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

Abstract

The present application relates to a Corynebacterium glutamicum mutant strain having improved L-lysine production ability and a method for producing L-lysine using the same, wherein the expression of a gene encoding enolase is increased or enhanced, or at the same time the expression of a glucose acid operon transcription inhibitor is reduced or weakened, the supply of precursors is increased, the sugar utilization ability is increased, and thus the production yield of L-lysine can be improved.

Description

Technical Field

[0001] This invention relates to a Corynebacterium glutamicum mutant strain with improved L-lysine production capacity and a method for producing L-lysine using the strain. Background Technology

[0002] L-Lysine is an essential amino acid that cannot be synthesized in the human or animal body and must be obtained from external sources. Generally, it is produced through fermentation using microorganisms such as bacteria or yeast. L-Lysine production can utilize wild-type strains obtained under natural conditions or mutant strains modified to enhance their L-lysine production capacity. In recent years, to improve L-lysine production efficiency, recombinant gene 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, resulting in the development of various recombinant strains or mutant strains with excellent L-lysine production capabilities, as well as L-lysine production methods utilizing them.

[0003] According to Korean Patent Nos. 10-0838038 and 10-2139806, L-lysine production capacity can be improved by altering the base sequence or amino acid sequence of a gene encoding a protein containing an enzyme related to L-lysine production, thereby increasing gene expression or removing unnecessary genes. Furthermore, Korean Patent Publication No. 10-2020-0026881 discloses a method for changing the existing promoter of a gene to a more active promoter to increase the expression of a gene encoding an enzyme involved in L-lysine production.

[0004] As mentioned above, various methods have been developed to increase L-lysine production capacity. However, there are dozens of types of proteins, such as enzymes, transcription factors, and transport proteins, that are directly or indirectly related to L-lysine production. Therefore, whether L-lysine production capacity increases based on changes in the activity of such proteins still requires extensive research.

[0005] Existing technical 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] The purpose of this invention is to provide a Corynebacterium glutamicum mutant strain with enhanced L-lysine production capacity.

[0011] In addition, the present invention aims to provide a method for producing L-lysine using the above-mentioned mutant strain.

[0012] The inventors of this invention conducted research to develop novel mutant strains with enhanced L-lysine production capacity using Corynebacterium glutamicum strains. The results confirmed that L-lysine production increased when the gene sequence of the Eno gene, which encodes enolase involved in the L-lysine biosynthesis pathway, was replaced, particularly the start codon. Furthermore, it was confirmed that L-lysine production increased even further when specific amino acids in the gntR gene, which encodes a transcriptional repressor factor of the gluconic acid operon, were replaced, thus completing this invention.

[0013] One aspect of the present invention provides a Corynebacterium glutamicum mutant strain with enhanced enolase activity and improved L-lysine production capacity.

[0014] The "enolase" used in this invention, also known as phosphopyruvate hydratase, is an enzyme that catalyzes the conversion of 2-phosphoglycerate (2-PG) to phosphoenolpyruvate (PEP) in order to supply pyruvate to the TCA cycle.

[0015] According to a specific embodiment of the present invention, the above-mentioned enolase may be derived from a strain of Corynebacterium.Specifically, the aforementioned Corynebacterium strains may include Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium callunae, Corynebacterium suranareeae, Corynebacterium lubricantis, Corynebacterium doosanense, Corynebacterium efficiens, Corynebacterium uterequi, Corynebacterium stationis, Corynebacterium pacaense, Corynebacterium singulare, and Corynebacterium humicum. Corynebacterium marinum, Corynebacterium halotolerans, Corynebacterium spheniscorum, Corynebacterium freiburgense, Corynebacterium striatum, Corynebacterium canis, Corynebacterium ammoniagenes, Corynebacterium renale, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium caspium, Corynebacterium testudinoris, Corynebacterium pseudocorynebacterium, or Corynebacterium flavovirens. (flavescens), but not limited to this.

[0016] In this invention, "activity enhancement" refers to the introduction or increase of new genes encoding target proteins such as enzymes, transcription factors, and transport proteins, resulting in increased expression levels compared to wild-type strains or pre-transformation strains. Such activity enhancement includes: situations where the activity of the protein itself increases compared to the activity of the original protein in the microorganism through nucleotide substitutions, insertions, deletions, or combinations thereof; and situations where the overall intracellular enzyme activity is higher than that of wild-type strains or pre-transformation strains due to increased expression or translation of the gene encoding it, including combinations thereof.

[0017] According to a specific embodiment of the present invention, the enhanced activity of the enolase described above may be caused by a position-specific mutation induced in the gene encoding the enolase.

[0018] According to a specific embodiment of the present invention, the gene encoding enolase described above can be represented by the base sequence of SEQ ID NO:1.

[0019] In addition, according to a specific embodiment of the present invention, the gene encoding enolase can be represented by the amino acid sequence of SE Q ID NO:2.

[0020] According to a specific embodiment of the present invention, the enhanced activity of the enolase may be achieved by the substitution of one or more bases in positions 1 to 100 of the base sequence of the gene encoding the enolase.

[0021] More specifically, the gene mutation in this invention may be the substitution of the first to 100th bases, preferably the first to 10th bases, in the base sequence of the gene encoding enolase, either continuously or discontinuously.

[0022] According to a specific embodiment of the present invention, the enhanced activity of the enolase may be due to the replacement of the start codon of the gene encoding the enolase with ATG.

[0023] According to one embodiment of the present invention, in the base sequence of SEQ ID NO:1 encoding the Eno gene encoding enolase of a strain of Corynebacterium glutamicum, the start codon is replaced by ATG instead of GTG, thereby obtaining a Corynebacterium glutamicum mutant strain with a novel start codon containing the Eno gene. Such a Corynebacterium glutamicum mutant strain may contain an enolase amino acid sequence gene encoded by the base sequence of SEQ ID NO:3 or the amino acid sequence of SEQ ID NO:4.

[0024] As described above, the L-lysine production capacity of *Corynebacterium glutamicum* mutant strains with mutations in the enolase gene can be improved.

[0025] In this invention, "increased production capacity" refers to an increase in L-lysine production compared to the parental strain. The aforementioned parental strain refers to a wild-type or mutant strain that becomes the target of mutation, including those directly targeted for mutation or transformed through recombinant vectors. In this invention, the parental strain can be a wild-type Corynebacterium glutamicum or a strain mutated from a wild-type strain.

[0026] According to a specific embodiment of the present invention, the aforementioned parental strain is a mutant strain in which mutations have been induced in the sequence of genes involved in lysine production (e.g., lysC, zwf, and hom genes), which may be the Corynebacterium glutamicum strain (hereinafter referred to as "Corynebacterium glutamicum DS1 strain") deposited at the Korean Culture Center of Microorganisms on April 2, 2021 with accession number KCCM12969P.

[0027] According to one embodiment of the present invention, the *Corynebacterium glutamicum* mutant strain with improved L-lysine production capacity increases the supply of PEP, an important precursor of L-lysine, through a start codon mutation containing the enolase Eno gene, and exhibits increased L-lysine production capacity compared to the parent strain. In particular, the L-lysine production is increased by more than 5% compared to the parent strain, specifically by 5% to 40% (preferably 10% to 30%), so that 55 to 85 g of L-lysine can be produced per 1 L of strain culture, preferably 60 to 80 g of L-lysine.

[0028] In addition, in order to further improve the L-lysine production capacity, mutations can be induced in other genes involved in L-lysine biosynthesis, except for the Eno gene encoding enolase.

[0029] According to a specific embodiment of the present invention, the mutant strain may further include weakened activity of the gluconate operon transcriptional repressor.

[0030] Microorganisms utilize operons, composed of regulatory genes, operators, promoters, and structural genes, for transcription, thereby regulating protein expression. A series of proteins that induce the activation of inactivated genes and promote successful transcription upon activation are called transcriptional activators. Conversely, a series of proteins generated to inactivate activated genes again are called transcriptional repressors. The "gluconate operon transcriptional repressor" used in this invention, as a regulatory protein involved in gluconate metabolism and glucose influx, binds to the operator gene of the gluconate operon to inhibit mRNA synthesis.

[0031] According to a specific embodiment of the present invention, the above-mentioned gluconate operon transcription repressor may be derived from a strain of the genus Corynebacterium.Specifically, the aforementioned Corynebacterium strains may include Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium callunae, Corynebacterium suranareeae, Corynebacterium lubricantis, Corynebacterium doosanense, Corynebacterium efficiens, Corynebacterium uterequi, Corynebacterium stationis, Corynebacterium pacaense, and Corynebacterium ovale. 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, and Corynebacterium pseudobacaterium. It can be pseudopelargi or Corynebacterium flavescens, but is not limited to these.

[0032] In this invention, "activity attenuation" refers to the suppression or knockout of the expression of genes encoding proteins such as enzymes, transcription factors, and transport proteins, resulting in a reduced expression level compared to the wild-type strain or the pre-transformation strain. Such activity attenuation includes: situations where the activity of the protein itself is reduced compared to the activity of the protein in the original microorganism through nucleotide substitutions, insertions, deletions, or combinations thereof; and situations where the overall intracellular enzyme activity is lower than that of the wild-type strain or the pre-transformation strain by inhibiting the expression of the gene encoding it or inhibiting translation, including combinations thereof.

[0033] According to one specific example of the invention, the weakening of the activity of the gluconate operon transcription repressor can be caused by a position-specific mutation in the gene encoding the gluconate operon transcription repressor.

[0034] According to a specific embodiment of the present invention, the gene encoding the gluconate operon transcriptional repressor can be represented by the base sequence of SEQ ID NO:5.

[0035] In addition, according to a specific embodiment of the present invention, the gene encoding the gluconate operon transcriptional repressor can be represented by the amino acid sequence of SEQ ID NO:6.

[0036] According to a specific embodiment of the present invention, the enhanced activity of the gluconate operon transcription repressor may be due to the substitution of one or more amino acids in the amino acid region from amino acid 10 to amino acid 100 of the gene encoding the gluconate operon transcription repressor.

[0037] More specifically, the gene mutation in this invention can be the continuous or discontinuous substitution of one or more amino acids in the amino acid sequence of the gene encoding the gluconate operon transcription repressor, preferably one, two, three, four or five amino acids in the 10th to 100th, 20th to 90th, or 30th to 80th amino acid regions.

[0038] According to one embodiment of the present invention, in the amino acid sequence of the gntR gene encoding the gluconic acid operon transcription repressor of *Corynebacterium glutamicum* strains, position 77 is replaced by phenylalanine (Phe), thereby obtaining a *Corynebacterium glutamicum* mutant strain with a novel amino acid sequence of the gntR gene. Such a *Corynebacterium glutamicum* mutant strain may contain the gluconic acid operon transcription repressor gntR gene encoded by the base sequence of SEQ ID NO:7 or the amino acid sequence of SEQ ID NO:8.

[0039] Here, the parental strain can be a Corynebacterium glutamicum mutant strain with a sequence-induced mutation in the Eno gene encoding enolase.

[0040] According to one embodiment of the present invention, the *Corynebacterium glutamicum* mutant strain with enhanced L-lysine production capacity exhibits increased sugar utilization capacity by simultaneously including a start codon mutation in the enolase Eno gene and an amino acid mutation in the gluconic acid operon transcription repressor gntR gene. Compared with the parent strain containing only a start codon mutation in the Eno gene encoding enolase, it shows increased L-lysine production capacity, particularly by more than 2%, specifically, by 2 to 20%, compared with the parent strain. Thus, 63 to 85 g of L-lysine can be produced per 1 L of strain culture, preferably 65 to 80 g of L-lysine.

[0041] According to a specific example of the present invention, the Corynebacterium glutamicum mutant strain can be realized by a recombinant vector containing a mutant in which the start codon sequence of the enolase Eno gene in the parent strain has been replaced and / or a mutant in which the amino acid sequence of the gluconic acid operon transcription repressor gntR gene has been partially replaced.

[0042] The term "part" as used in this invention refers to a portion that is not the entirety of an 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.

[0043] The "mutant" used in this invention refers to a mutant in which the start codon sequence of the enolase Eno gene, which is involved in the biosynthesis of L-lysine, is replaced by ATG and / or one or more amino acids in the amino acid region from amino acid 10 to 100 of the gluconic acid operon transcription repressor gntR gene are replaced.

[0044] According to a specific example of the present invention, the mutant in which the start codon of the above-mentioned enolase gene is replaced by ATG may have the base sequence of SEQ ID NO:3 or the amino acid sequence of SEQ ID NO:4.

[0045] In addition, according to a specific embodiment of the present invention, the mutant in which the 77th amino acid in the amino acid sequence of the above-mentioned gluconate operon transcription repressor gene is replaced may have the base sequence of SEQ ID NO:7 or the amino acid sequence of SEQ ID NO:8.

[0046] The "vector" used in this invention, as an expression vector capable of expressing a target protein in a suitable host cell, refers to a gene product that includes operably linked essential regulatory elements for expressing a gene insert. Here, "operably linked" means that the gene to be expressed and its regulatory sequence are functionally linked together in a manner that enables gene expression. "Regulatory elements" include promoters for carrying out transcription, arbitrary operon sequences for regulating transcription, sequences encoding suitable mRNA ribosome binding sites, and sequences regulating the termination of transcription and translation. Such vectors include, but are not limited to, plasmid vectors, granular vectors, phage vectors, viral vectors, etc.

[0047] 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 it can be sewn into the genome itself. In this case, the aforementioned "suitable host cell" can replicate the vector, which may include a replication originating from a specific base sequence that initiates replication.

[0048] In the above transformation, a suitable vector delivery technique is selected based on the host cell, thereby enabling the expression of the target gene within the host cell. For example, vector delivery can be performed via 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 combinations thereof. The transformed gene can be included as long as it can be expressed within the host cell, without limitation on whether it is inserted into or located extrachromosomally.

[0049] The aforementioned host cells include cells transfected, transformed, or infected in vivo or in vitro using the recombinant vector or polynucleotide of the present invention. Host cells containing the recombinant vector of the present invention are recombinant host cells, recombinant cells, or recombinant microorganisms.

[0050] Furthermore, the recombinant vector according to the present invention may include a selection marker, which is used to select transformants (host cells) transformed using the vector. In the culture medium treated with the selection marker, only cells expressing the selection marker can survive, thus enabling the selection of transformed cells. Representative examples of the selection marker include kanamycin, streptomycin, chloramphenicol, etc., but it is not limited to these.

[0051] Genes inserted into the recombinant vector for transformation of the present invention can be replaced into host cells such as Corynebacterium spp. due to homologous recombination crossover.

[0052] According to a specific embodiment of the present invention, the host cell can be a strain of Corynebacterium, for example, it can be Corynebacterium glutamicum DS1 strain.

[0053] In addition, another aspect of the present invention provides a method for producing L-lysine, comprising the steps of: a) culturing the above-mentioned Corynebacterium glutamicum mutant strain in a culture medium; and b) recovering L-lysine from the above-mentioned mutant strain or the culture medium in which the mutant strain is cultured.

[0054] The above-described culture can be carried out using suitable culture media and conditions known in the art, which can be easily adjusted by those skilled in the art. Specifically, the culture media can be liquid culture media, but is not limited thereto. Culture methods can include, for example, batch culture, continuous culture, fed-batch culture, or combinations thereof, but are not limited thereto.

[0055] According to a specific embodiment of the invention, the culture medium described above must be adapted to meet the requirements of a particular strain in a suitable manner, and may be appropriately modified by those skilled in the art. For information on culture media for Corynebacterium strains, reference may be made to well-known literature (Manual of Methods for General Bacteriology. American Society for Bacteriology. Washington DC, USA, 1981), but it is not limited thereto.

[0056] According to a specific embodiment of the invention, the culture medium may contain various 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 alone or in mixtures, but are not limited thereto. 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 alone or in mixtures, but are not limited thereto. Usable phosphorus sources may include potassium dihydrogen phosphate or dipotassium hydrogen phosphate or corresponding sodium-containing salts, but are not limited thereto. Furthermore, the culture medium may contain metal salts such as magnesium sulfate or ferric sulfate required for growth, but is not limited thereto. In addition, it may contain essential growth substances such as amino acids and vitamins. Furthermore, suitable culture medium precursors may be used. The aforementioned culture medium or individual components may be added to the culture medium in batches or continuously during the culture process, but are not limited to this.

[0057] According to one specific embodiment of the invention, during the cultivation process, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid can be added to the microbial culture medium in an appropriate manner to adjust the pH of the culture medium. Furthermore, during the cultivation process, antifoaming agents such as polyethylene glycol esters of fatty acids can be used to suppress bubble formation. Further, to maintain an aerobic state in the culture medium, oxygen or an oxygen-containing gas (e.g., air) can be injected into the culture medium. The temperature of the culture medium is typically between 20°C and 45°C, for example, between 25°C and 40°C. The cultivation time can continue until the desired production of the useful substance is achieved, for example, between 10 and 160 hours.

[0058] According to a specific embodiment of the present invention, in the above-described step of recovering L-lysine from the cultured mutant and the culture medium for the mutant, the produced L-lysine can be collected or recovered from the culture medium according to the culture method and using suitable methods known in the art. For example, centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, fractional dissolution (e.g., ammonium sulfate precipitation), chromatography (e.g., ion exchange, affinity, hydrophobicity and size exclusion), etc., can be used, but are not limited thereto.

[0059] According to a specific embodiment of the present invention, in the step of recovering lysine, the culture medium can be separated by low-speed centrifugation to remove biomass, and the resulting supernatant can be separated by ion exchange chromatography.

[0060] According to a specific embodiment of the present invention, the step of recovering L-lysine described above may include a step of purifying L-lysine.

[0061] The Corynebacterium glutamicum mutant strain of the present invention can increase the supply of precursors and increase sugar utilization by increasing or enhancing the expression of the gene encoding enolase, or simultaneously reducing or weakening the expression of the gluconate operon transcription repressor. Attached Figure Description

[0062] Figure 1 This describes the structure of a pCG1+eno(G1A) vector containing an enolase Eno gene whose start codon is replaced by ATG, according to an embodiment of the present invention.

[0063] Figure 2 This describes the structure of a pCG1+gntR(S77F) vector containing a gntR gene, according to an embodiment of the present invention, in which the 77th amino acid of the gluconic acid operon transcriptional repressor gntR gene is replaced by phenylalanine instead of serine. Detailed Implementation

[0064] The present invention will now be described in more detail. However, such description is merely illustrative and is intended to aid in understanding the invention, and the scope of the invention is not limited to such illustrative description.

[0065] Example 1. Production of Corynebacterium glutamicum mutant strain

[0066] To create a Corynebacterium glutamicum mutant with enhanced enolase activity, Corynebacterium glutamicum DS1 strain and E. coli DH5a (HIT Competent cells™, Cat No. RH618) were used.

[0067] The above-mentioned Corynebacterium glutamicum DS1 strain was cultured in CM-broth medium (pH 6.8) at a temperature of 30°C. The composition of the CM-broth medium was 5 g glucose, 2.5 g NaCl, 5.0 g yeast extract, 1.0 g urea, 10.0 g peptone and 5.0 g beef extract in 1 L of distilled water.

[0068] The above-mentioned E. coli DH5a was cultured on LB medium at a temperature of 37°C. The composition of the LB medium was 10.0 g tryptone, 10.0 g NaCl and 5.0 g yeast extract in 1 L of distilled water.

[0069] The antibiotics kanamycin and streptomycin were products manufactured by Sigma.

[0070] The DNA sequencing analysis was commissioned to Makro Gene Co., Ltd.

[0071] 1-1. Preparation of Recombinant Vectors

[0072] To increase the precursor supply for the TCA cycle, an enhancement of enolase was introduced into the strain. In the method used in this embodiment, a specific mutation was induced in the translation start codon of the Eno gene to increase the expression of the enolase. The translation start codon of the Eno gene was mutated from GTG to ATG. On the *Corynebacterium glutamicum* genome, a 430bp portion of the left arm and a 439bp portion of the right arm were amplified by PCR, centered on the center of the Eno gene. After ligation using overlap PCR, the amplified portion was cloned into the recombinant vector pCGI (reference [Kim et al., Journal of Microbiological Methods 84(2011)128-130]). The plasmid was named pCG1+eno(G1A) (reference...). Figure 1 To prepare the plasmids described above, the primers listed in Table 1 were used to amplify the various gene fragments.

[0073] Table 1

[0074]

[0075] PCR was performed using the primers described above under the following conditions. Using a thermal cycler (TP600, TAKARA BIO Inc., Japan), 100 μM of each deoxyribonucleotide triphosphate (dATP, dCTP, dGTP, dTTP) was added to a reaction solution. 1 pM of oligonucleotides and 10 ng of Corynebacterium glutamicum ATCC 13032 chromosomal DNA were used as a template, and 25–30 cycles were performed in the presence of 1 unit of pfu-X DNA polymerase mixture. The PCR conditions were: (i) denaturation at 94°C for 30 seconds, (ii) annealing at 58°C for 30 seconds, and (iii) extension at 72°C for 1–2 minutes (2 minutes of polymerization time per 1 kb).

[0076] Using self-assembly cloning, the gene fragment prepared as described above was cloned into a pCGI vector. This vector was then transformed into E. coli DH5α, plated on LB agar plates containing 50 μg / ml kanamycin, and incubated at 37°C for 24 hours. The resulting colonies were isolated, and after confirming the correct presence of the insert in the vector, the vector was isolated for recombination with Corynebacterium glutamicum.

[0077] As a common process in the above methods, gene amplification was performed using PCR on genomic DNA from Corynebacterium glutamicum ATCC 13032. Following a strategy, the DNA was inserted into a pCGI vector via self-assembled cloning and selected from E. coli DH5α. Chromosomal base substitution involved amplifying individual gene fragments separately, using stacked PCR to create the target DNA fragment. During gene manipulation, Ex Taq polymerase (Takara) and Pfu polymerase (Solgent) were used as PCR amplification enzymes, and various restriction enzymes and DNA-modifying enzymes were prepared using NEB products, following the provided buffers and protocols.

[0078] 1-2. Production of mutant strains

[0079] The DS7-1 strain, a mutant strain, was created using the pCG1+eno(G1A) described above. The vector was prepared at a final concentration of 1 μg / μl or higher, and one recombination was induced in Corynebacterium glutamicum DS1 strain using electroporation (see [Tauchet et al., FEMS Microbiology letters 123 (1994) 343-347]). The electroporated strain was then plated onto CM-agar plates containing 20 μg / μl kanamycin. After isolation, PCR and base sequence analysis confirmed the correct insertion into the induced position on the genome. To induce two more recombinations, the isolated strain was inoculated into CM-agar liquid medium containing streptomycin and cultured overnight. Afterward, it was plated onto agar medium containing the same concentration of streptomycin, and colonies were isolated. After confirming the presence of kanamycin resistance in the finally isolated colonies, base sequence analysis was used to confirm whether a mutation had been introduced into the Eno gene in strains without antibiotic resistance (reference [Schafer et al., Gene 145 (1994) 69-73]). Finally, a Corynebacterium glutamicum mutant strain (DS7-1) with the mutated Eno gene was obtained.

[0080] Experimental Example 1. Comparison of L-lysine production rates between parental strains and mutant strains

[0081] The L-lysine production rates of the parent strain Corynebacterium glutamicum DS1 and the DS7-1 strain, a mutant strain for lysine production manufactured in Example 1, were compared.

[0082] In 100 ml flasks containing 10 ml of lysine medium with the composition shown in Table 2 below, either the parental strain (DS1) or the mutant strain (DS7-1) was inoculated and cultured at 30°C with shaking at 180 rpm for 28 hours. After culture, the production of L-lysine was determined by HPLC (Shimadzu, Japan), and the results are shown in Table 3.

[0083] Table 2

[0084] composition Content (based on 1L distilled water) Glucose 100g Ammonium sulfate 55g <![CDATA[KH2PO4]]> 1.1g <![CDATA[MgSO4·H2O]]> 1.2g <![CDATA[MnSO4·H2O]]> 180mg <![CDATA[FeSO4·H2O]]> 180mg Thiamine·HCl 9mg Biotin 1.8mg <![CDATA[CaCO3]]> 5％ pH 7.0

[0085] Table 3

[0086] strain L-Lysine (g / L) L-lysine production per unit of bacterial cells (g / gDCW) Parental strain (DS1) 52.9 6.6 Mutant strain (DS7-1) 64.7 7.0

[0087] As shown in Table 3 above, it was confirmed that in the *Corynebacterium glutamicum* mutant strain DS7-1, the Eno gene was replaced with the optimal translation initiation sequence (ATG) to enhance the lysine biosynthesis pathway, resulting in an approximately 22% increase in L-lysine production compared to the parental strain *Corynebacterium glutamicum* DS1. These results indicate that enhanced expression of the Eno gene increases the metabolic carbon source flux, thereby improving the strain's L-lysine production capacity.

[0088] Example 2. Production of Corynebacterium glutamicum mutant strain

[0089] 2-1. Preparation of Recombinant Vectors

[0090] To further improve the lysine production capacity of the Corynebacterium glutamicum mutant strain produced in Example 1, a weakened gluconate operon transcriptional repressor that affects sugar utilization was introduced into the Corynebacterium glutamicum mutant strain DS7-1.

[0091] In the method used in this embodiment, a specific mutation was induced in the gntR gene to weaken the expression of the gluconic acid operon transcriptional repressor. The 77th amino acid in the gntR gene sequence was replaced with phenylalanine instead of serine. On the Corynebacterium glutamicum genome, focusing on the portion containing the 77th amino acid of the gntR gene, the left arm (510 bp) and right arm (540 bp) were amplified by PCR. After ligation using overlap PCR, the amplified amplified amplification was cloned into the recombinant vector pCGI (reference [Kim et al., Journal of Microbiological Methods 84(2011)128-130]). The plasmid was named pCG1+gntR(S77F) (reference...). Figure 2 To prepare the plasmids described above, the primers listed in Table 4 were used to amplify the various gene fragments.

[0092] Table 4

[0093]

[0094] The process of amplifying and cloning the corresponding gene was carried out using the same method as in Example 1-1.

[0095] 2-2. Production of mutant strains

[0096] The DS7-2 strain was created as a mutant strain using the aforementioned pCG1+gntR(S77F). Using pCG1+gntR(S77F) instead of pCG1+eno(G1A) as the vector, and using the Corynebacterium glutamicum mutant strain DS7-1 instead of Corynebacterium glutamicum DS1 as the host cell, the same methods as in Examples 1-2 were performed, thus finally obtaining the Corynebacterium glutamicum mutant strain (DS7-2) with the mutated gntR gene introduced.

[0097] Experimental Example 2. Comparison of L-lysine production rates between parental strains and mutant strains

[0098] The L-lysine production rates of the DS7-1 strain manufactured in Example 1 as a lysine-producing mutant strain and the DS7-2 strain manufactured in Example 2 as a lysine-producing mutant strain, with the DS7-1 strain used as the parent strain, were compared.

[0099] The L-lysine production was determined using the same method as in Experimental Example 1, and the results are shown in Table 5.

[0100] Table 5

[0101] strain L-Lysine (g / L) L-lysine production per unit of bacterial cells (g / gDCW) Parental strain (DS7-1) 63.8 6.9 Mutant strain (DS7-2) 65.3 7.4

[0102] As shown in Table 5 above, it was confirmed that in the *Corynebacterium glutamicum* mutant strain DS7-2, the Eno gene was replaced with the optimal translation initiation sequence (ATG) to enhance the lysine biosynthesis pathway. Furthermore, a specific position (amino acid 77) in the gntR gene amino acid sequence was replaced with the optimal base sequence, resulting in an approximately 2% increase in L-lysine production compared to the *Corynebacterium glutamicum* strain DS7-1 used as the parent strain. These results indicate that enhanced expression of the Eno gene and weakened expression of the gntR gene increase the metabolic carbon source flux, thereby improving the strain's L-lysine production capacity.

[0103] So far, the invention has been studied around its preferred embodiments. Those skilled in the art will understand that the invention can be implemented in modified forms without departing from its essential characteristics. Therefore, the disclosed embodiments should be considered illustratively rather than restrictively. The scope of the invention is shown in the claims rather than in the foregoing description and should be interpreted as including all differences within its equivalent scope.

Claims

1. A mutant strain of *Corynebacterium glutamicum* exhibits enhanced enolase activity and weakened gluconate operon transcriptional repressor activity, thereby increasing L-lysine production capacity. The mutant strain comprises: a nucleotide sequence represented by SEQ ID NO:3, wherein the start codon GTG of the gene encoding enolase is replaced with ATG; and an amino acid sequence of SEQ ID NO:8, wherein serine at position 77 of the amino acid sequence of the gluconate operon transcriptional repressor is replaced with phenylalanine.

2. A method for producing L-lysine, comprising the following steps: a) The step of culturing the Corynebacterium glutamicum mutant strain of claim 1 in a culture medium; as well as b) The step of recovering L-lysine from the mutant strain or the culture medium in which the mutant strain is cultured.

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