction of a strain with a substitution at the 109th amino acid in the amino acid sequence of the YggB protein As shown in Figure 4C, in order to expand the L-glutamate transport pathway, we presumed that substituting alanine for leucine at position 109 in the amino acid sequence of the YggB protein would remove the methyl group involved in the narrow inner part of the pore, increasing the diameter of the pore, and we created a mutant strain.

[0076] The chromosomal DNA of the I10 strain prepared in Example 2-2 was isolated, and PCR was performed using the chromosomal DNA as a template with primers 5 and 9 sets and primers 10 and 8 sets, respectively, at 95°C for 30 seconds, 57°C for 30 seconds, and 72°C for 60 seconds, repeating 30 cycles, and the resulting PCR product was purified. The purified PCR product was used as a template and amplified by crossover PCR using primers 5 and 8. The amplified product was confirmed for mutation by base sequence analysis, and then digested with XbaI and SalI restriction enzymes (Takara, Japan). The pK19mobSacB vector was digested with the same restriction enzyme and ligated with the amplified crossover PCR product to prepare a vector, which was named pKY109. The primer sequences used are listed in Table 8.

[0077] The pKY109 vector was introduced into the I10 strain in the same manner as in Example 2-2, and a transformed strain was selected and named I10-109. In the I10-109 mutant strain, the 109th amino acid, leucine, of the YggB protein is replaced with alanine.

[0078] Example 5. Confirmation of L-glutamic acid productivity of mutant strains Using the same method as in Example 1 and Example 3, the amount of L-glutamic acid produced and the time required for the I10, I10-93, and I10-109 strains were measured on a flask scale, and the results are shown in Table 9.

[0079] [Table 9]

[0080] As shown in Table 9, the I10-93 and I10-109 mutant strains, which had an expanded pore in the YggB protein, produced more L-glutamic acid than the I10 strain. In particular, the I10-109 strain produced significantly more L-glutamic acid than I10, and the fermentation time was shortened from 48 hours to 44 hours, allowing efficient production of L-glutamic acid.

Claims

1. A mutant strain of Corynebacterium sp. in which expression of the leuA (2-isopropylmalate synthase) gene is reduced, thereby weakening or inactivating the activity of citramalate synthase and improving L-glutamic acid productivity, a mutant of the YggB protein in which one or more leucines selected from the group consisting of leucine at the 93rd amino acid and leucine at the 109th amino acid in the amino acid sequence of the YggB protein represented by SEQ ID NO:15 are substituted with alanine or valine; or Transformed with a vector comprising a polynucleotide encoding the mutant YggB protein. A mutant strain of Corynebacterium sp.

2. 2. The mutant Corynebacterium strain according to claim 1, wherein the strain is Corynebacterium glutamicum.

3. (a) culturing the Corynebacterium mutant strain according to claim 1 in a medium; (b) recovering L-glutamic acid from the cultured mutant or from the culture medium.

4. An L-glutamic acid producing agent comprising the Corynebacterium mutant strain described in claim 1.

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