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

Abstract

The present invention relates to a Corynebacterium glutamicum mutant strain having improved L-lysine production ability and a method for producing L-lysine using the same, and the mutant strain can improve the L-lysine production yield compared to the parent strain by increasing or enhancing the expression of a gene encoding aspartate aminotransferase.

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 Patent Documents 1 and 2, 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 L-lysine production to increase the expression of the gene or removing unnecessary genes. Patent Document 3 discloses a method of changing the 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 L-lysine production.

[0004] Thus, various methods for increasing the L-lysine production ability have been developed. However, since the number of types of proteins such as enzymes, transcription factors, and transport proteins directly or indirectly related to L-lysine production reaches more than several tens, there is still a need for a lot of research on whether the L-lysine production ability is increased due to changes in the activity of such proteins.

Prior Art Documents

Patent Documents

[0005] [Patent Document 1] Korean Patent Registration No. 10-0838038 [Patent Document 2] Korean Patent Registration No. 10-2139806 [Patent Document 3] Korean Patent Publication No. 10-2020-0026881 [Non-Patent Document]

[0006] [Non-Patent Document 1] Manual of Methods for General Bacteriology. American Society for Bacteriology. Washington D.C., USA, 1981 [Non-Patent Document 2] Kim et al., Journal of Microbiological Methods 84 (2011) 128-130 [Non-Patent Document 3] Tauch et al., FEMS Microbiology letters 123 (1994) 343-347 [Non-Patent Document 4] Schafer et al., Gene 145 (1994) 69-73 [Summary of the Invention] [Problems to be Solved by the Invention]

[0007] An object of the present invention is to provide a Corynebacterium glutamicum mutant strain having improved L-lysine production ability. Another object of the present invention is to provide a method for producing L-lysine using the mutant strain. [Means for Solving the Problems]

[0008] As a result of research to develop a new mutant strain with improved L-lysine production ability using Corynebacterium glutamicum strains, the present inventors completed the present invention by confirming that the L-lysine production amount increases when the base sequence at a specific position in the promoter of the aspB gene encoding aspartate aminotransferase, which is a lysine precursor in the L-lysine biosynthetic pathway, is substituted.

[0009] One aspect of the present invention provides a Corynebacterium glutamicum mutant strain in which the activity of aspartate aminotransferase is enhanced and the L-lysine production ability is improved. The "aspartate aminotransferase" used in the present invention means an enzyme that catalyzes the conversion reaction of oxaloacetate to aspartate in the L-lysine biosynthetic pathway.

[0010] According to one specific example of the present invention, the aspartate aminotransferase may be derived from a strain belonging to the genus Corynebacterium. Specifically, the Corynebacterium genus strain may be Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium callunae, Corynebacterium suranareeae, Corynebacterium lubricantis, Corynebacterium doosanense, Corynebacterium efficiens, Corynebacterium uterequi, Corynebacterium stationis, Corynebacterium pacaense, Corynebacterium singulare, Corynebacterium humireducens, Corynebacterium marinum, Corynebacterium halotolerans, Corynebacterium spheniscorum, Corynebacterium freiburgense, Corynebacterium striatum, Corynebacterium canis, Corynebacterium ammoniagenes (CorynebacteriumIt may be, but is not limited to, Ammoniagenes, Corynebacterium renale, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium caspium, Corynebacterium testudinoris, Corynebacterium pseudopelargi or Corynebacterium flavescens.

[0011] As used in the present invention, "enhanced activity" means that the expression of a gene encoding a protein such as a target enzyme, transcription factor, transport protein, etc. is newly introduced or increased, and the expression level is increased compared to the wild-type strain or the strain before transformation. Such enhancement of activity includes cases where the activity of the protein itself increases compared to the activity of the protein originally possessed by the microorganism through substitution, insertion, deletion of nucleotides encoding the gene, or combinations thereof, and cases where the overall degree of enzyme activity in the cell is higher than that of the wild-type strain or the strain before transformation due to increased expression or increased translation of the gene encoding it, and also includes combinations thereof.

[0012] According to one specific example of the present invention, the enhancement of the activity of the aspartate aminotransferase may be one that induces a site-specific mutation in the promoter of the gene encoding the aspartate aminotransferase.

[0013] According to one specific example of the present invention, the promoter of the gene encoding the aspartate aminotransferase may be represented by the nucleotide sequence of SEQ ID NO: 1. The "promoter" used in the present invention refers to a specific site of DNA that contains a binding site for RNA polymerase, which initiates mRNA transcription of a target gene, and regulates gene transcription. Generally, it is located upstream with respect to the transcription start point. A promoter in prokaryotes is defined as a site around the transcription start point to which RNA polymerase binds, and generally consists of two short base sequences in which base pairs in the -10 region and -35 region are separated upstream from the transcription start point. The promoter mutation in the present invention is to improve it to have higher activity compared to the wild-type promoter, and by inducing a mutation within the promoter region located upstream of the transcription start point, the expression of a gene located downstream can be increased.

[0014] According to one specific example of the present invention, the enhancement of the activity of the aspartate aminotransferase may be one in which one or more bases in the region from -100 to -10 upstream from the transcription start point in the promoter sequence of the gene encoding aspartate aminotransferase are substituted.

[0015] More specifically, the promoter mutation in the present invention may be one in which 1, 2, 3, 4, or 5 bases are continuously or discontinuously substituted in one or more bases in the region from -100 to -10, preferably in the region from -95 to -15, -95 to -55, -55 to -40, or -30 to -15.

[0016] According to one embodiment of the present invention, a Corynebacterium glutamicum mutant strain having a new promoter sequence of the aspB gene was obtained by substituting the base sequence at the -22 region from C to G in the promoter sequence of the aspB gene encoding aspartate aminotransferase of the Corynebacterium glutamicum strain. Such a Corynebacterium glutamicum mutant strain may contain the mutated promoter of the aspB gene represented by the base sequence of SEQ ID NO: 2.

[0017] Also, according to one embodiment of the present invention, a Corynebacterium glutamicum mutant strain having a new promoter sequence of the aspB gene was obtained by substituting the base sequence in the -45 region of the promoter sequence of the aspB gene encoding aspartate aminotransferase of the Corynebacterium glutamicum strain from T to A. Such a Corynebacterium glutamicum mutant strain may contain the mutated promoter of the aspB gene represented by the base sequence of SEQ ID NO: 3.

[0018] According to one embodiment of the present invention, a Corynebacterium glutamicum mutant strain having a new promoter sequence of the aspB gene was obtained by substituting the base sequence in the -88 region of the promoter sequence of the aspB gene encoding aspartate aminotransferase of the Corynebacterium glutamicum strain from T to A. Such a Corynebacterium glutamicum mutant strain may contain the mutated promoter of the aspB gene represented by the base sequence of SEQ ID NO: 4.

[0019] 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, including those directly targeted for mutation or those transformed with a recombinant vector, etc. In the present invention, the parent strain may be a wild type Corynebacterium glutamicum strain or a strain mutated from the wild type.

[0020] 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 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.

[0021] Such a Corynebacterium glutamicum mutant with improved L-lysine production ability of the present invention may contain a mutated promoter sequence of the gene encoding aspartate aminotransferase.

[0022] According to a specific example of the present invention, the mutant may contain any one of the base sequences represented by SEQ ID NOs: 2 to 4 as the promoter sequence of the aspartate aminotransferase gene.

[0023] According to an embodiment of the present invention, the mutant contains a promoter mutation of the aspB gene encoding aspartate aminotransferase, and shows an increased L-lysine production ability compared to the parent strain. In particular, the L-lysine production amount is increased by 3% or more, specifically 3 to 40%, more specifically 5 to 30% compared to the parent strain, and 65 to 90 g of L-lysine can be produced per liter of the strain culture solution, preferably 70 to 80 g of L-lysine can be produced.

[0024] The Corynebacterium glutamicum mutant according to a specific example of the present invention is embodied through a recombinant vector containing a mutant in which a part of the promoter sequence of the gene encoding aspartate aminotransferase is substituted in the parent strain.

[0025] The "part" used in the present invention means not all of the 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.

[0026] The "mutant" used in the present invention means a promoter mutant in which one or more bases in the -100 to -10 region of the promoter sequence of the aspartate aminotransferase gene involved in the biosynthesis of L-lysine are substituted.

[0027] According to one specific example of the present invention, a mutant in which the base sequence in the -22 region within the promoter sequence of the aspartate aminotransferase gene is substituted with G may have the base sequence of SEQ ID NO: 2, a mutant in which the base sequence in the -45 region is substituted with A may have the base sequence of SEQ ID NO: 3, and a mutant in which the base sequence in the -88 region is substituted with A may have the base sequence of SEQ ID NO: 4.

[0028] The "vector" used in the present invention refers to an expression vector capable of expressing a target protein in an appropriate host cell, and means a genetic construct containing essential regulatory elements operably linked so that the gene insert is expressed. Here, "operably linked" means that the gene whose expression is required and its regulatory sequence are linked in a manner that enables gene expression by being functionally bound to each other, and the "regulatory element" includes a promoter for transcription, any operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence for regulating the termination of transcription and translation. Such vectors include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, viral vectors, and the like.

[0029] The "recombinant vector" used in the present invention is replicable independently of the host cell genome or integrated into the genome itself after being transformed into a suitable host cell. At this time, the "suitable host cell" can include an origin of replication which is a specific base sequence where replication is initiated as a vector-replicable one.

[0030] For the transformation, a vector introduction technique suitable for the host cell is selected so that the target gene can be expressed in the host cell. For example, the introduction of the vector can be carried out by electroporation, heat-shock, precipitation of calcium phosphate (CaPO 4 ) or calcium chloride (CaCl 2Precipitation of ), microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, lithium acetate-DMSO method, or a combination thereof. The transformed gene can be inserted into the host cell chromosome or located extrachromosomally without limitation as long as it can be expressed in the host cell.

[0031] 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. A host cell containing the recombinant vector of the present invention is a recombinant host cell, recombinant cell, or recombinant microorganism.

[0032] In addition, the recombinant vector according to the present invention can contain a selection marker, and the selection marker is for selecting a transformant (host cell) transformed with the vector. Only cells that express the selection marker in the medium treated with the selection marker can survive, so that the transformed cells can be selected. Representative examples of the selection marker include, but are not limited to, kanamycin, streptomycin, chloramphenicol, etc.

[0033] The gene inserted into the recombinant vector for transformation of the present invention is replaced into a host cell such as a Corynebacterium microorganism by homologous recombination crossover. According to one specific example of the present invention, the host cell may be a Corynebacterium strain, for example, a Corynebacterium glutamicum strain.

[0034] In addition, another aspect of the present invention provides a method for producing L-lysine, comprising: a) culturing the Corynebacterium glutamicum mutant strain in a medium; and b) recovering L-lysine from the mutant strain or the medium in which the mutant strain has been cultured.

[0035] The above-mentioned cultivation is carried out with an appropriate medium and cultivation conditions known in the art, and an ordinary technician can easily adjust and use the medium and cultivation conditions. Specifically, the medium may be a liquid medium, but is not limited thereto. The cultivation method may include, for example, batch culture, continuous culture, fed-batch culture or a combined culture thereof, but is not limited thereto.

[0036] According to a specific example of the present invention, the medium must meet the requirements of a specific strain in an appropriate manner and can be appropriately modified by an ordinary technician. For the culture medium for Corynebacterium strains, known literature (Manual of Methods for General Bacteriology. American Society for Bacteriology. Washington D.C., USA, 1981) can be referred to, but is not limited thereto.

[0037] According to one specific example of the present invention, the culture medium can contain various carbon sources, nitrogen sources, and trace element components. Examples of carbon sources that can be used 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 can be used individually or as a mixture, and are not limited thereto. Examples of nitrogen sources that can be used include peptone, yeast extract, gravy, 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. The nitrogen source can also be used individually or as a mixture, and is not limited thereto. Examples of phosphorus sources that can be used include potassium dihydrogen phosphate or dipotassium hydrogen phosphate or salts containing the corresponding sodium, and are not limited thereto. Further, the culture medium can contain metal salts such as magnesium sulfate or iron sulfate required for growth, and is not limited thereto. In addition, essential growth substances such as amino acids and vitamins can be included. Also, appropriate precursors can be used in the culture medium. The medium or individual components are added to the culture broth in a suitable manner, batchwise or continuously, during the culture process, but are not limited thereto.

[0038] According to one specific example of the present invention, during the culture, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid can be added to the microbial culture broth in a suitable manner to adjust the pH of the culture broth. Also, an antifoaming agent such as a fatty acid polyglycol ester can be used during the culture to suppress bubble formation. Further, in order to maintain the aerobic state of the culture broth, oxygen or an oxygen-containing gas (e.g., air) can be injected into the culture broth. The temperature of the culture broth is usually 20°C to 45°C, for example, 25°C to 40°C. The culture period can be continued until the desired production amount of the useful substance is obtained, for example, 10 to 160 hours.

[0039] According to one specific example of the present invention, in the step of recovering L-lysine from the cultured mutant strain and the medium in which the mutant strain was cultured, L-lysine produced from the medium can be collected or recovered by using an appropriate method known in the art according to the culture method. For example, methods such as 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) can be used, but are not limited thereto.

[0040] According to one specific example of the present invention, in the step of recovering lysine, the culture medium can be centrifuged at a low speed to remove biomass, and the resulting supernatant can be separated through ion exchange chromatography.

[0041] According to one specific example of the present invention, the step of recovering the L-lysine can include a step of purifying the L-lysine.

Advantages of the Invention

[0042] The Corynebacterium glutamicum mutant strain according to the present invention can improve the production yield of L-lysine compared to the parent strain by increasing or enhancing the expression of the gene encoding aspartate aminotransferase.

Brief Description of the Drawings

[0043]

Figure 1

Figure 2

Figure 3

Mode for Carrying Out the Invention

[0044] Hereinafter, the present invention will be described in more detail. However, such description is merely presented illustratively to assist in understanding the present invention, and the scope of the present invention is not limited by such illustrative description.

[0045] Example 1. Production of Corynebacterium glutamicum Mutant In order to produce a Corynebacterium glutamicum mutant with enhanced aspartate aminotransferase activity, random mutations were induced using Corynebacterium glutamicum DS1 strain.

[0046] 1-1. Induction of Mutation The Corynebacterium glutamicum DS1 strain was inoculated into a 50-ml flask containing CM liquid medium (containing 5 g of glucose, 2.5 g of NaCl, 5.0 g of yeast extract, 1.0 g of urea, 10.0 g of polypeptone, and 5.0 g of beef extract, pH 6.8). After adding N-methyl-N'-nitro-N-nitrosoguanidine (NTG), a mutagen, at a final concentration of 300 μg / ml, the mixture was cultured with shaking at 200 rpm at 30°C for 20 hours, and then exposed to UV for 20 minutes to induce additional mutations. After the culture was completed, the culture broth was centrifuged at 12,000 rpm for 10 minutes to remove the supernatant, washed once with saline, and further washed three times with phosphate buffer. This was suspended in 5 ml of phosphate buffer and then spread on CM solid medium (CM liquid medium further containing 15 g / l of agar and 8% of lysine), and cultured at 30°C for 30 hours to isolate 100 colonies.

[0047] 1-2. Selection of Mutants with Improved L-Lysine Productivity One hundred isolated colonies were each inoculated at 5% into flasks containing 10 ml of the production liquid medium shown in Table 1 below, and cultured with shaking at 30 °C and 200 rpm for 30 hours. The absorbance of each culture solution was measured at OD 610 nm, and 10 colonies that produced 75.0 g / l or more of L-lysine were selected by comparing the L-lysine production amounts, and the promoter mutation positions of the aspB gene were confirmed by analyzing the nucleotide sequences of these colonies. As a result of confirming the nucleotide sequences of these Corynebacterium glutamicum DS1 mutant strains in which mutations were induced, three types of mutations, C>g at position -22, T>A at position -45, and T>A at position -88, were confirmed.

[0048]

Table 1

[0049] Thereafter, an experiment was conducted to verify the increase in L-lysine productivity due to the three types of aspB gene promoter mutations. Example 2. Production of Corynebacterium glutamicum mutant strains In order to produce a Corynebacterium glutamicum mutant strain with enhanced aspartate aminotransferase activity, Corynebacterium glutamicum DS1 strain and E. coli DH5a (HIT Competent cells TM , Cat No. RH618) were used.

[0050] The Corynebacterium glutamicum DS1 strain was cultured at a temperature of 30 °C in a CM liquid medium or solid medium (agar 15 g / L was added as necessary) (pH 6.8) having a composition of 5 g of glucose, 2.5 g of NaCl, 5.0 g of yeast extract, 1.0 g of urea, 10.0 g of polypeptone, and 5.0 g of beef extract in 1 L of distilled water.

[0051] The E. coli DH5a was cultured at a temperature of 37 °C on an LB medium having a composition of 10.0 g of tryptone, 10.0 g of NaCl, and 5.0 g of yeast extract in 1 L of distilled water. The antibiotics kanamycin and streptomycin were products of Sigma, and the DNA sequencing analysis was commissioned to Macrogen, Inc. for analysis.

[0052] 2-1. Construction of Recombinant Vector In order to enhance the supply of aspartic acid, which is a lysine precursor, to the strain and increase lysine productivity, it was decided to enhance the activity of aspartate aminotransferase. The method used in this example was to induce specific mutations in the promoter of the aspB gene in order to increase the expression of the aspB gene encoding aspartate aminotransferase. The base sequence at the -22 position of the promoter of the aspB gene was substituted with G, and a 1256 bp fragment around the aspB gene on the genome of each mutant strain obtained in Example 1 was amplified by PCR and cloned into the recombinant vector pCGI (see [Kim et al., Journal of Microbiological Methods 84 (2011) 128-130]). The plasmid was named pCGI(DS7-2) (see Figure 1). The following primers in Table 2 were used to amplify the gene fragment for constructing the plasmid.

[0053]

Table 2

[0054] PCR was performed under the following conditions using the above primers. Using a Thermocycler (TP600, TAKARA BIO Inc., Japan), 1 pM of oligonucleotide and 10 ng of chromosomal DNA of the mutant Corynebacterium glutamicum DS1 strain (mutation occurred at the -22 position of the promoter) selected in Example 1 were used as templates in a reaction solution supplemented with 100 μM of each deoxynucleotide triphosphate (dATP, dCTP, dGTP, dTTP), and 25 to 30 cycles were performed in the presence of 1 unit of pfu-X DNA polymerase mixture (Solgent). The PCR execution 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 to 2 minutes (polymerization time of 2 minutes per 1 kb).

[0055] The gene fragment thus produced was cloned into the pCGI vector using self-assembly cloning. The vector was transformed into Escherichia coli DH5α, spread on an LB agar plate containing 50 μg / ml of kanamycin, and cultured at 37°C for 24 hours. After separating the finally formed colonies and confirming whether the insert was accurately present in the vector, this vector was separated and used for recombination of the Corynebacterium glutamicum strain.

[0056] 2-2. Production of Mutant Strains The mutant strain DS7-2 was produced using the pCGI(DS7-2) vector. The vector was prepared to have a final concentration of 1 μg / μl or more, and primary recombination was induced in Corynebacterium glutamicum DS1 strain using electroporation (see [Tauch et al., FEMS Microbiology letters 123 (1994) 343-347]). At this time, the electroporated strain was spread on CM solid medium containing 20 μg / μl of kanamycin to isolate colonies, and then it was confirmed through PCR and nucleotide sequence analysis whether it was appropriately inserted at the induced position on the genome. The strain thus isolated was inoculated into CM liquid medium to induce secondary recombination, cultured overnight or longer, and spread on agar medium containing 40 mg / L of streptomycin in the same manner to isolate colonies. After confirming the presence or absence of resistance to kanamycin in the finally isolated colonies, it was confirmed through nucleotide sequence analysis whether a mutation was introduced into the promoter of the aspB gene in the strain without antibiotic resistance (see [Schafer et al., Gene 145(1994)69-73]). Finally, a Corynebacterium glutamicum mutant strain (DS7-2) into which the mutant aspB promoter was introduced was obtained.

[0057] Example 3. Production of Corynebacterium glutamicum mutant strain A Corynebacterium glutamicum mutant strain was produced in the same manner as in Example 2, except that the nucleotide sequence at position -45 of the promoter of the aspB gene was substituted from T to A, and the mutant Corynebacterium glutamicum DS1 mutant strain (mutation occurred at position -45 of the promoter) selected in Example 1 was used as the DNA template.

[0058] Here, to produce the plasmid, the primers in Table 2 above were used to amplify the gene fragment, and the mutant strain DS7-1 was produced using the produced plasmid pCGI(DS7-1) vector (see Figure 2). Finally, a Corynebacterium glutamicum mutant strain (DS7-1) into which the mutant aspB gene was introduced was obtained.

[0059] Example 4. Production of Corynebacterium glutamicum mutant strains A Corynebacterium glutamicum mutant strain was produced in the same manner as in Example 2, except that the base sequence at position -88 of the promoter of the aspB gene was substituted from T to A, and the mutant Corynebacterium glutamicum DS1 strain (mutation occurred at position -88 of the promoter) selected in Example 1 was used as the DNA template.

[0060] Here, in order to construct the plasmid, the primers shown in Table 2 above were used to amplify the gene fragment, and the DS7 strain, a mutant strain, was produced using the constructed plasmid pCGI(DS7) vector (see Figure 3). Finally, a Corynebacterium glutamicum mutant strain (DS7) into which the mutant aspB gene was introduced was obtained.

[0061] Experimental Example 1. Comparison of L-lysine productivity of mutant strains The L-lysine productivities of the parent strain Corynebacterium glutamicum DS1 strain and the lysine-producing mutant strains DS7-2, DS7-1, and DS7 produced in Examples 2 to 4 were compared.

[0062] Each strain was inoculated into a 100 ml flask containing 10 ml of lysine medium having the composition as shown in Table 1 above, and shake-cultured at 30 °C for 28 hours under the conditions of 180 rpm. After the cultivation was completed, for the lysine analysis, the production amount of L-lysine was measured by HPLC (Shimazu, Japan), and the results are shown in Table 3.

[0063]

Table 3

[0064] As shown in Table 3 above, in Corynebacterium glutamicum mutant strains DS7-2, DS7-1, and DS7, for the enhancement of the lysine biosynthesis pathway, the specific positions (-22, -45, or -88 regions) of the promoter sequence of the aspB gene were replaced with the optimal base sequences, and the productivity of L-lysine increased by approximately 3.7%, 7.7%, and 16.9%, respectively, compared to the parent strain Corynebacterium glutamicum DS1 strain. In particular, it was confirmed that the DS7 strain had the highest productivity compared to other mutant strains. Through such results, it was found that enhancing the expression of the aspB gene improves the L-lysine production ability of the strain by strengthening the supply of lysine precursors.

[0065] So far, the present invention has been examined mainly with reference to its preferred embodiments. Those having ordinary knowledge in the technical field to which the present invention pertains will understand that the present invention can be embodied in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a limiting perspective. The scope of the present invention is disclosed not in the foregoing description but in the claims, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

Claims

1. A promoter in which the base at the -88 position of the promoter sequence of the gene encoding aspartate aminotransferase of SEQ ID NO: 1 is substituted from T to A.

2. A Corynebacterium glutamicum mutant with improved L-lysine-producing ability, in which the base at the -88 position of the promoter sequence of the gene encoding aspartate aminotransferase of SEQ ID NO: 1 is substituted from T to A.

3. The Corynebacterium glutamicum mutant according to claim 2, wherein the mutant contains the nucleotide sequence represented by SEQ ID NO:

4.

4. a) culturing the mutant according to claim 2 in a medium; and b) recovering L-lysine from the mutant or the medium in which the mutant has been cultured. A method for producing L-lysine comprising the steps.

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