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 pyruvate carboxylase.

Description

[Technical field]

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

[0002] L-lysine is an essential amino acid that cannot be synthesized in the human or animal body and must be supplied from an external source. It is generally produced by fermentation using microorganisms such as bacteria and yeast. L-lysine can be produced using wild-type strains obtained in natural conditions or mutant strains modified to improve L-lysine production. Recently, in order to improve the efficiency of L-lysine production, various recombinant strains or mutant strains with excellent L-lysine production ability and L-lysine production methods using them have been developed by applying gene recombination technology to microorganisms such as Escherichia coli and Corynebacterium, which are often used to produce L-amino acids and other useful substances.

[0003] According to Patent Documents 1 and 2, L-lysine production ability can be improved by increasing expression of a gene that encodes a protein including an enzyme involved in L-lysine production by modifying the nucleotide sequence or amino acid sequence of the gene, or by removing an unnecessary gene. Patent Document 3 discloses a method for modifying an existing promoter of a gene that encodes an enzyme involved in L-lysine production to a promoter with strong activity in order to increase expression of the gene.

[0004] As described above, various methods for increasing L-lysine production have been developed. However, since there are several dozen types of proteins, such as enzymes, transcription factors, and transport proteins, that are directly or indirectly related to L-lysine production, much research is still needed to determine whether L-lysine production can be increased by changing the activity of these proteins. [Prior art documents] [Patent documents]

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

[0006] [Non-Patent Document 1] Manual of Methods for General Bacteriology.American Society for Bacteriology.Washington DC,USA, 1981 [Non-Patent Document 2] Sambrook, J. et al. (2001) “Molecular Cloning: A Laboratory Manual”, Cold Spring Harbor Laboratory Press.volume 2.13.36~13.39 [Non-Patent Document 3] Shaw et al., 1991. Biochemistry.30(44):10806 [Non-Patent Document 4] Kim et al., Journal of Microbiological Methods 84(2011)128-130 [Non-Patent Document 5] Tauch et al., FEMS Microbiology letters 123(1994)343-347 [Non-Patent Document 6] Schafer et al., Gene 145(1994)69-73 Summary of the Invention [Problem 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 productivity.

[0008] Another object of the present invention is to provide a method for producing L-lysine using the mutant strain. [Means for solving the problem]

[0009] The present inventors conducted research to develop a new mutant strain with improved L-lysine production ability using Corynebacterium glutamicum strains, and as a result, they confirmed that when the base sequence at a specific position in the promoter of the pyc gene, which encodes pyruvate carboxylase, which is involved in the supply of the lysine precursor oxaloacetate in the L-lysine biosynthetic pathway, was replaced, the production of pyruvate-derived by-products was reduced while the amount of L-lysine produced was increased, thereby completing the present invention.

[0010] One aspect of the present invention provides a Corynebacterium glutamicum mutant having enhanced pyruvate carboxylase activity and thus improved L-lysine productivity.

[0011] As used herein, "pyruvate carboxylase" refers to an enzyme that catalyzes the reaction of inducing carboxylation of pyruvate in the L-lysine biosynthetic pathway to produce oxaloacetate (OAA), a precursor of lysine.

[0012] According to one embodiment of the present invention, the pyruvate carboxylase may be derived from a Corynebacterium strain. Specifically, the Corynebacterium genus strains include Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium callunae, Corynebacterium suranareeae, Corynebacterium lubricantis, Corynebacterium doosanense, Corynebacterium efficiens, Corynebacterium uteri, Corynebacterium spp. ... Corynebacterium uterequi, Corynebacterium stationis, Corynebacterium pacaense, Corynebacterium singulare, Corynebacterium humireducens, Corynebacterium marinum, Corynebacterium halotolerans, Corynebacterium spheniscorum, Corynebacterium freiburgense, Corynebacterium striatum striatum, Corynebacterium canis, Corynebacterium ammoniagenesammoniagenes, Corynebacterium renale, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium caspium, Corynebacterium testudinoris, Corynebacterium pseudopelargi, or Corynebacterium flavescens, but is not limited thereto.

[0013] As used herein, "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, resulting in an increased expression level compared to a wild-type strain or a strain before transformation. Such enhanced activity includes cases where the activity of a protein itself is increased compared to the activity of a protein originally possessed by a microorganism through substitution, insertion, deletion, or a combination of these of nucleotides encoding the gene, and cases where the overall level of enzyme activity in the cell is higher than that of a wild-type strain or a strain before transformation due to increased expression or translation of the gene encoding the protein, etc., in combination with these.

[0014] According to one embodiment of the present invention, the enhancement of pyruvate carboxylase activity may be achieved by site-specific mutagenesis of a promoter of a gene encoding pyruvate carboxylase.

[0015] According to one embodiment of the present invention, the promoter of the gene encoding pyruvate carboxylase may be represented by the nucleotide sequence of SEQ ID NO:1.

[0016] The term "promoter" as used herein means a specific site of DNA that contains a binding site for RNA polymerase that initiates mRNA transcription of a gene of interest and regulates gene transcription, and is generally located upstream of the transcription start point. A prokaryotic promoter is defined as a site around the transcription start point to which RNA polymerase binds, and generally consists of two short base sequences separated by a base pair in the -10 and -35 regions upstream from the transcription start point. The promoter mutation in the present invention is an improvement to have higher activity than a wild-type promoter, and the expression of a gene located downstream can be increased by inducing a mutation in the promoter region located upstream of the transcription start point.

[0017] According to one embodiment of the present invention, the enhanced activity of pyruvate carboxylase may be obtained by substituting one or more bases in the -90 to -30 region upstream from the transcription start site in the promoter sequence of a gene encoding pyruvate carboxylase.

[0018] More specifically, the promoter mutation in the present invention may be a consecutive or discontinuous substitution of one or more bases in the -90 to -30 region, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bases in the -90 to -30 region, -80 to -40 region, -75 to -45 region, -55 to -40 region, or -75 to -60 region.

[0019] According to one embodiment of the present invention, a Corynebacterium glutamicum mutant having a new promoter sequence of the pyc gene encoding pyruvate carboxylase of the Corynebacterium glutamicum strain was obtained by substituting the base sequence of the -73 to -61 region from ggggttacgatac to tgtggtatgatgg and the base sequence of the -51 to -38 region from gtgactgctatcac to acagctgctactgt. Such a Corynebacterium glutamicum mutant may include a mutated promoter of the pyc gene represented by the base sequence of SEQ ID NO:2.

[0020] According to one embodiment of the present invention, a Corynebacterium glutamicum mutant having a new promoter sequence of the pyc gene encoding pyruvate carboxylase of the Corynebacterium glutamicum strain was obtained by substituting the base sequence of the -73 to -61 region from ggggttacgatac to tgttgtatgattg and the base sequence of the -51 to -38 region from gtgactgctatcac to actgctgctactac. Such a Corynebacterium glutamicum mutant may include a mutated promoter of the pyc gene represented by the base sequence of SEQ ID NO:3.

[0021] As used herein, "improved productivity" means increased L-lysine productivity compared to a parent strain. The parent strain refers to a wild-type or mutant strain that is the subject of mutation, and includes a strain that is the subject of direct mutation or that is transformed with a recombinant vector or the like. In the present invention, the parent strain may be a wild-type Corynebacterium glutamicum strain or a strain mutated from the wild-type.

[0022] According to one embodiment of the present invention, the parent strain is a mutant strain in which a mutation has been induced in the sequence of a gene involved in lysine production (e.g., lysC, zwf, and hom genes), and may be a Corynebacterium glutamicum strain deposited at the Korean Culture Center of Microorganisms on April 2, 2021 under accession number KCCM12969P (hereinafter referred to as the "Corynebacterium glutamicum DS1 strain").

[0023] Such a Corynebacterium glutamicum mutant strain of the present invention having improved L-lysine production ability may contain a mutated promoter sequence of a gene encoding pyruvate carboxylase.

[0024] According to a specific example of the present invention, the mutant strain may contain any one of the base sequences shown in SEQ ID NOs: 2 to 3 as a promoter sequence of the pyruvate carboxylase gene.

[0025] According to one embodiment of the present invention, the mutant strain exhibits increased L-lysine productivity compared to the parent strain due to the inclusion of a promoter mutation in the pyc gene encoding pyruvate carboxylase, and in particular, the L-lysine production amount is increased by at least 3%, specifically 3 to 40%, and more specifically 5 to 30%, compared to the parent strain, and is capable of producing 65 to 75 g of L-lysine, and preferably 65 to 70 g of L-lysine per liter of strain culture medium.

[0026] According to one embodiment of the present invention, a Corynebacterium glutamicum mutant is produced through a recombinant vector comprising a mutant in which a promoter sequence of a gene encoding pyruvate carboxylase is partially substituted in a parent strain.

[0027] The term "part" used in the present invention means not the entire base sequence or polynucleotide sequence, and may be, but is not limited to, 1 to 300, preferably 1 to 100, and more preferably 1 to 50.

[0028] The term "mutant" as used in the present invention refers to a promoter mutant in which one or more bases in the -90 to -30 region within the promoter sequence of the pyruvate carboxylase gene involved in the biosynthesis of L-lysine have been substituted.

[0029] According to one specific example of the present invention, a mutant in which the nucleotide sequence of the -73 to -61 region in the promoter sequence of the pyruvate carboxylase gene is replaced by tgtggtatgatgg and the nucleotide sequence of the -51 to -38 region is replaced by acagctgctactgt may have the nucleotide sequence of SEQ ID NO: 2, and a mutant in which the nucleotide sequence of the -73 to -61 region is replaced by tgttgtatgattg and the nucleotide sequence of the -51 to -38 region is replaced by actgctgctactac may have the nucleotide sequence of SEQ ID NO: 3.

[0030] The term "vector" as used herein refers to an expression vector capable of expressing a target protein in a suitable host cell, and a gene construct containing essential regulatory elements operably linked to allow expression of a gene insert. Here, "operably linked" means that a gene to be expressed and its regulatory sequence are linked in such a manner that they are functionally linked to each other to allow gene expression, and "regulatory elements" include a promoter for transcription, an optional 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, and virus vectors.

[0031] The "recombinant vector" used in the present invention can replicate independently of the genome of a host cell after being transformed into the host cell, or can be integrated into the genome itself. In this case, the "suitable host cell" can include a replication origin, which is a specific base sequence from which replication of the vector is initiated, so that the vector can replicate.

[0032] For the transformation, a vector introduction technique suitable for the host cell is selected to allow the expression of the target gene in the host cell. For example, the vector can be introduced by electroporation, heat shock, calcium phosphate (CaPO 4 ) precipitate, calcium chloride (CaCl 2 ), microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, lithium acetate-DMSO method, or a combination thereof. The transformed gene may be, without limitation, one that is inserted into the chromosome of the host cell or located extrachromosomally, so long as it can be expressed in the host cell.

[0033] The host cell includes cells that have been transfected, transformed or infected in vivo or in vitro with a recombinant vector or polynucleotide of the invention. A host cell containing a recombinant vector of the invention may be a recombinant host cell, recombinant cell or recombinant microorganism.

[0034] In addition, the recombinant vector according to the present invention may contain a selection marker, which is used to select a transformant (host cell) transformed with the vector, and since only cells expressing the selection marker can survive in a medium treated with the selection marker, it is possible to select transformed cells. Representative examples of the selection marker include kanamycin, streptomycin, and chloramphenicol, but are not limited thereto.

[0035] The gene inserted into the recombinant vector for transformation of the present invention is replaced in a host cell such as a microorganism of the genus Corynebacterium by crossover of homologous recombination. According to one embodiment of the present invention, the host cell may be a strain of the genus Corynebacterium, for example a strain of Corynebacterium glutamicum.

[0036] Another aspect of the present invention provides a method for producing L-lysine, comprising: a) culturing the Corynebacterium glutamicum mutant in a medium; and b) recovering L-lysine from the mutant or the medium in which the mutant is cultured.

[0037] The culture is performed using an appropriate medium and culture conditions known in the art, and a person skilled in the art can easily adjust the medium and culture conditions. Specifically, the medium may be a liquid medium, but is not limited thereto. The culture method may include, but is not limited to, batch culture, continuous culture, fed-batch culture, or a combination thereof.

[0038] According to one embodiment of the present invention, the medium should meet the requirements of a specific strain in an appropriate manner, and can be appropriately modified by a person skilled in the art. Culture media for Corynebacterium strains can be found in the publicly known literature (Manual of Methods for General Bacteriology. American Society for Bacteriology. Washington DC, USA, 1981), but are not limited thereto.

[0039] According to one embodiment of the present invention, the medium may contain a variety of carbon sources, nitrogen sources and trace element components. Carbon sources that can be used include sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch, cellulose, oils and fats such as soybean oil, sunflower oil, castor oil, coconut oil, etc., fatty acids such as palmitic acid, stearic acid, linoleic acid, alcohols such as glycerol, ethanol, organic acids such as acetic acid. These substances can be used individually or in mixtures and are not limited thereto. Nitrogen sources that can be used include peptone, yeast extract, meat juice, 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 can also be used individually or in mixtures and are not limited thereto. Sources of phosphorus that can be used include, but are not limited to, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts. The culture medium may also contain metal salts necessary for growth, such as magnesium sulfate or ferrous sulfate, but are not limited thereto. In addition, essential growth substances such as amino acids and vitamins may be included. Also, suitable precursors may be used for the culture medium. The medium or individual components may be added to the culture medium in a suitable manner during the culture process, either batchwise or continuously, but is not limited thereto.

[0040] According to one embodiment of the present invention, the pH of the culture solution can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid to the microbial culture solution in an appropriate manner during the culture. Also, foam generation can be suppressed by using an antifoaming agent such as fatty acid polyglycol ester during the culture. Furthermore, oxygen or oxygen-containing gas (e.g., air) can be injected into the culture solution to maintain the aerobic state of the culture solution. The temperature of the culture solution can usually be 20°C to 45°C, for example, 25°C to 40°C. The culture period can be continued until the desired production amount of useful substances is obtained, and can be, for example, 10 to 160 hours.

[0041] According to one embodiment of the present invention, the step of recovering L-lysine from the cultured mutant and the medium in which the mutant is cultured can be carried out by collecting or recovering the produced L-lysine from the medium using a suitable method known in the art depending on the culture method, such as, but not limited to, centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, differential dissolution (e.g., ammonium sulfate precipitation), chromatography (e.g., ion exchange, affinity, hydrophobic, and size exclusion), etc.

[0042] According to one embodiment of the present invention, the step of recovering lysine can be carried out by subjecting the culture medium to low speed centrifugation, removing the biomass, and separating the resulting supernatant through ion exchange chromatography.

[0043] According to one embodiment of the present invention, the step of recovering L-lysine can include purifying L-lysine. Effect of the Invention

[0044] The Corynebacterium glutamicum mutant strain according to the present invention can improve the L-lysine production yield compared to the parent strain by increasing or enhancing the expression of the gene encoding pyruvate carboxylase. [Brief description of the drawings]

[0045] [Figure 1] This shows the structure of the pCGI(Pm1-pyc') vector according to one embodiment of the present invention, which contains a promoter in which the base sequence in the -73 to -61 region of the promoter sequence of the pyruvate carboxylase gene has been replaced with tgtggtatgatgg, and the base sequence in the -51 to -38 region has been replaced with acagctgctactgt. [Diagram 2]1 shows the structure of the pCGI(Pm2-pyc') vector containing a promoter in which the base sequence in the -73 to -61 region of the promoter sequence of the pyruvate carboxylase gene has been replaced with tgttgtatgattg and the base sequence in the -51 to -38 region has been replaced with actgctgctactac. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0046] The present invention will be described in more detail below. However, such description is merely presented as an example to help the understanding of the present invention, and the scope of the present invention is not limited by such an example description.

[0047] Example 1. Preparation of Corynebacterium glutamicum mutant strains To generate a Corynebacterium glutamicum mutant with enhanced pyruvate carboxylase activity, the Corynebacterium glutamicum DS1 strain was randomly mutagenized.

[0048] 1-1. Induction of mutations Corynebacterium glutamicum DS1 strain was inoculated into a flask containing 50 ml of seed culture CM liquid medium (1 L of distilled water containing 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, pH 6.8), and N-methyl-N'-nitro-N-nitrosoguanidine (NTG) was added to a final concentration of 300 μg / ml, followed by shaking culture at 30°C and 200 rpm for 20 hours. After the culture was completed, the culture was centrifuged at 12,000 rpm for 10 minutes to remove the supernatant, and the culture was washed once with saline and three times with phosphate buffer. This was suspended in 5 ml of phosphate buffer, then smeared on a seed culture solid medium (seed culture liquid medium further containing 15 g / l agar), and cultured at 30° C. for 30 hours to isolate 100 colonies.

[0049] 1-2. Selection of mutants with improved L-lysine production and construction of a mutant library 100 of the isolated colonies were inoculated at 5% into a flask containing 10 ml of the lysine production liquid medium shown in Table 1 below, and cultured at 30° C. for 30 hours with shaking at 200 rpm. The absorbance of each culture solution was measured at OD 610 nm, and the amount of L-lysine produced was compared. Ten colonies producing 75.0 g / L or more of L-lysine were selected, and their base sequences were analyzed to confirm the promoter mutation site of the pyc gene.

[0050] [Table 1]

[0051] Example 2. Improvement of the pyc promoter 2-1. Promoter improvement: introduction of mutations Thirty candidate sequences with up to 15 base sequence variations including the mutation sites on the pyc promoter selected in Example 1 were synthesized by the method of [Sambrook, J. et al. (2001) "Molecular Cloning: A Laboratory Manual", Cold Spring Harbor Laboratory Press. volume 2. 13.36-13.39], and the pyc promoter sequence of Corynebacterium glutamicum ATCC13032 (see SEQ ID NO: 1) and the synthesized pyc promoter region were cloned into the CAT (chloramphenicol acetyltransferase) reporter vector pSK1-CAT vector. During DNA cloning, the orientation and the occurrence of mutations were confirmed through DNA sequencing. The mutation libraries thus constructed were named pSK1-pyc1 to pSK1-pyc30. Finally, they were transformed into Corynebacterium glutamicum ATCC13032 and the promoter activities were compared.

[0052] 2-2. Transduction of pSK1-CAT construct into Corynebacterium glutamicum ATCC13032 The above-prepared pSK1-pyc1~pSK1-pyc30 confirmed through sequence analysis was transformed into Corynebacterium glutamicum ATCC13032 to prepare soluble cells. 100ml of cultured Corynebacterium glutamicum ATCC13032 was inoculated into 100ml of BHIS medium and cultured at 30℃ overnight, and then inoculated into 100ml of CM liquid medium to an OD600 of 0.3 and cultured at 18℃ and 120 rpm for about 28 hours until the OD600 reached 0.8. The culture was centrifuged at 6000 rpm at 4℃ for 10 minutes to recover the cells, suspended in 20ml of 10% glycerol solution, and the centrifugation process was repeated three times. The recovered cells were further suspended in 10% glycerol solution, dispensed into E-tubes in 100μl portions, and stored in a -70℃ deep freezer until use. 1 μg of DNA was added to 100 μl of Corynebacterium glutamicum ATCC13032 soluble cells, and the resulting mixture was placed in a cooled electroporation cuvette and electroporated using a Bio-Rad MicroPulser. Immediately after the pulse, 1 ml of CM liquid medium pre-warmed to 46°C was added to recover the cells, which were then incubated on ice for 2 minutes and then cultured at 180 rpm in an incubator at 30°C. 100 μl of the DNA was then smeared onto a BHIS agar plate supplemented with kanamycin (50 μg / ml) and cultured in an incubator at 30°C.

[0053] 2-3.CAT analysis The CAT assay (chloramphenicol acetyltransferase assay) of the pyc promoter mutants was performed by the Shaw method (Shaw et al., 1991. Biochemistry. 30(44):10806). Briefly, the transformed Corynebacterium glutamicum strains were cultured in CM liquid medium supplemented with kanamycin (50 μg / ml), and the cells were harvested to obtain protein lysates. 5 μg of each protein was added to 0.1 M Tris-HCl buffer (pH 7.8), 0.4 mg / ml 5,5'-dithiobis-2-nitrobenzoic acid (DTNB; Sigma D8130), 0.1 mM Acetyl CoA (Sigma A2056), and 0.1 mM chloramphenicol, and the mixture was incubated at RT for 15 min, after which the absorbance was measured at OD412 nm. Through this, two types, pSK-pyc3 and pSK-pyc23, which showed the highest improvement in CAT activity compared to the wild-type pyc promoter sequence of Corynebacterium glutamicum, were selected.

[0054] In pSK-pyc3, the base sequence in the -73 to -61 region of the promoter sequence, which is the upstream region of the start codon of the pyruvate carboxylase gene, was replaced with tgtggtatgatgg, and the base sequence in the -51 to -38 region was replaced with acagctgctactgt, while in pSK-pyc23, the base sequence in the -73 to -61 region was replaced with tgttgtatgattg, and the base sequence in the -51 to -38 region was replaced with actgctgctactac. Then, an experiment was carried out to verify the increase in L-lysine productivity due to the promoter mutation of the pyc gene using the Corynebacterium glutamicum DS1 strain.

[0055] Example 3. Preparation of Corynebacterium glutamicum mutants To prepare a mutant strain of Corynebacterium glutamicum with enhanced pyruvate carboxylase activity, we used the Corynebacterium glutamicum DS1 strain and E. coli DH5a (HIT Competent cells TM , Cat No. RH618) was used.

[0056] The Corynebacterium glutamicum DS1 strain was cultured at 30°C in CM-broth medium (pH 6.8) containing 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.

[0057] 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. Kanamycin and streptomycin products used were from Sigma, and DNA sequencing analysis was carried out by Macrogen Corporation.

[0058] 3-1. Construction of recombinant vector In order to increase the supply of oxaloacetate, a precursor of lysine, to the strain and thereby increase lysine productivity, an enhancement of pyruvate carboxylase was introduced. The method used in this example was to induce specific mutations in the promoter of the pyc gene, which encodes pyruvate carboxylase, to increase the expression of the pyc gene. In order to replace the base sequence at positions -73 to -61 of the pyc gene promoter from ggggttacgatac to tgtggtatgatgg and the base sequence at positions -51 to -38 from gtgactgctatcac to acagctgctactgt, primers containing the mutation sequences were prepared, and the 735 bp region of the left arm and the 730 bp region of the right arm were amplified by PCR from the mutation sites on both sides of the pyc gene promoter on the genome of the Corynebacterium glutamicum DS1 mutant strain selected in Example 1, and then linked by overlap PCR, and cloned into the recombinant vector pCGI (see literature [Kim et al., Journal of Microbiological Methods 84 (2011) 128-130]). The recombinant plasmid thus prepared was named pCGI (Pm1-pyc') (see FIG. 1). To construct the plasmid, the pyc promoter mutation 1 amplification primer and the pCGI vector amplification primer in Table 2 below were used to amplify each DNA fragment.

[0059] [Table 2]

[0060] The detailed description is as follows: PCR was performed using the genomic DNA of Corynebacterium glutamicum DS1 strain with the above primers under the following conditions.

[0061] Using a Thermocycler (TP600, TAKARA BIO Inc., Japan), 25 to 30 cycles were performed using 1 pM of oligonucleotide and 10 ng of the chromosomal DNA of the Corynebacterium glutamicum DS1 mutant strain confirmed in Example 1 or pCGI vector as a template in the reaction solution containing 100 μM of each deoxynucleotide triphosphate (dATP, dCTP, dGTP, dTTP) in the presence of 1 unit of pfu-X DNA polymerase mixture (Solgent). PCR was performed under the following conditions: (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 (2 minutes of polymerization time per 1 kb).

[0062] The gene fragment thus prepared was cloned into the pCGI vector using self-assembly cloning. The vector was transformed into E. coli DH5a, spread onto an LB agar plate containing 50 μg / ml kanamycin, and cultured at 37°C for 24 hours. The final colonies formed were isolated to confirm that the insert was correctly present in the vector, and the vector was then isolated and used for recombination of Corynebacterium glutamicum DS1 strain.

[0063] 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 NEB products, using the supplied buffers and protocols.

[0064] 3-2. Production of mutant strains The pCGI(Pm1-pyc') vector was used to prepare the DS5 strain. The vector was prepared so that the final concentration was 1 μg / μl or more, and the Corynebacterium glutamicum DS1 strain was subjected to electroporation (see Tauch et al., FEMS Microbiology letters 123 (1994) 343-347) to induce the first recombination. The electroporated strain was then spread on a CM agar plate containing 20 μg / μl kanamycin to separate colonies, and PCR and base sequence analysis were used to confirm whether the gene was properly inserted into the induced position on the genome. The strain thus separated was then inoculated into a CM agar liquid medium containing streptomycin in order to induce the second recombination, and cultured overnight or more, and then spread on an agar medium containing the same concentration of streptomycin to separate colonies. The final colonies were checked for resistance to kanamycin, and the strains without antibiotic resistance were examined by sequence analysis to see whether a mutation had been introduced into the promoter of the pyc gene (see reference [Schafer et al., Gene 145 (1994) 69-73]). Finally, the Corynebacterium glutamicum mutant DS5 strain, in which a mutation had been introduced into the promoter of the mutant pyc gene, was obtained.

[0065] Example 4. Preparation of Corynebacterium glutamicum mutants A Corynebacterium glutamicum mutant was produced in the same manner as in Example 3, except that the base sequence at positions -73 to -61 of the pyc gene promoter was replaced from ggggttacgatac to tgttgtatgattg, and the base sequence at positions -51 to -38 of the pyc gene promoter was replaced from gtgactgctatcac to actgctgctactac.

[0066] To prepare the plasmid, the pyc promoter mutation 2 amplification primer and the pCGI vector amplification primer in Table 3 were used to amplify each DNA fragment, and the plasmid pCGI(Pm2-pyc') vector (see Figure 2) was used. Finally, the Corynebacterium glutamicum mutant DS5-1 strain into which the mutant pyc gene was introduced was obtained.

[0067] [Table 3]

[0068] Experimental Example 1. Comparison of L-lysine productivity of mutant strains The L-lysine productivity of the parent strain Corynebacterium glutamicum DS1 and the lysine-producing mutant strains DS5 and DS5-1 prepared in Examples 3 and 4 was compared.

[0069] Each strain was inoculated into a 100 ml flask containing 10 ml of lysine medium having the composition shown in Table 1, and cultured with shaking at 180 rpm at 30° C. for 28 hours. After the culture was completed, the amount of L-lysine produced was measured by HPLC (Shimazu, Japan) for lysine analysis, and the results are shown in Table 4.

[0070] [Table 4]

[0071] As shown in Table 4, in the Corynebacterium glutamicum mutants DS5 and DS5-1, specific positions (-73 to -61 region and -51 to -38 region) of the promoter sequence of the pyc gene were replaced with optimal base sequences to enhance the supply of oxaloacetate, a precursor of lysine, and it was confirmed that the L-lysine productivity was increased by about 8.3% and 4.7%, respectively, compared to the parent strain, Corynebacterium glutamicum DS1. These results demonstrated that the enhanced expression of the pyc gene enhances the supply of lysine precursor, thereby improving the L-lysine production ability of the strains.

[0072] The present invention has been described above with reference to its preferred embodiments. It will be understood by those skilled in the art that the present invention can be embodied in modified forms without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered in an illustrative rather than restrictive manner. The scope of the present invention is disclosed in the claims rather than in the above description, and all differences within the scope of the equivalents should be interpreted as being included in the present invention.

Claims

1. In the promoter sequence of the gene encoding pyruvate carboxylase of SEQ ID NO: 1, the nucleotide sequence at positions 175 to 187 is substituted from ggggttacgatac to tgtggtatgatgg, and the nucleotide sequence at positions 197 to 210 is substituted from gtgactgctatcac to acagctgctactgt, the promoter.

2. In the promoter sequence of the gene encoding pyruvate carboxylase of SEQ ID NO: 1, the nucleotide sequence at positions 175 to 187 is substituted from ggggttacgatac to tgtggtatgatgg, and the nucleotide sequence at positions 197 to 210 is substituted from gtgactgctatcac to acagctgctactgt, a Corynebacterium glutamicum mutant with improved L-lysine production ability.

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

2.

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 is cultured, a method for producing L-lysine.

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