Corynebacterium glutamicum mutant strain with improved L-lysine production ability and method for producing L-lysine using the same

Abstract

The present invention relates to a Corynebacterium glutamicum mutant strain with improved L-lysine production ability and a method for producing L-lysine using the same. The above-mentioned Corynebacterium glutamicum mutant strain improves enzyme activity by inducing amino acid mutations in the gene encoding glyceraldehyde 3-phosphate dehydrogenase, thereby enabling an increase in the production yield of L-lysine.

Description

Technical Field

[0001] The present invention relates to a Corynebacterium glutamicum mutant strain with improved L-lysine production ability and a method for producing L-lysine using the same. Background Art

[0002] L-lysine is an essential amino acid that cannot be synthesized in the human body or animals and must be supplied from the outside. Generally, it is produced by fermentation using microorganisms such as bacteria or yeast. The production of L-lysine can use wild-type strains obtained in a natural state or mutant strains transformed in a way to improve their L-lysine production ability. In recent years, in order to improve the production efficiency of L-lysine, gene recombination technology has been applied to microorganisms such as Escherichia coli and Corynebacterium glutamicum, which are widely used in the production of L-amino acids and other useful substances, and various recombinant strains or mutant strains with excellent L-lysine production ability and methods for producing L-lysine using the same have been developed.

[0003] According to Korean Patent Nos. 10-0838038 and 10-2139806, by changing the base sequence or amino acid sequence of a gene encoding a protein such as an enzyme related to L-lysine production, the expression of the gene can be increased or unnecessary genes can be eliminated, thereby improving the L-lysine production ability. In addition, Korean Patent Publication No. 10-2020-0026881 discloses a method of changing the existing promoter of a gene to a promoter with strong activity in order to increase the expression of a gene encoding an enzyme involved in L-lysine production.

[0004] As described above, various methods for increasing the L-lysine production ability have been developed, but the number of proteins such as enzymes, transcription factors, and transport proteins directly or indirectly related to L-lysine production reaches dozens. Therefore, in fact, a large amount of research is still needed on whether the L-lysine production ability increases according to the activity changes of such proteins.

[0005] Prior Art Documents

[0006] Patent Documents

[0007] (Patent Document 1) Korean Patent No. 10-0838038

[0008] (Patent Document 2) Korean Patent No. 10-2139806

[0009] (Patent Document 3) Korean Patent Publication No. 10-2020-0026881 Summary of the Invention

[0010] An object of the present invention is to provide a mutant strain of Corynebacterium glutamicum with improved L-lysine production ability.

[0011] In addition, an object of the present invention is to provide a method for producing L-lysine using the above mutant strain.

[0012] The inventors of the present invention conducted research to develop a novel mutant strain with improved L-lysine production ability using Corynebacterium glutamicum strains. As a result, it was confirmed that when the amino acid at a specific position in the amino acid sequence of the gapA gene encoding glyceraldehyde 3-phosphate dehydrogenase involved in the L-lysine biosynthetic pathway was replaced, the L-lysine production increased, thereby completing the present invention.

[0013] One aspect of the present invention provides a mutant strain of Corynebacterium glutamicum with improved L-lysine production ability due to improved activity of glyceraldehyde 3-phosphate dehydrogenase.

[0014] The "glyceraldehyde 3-phosphate dehydrogenase (GAPDH)" used in the present invention refers to an enzyme that participates in glycolysis or gluconeogenesis in the energy metabolism process, catalyzes the reversible reaction of glyceraldehyde 3-phosphate and 1,3-bisphosphoglycerate, and at the same time reduces the coenzyme NAD+ or NADP+ to NADH or NADPH or oxidizes it conversely. GAPDH is divided into three subtypes, GapA, GapB, and GapN, according to the type of coenzyme. GapA not only catalyzes the reaction of producing 1,3-bisphosphoglycerate and NADH from glyceraldehyde 3-phosphate using NAD in glycolysis, but also catalyzes the reaction of producing glyceraldehyde 3-phosphate from 1,3-bisphosphoglycerate using NADH in gluconeogenesis. GapB is activated by both NAD and NADP and only acts in gluconeogenesis. GapN is NADP-dependent and catalyzes the irreversible oxidation of glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate without generating ATP.

[0015] In the present invention, in order to produce or supply a large amount of NADPH required for the lysine biosynthetic process, the activity of GapA was improved, and NADP was used instead of NAD in the conversion of glyceraldehyde 3-phosphate, thereby creating a mutant strain that produces NADPH.

[0016] According to a specific example of the present invention, the above glyceraldehyde 3-phosphate dehydrogenase may be GapA.

[0017] According to a specific example of the present invention, the above glyceraldehyde 3-phosphate dehydrogenase may be derived from a strain of the genus Corynebacterium.Specifically, the above-mentioned Corynebacterium strains may be Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium callunae, Corynebacterium suranareeae, Corynebacterium lubricantis, Corynebacterium doosanense, Corynebacterium efficiens, Corynebacterium uterequi, Corynebacterium stationis, Corynebacterium pacaense, Corynebacterium singulare, Corynebacterium humireducens, Corynebacterium marinum, Corynebacterium halotolerans, Corynebacterium spheniscorum, Corynebacterium freiburgense, Corynebacterium striatum, Corynebacterium canis, Corynebacterium ammoniagenes, Corynebacterium renale, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium caspium, Corynebacterium testudinoris, Corynebacaterium pseudopelargi or Corynebacterium flavescens, but not limited thereto.

[0018] "Activity improvement" as used in the present invention refers to a structural transformation of a protein such as a target enzyme, transcription factor, or transport protein, resulting in the production of a different product or protein complex from that of the wild-type strain or the strain before transformation, or an increase in the concentration of the product or protein complex compared to the wild-type strain or the strain before transformation. Here, the structural transformation refers to a physical change in the reaction site of the protein with a substrate or a binding protein, such as the active site of an enzyme. Such improvement in activity includes the following cases: the activity of the protein itself is different from or increased compared to the activity of the protein originally possessed by the microorganism through nucleotide substitution, insertion, deletion, or a combination thereof in the encoding gene, and the overall enzyme activity level in the cell is higher than that of the wild-type strain or the strain before transformation through an increase in the expression or translation of the gene encoding it, and also includes a combination thereof.

[0019] According to a specific example of the present invention, the improvement in the activity of the above glyceraldehyde 3-phosphate dehydrogenase may be inducing a site-specific mutation in the gene encoding glyceraldehyde 3-phosphate dehydrogenase.

[0020] According to a specific example of the present invention, the gene encoding the above glyceraldehyde 3-phosphate dehydrogenase may be represented by the amino acid sequence of SEQ ID NO:1.

[0021] In addition, according to a specific example of the present invention, the gene encoding the above glyceraldehyde 3-phosphate dehydrogenase may be represented by the base sequence of SEQ ID NO:2.

[0022] According to a specific example of the present invention, the improvement in the activity of the above glyceraldehyde 3-phosphate dehydrogenase may be substitution of one or more amino acids in the amino acid region from the 10th to the 130th amino acid in the amino acid sequence of the gene encoding glyceraldehyde 3-phosphate dehydrogenase.

[0023] More specifically, the gene mutation in the present invention may be substitution of one, two, three, four, or five amino acids in one or more amino acid regions from the 10th to the 130th amino acid, preferably in the amino acid regions from the 20th to the 120th, from the 30th to the 110th, from the 30th to the 50th, or from the 90th to the 110th amino acids, either continuously or discontinuously.

[0024] According to a specific example of the present invention, the improvement in the activity of the above glyceraldehyde 3-phosphate dehydrogenase may be substitution of one or more amino acids among the 36th, 37th, and 100th amino acids in the amino acid sequence of the gene encoding glyceraldehyde 3-phosphate dehydrogenase.

[0025] According to an embodiment of the present invention, in the amino acid sequence of the gapA gene encoding glyceraldehyde 3-phosphate dehydrogenase of Corynebacterium glutamicum, the 36th position is replaced from leucine (Leu) to serine (Ser), and the 37th position is replaced from threonine (Thr) to lysine (Lys), thereby obtaining a mutant strain of Corynebacterium glutamicum with a novel amino acid sequence of the gapA gene. Such a mutant strain of Corynebacterium glutamicum may contain the gapA gene represented by the amino acid sequence of SEQ ID NO:3.

[0026] In addition, according to an embodiment of the present invention, in the amino acid sequence of the gapA gene encoding glyceraldehyde 3-phosphate dehydrogenase of Corynebacterium glutamicum, the 36th position is replaced from leucine (Leu) to serine (Ser), the 37th position is replaced from threonine (Thr) to lysine (Lys), and the 100th position is replaced from phenylalanine (Phe) to valine (Val), thereby obtaining a mutant strain of Corynebacterium glutamicum with a novel amino acid sequence of the gapA gene. Such a mutant strain of Corynebacterium glutamicum may contain the gapA gene represented by the amino acid sequence of SEQ ID NO:5.

[0027] As described above, the L-lysine production ability of the mutant strain of Corynebacterium glutamicum having a mutation in the glyceraldehyde 3-phosphate dehydrogenase gene or the amino acid sequence encoding the same can be improved.

[0028] "The production ability is improved" as used in the present invention means that the productivity of L-lysine is increased compared to the parental strain. The above parental strain refers to the wild type or mutant strain that is the object of mutation, including the object directly mutated or the object transformed by a recombinant vector or the like. In the present invention, the parental strain may be a wild type Corynebacterium glutamicum strain or a strain mutated from the wild type.

[0029] According to an embodiment of the present invention, the above parental strain may be a Corynebacterium glutamicum strain (domestic application number 10-2021-0050318) in which the activity of citrate synthase involved in the lysine biosynthesis pathway is weakened (hereinafter referred to as "Corynebacterium glutamicum DS2 strain").

[0030] According to an embodiment of the present invention, the Corynebacterium glutamicum mutant strain with improved L-lysine production ability has an amino acid mutation in the gapA gene encoding glyceraldehyde 3-phosphate dehydrogenase, so that the supply of NADPH required for L-lysine biosynthesis is increased, and compared with the parental strain, it shows increased L-lysine production ability. In particular, compared with the parental strain, the L-lysine production amount is increased by more than 5%, specifically, increased by 5 to 20%, so that more than 70 g of L-lysine can be produced per 1 L of the strain culture solution. Preferably, 70 to 85 g of L-lysine can be produced.

[0031] The Corynebacterium glutamicum mutant strain according to a specific example of the present invention can be achieved by a recombinant vector containing a mutant in which a part of the amino acid sequence of the gapA gene encoding glyceraldehyde 3-phosphate dehydrogenase in the parental strain is replaced.

[0032] The "part" used in the present invention means not all of the amino acid sequence, base sequence or polynucleotide sequence, and can be 1 to 300, preferably 1 to 100, more preferably 1 to 50, but is not limited thereto.

[0033] The "mutant" used in the present invention refers to a mutant in which one or more amino acids in the amino acid region of positions 10 to 130 in the amino acid sequence of the glyceraldehyde 3-phosphate dehydrogenase gene involved in L-lysine biosynthesis are replaced.

[0034] According to a specific example of the present invention, the mutant in which the amino acids at positions 36 and 37 in the amino acid sequence of the glyceraldehyde 3-phosphate dehydrogenase gene are replaced may have the amino acid sequence of SEQ ID NO:3 or the base sequence of SEQ ID NO:4.

[0035] In addition, according to a specific example of the present invention, the mutant in which the amino acids at positions 36, 37 and 100 in the amino acid sequence of the glyceraldehyde 3-phosphate dehydrogenase gene are replaced may have the amino acid sequence of SEQ ID NO:5 or the base sequence of SEQ ID NO:6.

[0036] The "vector" used in the present invention, as an expression vector capable of expressing a target protein in a suitable host cell, refers to a gene product that includes essential regulatory elements operably linked for expressing a gene insert. Here, "operably linked" means that the gene to be expressed and its regulatory sequences are functionally bound to each other and linked in a manner that enables gene expression. The "regulatory elements" include a promoter for carrying out transcription, any operon sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence for regulating the termination of transcription and translation. Such vectors include plasmid vectors, cosmid vectors, phage vectors, viral vectors, etc., but are not limited thereto.

[0037] The "recombinant vector" used in the present invention can replicate independently of the genome of the host cell after being transformed into a suitable host cell, or can be integrated into the genome itself. At this time, the above-mentioned "suitable host cell" can replicate the vector and may include an origin of replication as a specific base sequence for starting replication.

[0038] In the above transformation, a suitable vector introduction technique is selected according to the host cell, so that the target gene can be expressed in the host cell. For example, vector introduction can be carried out by electroporation, heat-shock, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, lithium acetate-DMSO method, or a combination thereof. As long as the transformed gene can be expressed in the host cell, it can be included, and it is not limited to being inserted into the chromosome of the host cell or being located outside the chromosome.

[0039] The above-mentioned host cells include cells transfected, transformed, or infected with the recombinant vector or polynucleotide of the present invention in vivo or in vitro. A host cell containing the recombinant vector of the present invention is a recombinant host cell, recombinant cell, or recombinant microorganism.

[0040] In addition, the recombinant vector according to the present invention may include a selection marker. The above-mentioned selection marker is used to select transformants (host cells) transformed with the vector. Only cells expressing the selection marker can survive in the medium treated with the above-mentioned selection marker, so that transformed cells can be selected. As a representative example, the above-mentioned selection markers include kanamycin, streptomycin, chloramphenicol, etc., but are not limited thereto.

[0041] The gene inserted into the recombinant vector for transformation of the present invention can be replaced into a host cell such as a microorganism belonging to the genus Corynebacterium due to homologous recombination crossing.

[0042] According to a specific example of the present invention, the above-mentioned host cell may be a strain belonging to the genus Corynebacterium. For example, it may be Corynebacterium glutamicum DS2 strain.

[0043] In addition, another aspect of the present invention provides a method for producing L-lysine, which comprises the following steps: a) a step of culturing the above-mentioned Corynebacterium glutamicum mutant strain in a medium; and b) a step of recovering L-lysine from the above-mentioned mutant strain or the medium for culturing the mutant strain.

[0044] The above-mentioned culturing can be carried out according to suitable media and culturing conditions known in the art, and those skilled in the art can easily adjust the media and culturing conditions for use. Specifically, the above-mentioned medium may be a liquid medium, but is not limited thereto. The culturing method may include, for example, batch culture, continuous culture, fed-batch culture, or a combined culture thereof, but is not limited thereto.

[0045] According to a specific example of the present invention, the above-mentioned medium must meet the requirements of a specific strain in a suitable manner and can be appropriately changed by those skilled in the art. Regarding the medium for strains belonging to the genus Corynebacterium, reference can be made to known literature (Manual of Methods for General Bacteriology. American Society for Bacteriology. Washington D.C., USA, 1981), but is not limited thereto.

[0046] According to a specific example of the present invention, the culture medium may contain various carbon sources, nitrogen sources, and trace element components. As the carbon sources that can be used, sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch, and cellulose are included; 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 alone or in the form of a mixture, but are not limited thereto. As the nitrogen sources that can be used, 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 can be included. The nitrogen sources can also be used alone or in the form of a mixture, but are not limited thereto. As the source for supplying phosphorus that can be used, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts can be included, but are not limited thereto. In addition, the culture medium may contain metal salts such as magnesium sulfate or iron sulfate required for growth, but are not limited thereto. In addition to the above, essential growth substances such as amino acids and vitamins can be included. In addition, precursors of a suitable culture medium can be used. The above culture medium or individual components can be added to the culture solution in batches or continuously in a suitable manner during the culture process, but are not limited thereto.

[0047] According to a specific example of the present invention, during the culture process, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid can be added to the microbial culture solution in a suitable manner to adjust the pH of the culture solution. In addition, during the culture process, an antifoaming agent such as fatty acid polyethylene glycol ester can be used to inhibit the generation of bubbles. Further, in order to maintain an aerobic state of the culture solution, oxygen or an oxygen-containing gas (such as air) can be injected into the culture solution. The temperature of the culture solution can generally be 20°C to 45°C, for example, it can be 25°C to 40°C. The culture time can continue until the desired production amount of the useful substance is obtained, for example, it can be 10 to 160 hours.

[0048] According to a specific example of the present invention, in the step of recovering L-lysine from the above-mentioned cultured mutant strain and the culture medium for culturing the mutant strain, the produced L-lysine can be collected or recovered from the culture medium according to the culture method and using suitable methods well-known in the art. For example, methods such as centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, fractional dissolution (such as ammonium sulfate precipitation), chromatography (such as ion exchange, affinity, hydrophobicity, and size exclusion) can be used, but are not limited thereto.

[0049] According to a specific example of the present invention, in the step of recovering lysine, the culture medium can be centrifuged at a low speed to remove the biomass, and the obtained supernatant can be separated by ion exchange chromatography.

[0050] According to a specific example of the present invention, in the above step of recovering L-lysine, a process of purifying L-lysine may be included.

[0051] The Corynebacterium glutamicum mutant strain of the present invention improves the enzyme activity by inducing amino acid mutations in the gene encoding glyceraldehyde 3-phosphate dehydrogenase, thereby increasing the production yield of L-lysine. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 Shows the structure of the DS2-gapA-Pm1 vector containing the gapA gene in which the 36th amino acid in the amino acid sequence is replaced from leucine to serine and the 37th amino acid is replaced from threonine to lysine according to an embodiment of the present invention.

[0053] Figure 2 Shows the structure of the DS2-gapA-Pm2 vector containing the gapA gene in which the 36th amino acid in the amino acid sequence is replaced from leucine to serine, the 37th amino acid is replaced from threonine to lysine, and the 100th amino acid is replaced from phenylalanine to valine according to an embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0054] Hereinafter, the present invention will be described in more detail. However, such a description is provided illustratively only to assist in understanding the present invention, and the scope of the present invention is not limited to such illustrative description.

[0055] Example 1. Preparation of Corynebacterium glutamicum Mutant Strain

[0056] To prepare a Corynebacterium glutamicum mutant strain with improved glyceraldehyde 3-phosphate dehydrogenase activity, Corynebacterium glutamicum DS2 strain and E.coli DH5a (HIT Competent cells TM , Cat No.RH618) were used as parental strains.

[0057] The above Corynebacterium glutamicum DS2 strain was cultured at a temperature of 30 °C in a CM-broth medium (pH 6.8). The composition of the CM-broth medium was 5 g of glucose, 2.5 g of NaCl, 5.0 g of yeast extract, 1.0 g of urea, 10.0 g of polypeptone, and 5.0 g of beef extract in 1 L of distilled water.

[0058] The above-mentioned E. coli DH5α was cultured on LB medium at a temperature of 37°C. The composition of the above-mentioned LB medium is 10.0 g of tryptone, 10.0 g of NaCl, and 5.0 g of yeast extract in 1 L of distilled water.

[0059] The antibiotics kanamycin and streptomycin were products of Sigma Corporation.

[0060] DNA sequencing analysis was commissioned to Macrogen, Inc. for analysis.

[0061] 1-1. Preparation of Recombinant Vector

[0062] In order to expand the supply of NADPH required in the lysine biosynthesis process, improve the L-lysine productivity, and improve the activity of glyceraldehyde 3-phosphate dehydrogenase to produce NADPH instead of NADH. In the method used in this example, specific mutations were induced in the gapA gene encoding glyceraldehyde 3-phosphate dehydrogenase. The 36th amino acid in the amino acid sequence of the gapA gene was replaced by serine from leucine, and the 37th amino acid was replaced by lysine from threonine. On the Corynebacterium glutamicum genome, with the part containing the 36th and 37th amino acids of the gapA gene as the center, the 442-bp left arm part and the 552-bp right arm part were amplified by PCR, and after being ligated by the overlap PCR method, they were cloned into pCGI (reference [Kim et al., Journal of Microbiological Methods 84 (2011) 128-130]) as a recombinant vector. The above plasmid was named DS2-gapA-Pm1 (reference Figure 1 ). To prepare the above plasmid, the primer in Table 1 below was used to amplify each gene fragment.

[0063]

Table 1

[0064]

[0065] PCR was carried out using the above primers under the following conditions. Using a thermocycler (TP600, TAKARA BIO Inc., Japan), in a reaction solution supplemented with 100 μM of each deoxynucleotide triphosphate (dATP, dCTP, dGTP, dTTP), 1 pM of oligonucleotide and 10 ng of chromosomal DNA of Corynebacterium glutamicum ATCC 13032 were used as templates, and 25-30 cycles were carried out in the presence of 1 unit of pfu-X DNA polymerase mixture (Solgent). The PCR conditions were carried out under the conditions of (i) denaturation step: 30 seconds at 94°C, (ii) annealing step: 30 seconds at 58°C, and (iii) extension step: 1-2 minutes at 72°C (2 minutes of polymerization time was given per 1 kb).

[0066] Using self-assembly cloning, the gene fragment produced as described above was cloned into the pCGI vector. The above vector was transformed into E. coli DH5α, spread on an LB-agar plate containing 50 μg / ml of kanamycin, and cultured at 37°C for 24 hours. The finally formed colonies were isolated, and after confirming whether the insert was correctly present in the vector, the vector was isolated and used for recombination of Corynebacterium glutamicum strains.

[0067] As a process carried out in common in the above method, the amplification of the gene was carried out by PCR using the genomic DNA of Corynebacterium glutamicum ATCC 13032, inserted into the pCGI vector by the self-assembled cloning method according to the strategy, and selected in E. coli DH5α. Chromosomal base substitution was carried out by amplifying the genes of each fragment separately, and the target DNA fragment was produced by overlapping PCR. During gene manipulation, Ex Taq polymerase (Takara) and Pfu polymerase (Solgent) were used as PCR amplification enzymes, and various restriction enzymes and DNA modification enzymes were NEB products and were used according to the supplied buffers and protocols.

[0068] 1-2. Production of mutant strains

[0069] Using the above DS2-gapA-Pm1, the DS2-1 strain was produced as a mutant strain. Prepared in such a way that the final concentration of the above vector became 1 μg / μl or more, the electrotransformation method (reference [Tauchet al., FEMS Microbiology letters 123 (1994) 343-347]) was used for the Corynebacterium glutamicum DS2 strain to induce a single recombination. At this time, the electrotransformed strain was spread on a CM-agar plate containing 20 μg / μl of kanamycin, and after isolating colonies, it was confirmed by PCR and base sequence analysis whether it was correctly inserted into the induced position on the genome. In order to induce a second recombination in the strain isolated in this way, it was inoculated into a CM-agar liquid medium containing streptomycin, cultured overnight or more, and then spread on an agar medium containing the same concentration of streptomycin to isolate colonies. After confirming the presence of kanamycin resistance in the finally isolated colonies, it was confirmed by base sequence analysis whether a mutation was introduced into the gapA gene in the strain without antibiotic resistance (reference [Schafer et al., Gene 145 (1994) 69-73]). Finally, a Corynebacterium glutamicum mutant strain (DS2-1) into which the mutant gapA gene was introduced was obtained.

[0070] Example 2. Production of Corynebacterium glutamicum mutant strain

[0071] In order to expand the supply of NADPH required in the lysine biosynthesis process and improve the L-lysine productivity, the activity of glyceraldehyde 3-phosphate dehydrogenase was improved in such a way as to produce NADPH instead of NADH. In the method used in this example, a specific mutation was induced in the gapA gene encoding glyceraldehyde 3-phosphate dehydrogenase. The 36th amino acid in the amino acid sequence of the gapA gene was replaced with serine, the 37th amino acid was replaced with lysine, and the 100th amino acid was replaced with valine. On the Corynebacterium glutamicum genome, the 442 bp left arm part and the 360 bp right arm part were amplified by PCR with the part containing the 36th, 37th, and 100th amino acids of the gapA gene as the center, and after being ligated by the overlap PCR method, they were cloned into pCGI as a recombinant vector (reference [Kim et al., Journal of Microbiological Methods 84 (2011) 128-130]). The above plasmid was named DS2-gapA-Pm2 (reference Figure 2 ). In order to prepare the above plasmid, the primer in Table 2 below was used to amplify each gene fragment.

[0072]

Table 2

[0073]

[0074] Then, the same method as in Example 1 was implemented, and thus, the DS2-2 strain as a mutant strain was manufactured and obtained using the above-mentioned DS2-gapA-Pm2.

[0075] Experimental Example 1. Comparison of L-lysine productivity of parent strain and mutant strain

[0076] The L-lysine productivities of the parent strain Corynebacterium glutamicum DS2 strain and the DS2-1 and DS2-2 strains as mutant strains for producing lysine manufactured in Examples 1 and 2 were compared.

[0077] In a 100-ml flask containing 10 ml of lysine medium having the composition shown in Table 3 below, each strain was inoculated and shake-cultured at 30 °C under the condition of 180 rpm for 28 hours. After the cultivation was completed, the production amount of L-lysine was measured by HPLC for lysine analysis (Shimadzu, Japan), and the results are shown in Table 4.

[0078]

Table 3

[0079] Composition Content (based on 1 L distilled water) Glucose 100g Ammonium sulfate 55g <![CDATA[KH2PO4]]> 1.1g <![CDATA[MgSO4·H2O]]> 1.2g <![CDATA[MnSO4·H2O]]> 180 mg <![CDATA[FeSO4·H2O]]> 180 mg Thiamine·HCl 9 mg Biotin 1.8 mg <![CDATA[CaCO3]]> 5％ pH 7.0

[0080]

Table 4

[0081] Strain OD610 L-Lysine (g / L) L-Lysine production per unit cell (g / g DCW) Parent strain (DS2) 23.0 69.7 7.2 Mutant strain (DS2-1) 22.5 76.3 8.1 Mutant strain (DS2-2) 22.4 75.2 8.0

[0082] As shown in Table 4 above, it was confirmed that in the Corynebacterium glutamicum mutant strains DS2-1 and DS2-2, at specific positions (the 36th and 37th amino acids, or the 36th, 37th, and 100th amino acids) of the amino acid sequence of the gapA gene, the optimal amino acid residues were substituted. Compared with the parent strain Corynebacterium glutamicum DS2 strain, the productivities of L-lysine increased by about 12.5% and 11.1% respectively. From these results, it can be seen that the gapA gene mutation improves the activity of glyceraldehyde 3-phosphate dehydrogenase, thereby enhancing the L-lysine production ability of the strain.

[0083] So far, the present invention has been studied around its preferred embodiments. Those skilled in the art to which the present invention pertains can understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative perspective rather than a restrictive perspective. The scope of the present invention is shown in the claims rather than the above description, and should be interpreted to include all differences within the scope equivalent thereto in the present invention.

Claims

1. A mutant strain of Corynebacterium glutamicum with improved glyceraldehyde 3-phosphate dehydrogenase activity and enhanced L-lysine production ability, wherein said mutant strain contains a gene encoding the amino acid sequence shown in SEQ ID NO: 3 or 5.

2. The Corynebacterium glutamicum mutant strain according to claim 1, wherein, The nucleotide sequence encoding the amino acid sequence SEQ ID NO: 3 is as shown in SEQ ID NO:

4.

3. The Corynebacterium glutamicum mutant strain according to claim 1, wherein, The nucleotide sequence encoding the amino acid sequence SEQ ID NO: 5 is as shown in SEQ ID NO:

6.

4. A method for producing L-lysine, comprising the following steps: a) a step of culturing the mutant strain according to any one of claims 1 to 3 in a culture medium; and b) a step of recovering L-lysine from the mutant strain or the culture medium for culturing the mutant strain.

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