Novel variants of alkyl hydroperoxide reductase AHPD family core domain and method for producing L-glutamic acid using same

Abstract

The present invention relates to a novel variant of an alkyl hydroperoxide reductase AhpD family core domain and a method for producing L-glutamic acid using the same. The present invention relates to an alkyl hydroperoxide reductase AhpD family core domain variant capable of efficiently producing L-glutamic acid from a recombinant microorganism expressing the variant by replacing one or more amino acids in the amino acid sequence constituting the alkyl hydroperoxide reductase AhpD family core domain to change protein activity.

Description

TECHNICAL FIELD

[0001] The present application relates to a novel variant of a core domain of an alkyl hydroperoxide reductase AhpD family and a method for producing L-glutamic acid using the same. BACKGROUND

[0002] L-glutamic acid is a representative amino acid produced by microbial fermentation, and monosodium L-glutamate (MSG) as a salt form thereof balances and harmonizes the overall taste of food, thereby improving the preference of meat, fresh food, chicken, vegetables, sauce, soup, and sauce material, and can enhance the taste of low-salt food in which salt is reduced to 30%, and thus is widely used as a seasoning for household and processed food production.

[0003] Briefly, the fermentation pathway of L-glutamic acid is that glucose is mainly metabolized through a glycolytic pathway, but a part thereof is metabolized as two molecules of pyruvic acid through a pentose phosphate pathway. Among them, one molecule is fixed with CO2 to become oxaloacetic acid, and the other molecule is combined with acetyl CoA to become citric acid from pyruvic acid. The oxaloacetic acid and the citric acid again enter the TCA cycle to become α-ketoglutaric acid. Here, since there is a lack of an oxidative metabolic pathway from α-ketoglutaric acid to succinic acid, and isocitrate dehydrogenase and glutamate dehydrogenase are closely involved, reductive amination of α-ketoglutaric acid is efficiently performed, thereby producing L-glutamic acid.

[0004] Production of L-glutamic acid can be performed using a wild-type strain obtained in a natural state or a variant strain modified in a manner to improve the L-glutamic acid production ability thereof. In recent years, in order to improve the production efficiency of L-glutamic acid, microorganisms such as Escherichia coli and coryneform bacteria which are used for production of useful substances such as amino acids and nucleic acids have been targeted, and various recombinant strains or variant strains having excellent L-glutamic acid production ability and a method for producing L-glutamic acid using the same have been developed by applying genetic recombination techniques. In particular, attempts have been made to directly cause variation in genes of enzymes, transcription factors, transport proteins, and the like involved in the biosynthetic pathway of L-glutamic acid or to induce variation in promoters and the like that regulate expression thereof, so as to increase the production amount of L-glutamic acid. However, the types of proteins such as enzymes, transcription factors, and transport proteins that are directly or indirectly related to L-glutamic acid production are diverse, and thus it is still practically necessary to perform a large amount of research on whether the L-glutamic acid production ability is increased according to changes in the activity of such proteins.

[0005] Prior Art Documents

[0006] Patent Documents

[0007] U.S. Patent No. 6852516

[0008] U.S. Patent No. 6962805 SUMMARY

[0009] The present application provides a novel alkylhydroperoxide reductase AhpD family core domain variant.

[0010] In addition, the present application provides a polynucleotide encoding the above variant.

[0011] In addition, the present application provides a transformant containing the above variant or polynucleotide.

[0012] In addition, the present application provides a method for producing L-glutamic acid using the above transformant.

[0013] One embodiment of the present application provides an alkylhydroperoxide reductase AhpD family core domain variant consisting of the amino acid sequence of SEQ ID NO: 4 in which the tryptophan (W) at position 231 of the amino acid sequence of SEQ ID NO: 2 is replaced with arginine (R).

[0014] The "alkylhydroperoxidase AhpD family core domain" used in the present application is a constituent element of an alkylhydroperoxidase that functions to remove peroxide and other active oxygen species in a cell, and is known to function to regulate a stress response caused by H2O2. The alkylhydroperoxidase AhpD family core domain in the present application can be a polypeptide having the activity of an alkylhydroperoxidase AhpD family core domain encoded by the NCgl2349 or ahpDl gene, but is not limited thereto.

[0015] The nucleic acid and protein sequence information of the above alkylhydroperoxidase AhpD family core domain can be obtained through a publicly known sequence database (e.g., GenBank, UniProt).

[0016] According to an embodiment of the present application, the above alkylhydroperoxidase AhpD family core domain can be encoded by the base sequence of SEQ ID NO: 1, and can consist of the amino acid sequence of SEQ ID NO: 2.

[0017] The base sequence or amino acid sequence of the alkylhydroperoxidase AhpD family core domain according to the present application can include a base sequence or amino acid sequence having homology or identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% compared to each sequence. Here, the "homology" or "identity" refers to the rate of agreement (%) between two sequences when the base sequence or amino acid sequence as a reference and any other base sequence or amino acid sequence are aligned in a manner of corresponding to the maximum extent and analyzed.

[0018] According to an embodiment of the present application, the above alkylhydroperoxidase AhpD family core domain or the gene encoding the same can be derived from a wild-type Corynebacterium glutamicum.

[0019] The "variant" used in the present application means different from the original amino acid sequence of the protein due to a variation in the base sequence of the gene encoding the protein. Specifically, one or more bases or nucleotides in the gene sequence are different due to substitution, insertion, deletion, or the like, whereby the translated polypeptide or protein is a protein variant, one or more amino acids in the N-terminal, C-terminal, and / or internal of the amino acid sequence are conservatively substituted and / or modified, thereby being different from the amino acid sequence before the variation, but maintaining the functions or properties. Here, the "conservative substitution" means substitution of one amino acid to another amino acid similar in structure and / or chemical properties, and has little or can have no effect on the activity of the protein or polypeptide. As the above-mentioned amino acid, selected from alanine (Ala, A), isoleucine (Ile, I), valine (Val, V), leucine (Leu, L), methionine (Met, M), asparagine (Asn, N), cysteine (Cys, C), glutamine (Gln, Q), serine (Ser, S), threonine (Thr, T), phenylalanine (Phe, F), tryptophan (Trp, W), tyrosine (Tyr, Y), aspartic acid (Asp, D), glutamic acid (Glu, E), arginine (Arg, R), histidine (His, H), lysine (Lys, K), glycine (Gly, G), and proline (Pro, P).

[0020] In addition, the variant includes a variant in which one or more portions such as an N-terminal leader sequence or a transmembrane domain are removed, or a variant in which a portion from the N- and / or C-terminal of the mature protein is removed.

[0021] Such a variant can have an increased (enhanced), or unchanged, or decreased (weakened) ability compared to the protein before the variation. Here, "increased or enhanced" includes cases where the activity of the protein itself is increased compared to the protein before the variation; cases where the degree of overall enzyme activity in the cell is high compared to the wild-type strain or the strain expressing the protein before the variation, due to increased expression or translation of the gene encoding the protein, and the like; and combinations thereof. Furthermore, "decreased or weakened" includes cases where the activity of the protein itself is decreased compared to the protein before the variation; cases where the degree of overall enzyme activity in the cell is low compared to the wild-type strain or the strain expressing the protein before the variation, due to hindering of expression or translation of the gene encoding the protein, and the like; and combinations thereof. In the present application, the variant can be used interchangeably with variation, modification, variant polypeptide, variant protein, variation, and the like.

[0022] The alkylhydroperoxide reductase AhpD family core domain variant according to the present application can include an amino acid sequence having homology or identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% to the amino acid sequence of SEQ ID NO: 4 except for the variation position (amino acid residue at position 231), without limitation as long as the amino acid sequence maintains the function or property of the above-mentioned variant.

[0023] Another aspect of the present application provides a polynucleotide encoding the above-mentioned alkylhydroperoxide reductase AhpD family core domain variant.

[0024] The "polynucleotide" used in the present application refers to a polymer of nucleotides in which nucleotide monomers are covalently linked in a chain shape, and is a DNA or RNA chain of a certain length or more, and specifically refers to a polynucleotide fragment encoding the above-mentioned alkylhydroperoxide reductase AhpD family core domain variant.

[0025] According to an embodiment of the present application, the above-mentioned polynucleotide includes a base sequence encoding the amino acid sequence of SEQ ID NO: 4, and for example, can include the base sequence of SEQ ID NO: 3.

[0026] Another aspect of the present application provides a vector including the above-mentioned polynucleotide encoding the alkylhydroperoxide reductase AhpD family core domain variant.

[0027] In addition, another aspect of the present application provides a transformant including the above-mentioned alkylhydroperoxide reductase AhpD family core domain variant or polynucleotide.

[0028] The "vector" used in the present application refers to all types of nucleic acid sequence transport structures used as a means for delivering and expressing a gene of interest to a host cell. Unless otherwise specified, the above vector can refer to a vector that allows a nucleic acid sequence carried to be inserted into a host cell gene for expression and / or to be expressed independently. Such a vector includes necessary regulatory elements operably linked for insertion of a gene expression product, "operably linked" means that a gene of interest and its regulatory sequences are functionally combined with each other and linked in a manner capable of gene expression, and "regulatory sequences" include a promoter sequence for implementing transcription, any operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence regulating termination of transcription and translation.

[0029] The vector used in the present application is not particularly limited as long as it can be replicated in a host cell, and any vector known in the art can be used. As one example of the above vector, plasmids, cosmids, viruses, and bacteriophages in a natural state or a recombinant state can be cited. For example, as a bacteriophage vector or a cosmid vector, pWE15, M13, λMBL3, λMBL4, λIXII, λASHII, λAPII, λtlO, λtl 1, Charon 4A, Charon 21A, and the like can be cited, and as a plasmid vector, pBR series, pUC series, pBluescript II series, pGEM series, pTZ series, pCL series, and pET series, and the like can be cited, but are not limited thereto.

[0030] The above vector can be typically constructed as a vector for cloning or a vector for expression. The vector for expression can use a conventional vector used in the art for expressing a foreign gene or a protein in a plant, an animal, or a microorganism, and can be constructed by various methods known in the art.

[0031] The "recombinant vector" used in the present application can be constructed using a prokaryotic cell or a eukaryotic cell as a host, can replicate independently of the genome of the host cell, or can be integrated into the genome itself. The above-mentioned host cell can replicate the vector and can include a replication origin that is a specific base sequence for initiating replication. For example, when the vector used is an expression vector and a prokaryotic cell is used as a host, a strong promoter (e.g., pL lambda promoter, CMV promoter, trp promoter, lac promoter, tac promoter, T7 promoter) that can allow transcription to proceed, a ribosome binding site for the initiation of translation, and a transcription / translation termination sequence are generally included. In the case of a eukaryotic cell as a host, the replication origin included in the vector that initiates replication in a eukaryotic cell includes an f1 replication origin, an SV40 replication origin, a pMB1 replication origin, an adenovirus replication origin, an AAV replication origin, and a BBV replication origin, but is not limited thereto. In addition, a promoter derived from the genome of a mammalian cell (e.g., metallothionein promoter) or a promoter derived from a mammalian virus (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, HSV tk promoter) can be used, and a polyadenylation sequence is generally used as a transcription termination sequence.

[0032] The above-mentioned recombinant vector can include a selection marker for screening a transformant (host cell) transformed with the vector, and only cells expressing the selection marker can survive in a medium treated with the selection marker, so that the transformed cells can be screened. As representative examples, the selection marker includes kanamycin, streptomycin, chloramphenicol, etc., but is not limited thereto.

[0033] A transformant can be produced by inserting the above-mentioned recombinant vector into a host cell, and the 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 be used.

[0034] In order to transform a prokaryotic cell to produce a recombinant microorganism, as a host cell, Escherichia coli such as E. coli DH5α, 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; coryneform bacteria; Bacillus such as Bacillus subtilis and Bacillus thuringiensis; various enteric bacteria and strains such as Salmonella typhimurium, Serratia marcescens, and Pseudomonas can be used, but are not limited thereto.

[0035] In order to transform eukaryotic cells for the production of recombinant microorganisms, as host cells, yeasts (e.g., Saccharomyces cerevisiae), insect cells, plant cells, and animal cells, such as Sp2 / 0, CHO K1, CHO DG44, PER.C6, W138, BHK, COS7, 293, HepG2, Huh7, 3T3, RIN, MDCK cell lines, and the like, but are not limited thereto.

[0036] "Transformation" used in the present application means a phenomenon in which foreign DNA is introduced into a host cell to artificially cause a genetic change, and a "transformant" means a host cell into which foreign DNA is introduced and which stably maintains expression of a target gene.

[0037] In the above transformation, a suitable vector introduction technique is selected according to the host cell, so that a target gene or a recombinant vector including the same can be expressed in the host cell. For example, vector introduction can be performed by electroporation, heat shock, CaPO4 precipitation, CaCl2 precipitation, microinjection, PEG method, DEAE-dextran method, cationic liposome method, lithium acetate-DMSO method, or a combination thereof, but is not limited thereto. The transformed gene can be included as long as it can be expressed in the host cell, and is not limited to being inserted into the chromosome of the host cell or being located outside the chromosome.

[0038] The above transformant includes a cell transfected, transformed, or infected with the recombinant vector according to the present application in vivo or in vitro, and can be used as the same term as a recombinant host cell, a recombinant cell, or a recombinant microorganism.

[0039] According to an embodiment of the present application, the above transformant can be an Escherichia strain or a Corynebacterium strain.

[0040] As the above Escherichia strain, Escherichia coli, Escherichia albertii, Escherichia blattae, Escherichia fergusonii, Escherichia hermannii, Escherichia vulneris, and the like, but are not limited thereto.

[0041] As the above-mentioned Corynebacterium strain, 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 pollutisoli, Corynebacterium imitans, Corynebacterium caspium, Corynebacterium testudinoris, Corynebacterium pseudopelargi, Corynebacterium flavescens, and the like can be used, but are not limited thereto.

[0042] As an example, the transformant described above is a Corynebacterium strain, and specifically, it can be Corynebacterium glutamicum.

[0043] The transformant in the present application can be a strain containing the above-described alkyl hydroperoxide reductase AhpD family core domain variant or a polynucleotide encoding the same, or a strain containing a vector containing the same, a strain expressing the above-described alkyl hydroperoxide reductase AhpD family core domain variant or polynucleotide, or a strain having activity of the above-described alkyl hydroperoxide reductase AhpD family core domain variant, but is not limited thereto.

[0044] According to an embodiment of the present application, the transformant described above can be a transformant transformed to express the above-described alkyl hydroperoxide reductase AhpD family core domain variant, or a transformant transformed by introducing a polynucleotide encoding the above-described alkyl hydroperoxide reductase AhpD family core domain variant.

[0045] The transformant in the present application can contain other protein variants or genetic variations in addition to the above-described alkyl hydroperoxide reductase AhpD family core domain variant.

[0046] According to an embodiment of the present application, the transformant described above can have L-glutamic acid production ability.

[0047] The transformant described above can naturally have L-glutamic acid production ability, or can be artificially given L-glutamic acid production ability.

[0048] According to an embodiment of the present application, the transformant described above can have improved L-glutamic acid production ability due to changes in the activity of the alkyl hydroperoxide reductase AhpD family core domain.

[0049] Specifically, the transformant according to the present application can be expected to have improved ability to cope with oxidative stress due to the mutation of the alkyl hydroperoxide reductase AhpD family core domain, and thus to have improved L-glutamic acid production ability in the second half of fermentation.

[0050] "Improved production ability" used in the present application means that the productivity of L-glutamic acid is increased compared to a parent strain. The parent strain described above refers to a wild type or a variant strain that is the object of mutation, including a vector or the like that is directly the object of mutation or transformation by recombination. In the present application, the parent strain can be a wild type Escherichia strain or a wild type Corynebacterium strain, or an Escherichia strain or a Corynebacterium strain that is mutated from a wild type.

[0051] The transformant according to the present application exhibits increased L-glutamic acid production ability compared to a strain containing the protein before the mutation (parent strain) by introducing a variant of the core domain of the alkyl hydroperoxide reductase AhpD family, whereby the activity of the core domain of the alkyl hydroperoxide reductase AhpD family is changed. Specifically, the amount of L-glutamic acid produced by the above-mentioned transformant can be 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%, or can 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, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold, but is not limited thereto.

[0052] The composition containing the transformant according to the present application can be used as a composition for L-glutamic acid production.

[0053] Another aspect of the present application provides a method for producing L-glutamic acid, including the steps of culturing the above-mentioned transformant in a culture medium, and recovering L-glutamic acid from the above-mentioned transformant or the culture medium in which the transformant is cultured.

[0054] The above-mentioned culturing can be performed according to a suitable culture medium and culture conditions known in the art, and the culture medium and culture conditions can be easily adjusted by those skilled in the art. Specifically, the above-mentioned culture medium can be a liquid medium, but is not limited thereto. The culturing method can include, for example, batch culture, continuous culture, fed-batch culture, or a combination thereof, but is not limited thereto.

[0055] According to an embodiment of the present application, the above-mentioned culture medium must satisfy the requirements of a specific strain in a suitable manner, and can be appropriately changed by those skilled in the art. With regard to the culture medium for Bacillus strains, reference can be made to a well-known document (Manual of Methods for General Bacteriology. American Society for Bacteriology. Washington D.C., USA, 1981), but is not limited thereto.

[0056] According to an embodiment of the present application, various carbon sources, nitrogen sources, and trace element components can be included in the culture medium. As carbon sources that can be used, sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch, cellulose, oils and fats such as soybean oil, sunflower oil, castor oil, coconut oil, fatty acids such as palmitic acid, stearic acid, linoleic acid, alcohols such as glycerol, ethanol, and organic acids such as acetic acid can be included. These substances can be used individually or in a mixture, but are not limited thereto. As nitrogen sources that can be used, 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 can be included. The nitrogen sources can also be used individually or in a mixture, but are not limited thereto. As a source of phosphorus that can be used, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or a corresponding sodium-containing salt can be included, but is not limited thereto. In addition, the culture medium can contain metal salts such as magnesium sulfate or ferrous sulfate required for growth, but is not limited thereto. In addition thereto, essential growth substances such as amino acids and vitamins can be included. In addition, precursors suitable for the culture medium can be used. The above-described culture medium or individual components can be added to the culture solution in batches or continuously during the culture process by an appropriate method, but are not limited thereto.

[0057] According to an embodiment of the present application, 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, an antifoaming agent such as fatty acid polyethylene glycol ester can be used to inhibit the generation of bubbles. Further, 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 can generally be 20°C to 45°C, for example, 25°C to 40°C. The culture time can continue until the desired production amount of the useful substance is obtained, for example, 10 to 160 hours.

[0058] According to an embodiment of the present application, in the above-described step of recovering L-glutamic acid from the cultured transformant or the culture medium of the cultured transformant, the produced L-glutamic acid can be collected or recovered from the culture medium according to the culture method and using a suitable method known in the art. 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), and the like can be used, but are not limited thereto.

[0059] According to an embodiment of the present application, in the above-described step of recovering L-glutamic acid, the culture medium can be subjected to low-speed centrifugation to remove biomass, and the obtained supernatant can be separated by ion exchange chromatography.

[0060] According to an embodiment of the present application, the step of recovering L-glutamic acid can include a process of purifying L-glutamic acid.

[0061] The alkyl hydroperoxide reductase AhpD family core domain variant according to the present application is substituted with one or more amino acids in the amino acid sequence constituting the alkyl hydroperoxide reductase AhpD family core domain, changes the activity of the protein, and thus enables efficient production of L-glutamic acid from a recombinant microorganism expressing the variant. DETAILED DESCRIPTION

[0062] Hereinafter, the present application will be described in more detail. However, such description is merely illustrative in nature and is in no way intended to limit the scope of the present application to the illustrative examples.

[0063] Example 1. Preparation of a strain expressing an alkyl hydroperoxide reductase AhpD family core domain variant

[0064] To confirm the effect of the variant in which the tryptophan (W) at position 231 of the amino acid sequence of the alkyl hydroperoxide reductase AhpD family core domain (SEQ ID NO: 2) is changed to arginine (R) (SEQ ID NO: 4) on L-glutamic acid production, a vector expressing the alkyl hydroperoxide reductase AhpD family core domain variant and a strain into which the vector is introduced were prepared.

[0065] 1-1. Preparation of vector pK_ahpD1 (W231R) for transformation

[0066] Using the genomic DNA of Corynebacterium glutamicum ATCC13869 as a template, each PCR was performed using a primer pair of primers 1 and 2 and a primer pair of primers 3 and 4. The two PCR products of about 0.5 kb and 0.6 kb in size amplified by PCR were mixed as a template, and overlapping PCR was performed using a primer pair of primers 1 and 4, thereby ligating into one fragment. After treating the pK19msb vector (SEQ ID NO: 5) with a restriction enzyme smal (NEB), the above fragment was cloned using T4 ligase. The vector thus constructed was named pK_ahpD1 (W231R). PCR was performed using Pfu premix (bioneer) for 5 minutes at 95°C, and then repeating 30 times for 30 seconds at 95°C, 30 seconds at 55°C, and 1 minute at 72°C, and reacting for 5 minutes at 72°C. The primer sequences used to prepare the vector are shown in Table 1 below.

[0067]

[0068] 1-2. Preparation of L-glutamic acid-producing strain into which pK_ahpD1 (W231R) was introduced

[0069] To prepare the L-glutamic acid-producing strain, as the parent strain, Corynebacterium glutamicum U3 (Deposit number KCCM13218P) was used. The pK_ahpD1 (W231R) vector was prepared so that the final concentration was 1 pg / pl or more, and after electroporation into Corynebacterium glutamicum U3 (reference. Tauch et al., FEMS Microbiology letters 123 (1994) 343-347), 1 ml of a regeneration medium (containing 18.5 g / 1 of Brain Heart infusion and 91 g / 1 of sorbitol) was added, and heat treatment was performed at 46°C for 6 minutes. After the treatment, it was moved to a 15-ml capped tube, and incubated at 30°C for 2 hours, and spread on a selection medium (containing 5 g / 1 of tryptone, 5 g / 1 of NaCl, 2.5 g / 1 of yeast extract, 18.5 g / 1 of Brain Heart infusion powder, and 15 g / 1 of agar) containing 20 mg / 1 of kanamycin. Colonies generated by incubation at 30°C for 72 hours were induced twice by culturing in a BHI medium (18.5 g / 1 of Brain Heart infusion powder) for 15 hours, and diluted to 10 -2 ~10 -3 The colonies were isolated by spreading on a selection medium to which 10% of sucrose was added. The isolated colonies were cultured in two kinds of selection media to which kanamycin and sucrose were added, respectively, and a strain that grew in a medium without kanamycin resistance and containing sucrose was selected. The selected strain was named as ahpD1 (W231R).

[0070] Experimental Example 1. Evaluation of L-glutamic acid production ability

[0071] The L-glutamic acid production ability of the strain ahpD1 (W231R) expressing the AhpD family core domain variant of alkyl hydroperoxide reductase was evaluated compared to Corynebacterium glutamicum U3 as the parent strain.

[0072] Each strain was inoculated at 1% by volume in a 100-ml flask containing 10 ml of the glutamic acid production medium of Table 2 below, and incubated with shaking at 30°C, 200 rpm for 48 hours. After the incubation, the concentration of L-glutamic acid in the medium was measured using HPLC (Agilent), and the results are shown in Table 3 below.

[0073]

[0074]

[0075] As shown in Table 3 above, it was confirmed that the strain ahpD1 (W231R) expressing the alkyl hydroperoxide reductase AhpD family core domain variant, in which the amino acid at position 231, i.e., tryptophan, in the amino acid sequence of the alkyl hydroperoxide reductase AhpD family core domain was replaced with arginine, increased the production of L-glutamic acid by about 10.4% compared to the parent strain.

[0076] So far, the present application has been described around its preferred embodiments. It will be appreciated by those skilled in the art that the present application can be implemented in modified forms without departing from the essential characteristics of the present application. Therefore, the disclosed embodiments should be considered in the light of illustrative rather than limiting. The scope of the present application is shown in the claims rather than the above description, and all differences within the equivalent scope thereof should be construed as included in the present application.

[0077] [Deposit Information]

[0078] Name of Depository: Korean Culture Center of Microorganisms (KCCM)

[0079] Accession Number: KCCM 13218P

[0080] Date of Deposit: 20220629

[0081]

Claims

1. A variant of the alkyl hydroperoxide reductase AhpD family core domain, comprising the amino acid sequence of SEQ ID NO:4, wherein, In the amino acid sequence of SEQ ID NO:2, tryptophan (W) at position 231 is replaced by arginine (R).

2. A polynucleotide encoding the variant of claim 1.

3. A transformant, wherein, It comprises the variant of claim 1 or the polynucleotide of claim 2.

4. The transformant according to claim 3, wherein, The transformant is a strain of Escherichia or a strain of Corynebacterium.

5. The transformant according to claim 3, wherein, The transformant has the ability to produce L-glutamic acid.

6. A method for producing L-glutamic acid, wherein, include: The step of culturing the transformant of claim 3 in a culture medium; as well as The step of recovering L-glutamic acid from the transformant or the culture medium for culturing the transformant.

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