Corynebacterium genus microorganism having improved L-lysine productivity, and method for producing L-lysine using same

Abstract

The present invention relates to a microorganism belonging to the genus Corynebacterium and having improved L-lysine production capacity, and a method for producing L-lysine using the same, the microorganism being capable of producing L-lysine by enhancing the activity of a glucose-promoting diffusion transporter and a glucokinase. Therefore, the production yield of L-lysine can be improved compared with the condition of enhancing the parent strain, the glucose promoting diffusion transporter or the glucokinase alone.

Description

Technical Field

[0001] The present invention relates to a Corynebacterium microorganism having improved L-lysine productivity and a method for producing L-lysine using the same. Background Art

[0002] L-lysine is an essential amino acid that cannot be synthesized by humans or animals and must be supplied externally. It is generally produced through fermentation using microorganisms such as bacteria or yeast. L-lysine can be produced using naturally occurring wild-type strains or mutants modified to enhance their L-lysine production capacity.

[0003] In recent years, in order to improve the production efficiency of L-Methionin, with microorganisms such as Escherichia coli, coryneform bacteria that are used for the production of L-amino acid and other useful substances as object, applicable gene recombination technology has thereby developed various recombinant bacterial strains or mutants with excellent L-Methionin production capacity and utilized its L-Methionin production method.Particularly, carried out following attempt: with the gene of the enzyme, transcription factor, transporter etc. that participate in the biosynthetic pathway of L-Methionin as targeting or to the promoter induction variation of their expression of regulation and control, thereby expand the output of L-Methionin.But the type of protein such as enzyme, transcription factor, transporter that is directly or indirectly related to the production of L-Methionin has a lot of kinds, therefore about whether increase according to the L-Methionin production capacity of such proteinic activity variation, in fact still need a large amount of research.

[0004] Prior art literature

[0005] Patent Literature

[0006] Korean Patent No. 10-0838038

[0007] Korean Patent No. 10-2139806 Summary of the Invention

[0008] An object of the present invention is to provide a Corynebacterium microorganism having improved L-lysine production capacity.

[0009] Another object of the present invention is to provide a method for producing L-lysine using the above-mentioned Corynebacterium microorganism.

[0010] One embodiment of the present invention provides a Corynebacterium microorganism having enhanced activities of a glucose-facilitated diffusion transporter and glucokinase and improved L-lysine production capacity.

[0011] The "glucose facilitated diffusion protein" used in the present invention is a transporter protein present in the plasma membrane and involved in the facilitated diffusion of glucose. The glucose facilitated diffusion transporter in the present invention can be a polypeptide encoded by the glf gene and having glucose facilitated diffusion transporter activity, but is not limited thereto.

[0012] "Glucokinase" as used in the present invention is an enzyme that catalyzes the reaction of generating glucose 6-phosphate from glucose and ATP. It is equivalent to an enzyme that, along with other hexokinase enzymes, introduces glucose into the glycolytic pathway. The glucokinase in the present invention may be a polypeptide having glucokinase activity encoded by the glk gene, but is not limited thereto.

[0013] The nucleic acid sequence and protein sequence information of the glucose-facilitated diffusion transporter and glucokinase can be obtained from known sequence databases (eg, GenBank, UniProt).

[0014] As used in the present invention, "activity enhancement" refers to the increase in the expression level of a gene encoding a protein such as an enzyme, transcription factor, or transporter as a target, compared to the original microorganism, i.e., a wild-type strain or a strain before modification. Such activity enhancement includes the following situations: by modifying the nucleotides encoding the gene (e.g., substitution, insertion, deletion, or a combination thereof of a portion of the nucleotides in the target gene), the activity of the protein itself is increased compared to the activity of the protein possessed by the original microorganism; the copy number of the gene is increased; due to modification of the non-coding region of the non-coding gene such as the promoter (e.g., modification of all or part of the nucleotides in the promoter sequence, replacement with a strong promoter), the expression or translation of the target gene is increased, etc., and the overall protein activity level in the cell is higher than that of the wild-type strain or the strain before modification, and also includes combinations thereof.

[0015] The above-mentioned nucleotide modification refers to the difference between the original nucleotide sequence and the nucleotide sequence due to substitution, insertion, deletion or a combination thereof. The above-mentioned promoter modification refers to the difference between the original promoter sequence and the nucleotide sequence due to substitution, insertion, deletion or a combination thereof, which leads to an increase in the expression level or activity of the target gene. In addition, the above-mentioned promoter modification includes replacement with a promoter that has a stronger expression level or activity for the target gene than the promoter of the original gene. Among them, substitution refers to the change that a base, nucleotide, polynucleotide or nucleic acid is replaced by another base, nucleotide, polynucleotide or nucleic acid. Insertion refers to the change that adds another base, nucleotide, polynucleotide or nucleic acid. Deletion refers to the change that removes a base, nucleotide, polynucleotide or nucleic acid.

[0016] According to a specific example of the present invention, the enhancement of the activity of the glucose-facilitated diffusion transporter is achieved by introducing a gene encoding the glucose-facilitated diffusion transporter, increasing its copy number, modifying its promoter, or a combination thereof.

[0017] According to one embodiment of the present invention, the glucose facilitated diffusion transporter may be encoded by the glf gene derived from Zymomonas mobilis.

[0018] The glf gene derived from Zymomonas mobilis may comprise the base sequence of SEQ ID NO: 1.

[0019] In addition, according to one embodiment of the present invention, the glucose-facilitated diffusion transporter may be encoded by the iolT1 (NCgl0178) and iolT2 (NCgl2953) genes derived from Corynebacterium glutamicum.

[0020] The iolT1 and iolT2 genes from Corynebacterium glutamicum encode the myo-inositol transporters IolT1 and IolT2, respectively, which are known to be functionally similar to the glucose-facilitated diffusion transporter encoded by the glf gene of Zymomonas mobilis (FEMS Microbiol Lett. 2009 Jan; 290(2): 227-35).

[0021] The iolT1 gene derived from Corynebacterium glutamicum may comprise the base sequence of SEQ ID NO: 2, and the iolT2 gene derived from Corynebacterium glutamicum may comprise the base sequence of SEQ ID NO: 3.

[0022] As an example, the enhancement of the activity of the glucose-facilitated diffusion transporter can be achieved by introducing one or more of the glf gene from Zymomonas mobilis, the iolT1 gene from Corynebacterium glutamicum, and the iolT2 gene from Corynebacterium glutamicum, increasing their copy number, or a combination thereof.

[0023] According to one embodiment of the present invention, the enhancement of the glucokinase activity can be achieved by introducing a gene encoding glucokinase, increasing its copy number, modifying its promoter, or a combination thereof.

[0024] According to one embodiment of the present invention, the glucokinase may be encoded by the glk (NCgl2105) gene derived from Corynebacterium glutamicum.

[0025] The glk gene derived from Corynebacterium glutamicum may comprise the base sequence of SEQ ID NO:4.

[0026] The base sequence of the glucose-facilitated diffusion transporter or glucokinase according to the present invention may consist of or contain a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% homology or identity to the base sequence of SEQ ID NO: 1, 2, or 3, and may have the original function. Here, "homology" or "identity" refers to the percentage of identity between a base sequence serving as a reference and any other base sequence when the two sequences are aligned and analyzed to maximize correspondence.

[0027] As used herein, "increased productivity" means an increase in L-lysine productivity compared to the target strain (parent strain). The parent strain refers to the wild-type or variant strain that is the target strain for mutation, including those directly mutated or transformed using a recombinant vector, etc. In the present invention, the parent strain can be a microorganism or strain of the genus Corynebacterium that has or does not have the ability to produce L-lysine, a wild-type Corynebacterium, or a Corynebacterium that has been mutated from the wild-type.

[0028] According to a specific example of the present invention, the Corynebacterium microorganism can be Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium callunae, Corynebacterium suranareeae, Corynebacterium lubricantis, Corynebacterium doosanense, Corynebacterium efficiens, Corynebacterium uterequi, Corynebacterium stationis, Corynebacterium pacaense, Corynebacterium spp. singulare), Corynebacterium humireducens, Corynebacterium marinum, Corynebacterium halotolerans, Corynebacterium spheniscorum, Corynebacterium freiburgense, Corynebacterium striatum, Corynebacterium canis, Corynebacterium ammoniagenes, Corynebacterium renale, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium caspium, Corynebacterium testudinoris, Corynebacterium pseudocorynebacterium pseudopelargi), Corynebacterium flavescens, etc., but are not limited thereto.

[0029] As an example, the Corynebacterium microorganism may be Corynebacterium glutamicum.

[0030] The Corynebacterium microorganism according to the present invention can improve L-lysine production capacity by enhancing the activities of glucose-facilitated diffusion transporter and glucokinase.

[0031] Specifically, the L-lysine-producing ability of the Corynebacterium genus microorganism is improved, and the L-lysine production ability is increased compared to the parent strain. In particular, the L-lysine production amount can be increased by at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% compared to the parent strain. In some embodiments, the L-Lysine production can be 1.1 times, 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 5.5 times, 6 times, 6.5 times, 7 times, 7.5 times, 8 times, 8.5 times, 9 times, 9.5 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times or 100 times, but are not limited thereto. As an example, the L-Lysine production can be more than 3% for the Corynebacterium microorganisms that the activity of above-mentioned glucose facilitating diffusion transporter and glucokinase is enhanced compared with the parent strain, and specifically, can increase by 3 to 40% (preferably 5 to 30%).

[0032] The composition containing the Corynebacterium microorganism according to the present invention can be used as a composition for producing L-lysine.

[0033] The Corynebacterium microorganism according to one embodiment of the present invention can be realized by using a recombinant vector containing genes encoding a glucose-facilitated diffusion transporter and glucokinase, with the parent strain as the target.

[0034] " Vector " used in the present invention refers to all types of nucleic acid sequence transport structures used as a means for delivering and expressing a target gene to a variant object (host cell). Unless otherwise specified, the above-mentioned vector can represent the insertion of the carried nucleic acid sequence into a host cell gene for expression and / or independent expression. Such a vector includes the necessary regulatory elements that are operably connected in order to express a gene insert, and "operably linked" refers to that the target gene and its regulatory sequence are functionally combined with each other and connected in a manner that can carry out gene expression, and "regulatory elements" include a promoter for implementing transcription, an arbitrary operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence regulating the termination of transcription and translation.

[0035] The vectors used in the present invention are not particularly limited as long as they can replicate in the host cell, and any vector known in the art can be used. Examples of such vectors include plasmids, cosmids, viruses, and bacteriophages in their natural or recombinant states. For example, phage vectors or cosmid vectors include pWE15, M13, λMBL3, λMBL4, λIXII, λASHII, λAPII, λt10, λt11, Charon4A, and Charon21A. Plasmid vectors include, but are not limited to, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, and pET vectors.

[0036] Above-mentioned vector can be constructed as the vector for cloning or the vector for expression.The vector for expression can use the conventional vector for expressing foreign gene or protein in plant, animal or microorganism in this area, and can be constructed by the whole bag of tricks known in the art.

[0037] The "recombinant vector" used in the present invention can be constructed with prokaryotic cells or eukaryotic cells as hosts, can be replicated independently of the genome of the host cell, or can be sutured to the genome itself. The above-mentioned host cells are capable of replicating the vector and may include a replication origin as a specific base sequence for starting replication. For example, when the vector used is an expression vector and a prokaryotic cell is used as the host, it usually contains a strong promoter (e.g., pLλ promoter, CMV promoter, trp promoter, lac promoter, tac promoter, T7 promoter) that can enable transcription, a ribosome binding site for the initiation of translation, and a transcription / translation termination sequence. When eukaryotic cells are used as hosts, the replication origins initiated in the eukaryotic cells contained in the vector include f1 replication origin, SV40 replication origin, pMB1 replication origin, adenovirus replication origin, AAV replication origin, and BBV replication origin, but are not limited thereto. In addition, promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or promoters derived from mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, tk promoter of HSV) can be used, and usually have a polyadenylation sequence as a transcription termination sequence.

[0038] The recombinant vector may include a selection marker. The selection marker is used to screen transformants (host cells) transformed with the vector. In a culture medium treated with the selection marker, only cells expressing the selection marker can survive, thereby enabling screening of transformed cells. Representative examples of such selection markers include, but are not limited to, ampicillin, kanamycin, streptomycin, and chloramphenicol.

[0039] By inserting the above-mentioned recombinant vector into a host cell, a transformant can be prepared, and the above-mentioned transformant can be obtained by introducing the recombinant vector into a suitable host cell. The host cell is a cell that can stably and continuously clone or express the above-mentioned expression vector, and any host cell known in the art can also be used.

[0040] When prokaryotic cells are transformed to produce recombinant microorganisms, host cells that can be used include, but are not limited to, Escherichia coli such as E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli X1776, E. coli W3110, and E. coli XL1-Blue; Corynebacterium strains; Bacillus strains such as Bacillus subtilis and Bacillus thuringiensis; and various enteric bacteria and strains such as Salmonella typhimurium, Serratia marcescens, and Pseudomonas.

[0041] When eukaryotic cells are transformed to produce recombinant microorganisms, yeast (e.g., Saccharomyces cerevisiae), insect cells, plant cells, and animal cells can be used as host cells, for example, Sp2 / 0, CHO K1, CHO DG44, PER.C6, W138, BHK, COS7, 293, HepG2, Huh7, 3T3, RIN, MDCK cell lines, etc., but are not limited thereto.

[0042] As used herein, "transformation" refers to the phenomenon of introducing foreign DNA into host cells to cause artificial genetic changes, and "transformant" refers to a host cell into which foreign DNA has been introduced and which stably maintains the expression of the target gene.

[0043] In the above-mentioned transformation, a suitable vector introduction technology is selected according to the host cell, so that the target gene or a recombinant vector comprising the same can be expressed in the host cell. For example, the vector introduction can be implemented 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, but is not limited thereto. As long as the transformed gene can be expressed in the host cell, it can be included, without being limited to being inserted into the chromosome of the host cell or being located outside the chromosome.

[0044] The transformants mentioned above include cells transfected, transformed or infected in vivo or in vitro with the recombinant vector according to the present invention, and can be used as the same terms as recombinant host cells, recombinant cells or recombinant microorganisms.

[0045] The gene inserted into the recombinant vector for transformation of the present invention can be replaced into a host cell such as a microorganism belonging to the genus Corynebacterium by homologous recombination and crossover.

[0046] According to one embodiment of the present invention, the host cell may be a microorganism of the genus Corynebacterium. For example, the microorganism of the genus Corynebacterium may be Corynebacterium glutamicum.

[0047] Another embodiment of the present invention provides a method for producing L-lysine, comprising the steps of: culturing the aforementioned Corynebacterium microorganism in a culture medium; and recovering L-lysine from the culture medium in which the Corynebacterium microorganism is cultured or the Corynebacterium microorganism is cultured.

[0048] The above-mentioned culture can be carried out according to suitable culture medium and culture conditions known in the art, and those skilled in the art can easily adjust the culture medium and culture conditions for use. Specifically, the above-mentioned culture medium can be a liquid culture medium, but is not limited thereto. The culture method can include, for example, batch culture, continuous culture, fed-batch culture, or a combination thereof, but is not limited thereto.

[0049] According to one embodiment of the present invention, the culture medium must be adapted to meet the requirements of a specific strain and can be modified appropriately by a person skilled in the art. Regarding culture media for Corynebacterium strains, reference can be made to the well-known literature (Manual of Methods for General Bacteriology. American Society for Bacteriology. Washington DC, USA, 1981), but the invention is not limited thereto.

[0050] According to a specific embodiment of the present invention, the culture medium may contain various carbon sources, nitrogen sources, and trace element components. Possible carbon sources include sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch, and cellulose; oils and fats such as soybean oil, sunflower oil, castor oil, and coconut oil; fatty acids such as palmitic acid, stearic acid, and linoleic acid; alcohols such as glycerol and ethanol; and organic acids such as acetic acid. These substances may be used alone or in mixtures, but are not limited to these. Possible nitrogen sources include peptone, yeast extract, broth, malt extract, corn steep liquor, soybean meal, urea, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate. Nitrogen sources may also be used alone or in mixtures, but are not limited to these. Possible phosphorus sources include, but are not limited to, potassium dihydrogen phosphate or dipotassium hydrogen phosphate, or their corresponding sodium-containing salts. Furthermore, the culture medium may contain, but is not limited to, metal salts such as magnesium sulfate or ferrous sulfate, which are required for growth. In addition, essential growth substances such as amino acids and vitamins may be included. Furthermore, precursors suitable for the culture medium may be used. The above-mentioned culture medium or individual components may be added to the culture medium in batches or continuously during the culture process by appropriate means, but are not limited thereto.

[0051] According to a specific example of the present invention, during the culture process, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid can be added to the microbial culture solution in an appropriate manner to adjust the pH of the culture solution. In addition, during the culture process, a defoaming agent such as fatty acid polyethylene glycol ester can be used to suppress the generation of bubbles. Further, in order to maintain the aerobic state of the culture solution, oxygen or oxygen-containing gas (such as air) can be injected into the culture solution. The temperature of the culture solution can generally be 20°C to 45°C, for example, 25°C to 40°C. The culture time can continue until the desired production of useful substances is obtained, for example, it can be 10 to 160 hours.

[0052] According to one embodiment of the present invention, in the step of recovering L-lysine from the cultured transformant or the culture medium in which the transformant is cultured, the produced L-lysine can be collected or recovered from the culture medium according to the culture method and using appropriate methods known in the art. For example, centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, fractionated dissolution (e.g., ammonium sulfate precipitation), chromatography (e.g., ion exchange, affinity, hydrophobicity, and size exclusion) and the like can be used, but are not limited thereto.

[0053] According to one embodiment of the present invention, in the step of recovering L-lysine, the culture medium may be centrifuged at low speed to remove biomass, and the obtained supernatant may be separated by ion exchange chromatography.

[0054] According to one embodiment of the present invention, the step of recovering L-lysine may include a process of purifying L-lysine.

[0055] The Corynebacterium microorganism according to the present invention can improve the production yield of L-lysine by enhancing the activities of glucose facilitated diffusion transporter and glucokinase, compared with the case where the parent strain, glucose facilitated diffusion transporter or glucokinase is enhanced alone. DETAILED DESCRIPTION

[0056] The present invention will be described in more detail below. However, this description is provided for illustrative purposes only to help understand the present invention, and the scope of the present invention is not limited to this illustrative description.

[0057] Example 1. Preparation of strains with enhanced glucose-facilitated diffusion transporter and glucokinase activity

[0058] In order to prepare a strain with enhanced glucose facilitative diffusion transporter and glucokinase activity, Corynebacterium glutamicum DS1 (Deposit No. KCCM12969P) and E. coli DH5a (HIT Competent cells) were used. TM ,Cat No.RH618).

[0059] The above-mentioned Corynebacterium glutamicum DS1 was cultured at 30°C in a CM-broth medium (pH 6.8) containing 1 L of distilled water, 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.

[0060] The above-mentioned E. coli DH5a was cultured at 37°C in an LB medium containing 1 L of distilled water, 10.0 g of tryptone, 10.0 g of NaCl, and 5.0 g of yeast extract.

[0061] The antibiotic kanamycin (kanamycin) was a product of Sigma.

[0062] DNA sequencing analysis and gene synthesis were commissioned to MacroGene Co., Ltd.

[0063] 1-1. Recombinant vector

[0064] In order to import the glf gene (SEQ ID NO:1) that coding derives from the glucose facilitation diffusion transporter of zymomonas mobilis (Zymomonas mobilis), iolT1 gene (SEQ ID NO:2) and iolT2 gene (SEQ ID NO:3) and the glk gene (SEQ ID NO:4) of the inositol transporter (myo-inositol transporters) of coding derive from Corynebacterium glutamicum (Corynebacterium glutamicum), knocked out sequence 594bp between NCgl1668 gene and NCgl1669 gene in Corynebacterium glutamicum, imported target gene at this site.For expression of target gene, the promotor (SEQ ID NO:5) of the ddh gene of Corynebacterium glutamicum was utilized to make recombinant vector. On the Corynebacterium glutamicum genome, centered around the 594bp knockout sequence, a 762bp left arm and a 7906bp right arm, along with the promoter and target gene, were amplified by PCR. These fragments were then ligated by overlapping PCR and cloned into the pk19mobsacB (ATCC, 87098) vector. To create the five plasmids described above, the primers listed in Table 1 below were used to amplify each gene fragment. The glf base sequence was obtained by gene synthesis and used as a template.

[0065]

Table 1

[0066]

[0067]

[0068]

[0069]

[0070]

[0071]

[0072]

[0073] PCR was performed using the above primers under the following conditions. Using a thermocycler (TP600, TAKARABIO Inc., Japan), 1 pM of oligonucleotide and 10 ng of chromosomal DNA from Corynebacterium glutamicum ATCC 13032 or glf synthetic DNA from Zymomonas mobilis were used as templates in a reaction solution supplemented with 100 μM of each deoxyribonucleoside triphosphate (dATP, dCTP, dGTP, dTTP). 25 to 30 cycles were performed in the presence of 1 unit of pfu-X DNA polymerase mix (Solgent). PCR was performed under the following conditions: (i) denaturation step at 94°C for 30 seconds, (ii) annealing step at 58°C for 30 seconds, and (iii) extension step at 72°C for 1 to 2 minutes (giving a polymerization time of 2 minutes per kb).

[0074] The gene fragment prepared above was cloned into the pk19mobsacB vector using self-assembly cloning. This vector was transformed into E. coli DH5a, plated on LB-agar plates containing 50 μg / ml kanamycin, and cultured at 37°C for 24 hours. The resulting colonies were isolated, and after confirming the correct insertion of the insert into the vector, the vector was isolated and used for recombination in a Corynebacterium glutamicum strain.

[0075] As a common process in the above methods, the gene was amplified using PCR from genomic DNA from Corynebacterium glutamicum ATCC 13032 or synthetic DNA from Zymomonas mobilis. Depending on the strategy, the gene was inserted into the pk19mobsacB vector via self-assembled cloning and screened in E. coli DH5a. Chromosomal base substitution involved amplifying each gene fragment separately, and overlapping PCR was used to create the target DNA fragment. For gene manipulation, ExTaq polymerase (Takara) and Pfu polymerase (Solgent) were used as PCR amplification enzymes, and various restriction enzymes and DNA modifying enzymes were products from New England Biolabs (NEB), using the provided buffers and protocols.

[0076] Experimental Example 1. Evaluation of L-lysine production capacity

[0077] The L-lysine production capacity of the Corynebacterium glutamicum mutant strain prepared in Example 1 was evaluated in comparison with the parent strain.

[0078] Each strain (parent strain or variant) was inoculated into a 100 mL flask containing 10 mL of the lysine production medium listed in Table 2 below, and cultured at 30°C with shaking at 180 rpm for 28 hours. After completion of the culture, the L-lysine concentration in the culture medium was measured using HPLC (Shimadzu, Japan). The results are shown in Table 3 below.

[0079]

Table 2

[0080] Element Content (based on 1L distilled water) Glucose 100g Ammonium sulfate 55g <![CDATA[KH2PO4]]> 1.1g <![CDATA[MgSO4·H2O]]> 1.2g <![CDATA[MnSO4·H2O]]> 180mg <![CDATA[FeSO4·H2O]]> 180mg Thiamine·HCl 9mg Biotin 1.8mg <![CDATA[CaCO3]]> 5％ pH 7.0

[0081]

Table 3

[0082]

[0083] As shown in Table 3, when the activity of either the glucose-facilitated transporter or glucokinase was enhanced, the L-lysine production per unit strain was no different from that of the parent strain. However, when both the activity of the glucose-facilitated transporter and glucokinase was enhanced, the L-lysine production per unit strain increased compared to the parent strain, with the overall L-lysine production increasing by a minimum of approximately 6.6% and a maximum of approximately 17.2%. These results indicate that enhancing the activity of the glucose-facilitated transporter and glucokinase increases the sugar utilization capacity of the microorganism, thereby improving L-lysine production.

[0084] Thus far, the present invention has been studied around its preferred embodiments. Those skilled in the art will appreciate that the present invention can be implemented in various forms without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative perspective rather than a restrictive perspective. The scope of the present invention is shown in the claims rather than in the above description, and should be interpreted as including all differences within the scope of equivalence thereto.

[0085]

Collection Information

[0086] Name of depository institution: Korea Collection of Microorganisms (KCCM)

[0087] Accession number: KCCM12969P

[0088] Collection date: April 2, 2021

[0089] Classification and nomenclature of biological material: Corynebacterium glutamicum.

Claims

A Corynebacterium microorganism having enhanced L-lysine production capacity by enhancing the activities of a glucose-facilitated diffusion transporter and glucokinase.

2. The Corynebacterium microorganism according to claim 1, wherein The activity enhancement of the glucose-facilitated diffusion transporter is achieved by introducing a gene encoding the glucose-facilitated diffusion transporter, increasing its copy number, modifying its promoter, or a combination thereof.

3. The Corynebacterium microorganism according to claim 1, wherein The enhancement of the glucokinase activity is achieved by introducing a gene encoding glucokinase, increasing its copy number, modifying its promoter, or a combination thereof.

4. The Corynebacterium microorganism according to claim 1, wherein The Corynebacterium microorganism is Corynebacterium glutamicum.

5. A method for producing L-lysine, comprising the following steps: a step of culturing the Corynebacterium microorganism according to claim 1 in a culture medium; and A step of recovering L-lysine from the Corynebacterium microorganism or a culture medium for culturing the Corynebacterium microorganism.

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