Novel mutant of PTS transporter subunit EIIC and method for producing 5'-inosinic acid using the same

Abstract

The present invention relates to a novel mutant of the PTS transporter subunit EIIC and a method for producing 5'-inosinic acid using the same. In the PTS transporter subunit EIIC mutant, one or more amino acids in the amino acid sequence constituting the PTS transporter subunit EIIC are substituted, resulting in a change in the activity of the protein, and a recombinant microorganism containing the same can efficiently produce 5'-inosinic acid.

Description

Technical Field

[0001] The present invention relates to a novel mutant of the PTS transporter subunit EIIC and a method for producing 5'-inosinic acid using the same.

Background Art

[0002] 5'-inosinic acid (5'-inosinic acid or inosine monophosphate, IMP) is an intermediate in the nucleic acid biosynthesis metabolism system, which not only plays a physiologically important role in the bodies of animals and plants, but also is used in various fields such as food, pharmaceuticals and various medical applications. In particular, when used together with monosodium glutamate (MSG), it has a large synergistic effect on taste and is one of the nucleic acid-based seasonings attracting attention as a flavor seasoning.

[0003] Methods for producing 5'-inosinic acid include an enzymatic decomposition method of ribonucleic acid extracted from yeast cells, a chemical phosphorylation method of inosine produced by fermentation, etc. Recently, a method of culturing microorganisms that produce 5'-inosinic acid and recovering the 5'-inosinic acid accumulated in the medium has been mainly used.

[0004] In the production of 5'-inosinic acid using microorganisms, in order to improve the production efficiency of 5'-inosinic acid, genetic recombination technology has been applied to microorganisms such as Escherichia coli and Corynebacterium that are widely used in the production of useful substances such as nucleic acids and L-amino acids, and various recombinant strains or mutant strains having excellent 5'-inosinic acid production ability and a method for producing 5'-inosinic acid using the same have been developed. In particular, attempts have been made to target genes such as enzymes, transcription factors, and transport proteins involved in the biosynthetic pathway of 5'-inosinic acid, or to induce mutations in promoters that regulate their expression to increase the production amount of 5'-inosinic acid. However, since the number of proteins such as enzymes, transcription factors, and transport proteins that are directly or indirectly related to the production of 5'-inosinic acid reaches dozens to hundreds, there is still a great need for many studies on whether the production ability of 5'-inosinic acid can be increased by changes in the activity of such proteins at present.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0006] An object of the present invention is to provide a novel PTS transporter subunit EIIC 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 5'-inosinic acid using the transformant.

Means for Solving the Problems

[0010] One aspect of the present invention provides a PTS transporter subunit EIIC variant consisting of the amino acid sequence of SEQ ID NO: 2, in which alanine at position 281 in the amino acid sequence of SEQ ID NO: 4 is substituted with valine.

[0011] The "PTS transporter subunit EIIC" used in the present invention is a component of the phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS), which is a major carbohydrate active transport system, and catalyzes the phosphorylation of the sugar substrate that has flowed in together with the translocation of the sugar substrate across the cell membrane. It may be a polypeptide or protein consisting of the amino acid sequence of SEQ ID NO: 4 and having the activity of the PTS transporter subunit EIIC.

[0012] The nucleic acid and protein sequence information of the PTS transporter subunit EIIC can be obtained through known sequence databases (for example, GenBank, UniProt).

[0013] According to one specific example of the present invention, the PTS transporter subunit EIIC may be encoded by the nucleotide sequence of SEQ ID NO: 3.

[0014] The amino acid sequence of the PTS transporter subunit EIIC according to the present invention or the nucleotide sequence encoding the same may include a nucleotide sequence or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% homology or identity as compared with the amino acid sequence of SEQ ID NO: 4 or the nucleotide sequence of SEQ ID NO: 3. Here, "homology" or "identity" means the percentage of coincidence between two sequences when the reference nucleotide sequence or amino acid sequence and any other nucleotide sequence or amino acid sequence are aligned and analyzed so as to correspond maximally.

[0015] According to one specific example of the present invention, the PTS transporter subunit EIIC may be derived from wild-type Corynebacterium stationis.

[0016] As used in the present invention, a "variant" means that one or more amino acids in the amino acid sequence are conservatively substituted and / or modified at the N-terminus, C-terminus, and / or internally by mutations in the nucleotide sequence of the gene encoding the protein, resulting in a sequence different from the amino acid sequence before mutation, but the functions or properties are maintained. Here, "conservative substitution" means substituting one amino acid with another amino acid having similar structural and / or chemical properties, which may have little or no effect on the activity of the protein or polypeptide. Also, "modification" means substitution, insertion, deletion, etc. of amino acids. The amino acids are 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).

[0017] In addition, variants include 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.

[0018] Such mutants may have increased (enhanced), unchanged, or decreased (attenuated) abilities compared to the protein before the mutation. Here, "increased or enhanced" includes cases where the activity of the protein itself increases compared to the protein before the mutation, cases where the overall activity level of the protein in the cell is higher than that of the wild-type strain or the strain expressing the protein before the mutation due to increased 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 decreases compared to the protein before the mutation, cases where the overall activity level of the protein 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 protein, and combinations thereof. In the present invention, the mutant can be used interchangeably with mutant type, variant, mutant polypeptide, mutated protein, mutation, etc.

[0019] The PTS transporter subunit EIIC mutant according to the present invention can include an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% homology or identity compared to the amino acid sequence of SEQ ID NO: 2.

[0020] Another aspect of the present invention provides a polynucleotide encoding the PTS transporter subunit EIIC mutant.

[0021] The "polynucleotide" used in the present invention is a polymer of nucleotides in which nucleotide monomers are linked in a long chain by covalent bonds and is a DNA or RNA strand having a certain length or more, and more specifically, means a polynucleotide fragment encoding the mutant.

[0022] According to one specific example of the present invention, the polynucleotide may include a base sequence encoding the amino acid sequence of SEQ ID NO: 2.

[0023] More specifically, the polynucleotide can include the nucleotide sequence of SEQ ID NO: 1 in which the 842nd base c is substituted with t in the nucleotide sequence of SEQ ID NO: 3 encoding the PTS transporter subunit EIIC.

[0024] Another aspect of the present invention provides a vector comprising a polynucleotide encoding the PTS transporter subunit EIIC variant.

[0025] In addition, another aspect of the present invention provides a transformant comprising the PTS transporter subunit EIIC variant or polynucleotide.

[0026] As used in the present invention, the "vector" 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 that inserts and expresses the carried nucleic acid sequence into the host cell genome and / or one that expresses independently. Such a vector includes essential regulatory elements operably linked so that the gene insert is expressed. "Operably linked" means that the target gene and its regulatory sequences are linked in a manner that enables genetic 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.

[0027] 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.

[0028] The vector can typically be constructed as a vector for cloning or a vector for expression. As a vector for expression, those commonly used for expressing foreign genes or proteins in plants, animals, or microorganisms in the art can be used, and can be constructed by various methods known in the art.

[0029] The "recombinant vector" used in the present invention can be replicated regardless of the host cell genome or integrated into the genome itself after being transformed into a suitable host cell. At this time, the "suitable host cell" can be one in which the vector can replicate and can contain an origin of replication which is a specific base sequence at which replication starts. For example, when the vector used is an expression vector and a prokaryotic cell is used as the host, 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 a eukaryotic cell is used as the host, 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. In addition, a promoter derived from the genome of a mammalian cell (for example, metallothionein promoter) or a promoter derived from a mammalian virus (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.

[0030] The recombinant vector can contain a selection marker, and the selection marker is for selecting a transformant (host cell) transformed with the vector. Since only cells expressing the selection marker can survive in a medium treated with the selection marker, it is possible to select the transformed cells. Representative examples of the selection marker include, but are not limited to, kanamycin, streptomycin, chloramphenicol, etc.

[0031] Transformants can be created by inserting a recombinant vector into a host cell, and the transformant 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.

[0032] When transforming a prokaryotic cell to produce a recombinant microorganism, as the host cell, strains of the genus Escherichia such as E. coli DH5α, 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, strains of the genus Bacillus such as Bacillus subtilis, Bacillus thuringiensis, strains of the genus Corynebacterium such as Corynebacterium glutamicum, Corynebacterium stationis, various enterobacteria and strains such as Salmonella typhimurium, Serratia marcescens and Pseudomonas species may be used, but are not limited thereto.

[0033] When transforming a eukaryotic cell to produce a recombinant microorganism, as the host cell, yeast (e.g., Saccharomyces cerevisiae), insect cells, plant cells and animal cells, e.g., 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.

[0034] "Transformation" as used in the present invention means a phenomenon in which foreign DNA is introduced into a host cell to artificially cause a genetic change, and "transformant" means a host cell into which foreign DNA has been introduced and which stably maintains the expression of the target gene.

[0035] For the above transformation, a vector introduction technique suitable for the host cell is selected so that 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 may be included without limitation whether it is inserted into the host cell chromosome or located extrachromosomally.

[0036] 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.

[0037] The gene inserted into the recombinant vector of the present invention may be introduced into a host cell such as a Corynebacterium strain by homologous recombination crossover.

[0038] According to one specific example of the present invention, the above transformant may be a microorganism belonging to the genus Corynebacterium.

[0039] 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, and is not limited thereto.

[0040] The transformant in the present invention may be a strain containing the aforementioned PTS transporter subunit EIIC mutant or a polynucleotide encoding the same, or a vector containing the same, a strain expressing the PTS transporter subunit EIIC mutant or polynucleotide, or a strain having activity against the PTS transporter subunit EIIC mutant, but is not limited thereto.

[0041] In addition to the PTS transporter subunit EIIC mutant, the transformant in the present invention may contain other protein mutants or gene mutations.

[0042] According to one specific example of the present invention, the transformant may have the ability to produce 5'-inosinic acid.

[0043] The 5'-inosinic acid is a nucleic acid compound that imparts flavor, particularly umami, to food, and is used in the same meaning as inosine monophosphate (IMP).

[0044] The transformant may naturally have the ability to produce 5'-inosinic acid or may be artificially endowed with the ability to produce 5'-inosinic acid.

[0045] According to one specific example of the present invention, the transformant may have a changed activity of the PTS transporter subunit EIIC and an improved ability to produce 5'-inosinic acid.

[0046] "The productivity has been improved" used in the present invention means that the productivity of 5'-inosinic acid has increased compared to the parent strain. The parent strain means a wild type or mutant strain to be mutated, including those directly to be mutated or those to be transformed with a recombinant vector or the like. In the present invention, the parent strain may be a wild type Corynebacterium strain or a Corynebacterium strain mutated from the wild type.

[0047] The transformant according to the present invention shows a changed activity of the PTS transporter subunit EIIC and an increased ability to produce 5'-inosinic acid compared to the parent strain by introducing a PTS transporter subunit EIIC mutant. More specifically, the transformant may have a production amount of 5'-inosinic 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% compared to the parent strain, or increased by 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, or 10 times, but is not limited thereto. As an example, the transformant containing the PTS transporter subunit EIIC mutant may have a production amount of 5'-inosinic acid increased by 5% or more, specifically 5 to 50% (preferably 10 to 40%) compared to the parent strain.

[0048] Another aspect of the present invention provides a method for producing 5'-inosinic acid, which includes the steps of culturing the transformant in a medium and recovering 5'-inosinic acid from the transformant or the medium in which the transformant has been cultured.

[0049] The above-mentioned culture may be carried out using an appropriate medium and culture conditions known in the art, and an ordinary technician can easily adjust and use the medium and culture conditions. Specifically, the medium may be a liquid medium, but is not limited thereto. The culture method can include, for example, batch culture, continuous culture, fed-batch culture, or a combined culture thereof, but is not limited thereto.

[0050] According to a specific example of the present invention, the medium must meet the requirements of a specific strain in an appropriate manner and can be appropriately modified by an ordinary technician. For the culture medium for Escherichia strains, known literature (Manual of Methods for General Bacteriology. American Society for Bacteriology. Washington D.C., USA, 1981) can be referred to, but is not limited thereto.

[0051] According to one specific example of the present invention, the culture medium can contain various carbon sources, nitrogen sources, and trace element components. Examples of available 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. Examples of available 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. The nitrogen source can also be used individually or as a mixture, but is not limited thereto. The available source of phosphorus may include, but is not limited to, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts. Further, the culture medium can contain metal salts such as magnesium sulfate or iron sulfate required for growth, but is not limited thereto. In addition, essential growth substances such as amino acids and vitamins may be included. Also, 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 a suitable manner during the culture process, but are not limited thereto.

[0052] According to one specific example of the present invention, during cultivation, 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 cultivation, an antifoaming agent such as a fatty acid polyglycol ester can be used to suppress bubble generation. 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 cultivation period can be continued until the useful substance is obtained in the desired production amount, for example, it may be 10 to 160 hours.

[0053] According to one specific example of the present invention, the step of recovering 5'-inosinic acid from the cultured transformant or the medium in which the transformant was cultured can collect or recover the 5'-inosinic acid produced from the medium using a suitable method known in the art depending on the cultivation 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.

[0054] According to one specific example of the present invention, the step of recovering the 5'-inosinic acid can separate the supernatant obtained by subjecting the culture medium to low-speed centrifugation to remove biomass by ion exchange chromatography.

[0055] According to one specific example of the present invention, the step of recovering the 5'-inosinic acid can include a step of purifying the 5'-inosinic acid.

Advantages of the Invention

[0056] The PTS transporter subunit EIIC mutant according to the present invention has a change in the activity of the protein due to substitution of one or more amino acids in the amino acid sequence constituting the PTS transporter subunit EIIC, and a recombinant microorganism containing this can efficiently produce 5'-inosinic acid.

Brief Description of the Drawings

[0057]

Figure 1

Modes for Carrying Out the Invention

[0058] Hereinafter, the present invention will be described in more detail. However, such description is merely presented as an example for understanding the present invention, and the scope of the present invention is not limited by such illustrative description.

[0059] Example 1. Preparation of a Strain Expressing a PTS Transporter Subunit EIIC Mutant In order to confirm the effect of a mutant (SEQ ID NO: 2) in which alanine (A) at position 281 is substituted with valine (V) in the amino acid sequence of the PTS transporter subunit EIIC (SEQ ID NO: 4) on the production of 5'-inosinic acid, a vector expressing the PTS transporter subunit EIIC mutant and a strain into which the vector was introduced were prepared.

[0060] 1-1. Preparation of a Vector for Expressing the PTS Transporter Subunit EIIC Mutant Using the genomic DNA of wild-type Corynebacterium stationis ATCC6872 as a template, PCR was performed using the primer pairs of primer 1 and 2 and the primer pairs of primer 3 and 4, respectively. Thereafter, using the two PCR products as templates respectively, overlapping PCR was performed with the primer pair of primer 1 and 4 and ligated into one fragment. The PCR fragment and the pK19msb plasmid (SEQ ID NO: 5) were treated with the restriction enzyme smaI (NEB) and ligated using T4 ligase. The constructed plasmid was named pK_FP.

[0061] For PCR amplification, pfu premix (bioneer) was used. After denaturation at 95°C for 5 minutes, cycles of 95°C for 30 seconds, 58°C for 30 seconds, and 72°C for 1 minute 30 seconds were repeated 30 times, followed by a reaction at 72°C for 5 minutes.

[0062] The primer sequences used for plasmid preparation are as shown in Table 1 below.

[0063]

Table 1

[0064] 1-2. Preparation of Mutant Strains with Introduced Mutations in the PTS Transporter Subunit EIIC As a method for the transformation of Corynebacterium stationis KCCM13339P, a modified method for manufacturing electrocompetent cells based on the method of van der Rest et al. was used.

[0065] First, Corynebacterium stationis KCCM13339P was pre-cultured in 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 to prepare a seed culture solution. To 100 ml of 2YT medium without glucose, isonicotinic acid hydrazine at a concentration of 1 mg / ml and 2.5% glycine were added. Then, after inoculating the seed culture solution so that the OD 610 value reached 0.3, the culture was incubated at 30°C and 180 rpm for 5 - 8 hours until the OD 610The value was set to be between 0.6 and 0.7. After leaving the culture solution standing on ice for 30 minutes, it was centrifuged at 4°C and 3500 rpm for 10 minutes. Then, the supernatant was discarded, and the precipitated Corynebacterium stationis KCCM13339P was washed 4 times with a 10% glycerol solution. Finally, it was resuspended in 0.5 ml of a 10% glycerol solution to prepare competent cells. Electroporation was performed using an electroporator from Bio-Rad. After adding the prepared competent cells and the produced pK_FP 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, 1 ml of RG medium (containing 18.5 g / l of Brain Heart infusion and 0.5 M of sorbitol) was added, and heat treatment was performed 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 the medium until the stationary phase to induce secondary recombination, and then diluted to 10 -5 ~10 -7 and spread on a plate medium without antibiotics (containing 10% sucrose) to select a strain that was not resistant to kanamycin and was growth-proficient in a medium containing 10% sucrose, and this was named IFP-1.

[0066] Experimental Example 1. Evaluation of the 5'-inosinic acid production ability of a strain expressing a PTS transporter subunit EIIC mutant The 5'-inosinic acid production abilities of the parent strain KCCM13339P and the mutant strain IFP-1 into which a PTS transporter subunit EIIC mutant was introduced were compared.

[0067] Into a 100 mL flask containing 10 mL of the medium for 5'-inosinic acid production shown in Table 2 below, each strain (parent strain or mutant strain) was inoculated at 1% based on volume, and shake-cultured at 34 °C and 200 rpm for 45 hours. After the cultivation was completed, the concentration of 5'-inosinic acid in the medium was measured using HPLC (Agilent), and the results are shown in Table 3 below.

[0068]

Table 2

[0069]

Table 3

[0070] As shown in Table 3 above, it was confirmed that in the mutant strain into which the PTS transporter subunit EIIC mutant was introduced, the production amount of 5'-inosinic acid was improved by about 18% compared to the parent strain due to the substitution of alanine at the 281st position with valine. Such results suggest that the introduction of point mutations in the PTS transporter subunit EIIC provides an effective effect on the productivity of 5'-inosinic acid.

[0071] So far, 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 should be construed as being included in the present invention.

[0072] [Deposit Number] Name of the depository institution: Korean Culture Center of Microorganisms (KCCM) Deposit Number: KCCM13339P Date of deposit: 20230329

Claims

**Claim 1** A PTS transporter subunit EIIc variant consisting of the amino acid sequence of SEQ ID NO: 2, in which alanine at position 281 in the amino acid sequence of SEQ ID NO: 4 is replaced by valine. **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 5'-inosinic acid. **Claim 6** A method for producing 5'-inosinic acid, comprising culturing the transformant according to claim 3 in a medium, and recovering 5'-inosinic acid from the transformant or the medium in which the transformant has been cultured.

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