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

Abstract

The problem that this invention aims to solve is to provide a Corynebacterium glutamicum mutant strain with improved L-lysine production ability, and a method for producing L-lysine using the mutant strain. [Solution] 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 Corynebacterium glutamicum mutant strain can increase the supply of precursors and increase sugar utilization, thereby improving the L-lysine production yield, by increasing or enhancing the expression of genes that encode enolase, or simultaneously decreasing or weakening the expression of gluconate operon transcription repressors.

Description

Technical Field

[0001] The present invention relates to a Corynebacterium glutamicum mutant 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 for 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 containing an enzyme related to the production of L-lysine to increase the expression of the gene or removing unnecessary genes. In addition, Korean Published Patent 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 tens, there is still a great need for much research on the presence or absence of an increase in the L-lysine production ability due to changes in the activity of such proteins at present.

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 conducted research using Corynebacterium glutamicum strains to develop new mutant strains with improved L-lysine production capacity. As a result, they confirmed that L-lysine production increases when the gene sequence of the Eno gene, which encodes enolase involved in the L-lysine biosynthesis pathway, is substituted, particularly the start codon. Furthermore, they confirmed that L-lysine production increases even further when amino acids at specific positions in the amino acid sequence of the gntR gene, which encodes a gluconate operon transcription repressor, are substituted. This led to the completion of the present invention.

[0009] One aspect of the present invention provides a Corynebacterium glutamicum mutant strain in which enolase activity is enhanced and L-lysine production capacity is improved.

[0010] The term "enolase" used in this invention is also called phosphopyruvate hydratase, and refers to an enzyme that catalyzes the reaction in which 2-phosphoglycerate (2-PG) is converted to phosphoenolpyruvate (PEP) in order to supply pyruvate to the TCA cycle.

[0011] According to one specific example of the present invention, the enolase 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.

[0012] In this invention, "enhanced activity" means that the expression of genes that encode target proteins such as enzymes, transcription factors, and transport proteins is newly introduced or increased, resulting in an increase in expression levels compared to the wild-type strain or the strain before deformation. Such enhanced activity includes cases where the activity of the protein itself increases compared to the activity of the protein originally possessed by the microorganism due to nucleotide substitution, insertion, deletion, or a combination thereof that encodes the gene, as well as cases where the overall level of enzyme activity within the cell is higher than that of the wild-type strain or the strain before deformation due to increased expression or increased translation of the gene that encodes it, and combinations thereof.

[0013] According to one specific example of the present invention, the enhancement of enolase activity may involve inducing a site-directed mutation in the gene that encodes enolase.

[0014] According to one specific example of the present invention, the gene that encodes the enolase may be represented by the base sequence of Sequence ID No. 1.

[0015] Furthermore, according to one specific example of the present invention, the gene that encodes the enolase may be represented by the amino acid sequence of SEQ ID NO: 2.

[0016] According to one specific example of the present invention, the enhancement of enolase activity may be achieved by substituting one or more bases among the 1 to 100 bases in the base sequence of the gene that encodes enolase.

[0017] More specifically, the gene mutation in the present invention may be a result of the sequential or discontinuous substitution of 1 to 100 bases, preferably 1 to 10 bases, in the base sequence of the gene that encodes enolase.

[0018] According to one specific example of the present invention, the enhancement of enolase activity may be achieved by substituting the start codon of the gene encoding enolase with ATG.

[0019] According to one embodiment of the present invention, a Corynebacterium glutamicum mutant strain having a new start codon for the Eno gene was obtained by substituting the start codon from GTG to ATG in the nucleotide sequence of Sequence ID No. 1, which encodes the enolase of the Corynebacterium glutamicum strain. Such a Corynebacterium glutamicum mutant strain may contain an enolase amino acid sequence gene encoded in the nucleotide sequence of Sequence ID No. 3 or the amino acid sequence of Sequence ID No. 4.

[0020] Thus, Corynebacterium glutamicum mutant strains possessing mutations in the enolase gene can exhibit improved L-lysine production capacity.

[0021] In this invention, "improved production capacity" means an increase in L-lysine production compared to the parent strain. The parent strain refers to the wild type or mutant strain that is the target of mutation, and includes strains that are directly subjected to mutation or transformed with recombinant vectors, etc. In this invention, the parent strain may be a wild-type Corynebacterium glutamicum strain or a strain that has mutated from the wild type.

[0022] According to one specific example of the present invention, the parent strain is a mutant strain in which mutations are induced in the sequences of genes involved in lysine production (for example, lysC, zwf, and hom genes), and it may be a Corynebacterium glutamicum strain (hereinafter referred to as "Corynebacterium glutamicum DS1 strain") deposited with the Korean Culture Center of Microorganisms under the accession number KCCM12969P on April 2, 2021.

[0023] According to one embodiment of the present invention, the Corynebacterium glutamicum mutant strain with improved L-lysine production ability contains a start codon mutation of the enolase Eno gene, thereby increasing the supply amount of PEP, an important precursor of L-lysine, 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 40% (preferably 10 to 30%) compared to the parent strain, and 55 to 85 g of L-lysine can be produced per liter of the strain culture solution, preferably 60 to 80 g of L-lysine can be produced.

[0024] In addition, in the present invention, in order to further improve the L-lysine production ability, in addition to the Eno gene encoding enolase, mutations can be induced in other genes involved in L-lysine biosynthesis.

[0025] According to one specific example of the present invention, the mutant strain may further include attenuation of the activity of the glucuronate operon transcriptional repressor.

[0026] In microorganisms, transcription proceeds and protein expression is regulated by an operon composed of regulatory genes, activator genes, promoters, and structural genes. When transcriptional activation is determined, a series of proteins that induce the activation of inactivated genes and promote smooth transcription are called transcriptional activators, while a series of proteins produced to reactivate activated genes are called transcriptional repressors. The "gluconate operon transcriptional repressor" used in this invention is a regulatory protein involved in gluconate metabolism and glucose influx, and plays a role in suppressing mRNA synthesis by binding to the activator gene of the gluconate operon.

[0027] According to one specific example of the present invention, the gluconate operon transcription repressor may be derived from a strain of 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.

[0028] As used in this invention, "weakened activity" means that the expression of the gene encoding the target protein, such as the enzyme, transcription factor, or transport protein, is suppressed or removed, resulting in a decrease in expression compared to the wild-type strain or the strain before deformation. Such weakened activity includes cases where the activity of the protein itself is reduced compared to the activity of the protein originally possessed by the microorganism due to nucleotide substitution, insertion, deletion, or a combination thereof that encodes the gene, and cases where the overall level of enzyme activity within the cell is lower than that of the wild-type strain or the strain before deformation due to inhibition of gene expression or translation inhibition, or a combination thereof.

[0029] According to one specific example of the invention, the weakening of the activity of the gluconate operon transcription repressor may involve inducing a site-directed mutation in the gene encoding the gluconate operon transcription repressor.

[0030] According to one specific example of the present invention, the gene that encodes the gluconate operon transcription repressor may be represented by the nucleotide sequence of Sequence ID No. 5.

[0031] Furthermore, according to one specific example of the present invention, the gene that encodes the gluconate operon transcription repressor may be represented by the amino acid sequence of SEQ ID NO: 6.

[0032] According to one specific example of the present invention, the weakening of the activity of the gluconate operon transcription repressor may be achieved by substituting one or more amino acids in the amino acid region between positions 10 and 100 within the amino acid sequence of the gene encoding the gluconate operon transcription repressor.

[0033] More specifically, the gene mutation in the present invention may be a mutation in which 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 amino acid regions of positions 10-100, 20-90, or 30-80, are substituted consecutively or discontinuously.

[0034] According to one embodiment of the present invention, a Corynebacterium glutamicum mutant strain having a novel amino acid sequence of the gntR gene was obtained by substituting the 77th position of the gntR gene, which encodes a gluconate operon transcription repressor, from serine (Ser) to phenylalanine (Phe). Such a Corynebacterium glutamicum mutant strain may contain the gntR gene, which encodes a gluconate operon transcription repressor, in the nucleotide sequence of SEQ ID NO: 7 or the amino acid sequence of SEQ ID NO: 8.

[0035] Here, the parent strain may be a Corynebacterium glutamicum mutant strain in which a mutation has been induced in the sequence of the Eno gene that encodes enolase.

[0036] According to one embodiment of the present invention, the Corynebacterium glutamicum mutant strain with improved L-lysine production ability, by including an amino acid mutation in the gluconate operon transcription repressor gntR gene along with a start codon mutation in the enolase Eno gene, increases the sugar utilization ability of the strain and exhibits increased L-lysine production ability compared to the parent strain containing only the start codon mutation in the Eno gene that encodes enolase. In particular, the amount of L-lysine produced is increased by 2% or more, specifically 2-20%, compared to the parent strain, and it is possible to produce 63-85g of L-lysine per liter of strain culture medium, preferably 65-80g.

[0037] A specific example of the present invention, a Corynebacterium glutamicum mutant, can be realized by a recombinant vector containing a mutant in which the start codon sequence of the enolase Eno gene is substituted in the parent strain and / or a mutant in which the amino acid sequence of the gluconate operon transcription repressor gntR gene is partially substituted.

[0038] The term "part" as used in this invention means that it does not refer to the entirety of an 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.

[0039] The term "mutant" as used in this invention refers to a mutant in which the start codon sequence of the enolase Eno gene, which is involved in L-lysine biosynthesis, is replaced from GTG to ATG, and / or in which one or more amino acids in the amino acid region between positions 10 and 100 of the amino acid sequence of the gluconate operon transcription repressor gntR gene are replaced.

[0040] According to one specific example of the present invention, the mutant in which the start codon of the enolase gene is replaced with ATG may have the base sequence of SEQ ID NO: 3 or the amino acid sequence of SEQ ID NO: 4.

[0041] Furthermore, according to one specific example of the present invention, a mutant in which the 77th amino acid in the amino acid sequence of the gluconate operon transcription repressor gene is substituted may have the base sequence of SEQ ID NO: 7 or the amino acid sequence of SEQ ID NO: 8.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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 DS1 strain.

[0049] Another aspect of the present invention provides a method for producing L-lysine, comprising the steps of: a) culturing the Corynebacterium glutamicum mutant strain in a culture medium; and b) recovering L-lysine from the mutant strain or the culture medium in which the mutant strain was cultured.

[0050] 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.

[0051] 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).

[0052] 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 iron sulfate necessary for growth. Other essential growth substances such as amino acids and vitamins may also be included. Appropriate precursors can also 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 is not limited to this.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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]

[0057] The Corynebacterium glutamicum mutant strain according to the present invention can increase the supply of precursors and improve sugar utilization capacity, thereby improving the production yield of L-lysine, by increasing or enhancing the expression of genes that encode enolase, or simultaneously decreasing or weakening the expression of gluconate operon transcription repressors. [Brief explanation of the drawing]

[0058] [Figure 1] This figure shows the structure of a pCG1+eno(G1A) vector containing the enolase Eno gene in which the start codon has been substituted from GTG to ATG according to one embodiment of the present invention. [Figure 2]This figure shows the structure of a pCG1+gntR(S77F) vector containing a gntR gene in which the 77th amino acid in the amino acid sequence of the gntR gene, a gluconate operon transcription repressor, is substituted from serine to phenylalanine, according to one embodiment of the present invention. [Modes for carrying out the invention]

[0059] 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.

[0060] Example 1. Production of Corynebacterium glutamicum mutant strain To produce Corynebacterium glutamicum mutant strains with enhanced enolase activity, Corynebacterium glutamicum DS1 strain and E. coli DH5a (HIT Competent cells) were used. TM Cat No. RH618 was used.

[0061] The Corynebacterium glutamicum DS1 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. 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.

[0062] The antibiotics kanamycin and streptomycin were products of Sigma.

[0063] DNA sequencing analysis was commissioned to Macrogen Co., Ltd.

[0064] 1-1. Preparation of recombinant vectors Enolase enhancement was introduced to increase the supply of precursors to the TCA cycle in the bacterial strain. The method used in this example involved inducing a specific mutation in the start codon of the Eno gene, which encodes enolase, in order to increase the expression of the gene. A mutation was introduced in the start codon of the Eno gene from GTG to ATG, and the left arm 430bp and right arm 439bp portions of the Eno gene, centered on the middle, 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 pCG1+eno(G1A) (see Figure 1). The primers shown in Table 1 below were used to amplify each gene fragment to construct the plasmid.

[0065] [Table 1]

[0066] 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).

[0067] 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.

[0068] 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.

[0069] 1-2. Production of mutant strains The mutant strain DS7-1 was prepared using the aforementioned pCG1+eno(G1A). The vector was prepared to a final concentration of 1 μg / μl or higher, and primary recombination was induced in the Corynebacterium glutamicum DS1 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, 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 non-antibiotic-resistant strains were examined by nucleotide sequence analysis to determine if mutations had been introduced into the Eno gene (see reference [Schafer et al., Gene145(1994)69-73]). Finally, a Corynebacterium glutamicum mutant strain (DS7-1) with a mutated Eno gene was obtained.

[0070] Experimental Example 1. Comparison of L-lysine productivity between parent strain and mutant strain. The L-lysine productivity of the parent strain Corynebacterium glutamicum DS1 and the lysine-producing mutant strain DS7-1, produced in Example 1, were compared.

[0071] The parent strain (DS1) or mutant strain (DS7-1) was inoculated into a 100 ml flask containing 10 ml of lysine medium with the composition shown in Table 2 below, and cultured with shaking at 30°C for 28 hours at 180 rpm. After the culture was completed, lysine analysis was performed by measuring the amount of L-lysine produced by HPLC (Shimazu, Japan), and the results are shown in Table 3 below.

[0072] [Table 2]

[0073] [Table 3]

[0074] As shown in Table 3 above, the Corynebacterium glutamicum mutant DS7-1 was found to have approximately 22% increased L-lysine productivity compared to the parent Corynebacterium glutamicum DS1 strain, due to the substitution of the Eno gene with the optimal translation initiation sequence (ATG) to enhance the lysine biosynthesis pathway. This result indicates that enhancing Eno gene expression improves the L-lysine production capacity of the strain by increasing the flow of metabolic carbon sources.

[0075] Example 2. Production of Corynebacterium glutamicum mutant strains 2-1. Preparation of recombinant vectors To further improve the lysine production capacity of the Corynebacterium glutamicum mutant strain produced in Example 1, a weakening of the gluconate operon transcription repressor affecting sugar utilization was introduced into the Corynebacterium glutamicum mutant strain DS7-1.

[0076] The method used in this example induced a gntR gene-specific mutation to weaken the expression of the gntR gene, which encodes a gluconate operon transcription repressor. The 77th amino acid in the gntR gene sequence was replaced from serine to phenylalanine. The 510bp left arm and 540bp right arm regions, centered on the portion containing the 77th amino acid of the gntR gene, were amplified by PCR on the Corynebacterium glutamicum genome. These regions were then ligated using overlap PCR and cloned into the recombinant vector pCGI (see reference [Kim et al., Journal of Microbiological Methods 84(2011)128-130]). The plasmid was named pCG1+gntR(S77F) (see Figure 2). The primers listed in Table 4 below were used to amplify each gene fragment to construct the plasmid.

[0077] [Table 4]

[0078] The process of amplifying and cloning the gene was carried out in the same manner as in Example 1-1.

[0079] 2-2. Production of mutant strains The mutant strain DS7-2 was produced using the aforementioned pCG1+gntR(S77F). The same procedure as in Example 1-2 was followed, except that pCG1+gntR(S77F) was used as the vector instead of pCG1+eno(G1A), and the Corynebacterium glutamicum mutant strain DS7-1 was used as the host cell instead of the Corynebacterium glutamicum DS1 strain. Ultimately, a Corynebacterium glutamicum mutant strain (DS7-2) with the mutant gntR gene introduced was obtained.

[0080] Experimental Example 2. Comparison of L-lysine productivity between parent strain and mutant strain. The L-lysine productivity of the DS7-1 strain, a lysine-producing mutant strain produced in Example 1, and the DS7-2 strain, a lysine-producing mutant strain produced in Example 2 using the DS7-1 strain as the parent strain, were compared.

[0081] L-lysine production was measured using the same method as in Experimental Example 1, and the results are shown in Table 5.

[0082] [Table 5]

[0083] As shown in Table 5 above, the Corynebacterium glutamicum mutant DS7-2 showed an approximately 2% increase in L-lysine productivity compared to the parent strain Corynebacterium glutamicum DS7-1. This was achieved not only by the substitution of the Eno gene with the optimal translation initiation sequence (ATG) to enhance the lysine biosynthesis pathway, but also by the substitution of a specific position (amino acid 77) in the amino acid sequence of the gntR gene with the optimal base sequence. Through these results, it was found that enhancing Eno gene expression and weakening gntR gene expression improve the L-lysine production capacity of the strain by increasing the flow of metabolic carbon sources.

[0084] 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. A Corynebacterium glutamicum mutant strain in which enolase activity is enhanced and L-lysine production capacity is improved, wherein the enhancement of enolase activity is achieved by substituting the start codon GTG of the gene encoding enolase with ATG.

2. The Corynebacterium glutamicum mutant strain according to claim 1, wherein the mutant strain contains the nucleotide sequence of Sequence ID No.

3.

3. The Corynebacterium glutamicum mutant according to claim 1, further comprising weakened activity of a gluconate operon transcriptional repressor.

4. The Corynebacterium glutamicum mutant strain according to claim 3, wherein the mutant strain contains the amino acid sequence of SEQ ID NO:

8.

5. a) The step of culturing the Corynebacterium glutamicum mutant strain described in any one of claims 1 to 4 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 containing [the specified ingredient].

Images