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

Abstract

The present invention relates to a Corynebacterium glutamicum mutant having improved L-lysine-producing ability and a method for producing L-lysine using the same. The Corynebacterium glutamicum mutant has an amino acid mutation induced in a gene encoding glyceraldehyde-3-phosphate dehydrogenase, and thus the enzyme activity is improved, thereby improving the L-lysine production yield.

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 or animal body and must be supplied from the outside. Generally, it is produced by fermentation using microorganisms such as bacteria and yeast. For the production of L-lysine, wild-type strains obtained in a natural state or mutant strains modified to improve their L-lysine production ability can be used. Recently, in order to improve the production efficiency of L-lysine, gene recombination technology has been applied to microorganisms such as Escherichia coli and Corynebacterium, which are widely used in the production of L-amino acids and other useful substances, to develop various recombinant strains or mutant strains having excellent L-lysine production ability and a method for producing L-lysine using the same.

[0003] According to Korean Registered Patents Nos. 10-0838038 and 10-2139806, the L-lysine production ability can be improved by changing the nucleotide sequence or amino acid sequence of a gene encoding a protein including an enzyme related to the production of L-lysine to increase the expression of the gene or removing unnecessary genes. In addition, Korean Patent Publication No. 10-2020-0026881 discloses a method of changing an existing promoter of a gene to a promoter having strong activity in order to increase the expression of a gene encoding an enzyme involved in the production of L-lysine.

[0004] Although various methods for increasing the L-lysine production ability have been developed in this way, since the number of types of proteins such as enzymes, transcription factors, and transport proteins directly or indirectly related to the production of L-lysine reaches more than several dozen, there is still a great need for many studies on the presence or absence of an increase in the L-lysine production ability due to changes in the activity of such proteins.

Prior Art Documents

[0005] [Patent Document 1] Korean Registered Patent No. 10-0838038 [Patent Document 2] Korean Registered Patent No. 10-2139806 [Patent Document 3] Korean Published Patent No. 10-2020-0026881 [Overview of the project] [Problems that the invention aims to solve]

[0006] The present invention aims to provide a Corynebacterium glutamicum mutant strain with improved L-lysine production ability.

[0007] Furthermore, the present invention aims to provide a method for producing L-lysine using the aforementioned mutant strain. [Means for solving the problem]

[0008] The inventors of the present invention completed their research to develop a new mutant strain of Corynebacterium glutamicum with improved L-lysine production ability. As a result, they confirmed that substituting an amino acid at a specific position in the amino acid sequence of the gapA gene, which encodes glyceraldehyde 3-phosphate dehydrogenase involved in the L-lysine biosynthesis pathway, increases L-lysine production.

[0009] One aspect of the present invention provides a Corynebacterium glutamicum mutant strain in which the activity of glyceraldehyde 3-phosphate dehydrogenase is improved, thereby enhancing L-lysine production capacity.

[0010] The "glyceraldehyde 3-phosphate dehydrogenase (GAPDH)" used in this invention refers to an enzyme that is involved in sugar breakdown or sugar synthesis in energy metabolism, catalyzing the reversible reaction between glyceraldehyde 3-phosphate and 1,3-bisphosphoglycerate, while simultaneously reducing coenzymes NAD+ or NADP+ to NADH or NADPH, or conversely, oxidizing them. GAPDH is classified into three subtypes, GapA, GapB, and GapN, depending on the type of coenzyme. GapA not only catalyzes the production of 1,3-bisphosphoglycerate and NADH from glyceraldehyde 3-phosphate using NAD during glycolysis, but also catalyzes the production of glyceraldehyde 3-phosphate from 1,3-bisphosphoglycerate using NADH during glucoseogenesis. GapB is activated by both NAD and NADP and acts only in glucoseogenesis. GapN is NADP-dependent and catalyzes the irreversible oxidation of glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate without ATP production.

[0011] In this invention, in order to produce or supply large quantities of NADPH necessary for the lysine biosynthesis process, we have created a mutant strain that improves GapA activity and produces NADPH using NADP instead of NAD in the conversion of glyceraldehyde 3-phosphate.

[0012] According to one specific example of the present invention, the glyceraldehyde 3-phosphate dehydrogenase may be GapA.

[0013] According to one specific example of the present invention, the glyceraldehyde 3-phosphate dehydrogenase may be derived from a strain of the genus Corynebacterium. Specifically, the Corynebacterium strains mentioned above include Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium callunae, Corynebacterium suranareeae, Corynebacterium lubricantis, Corynebacterium doosanense, Corynebacterium efficiens, and Corynebacterium uterechii. Corynebacterium uterequi), Corynebacterium stationis, Corynebacterium pacaense, Corynebacterium singulare, Corynebacterium humireducens, Corynebacterium marinum, Corynebacterium halotolerans, Corynebacterium spheniscorum, Corynebacterium freiburgense, Corynebacterium striatum (Corynebacterium Corynebacterium striatum, Corynebacterium canis, Corynebacterium ammoniagenesThis may also be, but is not limited to, Corynebacterium ammoniagenes, Corynebacterium renale, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium caspium, Corynebacterium testudinoris, Corynebacterium pseudopelargi, or Corynebacterium flavescens.

[0014] In this invention, "improved activity" means that a target protein, such as an enzyme, transcription factor, or transport protein, is structurally modified to produce a different product or protein complex than that of the wild-type strain or the strain before modification, or that the concentration of the product or protein complex increases compared to the wild-type strain or the strain before modification. Here, structural modification refers to a physical alteration of the reaction site of the protein to its substrate or binding protein, for example, the active site of an enzyme. Such improvement in activity includes cases where the activity of the protein itself differs from or increases compared to the activity of the protein originally possessed by the microorganism due to intracellular nucleotide substitution, insertion, deletion, or a combination thereof in the gene encoding the protein, as well as cases where the overall level of enzyme activity in the cell is higher than that of the wild-type strain or the strain before modification due to increased expression or increased translation of the gene encoding the protein, and combinations thereof.

[0015] According to one specific example of the present invention, the improvement of the activity of glyceraldehyde 3-phosphate dehydrogenase may be achieved by inducing a site-directed mutation in the gene that encodes glyceraldehyde 3-phosphate dehydrogenase.

[0016] According to one specific example of the present invention, the gene encoding the glyceraldehyde 3-phosphate dehydrogenase may be represented by the amino acid sequence of Sequence ID No. 1.

[0017] Furthermore, according to one specific example of the present invention, the gene encoding the glyceraldehyde 3-phosphate dehydrogenase may be represented by the base sequence of Sequence ID No. 2.

[0018] According to one specific example of the present invention, the improvement in the activity of glyceraldehyde 3-phosphate dehydrogenase may be achieved by substituting one or more amino acids in the amino acid region between positions 10 and 130 within the amino acid sequence of the gene encoding glyceraldehyde 3-phosphate dehydrogenase.

[0019] More specifically, the gene mutation in the present invention may be a mutation in which one or more amino acids in the amino acid region between positions 10 and 130, preferably one, two, three, four, or five amino acids in the amino acid region between positions 20 and 120, 30 and 110, 30 and 50, or 90 and 110, are substituted consecutively or discontinuously.

[0020] According to one specific example of the present invention, the improvement in the activity of glyceraldehyde 3-phosphate dehydrogenase may be achieved by substituting 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.

[0021] According to one embodiment of the present invention, in the amino acid sequence of the gapA gene encoding glyceraldehyde 3-phosphate dehydrogenase of Corynebacterium glutamicum strain, the 36th site was substituted from leucine (Leu) to serine (Ser), and the 37th site was substituted from threonine (Thr) to lysine (Lys), to obtain a Corynebacterium glutamicum mutant having a new amino acid sequence of the gapA gene. Such a Corynebacterium glutamicum mutant may contain the gapA gene represented by the amino acid sequence of SEQ ID NO: 3.

[0022] Also, according to one embodiment of the present invention, in the amino acid sequence of the gapA gene encoding glyceraldehyde 3-phosphate dehydrogenase of Corynebacterium glutamicum strain, the 36th site was substituted from leucine (Leu) to serine (Ser), the 37th site was substituted from threonine (Thr) to lysine (Lys), and the 100th site was substituted from phenylalanine (Phe) to valine (Val), to obtain a Corynebacterium glutamicum mutant having a new amino acid sequence of the gapA gene. Such a Corynebacterium glutamicum mutant may contain the gapA gene represented by the amino acid sequence of SEQ ID NO: 5.

[0023] Thus, a Corynebacterium glutamicum mutant having a mutation in the glyceraldehyde 3-phosphate dehydrogenase gene or the amino acid sequence encoding the same can improve the L-lysine production ability.

[0024] As used in the present invention, "improved production ability" means that the productivity of L-lysine has increased compared to the parent strain. The parent strain means a wild-type or mutant strain that is the target of mutation, and includes those that are directly the target of mutation or those that are transformed with a recombinant vector or the like. In the present invention, the parent strain may be a wild-type Corynebacterium glutamicum strain or a strain mutated from the wild-type.

[0025] According to one embodiment of the present invention, the parent strain may be a Corynebacterium glutamicum strain (Korean Application No. 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").

[0026] According to one embodiment of the present invention, the Corynebacterium glutamicum mutant with improved L-lysine production ability contains an amino acid mutation in the gapA gene encoding glyceraldehyde 3-phosphate dehydrogenase, thereby increasing the supply amount of NADPH required for L-lysine biosynthesis and showing increased L-lysine production ability compared to the parent strain. In particular, the L-lysine production amount is increased by 5% or more, specifically 5 to 20%, compared to the parent strain, and 70 g or more, specifically 70 to 85 g, of L-lysine can be produced per liter of the strain culture solution.

[0027] The Corynebacterium glutamicum mutant according to one specific example of the present invention can be realized 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 parent strain is substituted.

[0028] As used in the present invention, "part" means not all of the amino acid sequence, base sequence, or polynucleotide sequence, and may be 1 to 300, preferably 1 to 100, more preferably 1 to 50, but is not limited thereto.

[0029] The term "mutant" used in this invention refers to a mutant in which one or more amino acids in the amino acid region between positions 10 and 130 of the amino acid sequence of the glyceraldehyde 3-phosphate dehydrogenase gene, which is involved in the biosynthesis of L-lysine, are substituted.

[0030] According to one specific example of the present invention, the mutant in which the 36th and 37th amino acids in the amino acid sequence of the glyceraldehyde 3-phosphate dehydrogenase gene are substituted may have the amino acid sequence of SEQ ID NO: 3 or the nucleotide sequence of SEQ ID NO: 4.

[0031] Furthermore, according to one specific example of the present invention, a mutant in which the 36th, 37th, and 100th amino acids in the amino acid sequence of the glyceraldehyde 3-phosphate dehydrogenase gene are substituted may have the amino acid sequence of SEQ ID NO: 5 or the base sequence of SEQ ID NO: 6.

[0032] As used in the present invention, “vector” means an expression vector capable of expressing a target protein in a suitable host cell, and a gene product comprising essential regulatory elements that are operably linked to enable the expression of a gene insert. Here, “operably linked” means that the gene to be expressed and its regulatory sequence are linked in a manner that allows gene expression to occur, and “regulatory elements” include a promoter for transcription, an optional operator sequence for regulating transcription, a sequence for encoding a suitable mRNA-ribosome binding site, and a sequence for regulating the termination of transcription and decoding. Such vectors include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, viral vectors, and the like.

[0033] The "recombinant vector" used in this invention, after being transformed into a suitable host cell, can replicate independently of the host cell's genome or can be stitched into the genome itself. In this case, the "suitable host cell" is one in which the vector can replicate and may include an origin of replication, which is a specific base sequence from which replication begins.

[0034] The transformation can be performed by selecting a suitable vector introduction technique depending on the host cell, thereby enabling the expression of the target gene within the host cell. For example, vector introduction can be performed by electroporation, heat shock, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cation liposome method, lithium acetate-DMSO method, or a combination thereof. The transformed gene can be any gene, whether intrachromosomal or extrachromosomal, as long as it can be expressed within the host cell.

[0035] The host cells include cells that have been transfected, transformed, or infected with the recombinant vector or polynucleotide of the present invention in vivo or in vitro. Host cells containing the recombinant vector of the present invention are recombinant host cells, recombinant cells, or recombinant microorganisms.

[0036] Furthermore, the recombinant vector according to the present invention may include a selection marker, which is used to select transformants (host cells) transformed by the vector. Since only cells expressing the selection marker can survive in a culture medium treated with the selection marker, it is possible to select transformed cells. Typical examples of the selection marker include kanamycin, streptomycin, and chloramphenicol, but the invention is not limited to these.

[0037] The genes inserted into the recombinant transformation vector of the present invention are replaced in host cells, such as those of the genus Corynebacterium, by homologous recombination cross-reactivity.

[0038] According to one specific example of the present invention, the host cell may be a strain of the genus Corynebacterium, or for example, the Corynebacterium glutamicum DS2 strain.

[0039] Another aspect of the present invention provides a method for producing L-lysine, comprising the steps of: a) culturing the Corynebacterium glutamicum mutant in a culture medium; and b) recovering L-lysine from the mutant or the culture medium in which the mutant was cultured.

[0040] The culture may be carried out using appropriate culture media and conditions known in the art, and a skilled technician can easily adjust and use the culture media and conditions. Specifically, the culture media may be, but is not limited to, a liquid culture. The culture method may include, but is not limited to, batch culture, continuous culture, fed-batch culture, or a combination thereof.

[0041] According to one specific example of the present invention, the culture medium must meet the requirements of a specific bacterial strain in an appropriate manner and can be modified as appropriate by an ordinary technician. Culture media for Corynebacterium strains can be found in, but are not limited to, known literature (Manual of Methods for General Bacteriology. American Society for Bacteriology. Washington DC, USA, 1981).

[0042] According to one specific example of the present invention, the culture medium may contain a variety of carbon sources, nitrogen sources, and trace element components. Usable carbon sources include sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch, and cellulose; oils and fats such as soybean oil, sunflower oil, castor oil, and coconut oil; fatty acids such as palmitic acid, stearic acid, and linoleic acid; alcohols such as glycerol and ethanol; and organic acids such as acetic acid. These substances may be used individually or in mixtures, but are not limited thereto. Usable nitrogen sources include peptone, yeast extract, meat juice, malt extract, corn maceration, soybean barley, and urea, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate. Nitrogen sources may also be used individually or in mixtures, but are not limited thereto. Usable phosphorus sources may include, but are not limited to, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or corresponding sodium-containing salts. Furthermore, the culture medium may, but is not limited to, contain metal salts such as magnesium sulfate or ferrous sulfate necessary for growth. Other essential growth substances, such as amino acids and vitamins, may also be included. Additionally, appropriate precursors can be used in the culture medium. The medium or individual components are added to the culture medium in a batch or continuous manner by an appropriate method during the culturing process, but are not limited to this.

[0043] According to one specific example of the present invention, the pH of the microbial culture medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid to the culture medium in an appropriate manner during cultivation. Furthermore, the formation of bubbles can be suppressed during cultivation using an antifoaming agent such as fatty acid polyglycol ester. Additionally, oxygen or an oxygen-containing gas (e.g., air) can be injected into the culture medium to maintain an aerobic state. The temperature of the culture medium is typically 20°C to 45°C, for example, 25°C to 40°C. The cultivation period can be continued until the useful substance is obtained in the desired yield, and may be, for example, 10 to 160 hours.

[0044] According to one specific example of the present invention, the step of recovering L-lysine from the cultured mutant and the culture medium in which the mutant was cultured can be performed by collecting or recovering the L-lysine produced from the culture medium using a suitable method known in the art, depending on the culture method. For example, methods such as centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, fractional dissolution (e.g., ammonium sulfate precipitation), and chromatography (e.g., ion exchange, affinity, hydrophobicity, and size exclusion) can be used, but are not limited thereto.

[0045] According to one specific example of the present invention, the step of recovering lysine can be performed by removing biomass by slow centrifugation of the culture medium and separating the resulting supernatant by ion exchange chromatography.

[0046] According to one specific example of the present invention, the step of recovering L-lysine may include a step of purifying L-lysine. [Effects of the Invention]

[0047] The Corynebacterium glutamicum mutant strain according to the present invention induces amino acid mutations in the gene encoding glyceraldehyde 3-phosphate dehydrogenase, thereby improving enzyme activity and increasing the production yield of L-lysine. [Brief explanation of the drawing]

[0048] [Figure 1] This figure shows the structure of the DS2-gapA-Pm1 vector, which contains 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 one embodiment of the present invention. [Figure 2]This figure 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 from threonine to lysine, and the 100th amino acid from phenylalanine to valine, according to one embodiment of the present invention. [Modes for carrying out the invention]

[0049] The present invention will be described in more detail below. However, this description is merely illustrative for the purpose of understanding the present invention, and the scope of the present invention is not limited by such illustrative description.

[0050] Example 1. Production of Corynebacterium glutamicum mutant strain To produce a Corynebacterium glutamicum mutant strain with improved glyceraldehyde 3-phosphate dehydrogenase activity, Corynebacterium glutamicum DS2 strain and E. coli DH5a (HIT Competent cells) were used as parent strains. TM Cat No. RH618 was used.

[0051] The Corynebacterium glutamicum DS2 strain was cultured at 30°C in CM-broth medium (pH 6.8) with a composition of 5 g glucose, 2.5 g NaCl, 5.0 g yeast extract, 1.0 g urea, 10.0 g polypeptone, and 5.0 g beef extract in 1 L of distilled water.

[0052] The aforementioned E. coli DH5a was cultured at 37°C on LB medium containing 10.0 g of tryptone, 10.0 g of NaCl, and 5.0 g of yeast extract in 1 L of distilled water.

[0053] The antibiotics kanamycin and streptomycin were products of Sigma.

[0054] DNA sequencing analysis was commissioned to Macrogen Co., Ltd.

[0055] 1-1. Preparation of recombinant vectors To increase L-lysine productivity by expanding the supply of NADPH necessary for lysine biosynthesis, the activity of glyceraldehyde 3-phosphate dehydrogenase was improved to produce NADPH instead of NADH. The method used in this example induced specific mutations in the gapA gene, which encodes glyceraldehyde 3-phosphate dehydrogenase. The 36th amino acid of the gapA gene was replaced from leucine to serine, and the 37th amino acid was replaced from threonine to lysine. The left arm (442 bp) and right arm (552 bp) of the gapA gene, centering on the regions containing the 36th and 37th amino acids, were amplified by PCR on the Corynebacterium glutamicum genome. After ligation using overlap PCR, the resulting plasmid was cloned into a recombinant vector pCGI (see reference [Kim et al., Journal of Microbiological Methods 84 (2011) 128-130]). The plasmid was named DS2-gapA-Pm1 (see Figure 1). The primers listed in Table 1 below were used to amplify each gene fragment in order to construct the aforementioned plasmid.

[0056] [Table 1]

[0057] PCR was performed using the above primers under the following conditions. Using a thermocycler (TP600, TAKARA BIO Inc., Japan), 100 μM of each deoxynucleotide triphosphate (dATP, dCTP, dGTP, dTTP) was added to the reaction mixture, and 1 pM of oligonucleotide and 10 ng of Corynebacterium glutamicum ATCC13032 chromosomal DNA were used as templates. The PCR was performed for 25-30 cycles in the presence of 1 unit of pfu-X DNA polymerase mixture (Solgent). The PCR conditions were as follows: (i) denaturation step: 94°C for 30 seconds, (ii) annealing step: 58°C for 30 seconds, and (iii) extension step: 72°C for 1-2 minutes (polymerization time of 2 minutes per kb).

[0058] The gene fragments thus produced were cloned into a pCGI vector using self-assembly cloning. The vector was used to transform E. coli DH5a, which was then streaked onto an LB-agar plate containing 50 μg / ml kanamycin and incubated at 37°C for 24 hours. After isolating the final formed colonies to confirm the accurate presence of the insert in the vector, the vector was isolated and used for recombination of Corynebacterium glutamicum strains.

[0059] A common process in the aforementioned methods was the amplification of the gene in question, which was performed by PCR from the genomic DNA of Corynebacterium glutamicum ATCC13032, inserted into a pCGI vector using a self-assembled cloning method according to the strategy, and selected from E. coli DH5a6. For chromosomal base substitution, the genes of each fragment were amplified individually, and the target DNA fragment was produced by overlap PCR. Ex Taq polymerase (Takara) and Pfu polymerase (Solgent) were used as PCR amplification enzymes during gene recombination, and various restriction enzymes and DNA modifying enzymes were NEB products, used according to the supplied buffers and protocols.

[0060] 1-2. Production of mutant strains A mutant strain, DS2-1, was prepared using the aforementioned DS2-gapA-Pm1. The vector was prepared to a final concentration of 1 μg / μl or higher, and primary recombination was induced in the Corynebacterium glutamicum DS2 strain using electroporation (see reference [Tauch et al., FEMS Microbiology letters 123 (1994) 343-347]). At this time, the electroporated strain was smeared onto a CM-agar plate containing 20 μg / μl of kanamycin, and colonies were isolated. Then, PCR and nucleotide sequence analysis were used to confirm whether the induced position on the genome was properly inserted. The isolated strains were then inoculated into a CM-agar liquid medium containing streptomycin to induce secondary recombination again, cultured overnight or longer, and then smeared onto agar medium containing the same concentration of streptomycin to isolate colonies. After confirming the presence or absence of resistance to kanamycin in the finally isolated colonies, the absence of antibiotic resistance in the strains was confirmed by nucleotide sequence analysis to determine whether a mutation had been introduced into the gapA gene (see reference [Schafer et al., Gene145(1994)69-73]). Finally, a Corynebacterium glutamicum mutant strain (DS2-1) with a mutated gapA gene was obtained.

[0061] Example 2. Production of Corynebacterium glutamicum mutant strains To increase L-lysine productivity by expanding the supply of NADPH necessary for lysine biosynthesis, the activity of glyceraldehyde 3-phosphate dehydrogenase was improved to produce NADPH instead of NADH. The method used in this example induced specific mutations in the gapA gene, which encodes glyceraldehyde 3-phosphate dehydrogenase. The 36th amino acid of the gapA gene was replaced from leucine to serine, the 37th amino acid from threonine to lysine, and the 100th amino acid from phenylalanine to valine. The left arm (442 bp) and right arm (360 bp) of the gapA gene, centering on the regions containing the 36th, 37th, and 100th amino acids, were amplified by PCR on the Corynebacterium glutamicum genome. After ligation using overlap PCR, the resulting molecules were cloned into the recombinant vector pCGI (see reference [Kim et al., Journal of Microbiological Methods 84 (2011) 128-130]). The plasmid was named DS2-gapA-Pm2 (see Figure 2). The primers listed in Table 2 below were used to amplify each gene fragment in order to construct the plasmid.

[0062] [Table 2]

[0063] Subsequently, the mutant strain DS2-2 was produced and obtained using DS2-gapA-Pm2 in the same manner as in Example 1.

[0064] Experimental Example 1. Comparison of L-lysine productivity between parent strain and mutant strain. The L-lysine productivity of the parent strain Corynebacterium glutamicum DS2 and the lysine-producing mutant strains DS2-1 and DS2-2, produced in Examples 1 and 2, was compared.

[0065] Each bacterial strain was inoculated into a 100 ml flask containing 10 ml of lysine medium with the composition shown in Table 3 below, and cultured with shaking at 30°C for 28 hours at 180 rpm. After the culture was complete, lysine analysis was performed by measuring the amount of L-lysine produced using HPLC (Shimazu, Japan), and the results are shown in Table 4 below.

[0066] [Table 3]

[0067] [Table 4]

[0068] As shown in Table 4 above, Corynebacterium glutamicum mutants DS2-1 and DS2-2 were found to have increased L-lysine production by approximately 12.5% ​​and 11.1%, respectively, compared to the parent Corynebacterium glutamicum DS2 strain, due to substitution of specific amino acid residues (amino acids 36 and 37, or 36, 37, and 100) in the amino acid sequence of the gapA gene to enhance the lysine biosynthesis pathway. Through these results, it was found that gapA gene mutations improve the L-lysine production capacity of the strain by improving the activity of glyceraldehyde 3-phosphate dehydrogenase.

[0069] The present invention has been described above, focusing on its preferred embodiments. Those with ordinary skill in the art to which the present invention pertains will understand that the present invention can be realized in modified forms without departing from its essential characteristics. Therefore, the disclosed embodiments should be considered in an explanatory rather than restrictive manner. The scope of the present invention is shown in the claims, not in the above description, and all differences within an equivalent scope should be interpreted as being included in the present invention.

Claims

1. This is a mutant strain of Corynebacterium glutamicum in which the activity of glyceraldehyde 3-phosphate dehydrogenase is improved, and the ability to produce L-lysine is enhanced. The mutant strain is a mutant strain that contains glyceraldehyde 3-phosphate dehydrogenase containing the amino acid sequence of SEQ ID NO: 3 or 5.

2. Corynebacterium glutamicum mutant strain according to claim 1, wherein the gene that encodes the amino acid sequence of SEQ ID NO: 3 contains the nucleic acid sequence of SEQ ID NO:

4.

3. Corynebacterium glutamicum mutant strain according to claim 1, wherein the gene that encodes the amino acid sequence of SEQ ID NO: 5 contains the nucleic acid sequence of SEQ ID NO:

6.

4. a) A step of culturing the Corynebacterium glutamicum mutant strain described in any one of claims 1 to 3 in a culture medium, b) The step of recovering L-lysine from the mutant strain or the culture medium in which the mutant strain was cultured. A method for producing L-lysine, including [the specified ingredient].

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