Corynebacterium mutant microorganism producing L-glutamic acid and method for producing L-glutamic acid using the same

Abstract

The present invention relates to a mutant Corynebacterium microorganism that produces L-glutamic acid and a method for producing L-glutamic acid using the same, and more specifically, to a novel mutant of NADP-dependent malic enzyme involved in the biosynthetic pathway of L-glutamic acid, a polynucleotide and a transformant, and a method for producing L-glutamic acid using the same. The NADP-dependent malic enzyme mutant according to the present invention has one or more amino acids in the amino acid sequence constituting the NADP-dependent malic enzyme substituted, resulting in a change in the activity of the enzyme, and a recombinant microorganism containing the same can efficiently produce L-glutamic acid.

Description

Technical Field

[0001] The present invention relates to a mutant microorganism of the genus Corynebacterium that produces L-glutamic acid and a method for producing L-glutamic acid using the same. More specifically, the present invention relates to a novel mutant of NADP-dependent malic enzyme involved in the biosynthetic pathway of L-glutamic acid, a polynucleotide, a transformant, and a method for producing L-glutamic acid using the same.

Background Art

[0002] L-Glutamic acid is a typical amino acid produced by microbial fermentation. Monosodium L-glutamate (MSG) enhances the preference of foods such as meat, fish, chicken, vegetables, sauces, soups, and seasonings by balancing and harmonizing the overall taste of foods, and can enhance the taste of low-salt foods with up to 30% reduction in salt. It is widely used as a seasoning for household and processed food production.

[0003] Looking briefly at the fermentation pathway of L-glutamic acid, glucose mainly undergoes the glycolytic pathway, but a part of it is metabolized to two molecules of pyruvic acid via the pentose phosphate pathway. One of these molecules fixes CO2 to become oxaloacetic acid, and the other molecule combines with acetyl CoA from pyruvic acid to become citric acid. Furthermore, oxaloacetic acid and citric acid enter the TCA cycle to become α-ketoglutaric acid. Here, the oxidative metabolic pathway from α-ketoglutaric acid to succinic acid is lacking, and since isocitrate dehydrogenase and glutamate dehydrogenase are closely involved, the reductive amination reaction of α-ketoglutaric acid proceeds efficiently to produce L-glutamic acid.

[0004] For the production of L-glutamic acid, wild-type strains obtained in a natural state or mutant strains modified to improve their glutamic acid production ability can be used. Recently, in order to improve the production efficiency of L-glutamic acid, gene recombination technology has been applied to microorganisms such as Escherichia coli and Corynebacterium, which are widely used in the production of useful substances such as amino acids and nucleic acids, and various recombinant strains or mutant strains with excellent L-glutamic acid production ability and methods for producing L-glutamic acid using them have been developed. In particular, there have been attempts to target genes such as enzymes, transcription factors, and transport proteins involved in the biosynthetic pathway of L-glutamic acid, or to induce mutations in promoters that regulate their expression to increase the production amount of L-glutamic acid. However, since the number of types of proteins such as enzymes, transcription factors, and transport proteins directly or indirectly related to the production of L-glutamic acid reaches more than several dozen, there is still a great need for many studies on whether the production ability of L-glutamic acid increases due to changes in the activity of such proteins.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0006] An object of the present invention is to provide a novel NADP-dependent malic enzyme variant.

[0007] Another object of the present invention is to provide a polynucleotide encoding the variant.

[0008] Furthermore, an object of the present invention is to provide a transformant containing the variant or polynucleotide.

[0009] Another object of the present invention is to provide a method for producing L-glutamic acid using the transformant.

Means for Solving the Problems

[0010] One aspect of the present invention provides a NADP-dependent malic enzyme variant consisting of the amino acid sequence of SEQ ID NO: 2, in which the aspartic acid at position 372 in the amino acid sequence of SEQ ID NO: 4 is substituted with asparagine.

[0011] The "NADP-dependent malic enzyme" used in the present invention is an enzyme that catalyzes the oxidative decarboxylation of malic acid to produce CO2 and NADPH together with pyruvic acid, and plays a role in supplying the carbon source required for the citric acid cycle. The NADP malic enzyme may be a gene encoding NADP-dependent malic enzyme or a sequence having substantial identity thereto. Here, "substantial identity" means that when each gene sequence, that is, the base sequence or nucleotide sequence, is aligned and analyzed so as to maximally correspond to any other nucleotide sequence, the any other nucleotide sequence has a sequence homology of 70% or more, 80% or more, 90% or more, or 98% or more with each nucleotide sequence.

[0012] The NADP-dependent malic enzyme in the present invention contains the amino acid sequence of SEQ ID NO: 4.

[0013] According to a specific example of the present invention, the amino acid sequence of SEQ ID NO: 4 may be derived from a microorganism belonging to the genus Corynebacterium of wild type.

[0014] More specifically, the microorganism belonging to the genus Corynebacterium may be Corynebacterium glutamicum.

[0015] The "variant" used in the present invention means a polypeptide in which one or more amino acids are conservatively substituted, deleted, modified or added at the N-terminus, C-terminus and / or internally in the amino acid sequence of a specific gene, which is different from the amino acid sequence before the mutation of the variant, but maintains its functions or properties. Here, "conservative substitution" means substituting one amino acid with another amino acid having similar structural and / or chemical properties, which has little or no effect on the activity of the protein or polypeptide. The amino acids are selected from alanine (Ala), isoleucine (Ile), valine (Val), leucine (Leu), methionine (Met), asparagine (Asn), cysteine (Cys), glutamine (Gln), serine (Ser), threonine (Thr), phenylalanine (Phe), tryptophan (Trp), tyrosine (Tyr), aspartic acid (Asp), glutamic acid (Glu), arginine (Arg), histidine (His), lysine (Lys), glycine (Gly) and proline (Pro).

[0016] In addition, the variant includes those in which one or more parts such as the N-terminal leader sequence or transmembrane domain are removed, or a part is removed from the N- and / or C-terminus of the mature protein.

[0017] Such mutants may have increased (enhanced), unchanged, or decreased (attenuated) capabilities compared to the protein or polypeptide before the mutation. Here, "increased or enhanced" includes cases where the activity of the protein itself has increased compared to the protein before the mutation, cases where the overall protein activity level in the cell is higher than that of the wild-type strain or the strain expressing the protein before the mutation due to increased gene expression or increased translation of the gene encoding the protein, and combinations thereof. Also, "decreased or attenuated" includes cases where the activity of the protein itself has decreased compared to the protein before the mutation, cases where the overall protein activity level in the cell is lower than that of the wild-type strain or the strain expressing the protein before the mutation due to inhibition of gene expression or inhibition of translation of the gene encoding the protein, and combinations thereof. In the present invention, the mutant can be used interchangeably with mutant form, variant, mutant polypeptide, mutated protein, mutation, etc.

[0018] The mutant in the present invention is an NADP-dependent malic enzyme in which the aspartic acid, which is the amino acid located at position 372 in the amino acid sequence of SEQ ID NO: 4, is substituted with asparagine, and may consist of the amino acid sequence of SEQ ID NO: 2.

[0019] Another aspect of the present invention provides a polynucleotide encoding the NADP-dependent malic enzyme mutant.

[0020] The "polynucleotide" used in the present invention is a polymer of nucleotides in which nucleotide monomers are covalently linked in a long chain, and is a DNA or RNA strand having a certain length or more, and more specifically, means a polynucleotide fragment encoding the mutant.

[0021] The polynucleotide may include a base sequence encoding the amino acid sequence of SEQ ID NO: 2.

[0022] According to one specific example of the present invention, the polynucleotide may contain the nucleotide sequence represented by SEQ ID NO: 1.

[0023] Another aspect of the present invention provides a vector containing a polynucleotide encoding the NADP-dependent malic enzyme variant.

[0024] Also, another aspect of the present invention provides a transformant containing the NADP-dependent malic enzyme variant or polynucleotide.

[0025] The "vector" used in the present invention means all types of nucleic acid sequence carrier structures used as a means for transferring and expressing a target gene in a host cell. Unless otherwise specified, the vector can mean one in which the carried nucleic acid sequence is inserted into the host cell genome and expressed and / or expressed independently. Such a vector contains essential regulatory elements operably linked so that the gene insert is expressed, and "operably linked" means that the target gene and its regulatory sequences are linked in a manner that enables gene expression by being functionally bound to each other. The "regulatory element" includes a promoter for transcription, any operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence for regulating the termination of transcription and translation.

[0026] The vector used in the present invention is not particularly limited as long as it can replicate in a host cell, and any vector known in the art can be used. Examples of the vector 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, Charon21A, etc., and plasmid vectors include pBR series, pUC series, pBluescriptII series, pGEM series, pTZ series, pCL series, and pET series, etc., but are not limited thereto.

[0027] The vector can typically be constructed as a vector for cloning or a vector for expression. Vectors for expression can use those commonly used in the art to express foreign genes or proteins in plants, animals, or microorganisms, and can be constructed by various methods known in the art.

[0028] The "recombinant vector" used in the present invention, after being transformed into a suitable host cell, can be replicated regardless of the host cell genome or integrated into the genome itself. At this time, the "suitable host cell" can be one in which the vector can replicate and contains an origin of replication which is a specific base sequence at which replication is initiated. For example, when the vector used is an expression vector and the host is a prokaryotic cell, it generally contains a strong promoter capable of promoting transcription (for example, pLλ promoter, CMV promoter, trp promoter, lac promoter, tac promoter, T7 promoter), a ribosome binding site for the start of translation, and a transcription / translation termination sequence. When the host is a eukaryotic cell, the origin of replication that functions in eukaryotic cells contained in the vector includes, but is not limited to, f1 origin of replication, SV40 origin of replication, pMB1 origin of replication, adenovirus origin of replication, AAV origin of replication, and BBV origin of replication. Also, a promoter derived from the genome of mammalian cells (for example, metallothionein promoter) or a promoter derived from mammalian viruses (for example, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, HSV tk promoter) may be used, and it generally has a polyadenylation sequence as a transcription termination sequence.

[0029] The recombinant vector can contain a selection marker. The selection marker is for screening transformants (host cells) transformed with the vector. Since only cells expressing the selection marker can survive in a medium treated with the selection marker, transformed cells can be screened. Representative examples of the selection marker include, but are not limited to, kanamycin, streptomycin, chloramphenicol, etc.

[0030] Transformants can be created by inserting a recombinant vector into a host cell, and the transformants may be obtained by introducing the recombinant vector into an appropriate host cell. The host cell is a cell capable of stably and continuously cloning or expressing the expression vector, and any host cell known in the art can be used.

[0031] When transforming a prokaryotic cell to produce a recombinant microorganism, host cells such as Escherichia coli strains like E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, E. coli XL1-Blue, Bacillus subtilis, Bacillus thuringiensis strains like those of the genus Bacillus, Corynebacterium strains, Salmonella typhimurium, Serratia marcescens, and various enterobacteria and strains such as Pseudomonas species may be used, but are not limited thereto.

[0032] When transforming a eukaryotic cell to produce a recombinant microorganism, host cells such as yeast (e.g., Saccharomyces cerevisiae), insect cells, plant cells, and animal cells such as the Sp2 / 0, CHO K1, CHO DG44, PER.C6, W138, BHK, COS7, 293, HepG2, Huh7, 3T3, RIN, MDCK cell lines, etc. may be used, but are not limited thereto.

[0033] "Transformation" as used in the present invention means a phenomenon of artificially causing a genetic change by introducing foreign DNA into a host cell, and "transformant" means a host cell into which foreign DNA has been introduced and stably maintains the expression of the target gene.

[0034] For the above transformation, a suitable vector introduction technique is selected by the host cell, and the target gene or the recombinant vector containing the same can be expressed in the host cell. For example, the introduction of the vector may 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, but is not limited thereto. As long as the transformed gene can be expressed in the host cell, it is inclusively and unrestrictedly included whether it is inserted into the host cell chromosome or located extrachromosomally.

[0035] The above transformant includes cells that have been transfected, transformed, or infected with the recombinant vector according to the present invention in vivo or in vitro, and can be used interchangeably with the terms recombinant host cell, recombinant cell, or recombinant microorganism.

[0036] According to one specific example of the present invention, the above transformant may be a microorganism belonging to the genus Corynebacterium.

[0037] More specifically, examples of the Corynebacterium genus microorganisms include Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium callunae, Corynebacterium suranareeae, Corynebacterium lubricantis, Corynebacterium doosanense, Corynebacterium efficiens, Corynebacterium uterequi, Corynebacterium stationis, Corynebacterium pacaense, Corynebacterium singulare, Corynebacterium humireducens, Corynebacterium marinum, Corynebacterium halotolerans, Corynebacterium spheniscorum, Corynebacterium freiburgense, Corynebacterium striatum, Corynebacterium canis, Corynebacterium ammoniagenes, Corynebacterium renale, Corynebacterium portisoli, Corynebacteriumpollutisoli), Corynebacterium imitans, Corynebacterium caspium, Corynebacterium testudinoris, Corynebacterium pseudopelargi or Corynebacterium flavescens, etc., but not limited thereto.

[0038] The transformant in the present invention may be a strain containing the aforementioned NADP-dependent malic enzyme variant or a polynucleotide encoding the same, or a vector containing the same, a strain expressing the NADP-dependent malic enzyme variant or polynucleotide, or a strain having an activity against the NADP-dependent malic enzyme variant, but is not limited thereto.

[0039] According to one specific example of the present invention, the transformant may have the ability to produce L-glutamic acid.

[0040] The transformant may naturally have the ability to produce L-glutamic acid or may be artificially endowed with the ability to produce L-glutamic acid.

[0041] According to one specific example of the present invention, the transformant may have a changed activity of NADP-dependent malic enzyme and an improved ability to produce L-glutamic acid.

[0042] "Improved production ability" used in the present invention means that the productivity of L-glutamic acid has increased compared to the parent strain. The parent strain means a wild type or mutant strain to be mutated, including those directly targeted for mutation or those transformed with a recombinant vector, etc. In the present invention, the parent strain may be a wild type Corynebacterium genus microorganism or a Corynebacterium genus microorganism mutated from the wild type.

[0043] The transformant according to the present invention has a changed activity of NADP-dependent malic enzyme due to the introduction of a NADP-dependent malic enzyme variant, and exhibits an increased L-glutamic acid-producing ability as compared with the parent strain. More specifically, the transformant may have a production amount of L-glutamic acid increased by at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% as compared with the parent strain, or may be increased by 1.1-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, or 10-fold, but is not limited thereto. As an example, the transformant containing the NADP-dependent malic enzyme variant may have a production amount of L-glutamic acid increased by 5% or more, specifically 5 to 50% (preferably 7 to 30%) as compared with the parent strain.

[0044] Another aspect of the present invention provides a method for producing L-glutamic acid, comprising the steps of culturing the transformant in a medium and recovering L-glutamic acid from the transformant or the medium in which the transformant has been cultured.

[0045] The culturing may be carried out using an appropriate medium and culturing conditions known in the art, and a normal technician can easily adjust and use the medium and culturing conditions. Specifically, the medium may be a liquid medium, but is not limited thereto. The culturing method may include, for example, batch culture, continuous culture, fed-batch culture, or a combined culture thereof, but is not limited thereto.

[0046] According to a specific example of the present invention, the medium must meet the requirements of specific strains in an appropriate manner and can be appropriately modified by an ordinary technician. For the culture medium for microorganisms of the genus Corynebacterium, known literature (Manual of Methods for General Bacteriology. American Society for Bacteriology. Washington D.C., USA, 1981) can be referred to, but it is not limited thereto.

[0047] According to a specific example of the present invention, the medium can contain various carbon sources, nitrogen sources, and trace element components. Usable carbon sources include sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch, and cellulose; oils and fats such as soybean oil, sunflower oil, castor oil, and coconut oil; fatty acids such as palmitic acid, stearic acid, and linoleic acid; alcohols such as glycerol and ethanol; and organic acids such as acetic acid. These substances can be used individually or as a mixture, but are not limited thereto. Usable nitrogen sources include peptone, yeast extract, gravy, malt extract, corn steep liquor, soybean meal, and urea, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate may also be included. The nitrogen source can also be used individually or as a mixture, but is not limited thereto. Usable phosphorus sources may include potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts, but are not limited thereto. In addition, the culture medium can contain metal salts such as magnesium sulfate or iron sulfate necessary for growth, but is not limited thereto. Other essential growth substances such as amino acids and vitamins may also be included. Furthermore, appropriate precursors can be used in the culture medium. The medium or individual components may be added batchwise or continuously to the culture broth in an appropriate manner during the culture process, but are not limited thereto.

[0048] According to one specific example of the present invention, during culturing, 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. Also, during culturing, an antifoaming agent such as a fatty acid polyglycol ester can be used to suppress the generation of bubbles. Additionally, in order to maintain the aerobic state of the culture solution, oxygen or an oxygen-containing gas (e.g., air) can be injected into the culture solution. The temperature of the culture solution is usually 20 to 45 °C, for example, it may be 25 to 40 °C. The culturing period can be continued until the useful substance is obtained in the desired production amount, and for example, it may be 10 to 160 hours.

[0049] According to one specific example of the present invention, the step of recovering L-glutamic acid from the cultured transformant or the medium in which the transformant was cultured can collect or recover the L-glutamic acid produced from the medium using a suitable method known in the art according to the culturing method. For example, methods such as centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, fractional dissolution (e.g., ammonium sulfate precipitation), chromatography (e.g., ion exchange, affinity, hydrophobicity, and size exclusion) can be used, but it is not limited thereto.

[0050] According to one specific example of the present invention, the step of recovering the L-glutamic acid can remove the biomass by subjecting the culture medium to low-speed centrifugation, and the obtained supernatant can be separated by ion exchange chromatography.

[0051] According to one specific example of the present invention, the step of recovering the L-glutamic acid can include a step of purifying the L-glutamic acid.

Effects of the Invention

[0052] The NADP-dependent malic enzyme variant according to the present invention has one or more amino acids substituted in the amino acid sequence constituting the NADP-dependent malic enzyme, resulting in a change in the enzyme activity, and a recombinant microorganism containing this can efficiently produce L-glutamic acid.

Brief Description of Drawings

[0053]

Figure 1

Modes for Carrying Out the Invention

[0054] Hereinafter, the present invention will be described in more detail. However, such a description is only presented as an example for understanding the present invention, and the scope of the present invention is not limited by such an exemplary description.

[0055] Example 1. Preparation of a Vector for Expression of NADP-Dependent Malic Enzyme Mutant A vector was prepared that expresses a mutant in which aspartic acid (D) located at position 372 in the amino acid sequence of NADP-dependent malic enzyme (SEQ ID NO: 4) was substituted with asparagine (N).

[0056] Using the gDNA of wild-type Corynebacterium glutamicum ATCC13869 as a template, PCR was performed using the primer pairs of primer 1 and primer 2 and the primer pairs of primer 3 and primer 4, respectively. Thereafter, using the mixture obtained by mixing the two PCR products as a template, overlapping PCR was performed again using primer 1 and primer 4 to obtain a fragment. Here, Takara PrimeSTAR Max DNA polymerase was used as the polymerase, and the PCR amplification conditions were denaturation at 95°C for 5 minutes, followed by 30 cycles of 95°C for 30 seconds, 58°C for 30 seconds, and 72°C for 1 minute 30 seconds, and reaction at 72°C for 5 minutes. The pK19msb vector was treated with smaI and ligated with the obtained PCR product (fragment), and the plasmid obtained here was named pK_malE(D372N).

[0057] The primer sequences used for the preparation of the vector are as shown in Table 1 below.

[0058]

Table 1

[0059] Example 2. Preparation of Mutant Strains into Which NADP-Dependent Malic Enzyme Mutants Have Been Introduced Corynebacterium glutamicum U3 (KCCM13218P) was used as the parent strain for introducing the NADP-dependent malic enzyme mutant, and the method for producing electrocompetent cells modified based on the method of van der Rest et al. was used as the method for transforming the U3 strain.

[0060] First, 10 ml of 2YT medium (containing 16 g / l of tryptone, 10 g / l of yeast extract, and 5 g / l of sodium chloride) supplemented with 2% glucose was used to primary culture the U3 strain to prepare a seed culture solution. Subsequently, 1 mg / ml of isonicotinic acid hydrazine and 2.5% glycine were added to 100 ml of 2YT medium without glucose, and the seed culture solution was inoculated so that the OD610 value became 0.3. Then, it was cultured at 18°C and 180 rpm for 12 - 16 hours to make the OD610 value reach 1.2 - 1.4. After the culture solution was left on ice for 30 minutes, it was centrifuged at 4°C and 4000 rpm for 15 minutes. Then, the supernatant was discarded, and the precipitated U3 strain was washed 4 times with a 10% glycerol solution and finally resuspended in 0.5 ml of 10% glycerol solution to prepare competent cells. Electroporation was performed using an electroporator from Bio - Rad. After adding the prepared competent cells and the pK_malE(D372N) vector to an electroporation cuvette (0.2 mm), an electrical shock was applied under the conditions of 2.5 kV, 200 Ω, and 12.5 μF. Immediately after the electrical shock ended, 1 ml of regeneration medium (containing 18.5 g / l of Brain Heart infusion and 0.5 M of sorbitol) was added and heat - treated at 46°C for 6 minutes. Then, after cooling at room temperature, it was transferred to a 15 - ml capped tube and cultured at 30°C for 2 hours, and then spread on a selection medium (containing 5 g / l of tryptone, 5 g / l of NaCl, 2.5 g / l of yeast extract, 18.5 g / l of Brain Heart infusion powder, 15 g / l of agar, 91 g / l of sorbitol, and 20 μg / l of kanamycin). The colonies generated after culturing at 30°C for 72 hours were cultured in BHI medium until the stationary phase to induce secondary recombination, and -5 ~10 -7 diluted to 10 ~10 -5 and spread on a 2YT plate medium without antibiotics (containing 10% sucrose) to select strains that were not kanamycin - resistant and were growth - viable on a medium containing 10% sucrose, and this was named NMD1.

[0061] Experimental Example 1. Evaluation of L-Glutamic Acid Production Ability of Mutant Strains into Which NADP-Dependent Malic Enzyme Variants Have Been Introduced The L-glutamic acid production abilities of the parental strain U3 and the mutant strain NMD1 into which the NADP-dependent malic enzyme variant had been introduced were compared.

[0062] Each strain (parental strain or mutant strain) was inoculated at 1% by volume into a 100 mL flask containing 10 mL of the glutamic acid production medium shown in Table 2 below, and shake-cultured at 30 °C and 200 rpm for 48 hours. After the cultivation was completed, the concentration of L-glutamic acid in the medium was measured using HPLC (Agilent), and the results are shown in Table 3 below, respectively.

[0063] [Table 2]

[0064] [Table 3]

[0065] As shown in Table 3 above, it was confirmed that in the mutant strain into which the NADP-dependent malic enzyme variant had been introduced, the production amount of L-glutamic acid was improved by about 8.7% compared to the parental strain. Such results suggest that the productivity of L-glutamic acid increases due to an increase in the flow of the carbon source in the glutamic acid production pathway by the NADP-dependent malic enzyme mutation.

[0066] As described above, the present invention has been described mainly with reference to its preferred embodiments. Those having ordinary knowledge in the technical field to which the present invention pertains will understand that the present invention can be realized in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a limiting perspective. The scope of the present invention is shown not in the above description but in the claims, and all differences within the equivalent scope thereof should be construed as being included in the present invention.

[0067] [Deposit Number] Depositing Institution: Korea Culture Center of Microorganisms (KCCM) Deposit Number: KCCM13218P Date of Deposit: 20220629

Claims

Claim 1 An NADP-dependent malic enzyme variant consisting of the amino acid sequence of SEQ ID NO: 2, in which the 372nd aspartic acid in the amino acid sequence of SEQ ID NO: 4 is substituted with asparagine. Claim 2 A polynucleotide encoding the variant according to Claim 1. Claim 3 A transformant comprising the variant according to Claim 1 or the polynucleotide according to Claim 2. Claim 4 The transformant according to Claim 3, wherein the transformant is a microorganism belonging to the genus Corynebacterium. Claim 5 The transformant according to Claim 3, wherein the transformant has the ability to produce L-glutamic acid. Claim 6 A method for producing L-glutamic acid, comprising culturing the transformant according to Claim 3 in a medium, and recovering L-glutamic acid from the transformant or the medium in which the transformant has been cultured. ​

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