Corynebacterium glutamicum mutant strain 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 transketolase.

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, and is generally produced by fermentation using microorganisms such as bacteria and yeast. Wild-type strains obtained in natural conditions and mutant strains modified to improve L-lysine production are used for 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 capabilities and L-lysine production methods using them have been developed by applying genetic 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 deleting 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 into a promoter with high activity in order to increase expression of the gene.

[0004] As described above, various methods for improving L-lysine production have been developed. However, since there are several dozen types of proteins, including enzymes, transcription factors, and transport proteins, that are directly or indirectly involved in L-lysine production, much research is still needed to determine whether L-lysine production can be improved 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 [Patent Document 4] Korean Patent No. 10-1504900 [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. Another object of the present invention is to provide a method for producing L-lysine using the mutant strain. [Means for solving the problem]

[0008] The inventors conducted extensive research to develop a new mutant strain with improved L-lysine production ability using Corynebacterium glutamicum strains, and as a result, they confirmed that L-lysine production increases when the base sequence at a specific position in the promoter of the tkt gene, which encodes transketolase involved in the L-lysine biosynthetic pathway, is replaced, thereby completing the present invention.

[0009] One aspect of the present invention provides a Corynebacterium glutamicum mutant having enhanced transketolase activity and thus improved L-lysine productivity. In the present invention, "transketolase" refers to an enzyme that converts a ketol group (HOCHCO) from xylulose phosphate to ribose phosphate in the pentose phosphate pathway. - It means an enzyme that catalyzes the reaction of transferring sedoheptulose phosphate to form sedoheptulose phosphate. The gene encoding such a transketolase constitutes an operon together with the genes encoding transaldolase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconolactonase, and the gene expression of the operon is regulated by a single promoter.

[0010] According to one embodiment of the invention, the transketolase may be derived from a strain of the genus Corynebacterium. 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.

[0011] In the present invention, "enhancing activity" means increasing the expression level compared to a wild-type strain or a strain before modification by newly introducing or increasing the expression of a gene encoding a protein such as a target enzyme, transcription factor, transport protein, etc. Such enhancement of activity includes improving the activity of a protein itself compared to the activity of the protein originally possessed by a microorganism by substitution, insertion, deletion, or a combination thereof of nucleotides encoding the gene, increasing the overall level of enzyme activity in the cell compared to a wild-type strain or a strain before modification by increasing the expression or translation of the gene encoding it, or a combination thereof.

[0012] According to one embodiment of the present invention, the enhancement of transketolase activity may be achieved by inducing site-specific mutagenesis in the promoter of a gene encoding transketolase. According to one embodiment of the present invention, the promoter of the gene encoding transketolase may be represented by the base sequence of SEQ ID NO:1.

[0013] In the present invention, the term "promoter" refers to a specific site of DNA that initiates mRNA transcription of a target gene, contains a binding site for RNA polymerase, and regulates gene transcription, and is generally located upstream of the transcription start point. A promoter in a prokaryote is defined as a site near the transcription start point to which RNA polymerase binds, and generally consists of two short base sequences separated by base pairs located in the -10 and -35 regions upstream of the transcription start point. The promoter mutation in the present invention is an improvement to improve activity compared to a wild-type promoter, and by inducing a mutation in 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 transketolase activity may be achieved by substituting at least one base in the -300 to -10 region upstream of the transcription start point in the promoter sequence of a gene encoding transketolase.

[0015] More specifically, the promoter mutation in the present invention may be a continuous or discontinuous substitution of at least one base in the -300 to -10 region, preferably one, two, three, four, five, six, seven, eight, nine, or ten bases in the -250 to -50 region, the -230 to -200 region, the -190 to -160 region, or the -90 to -60 region.

[0016] According to one embodiment of the present invention, a Corynebacterium glutamicum mutant having a new promoter sequence of the tkt gene encoding transketolase was obtained by replacing the base sequence of the -83 to -74 region of the promoter sequence of the tkt gene encoding transketolase from ccaattaacc to tgtgctgtca. Such a Corynebacterium glutamicum mutant may contain a mutated promoter of the tkt gene represented by the base sequence of SEQ ID NO:2.

[0017] According to one embodiment of the present invention, a Corynebacterium glutamicum mutant having a new promoter sequence of the tkt gene encoding transketolase of the Corynebacterium glutamicum strain was obtained by substituting the base sequence of the -83 to -74 region of the promoter sequence of the tkt gene from ccaattaacc to tgtggtatca. Such a Corynebacterium glutamicum mutant may contain a mutated promoter of the tkt gene represented by the base sequence of SEQ ID NO:3.

[0018] In the present invention, "improved productivity" means that the productivity of L-lysine is improved compared to that of a parent strain. The parent strain means a wild-type or mutant strain to be mutated, and includes those to be directly mutated and those to be 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.

[0019] According to one embodiment of the present invention, the parent strain may be 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").

[0020] 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 transketolase.

[0021] According to a specific example of the present invention, the mutant strain may contain any of the nucleotide sequences shown in SEQ ID NOs: 2 to 4 as a promoter sequence of the transketolase gene. According to one embodiment of the present invention, the mutant strain exhibits improved L-lysine productivity as compared to the parent strain by including a promoter mutation in the tkt gene encoding transketolase, and in particular, the L-lysine production amount is increased by 5% or more, specifically 5-40%, more specifically 10-30%, compared to the parent strain, so that 65-90 g, preferably 70-80 g, of L-lysine can be produced per liter of the strain culture medium. In addition, the mutant strain exhibits an L-lysine production amount increased by about 4% or more compared to a conventional promoter mutant strain of the tkt gene having a different mutation position, so that L-lysine can be produced at a high yield.

[0022] According to one embodiment of the present invention, a Corynebacterium glutamicum mutant is realized by a recombinant vector containing a mutant in which a promoter sequence of a gene encoding transketolase is partially replaced in a parent strain.

[0023] In the present invention, a "part" does not mean the entire base sequence or polynucleotide sequence, and is 1 to 300, preferably 1 to 100, and more preferably 1 to 50, but is not limited thereto.

[0024] In the present invention, the term "mutant" refers to a promoter mutant in which at least one base in the -300 to -10 region in the promoter sequence of a transketolase gene involved in the biosynthesis of L-lysine is substituted.

[0025] According to one specific example of the present invention, a mutant in which the base sequence of the -83 to -74 region in the promoter sequence of the transketolase gene is replaced with tgtgctgtca may have the base sequence of SEQ ID NO: 2, and a mutant in which the base sequence is replaced with tgtggtatca may have the base sequence of SEQ ID NO: 3.

[0026] In the present invention, the term "vector" refers to an expression vector that expresses a target protein in a suitable host cell, and is a genetic construct that includes the necessary regulatory elements operably linked to express a gene insert. Here, "operably linked" refers to a gene that requires expression and its regulatory sequence that are functionally linked to each other to enable 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 viral vectors.

[0027] The "recombinant vector" of the present invention, when transformed into a suitable host cell, is replicated independently of the genome of the host cell or is integrated into the genome itself. Here, the "suitable host cell" may be one in which the vector is replicable and which contains an origin of replication, which is a specific base sequence from which replication is initiated.

[0028] The transformation can be carried out by selecting a suitable vector introduction technique depending on the host cell, thereby expressing a gene of interest in the host cell. For example, the vector introduction can be carried out by electroporation, heat shock, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, lithium acetate-DMSO method, or a combination thereof. The transformed gene may be any gene that is expressed in the host cell, regardless of whether it is inserted and located within the chromosome of the host cell or located extrachromosomally.

[0029] The host cell includes cells transfected, transformed or infected in vivo or in vitro with a recombinant vector or polynucleotide of the invention. A host cell comprising a recombinant vector of the invention may be a recombinant host cell, recombinant cell or recombinant microorganism.

[0030] In addition, the recombinant vector according to the present invention may contain a selection marker. The selection marker is used to select a transformant (host cell) transformed with the vector, and only cells expressing the selection marker survive in a medium treated with the selection marker, so that transformed cells can be selected. Representative examples of the selection marker include, but are not limited to, kanamycin, streptomycin, and chloramphenicol.

[0031] 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 homologous recombination crossing over. According to one embodiment of the invention, the host cell may be a strain of the genus Corynebacterium, for example a strain of Corynebacterium glutamicum.

[0032] Another aspect of the present invention provides a method for producing L-lysine, comprising the steps of: a) culturing the Corynebacterium glutamicum mutant in a medium; and b) recovering L-lysine from the mutant or from the medium in which the mutant is cultured.

[0033] The culture can be performed in a suitable medium and under suitable culture conditions known in the art, and a person skilled in the art can easily adjust the medium and the culture conditions. Specifically, the medium is a liquid medium, but is not limited thereto. The culture method can be, for example, batch culture, continuous culture, fed-batch culture, or a combination thereof, but is not limited thereto.

[0034] According to one embodiment of the present invention, the medium should meet the requirements of the particular strain in a suitable manner and may be modified by a person skilled in the art. Culture media for Corynebacterium strains are disclosed in a known literature (Non-Patent Document 1), but are not limited thereto.

[0035] According to one embodiment of the present invention, the medium may contain various 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, etc., fats and oils such as soybean oil, sunflower oil, castor oil, coconut oil, etc., fatty acids such as palmitic acid, stearic acid, linoleic acid, alcohols such as glycerin, ethanol, organic acids such as acetic acid, etc. These substances can be used alone or in mixtures, but are not limited to these. Nitrogen sources that can be used include peptone, yeast extract, meat juice, malt extract, corn steep liquor, soybean flour and urea, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. Nitrogen sources can also be used alone or in mixtures, but are not limited to these. Phosphorus sources that can be used include, but are not limited to, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or the sodium-containing salts corresponding thereto. The culture medium may also contain metal salts necessary for growth, such as, but not limited to, magnesium sulfate, iron sulfate, etc. Other essential growth substances, such as amino acids, vitamins, etc., may also be used. Also, suitable precursors for the culture medium may be used. The medium or individual components are added to the culture solution in a suitable manner during the culture process, either batchwise or continuously, but not limited to, the above.

[0036] According to a specific example of the present invention, a compound such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, or sulfuric acid may be added to the microbial culture solution during the culture in a suitable manner to adjust the pH of the culture solution. Also, foam generation may be suppressed during the culture using an antifoaming agent such as fatty acid polyglycol ester. Furthermore, oxygen or an oxygen-containing gas (e.g., air) may be injected into the culture solution to maintain the aerobic state of the culture solution. The temperature of the culture solution is usually 20°C to 45°C, for example, 25°C to 40°C. The culture period is continued until a desired production amount of a useful substance is obtained, and may be, for example, 10 to 160 hours.

[0037] According to one embodiment of the present invention, the step of recovering L-lysine from the mutant strain cultured as described above and from the medium in which the mutant strain was cultured may involve collecting or recovering the produced L-lysine from the medium using any 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.

[0038] According to one embodiment of the invention, the step of recovering lysine may involve centrifuging the culture medium at low speed to remove the biomass and separating the resulting supernatant by ion exchange chromatography.

[0039] According to one embodiment of the invention, the step of recovering L-lysine may comprise a step of purifying L-lysine. Effect of the Invention

[0040] The Corynebacterium glutamicum mutant strain according to the present invention can increase or enhance the expression of the gene encoding transketolase, thereby improving the L-lysine production yield compared to the parent strain. [Brief description of the drawings]

[0041] [Figure 1] FIG. 2 is a diagram showing the structure of a pCGI (Ptkt83-1) vector containing a mutation in which the base sequence in the −83 to −74 region of the transketolase promoter is replaced with tgtgctgtca according to one embodiment of the present invention. [Diagram 2] FIG. 1 shows the structure of the pCGI (Ptkt83-7) vector containing a mutation in which the nucleotide sequence in the −83 to −74 region of the transketolase promoter is replaced with tgtggtatca. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042] The present invention will be described in more detail below. However, these descriptions are merely illustrative to aid in understanding the present invention, and the present invention is not limited thereto. [Example 1] Preparation of Corynebacterium glutamicum mutants To generate a mutant strain of Corynebacterium glutamicum with enhanced transketolase activity, random mutagenesis was performed using the Corynebacterium glutamicum DS1 strain.

[0043] 1-1. Induction of mutations Corynebacterium glutamicum DS1 strain was inoculated into a flask containing 50 ml of CM liquid medium (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), a mutagen, was added to a final concentration of 300 μg / ml. The medium was then shake-cultured 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 medium was washed once with saline and three times with phosphate buffer. The bacteria was suspended in 5 ml of phosphate buffer, then smeared onto a solid seed culture medium (CM liquid medium further supplemented with 15 g / l agar and 8% lysine), and cultured at 30°C for 30 hours to isolate 100 colonies.

[0044] 1-2. Selection of mutants with improved L-lysine production and construction of a mutant library One hundred of the isolated colonies were inoculated at 5% into flasks containing 10 ml of the lysine production liquid medium shown in Table 1, and cultured at 30°C and 200 rpm for 30 hours with shaking. The degree of growth of the cells was confirmed by measuring the absorbance at OD 610 nm for each culture, and the amount of L-lysine produced was measured by HPLC (Shimazu, Japan). By comparing the amounts of L-lysine produced in this way, colonies producing 67.0 g / l or more of L-lysine were selected, and the base sequences of the lysine-related main genes and promoter regions were analyzed to confirm that the strains had a mutation in the tkt gene promoter.

[0045] [Table 1] [Example 2] Improvement of the tkt promoter 2-1. Promoter improvement: introduction of mutations Thirty candidate sequences with up to 15 base sequence variations including the positions on the tkt promoter selected in Example 1 were synthesized by the method of Non-Patent Document 2, and the tkt promoter sequence of Corynebacterium glutamicum ATCC13032 (see SEQ ID NO: 1) and the synthesized tkt promoter region were each cloned into the CAT (chloramphenicol acetyltransferase) reporter vector pSK1-CAT vector. The orientation during DNA cloning and the presence or absence of mutations were confirmed by DNA sequencing. The mutant libraries thus created were named pSK1-tkt1 to pSK1-tkt30. Finally, they were transformed into Corynebacterium glutamicum ATCC13032, and the promoter activities were compared and examined.

[0046] 2-2. Transduction of pSK1-CAT construct into Corynebacterium glutamicum ATCC13032 Each pSK-tkt clone confirmed by sequence analysis was prepared as described above, and soluble cells were prepared for transformation into Corynebacterium glutamicum ATCC13032. 100 ml of BHIS medium was inoculated with 10 ml of cultured Corynebacterium glutamicum ATCC13032 and cultured at 30°C overnight, and then inoculated into 100 ml of CM liquid medium to an OD 600 of 0.3 and cultured at 18°C ​​and 120 rpm for approximately 28 hours until the OD 600 reached 0.8. The culture was centrifuged at 6000 rpm at 4°C for 10 minutes to recover the cells, which were then suspended in 20 ml of 10% glycerol solution and centrifuged three times. The recovered cells were again suspended in 10% glycerol solution and dispensed into E tubes in 100 μl portions and stored in a -70°C deep freezer until use. 1 μg of DNA was added to 100 μl of Corynebacterium glutamicum ATCC13032 soluble cells, and the mixture was added to 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 a 30°C incubator. 100 μl of the DNA was also smeared on a BHIS agar plate supplemented with kanamycin (50 μg / ml) and then cultured in an incubator at 30°C.

[0047] 2-3.CAT analysis CAT analysis (chloramphenicol acetyltransferase assay) of the tkt promoter mutants was performed by the Shaw method (Non-Patent Document 3). Briefly, the transformed Corynebacterium glutamicum strains were cultured in CM liquid medium supplemented with kanamycin (50 μg / ml), and then 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 of 5,5'-dithiobis-2-nitrobenzoic acid (DTNB; Sigma D8130), 0.1 mM Acetyl CoA (Sigma A2056), and 0.1 mM chloramphenicol, and the reaction was allowed to proceed at RT for 15 minutes, after which the absorbance was measured at OD 412 nm. Thus, Ptkt83-1 and Ptkt83-7, which show improved CAT activity compared to the wild-type tkt promoter sequence, were selected.

[0048] In Ptkt83-1 and Ptkt83-7, the base sequence in the -83 to -74 region of the promoter sequence of the tkt gene encoding transketolase was replaced with tgtgctgtca and tgtggtatca, respectively. Below, we conducted an experiment to verify the improvement of L-lysine productivity by the promoter mutation of the tkt gene using the Corynebacterium glutamicum DS1 strain.

[0049] [Example 3] Construction of promoter mutant strain of Corynebacterium glutamicum DS1 To generate a Corynebacterium glutamicum mutant strain with enhanced transketolase activity, Corynebacterium glutamicum DS1 strain and E. coli DH5a (HIT Competent cells™, Cat No. RH618) were used.

[0050] The Corynebacterium glutamicum DS1 strain was cultured at 30°C in CM-broth medium (pH 6.8) 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 in 1 L of distilled water.

[0051] The E. coli DH5a was cultured at 37° C. in 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 were used as products of Sigma Co., Ltd. DNA sequencing analysis was conducted by Macrogen Co., Ltd.

[0052] 3-1. Construction of recombinant vector In order to enhance the activity of transketolase involved in the pentose metabolism of the strain and improve lysine productivity, transketolase enhancement was introduced. The method used in this example was to induce specific mutations in the promoter of the tkt gene to increase the expression of the tkt gene encoding transketolase. Primers containing a mutation sequence were prepared to replace the base sequence of the -83 to -74 region of the tkt gene promoter from ccaattaacc to tgtgctgtca, and the left arm 696 bp region and the right arm 697 bp region were amplified by PCR centered on the mutation part of the tkt gene promoter in the genome of the Corynebacterium glutamicum DS1 mutant strain selected in Example 1, and then linked by overlap PCR, and then cloned into the recombinant vector pCGI (see Non-Patent Document 4). The plasmid was named pCGI (Ptkt83-1) (see FIG. 1). To prepare the plasmid, the primers in Table 2 were used to amplify each gene fragment.

[0053] [Table 2] The following is a detailed description. Using the primers, PCR was performed from the genomic DNA of Corynebacterium glutamicum DS1 strain under the following conditions. Using a Thermocycler (TP600, TAKARA BIO Inc., Japan), 100 μM of each deoxynucleoside triphosphate (dATP, dCTP, dGTP, dTTP) was added to the reaction solution, and 25 to 30 cycles were performed in the presence of 1 pM of oligonucleotide and 10 ng of chromosomal DNA of Corynebacterium glutamicum DS1 strain as a template in the presence of 1 unit of pfu-X DNA polymerase mixture (Solgent, Korea). The PCR conditions were: (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 (allowing 2 minutes of polymerization time per 1 kb).

[0054] The gene fragment thus prepared was cloned into the pCGI vector using BamHI and EcoRI restriction enzymes and the DNA Ligation kit Ver2.1 (Takara, Japan). The vector was transformed into Escherichia coli (E. coli) DH5a, spread on an LB agar plate containing 50 μg / ml kanamycin, and cultured at 37°C for 24 hours. The finally formed colonies were isolated to confirm whether the insert was correctly present in the vector, and then the vector was isolated and used for recombination of Corynebacterium glutamicum strains.

[0055] 3-2. Creation of mutant strains The pCGI (Ptkt83-1) vector was used to prepare a mutant strain, DS1-Ptkt83-1 strain. The final concentration of the vector was adjusted to 1 μg / μl or more, and the first recombination was induced in the Corynebacterium glutamicum DS1 strain by electroporation (see Non-Patent Document 5). Here, the electroporated strain was smeared on a CM agar plate containing 20 μg / μl of kanamycin to separate colonies, and then PCR and base sequence analysis were used to confirm whether the gene was suitably inserted at the induction position in the genome. In order to further induce a second recombination in the strain thus isolated, the strain was inoculated into a CM agar liquid medium containing streptomycin, cultured overnight or more, and then smeared on an agar medium containing the same concentration of streptomycin to separate colonies. The presence or absence of resistance to kanamycin in the finally isolated colonies was confirmed, and then, among the strains not resistant to antibiotics, whether a mutation was introduced into the tkt gene was confirmed by base sequence analysis (see Non-Patent Document 6). Finally, a Corynebacterium glutamicum mutant strain (DS1-Ptkt83-10) carrying the mutant tkt gene was obtained.

[0056] [Example 4] Preparation of Corynebacterium glutamicum mutants A Corynebacterium glutamicum mutant was prepared in the same manner as in Example 3, except that the base sequence in the -83 to -74 region of the tkt gene promoter was replaced from ccaattaacc to tgtggtatca.

[0057] Here, to prepare the plasmid, the primers in Table 3 were used to amplify each gene fragment, and the resulting plasmid pCGI(Ptkt83-7) vector was used to prepare the mutant strain DS1-Ptkt83-7. Finally, a Corynebacterium glutamicum mutant (DS1-Ptkt83-7) carrying the mutant tkt gene was obtained.

[0058] [Table 3] Comparative Example 1. Corynebacterium glutamicum mutant strain A mutant strain (Ptkt217) in which a mutation from ATTGATCACACC to CCCTGACTACAAA was induced in the -217 to -206 region in the tkt gene promoter sequence of a wild-type Corynebacterium glutamicum strain, as disclosed in Patent Document 4, was used.

[0059] Experimental Example 1. Comparison of L-lysine productivity of mutant strains The L-lysine productivity of the parent strain Corynebacterium glutamicum DS1, the lysine-producing mutants DS1-Ptkt83-1 and DS1-Ptkt83-7 prepared in Examples 3 and 4, and the conventional lysine-producing mutant Ptkt217 of Comparative Example 1 was compared in the same manner as in Example 1-2. The results are shown in Table 4.

[0060] [Table 4] As shown in Table 4, the Corynebacterium glutamicum mutants DS1-Ptkt83-1 and DS1-Ptkt83-7 strains were confirmed to have approximately 14% and 17% higher L-lysine productivity, respectively, than the parent Corynebacterium glutamicum DS1 strain, due to the substitution of a specific base sequence (-83 to -74 region) in the promoter sequence of the tkt gene with an optimal base sequence to enhance the lysine biosynthetic pathway.

[0061] In addition, the Corynebacterium glutamicum mutants DS1-Ptkt83-1 and DS1-Ptkt83-7 were confirmed to have L-lysine productivity that was increased by approximately 4% and 7%, respectively, compared to the conventional mutant Ptkt217, which has a different mutation position (-217 to -206 region) and sequence (CCCTGACTACAAA).

[0062] These results indicate that enhanced expression of the tkt gene caused by mutations in the -83 to -74 region of the promoter enhances the lysine biosynthetic ability, thereby improving the L-lysine producing ability of the strain.

[0063] The present invention has been described above with reference to preferred embodiments. Those skilled in the art will understand that the present invention can be modified without departing from its essential characteristics. Therefore, the disclosed embodiments are illustrative of the present invention and do not limit the present invention. The scope of the present invention is defined by the claims, not by these descriptions, and all differences within the scope of the equivalents should be interpreted as being included in the present invention.

Claims

1. A promoter in which the base sequence in the -83 to -74 region of the promoter sequence of the gene encoding the transketolase of SEQ ID NO: 1 has been replaced from ccaattaacc to tgtggtatca.

2. A mutant strain of Corynebacterium glutamicum in which the base sequence in the -83 to -74 region of the promoter sequence of the gene encoding the transketolase of SEQ ID NO: 1 is replaced with tgtggtatca from ccaattaacc, thereby improving L-lysine production ability.

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

3.

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

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