Novel Mutant of Phosphoenolpyruvate Carboxylase and Method for Producing 5'-Inosinic Acid Using the Same

Abstract

The present invention relates to a novel mutant of phosphoenolpyruvate carboxylase and a method for producing 5'-inosinic acid using the same. In the phosphoenolpyruvate carboxylase mutant, one or more amino acids in the amino acid sequence constituting phosphoenolpyruvate carboxylase 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 phosphoenolpyruvate carboxylase 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. It not only plays a physiologically important role in the bodies of animals and plants, but is also 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 method of chemically phosphorylating inosine produced by fermentation, etc. Recently, a method of culturing a microorganism that produces 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, gene 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 types 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.

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 phosphoenolpyruvate carboxylase mutant.

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

[0008] Furthermore, an object of the present invention is to provide a transformant containing the mutant 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 phosphoenolpyruvate carboxylase mutant in which one or more of the 472nd and 673rd amino acids in the amino acid sequence of SEQ ID NO: 4 are substituted with other amino acids.

[0011] The "phosphoenolpyruvate caboxylase" used in the present invention catalyzes the reaction of removing carbon dioxide from phosphoenolpyruvate, and may be a polypeptide or protein having the activity of phosphoenolpyruvate carboxylase and consisting of the amino acid sequence of SEQ ID NO: 4.

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

[0013] According to one specific example of the present invention, the phosphoenolpyruvate carboxylase may be encoded by the base sequence of SEQ ID NO: 3.

[0014] The amino acid sequence of the phosphoenolpyruvate carboxylase according to the present invention or the base sequence encoding the same may include a base 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 base sequence of SEQ ID NO: 3. Here, "homology" or "identity" means the coincidence rate (%) between two sequences when the reference base sequence or amino acid sequence and any other base sequence or amino acid sequence are aligned and analyzed so as to correspond to each other to the maximum extent.

[0015] According to one specific example of the present invention, the phosphoenolpyruvate carboxylase may be derived from wild-type Corynebacterium stationis.

[0016] The "variant" used in the present invention means that due to mutations in the nucleotide sequence of the gene encoding the protein, one or more amino acids among the amino acid sequence are conservatively substituted and / or modified at the N-terminus, C-terminus and / or internally, which are 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 hardly affect or not affect at all 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, 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.

[0018] Such mutants may have increased (enhanced), unchanged, or decreased (attenuated) capabilities compared to the protein before the mutation. Here, "increase or enhancement" 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 compared to 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, "decrease or attenuation" 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 compared to 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] According to one specific example of the present invention, the mutant may consist of the amino acid sequence of SEQ ID NO: 2, in which proline at position 472 is substituted with serine and glycine at position 673 is substituted with aspartic acid in the amino acid sequence of SEQ ID NO: 4.

[0020] More specifically, the phosphoenolpyruvate carboxylase mutant 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.

[0021] Another aspect of the present invention provides a polynucleotide encoding the phosphoenolpyruvate carboxylase mutant.

[0022] 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 chain having a certain length or more. More specifically, it means a polynucleotide fragment encoding the mutant.

[0023] According to one specific example of the present invention, the polynucleotide may contain a base sequence encoding the amino acid sequence of SEQ ID NO: 2.

[0024] More specifically, the polynucleotide can contain the base sequence of SEQ ID NO: 1 in which the 1414th base c is substituted with t and the 2018th base g is substituted with a in the base sequence of SEQ ID NO: 3 encoding phosphoenolpyruvate carboxylase.

[0025] Another aspect of the present invention provides a vector containing a polynucleotide encoding the phosphoenolpyruvate carboxylase mutant.

[0026] Also, another aspect of the present invention provides a transformant containing the phosphoenolpyruvate carboxylase mutant or polynucleotide.

[0027] The "vector" used in the present invention means all types of nucleic acid sequence carrier structures used as 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 itself independently. Such a vector contains 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. "Regulatory elements" include 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.

[0028] 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.

[0029] The vector can typically be constructed as a vector for cloning or a vector for expression. The vector for expression can use those commonly used to express foreign genes or proteins in plants, animals, or microorganisms in the art and can be constructed by various methods known in the art.

[0030] 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 decoding, and a transcription / decoding 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. Also, 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.

[0031] The recombinant vector can contain a selection marker, and 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, it is possible to screen transformed cells. Representative examples of the selection marker include, but are not limited to, kanamycin, streptomycin, chloramphenicol, etc.

[0032] A transformant can be produced by inserting a recombinant vector into a host cell, and the transformant is 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.

[0033] 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.

[0034] 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, for example, cell lines such as Sp2 / 0, CHO K1, CHO DG44, PER.C6, W138, BHK, COS7, 293, HepG2, Huh7, 3T3, RIN, MDCK may be used, but are not limited thereto.

[0035] "Transformation" used in the present invention means a phenomenon of artificially causing genetic changes by introducing external DNA into a host cell, and "transformant" means a host cell into which external DNA has been introduced and which stably maintains the expression of the target gene.

[0036] For the above-mentioned 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.

[0037] The above-mentioned transformant includes cells that have been transduced, 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.

[0038] 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.

[0039] According to a specific example of the present invention, the above-mentioned transformant may be a microorganism belonging to the genus Corynebacterium.

[0040] 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, or the like, and is not limited thereto.

[0041] The transformant in the present invention may be a strain containing the aforementioned phosphoenolpyruvate carboxylase mutant or a polynucleotide encoding the same, or a vector containing the same, a strain expressing the phosphoenolpyruvate carboxylase mutant or polynucleotide, or a strain having an activity against the phosphoenolpyruvate carboxylase mutant, but is not limited thereto.

[0042] In addition to the phosphoenolpyruvate carboxylase mutant, the transformant in the present invention can contain other protein mutants or gene mutations.

[0043] According to one specific example of the present invention, the transformant may have the ability to produce 5'-inosinic acid.

[0044] The 5'-inosinic acid is a nucleic acid compound that imparts the flavor, especially umami, of food, and is used in the same meaning as inosine monophosphate (IMP).

[0045] 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.

[0046] According to one specific example of the present invention, the transformant may have a changed activity of phosphoenolpyruvate carboxylase and an improved ability to produce 5'-inosinic acid.

[0047] "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 that is the target of mutation, including those that are directly the target of mutation or those that are 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.

[0048] The transformant according to the present invention shows a changed activity of phosphoenolpyruvate carboxylase and an increased ability to produce 5'-inosinic acid compared to the parent strain by introducing a phosphoenolpyruvate carboxylase mutant. More specifically, the transformant may have a production amount of 5'-inosinic acid that is at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% increased compared to the parent strain, or may be 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 phosphoenolpyruvate carboxylase mutant may have a production amount of 5'-inosinic acid that is increased by 5% or more, specifically 5 to 50% (preferably 10 to 40%) compared to the parent strain.

[0049] Another aspect of the present invention provides a method for producing 5'-inosinic acid, including 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.

[0050] The above-mentioned cultivation may be carried out using an appropriate medium and cultivation conditions known in the art, and an ordinary technician can easily adjust and use the medium and cultivation conditions. Specifically, the medium may be a liquid medium, but is not limited thereto. The cultivation method can include, for example, batch culture, continuous culture, fed-batch culture, or a combined culture thereof, but is not limited thereto.

[0051] 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.

[0052] 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 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. Examples of 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. The nitrogen source can also be used individually or as a mixture, but is not limited thereto. The usable 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, suitable 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 culturing process, but are not limited thereto.

[0053] According to one specific example of the present invention, during the culture, 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. Further, during the culture, a defoaming 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 culture period can be continued until the useful substance is obtained in the desired production amount, for example, it may be 10 to 160 hours.

[0054] 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 is cultured can collect or recover the 5'-inosinic acid produced from the medium using a suitable method known in the art depending on the culture 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.

[0055] 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.

[0056] 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.

Effects of the Invention

[0057] The phosphoenolpyruvate carboxylase mutant according to the present invention has one or more amino acids in the amino acid sequence constituting phosphoenolpyruvate carboxylase substituted, resulting in a change in the activity of the protein, and a recombinant microorganism containing the same can efficiently produce 5'-inosinic acid.

Brief Description of the Drawings

[0058]

Figure 1

Modes for Carrying Out the Invention

[0059] 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.

[0060] Example 1. Preparation of a Strain Expressing a Phosphoenolpyruvate Carboxylase Mutant In order to confirm the effect of a mutant (SEQ ID NO: 2) in which proline (P) at position 472 is substituted with serine (S) and glycine (G) at position 673 is substituted with aspartic acid (D) in the amino acid sequence of phosphoenolpyruvate carboxylase (SEQ ID NO: 4) on the production of 5'-inosinic acid, a vector expressing the phosphoenolpyruvate carboxylase mutant and a strain into which the vector was introduced were prepared.

[0061] 1-1. Preparation of a Vector for Expressing a Phosphoenolpyruvate Carboxylase Mutant Using the genomic DNA of wild-type Corynebacterium stationis ATCC6872 as a template, PCR was performed using the primer pair of Primer 1 and Primer 2. 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_PC.

[0062] For PCR amplification, pfu premix (bioneer) was used. After denaturation at 95°C for 5 minutes, the cycle of 95°C for 30 seconds, 58°C for 30 seconds, and 72°C for 1 minute 30 seconds was repeated 30 times, followed by a reaction at 72°C for 5 minutes.

[0063] The primer sequences used for plasmid preparation are as shown in Table 1 below.

[0064]

Table 1

[0065] 1-2. Preparation of Mutant Strains with Phosphoenolpyruvate Carboxylase Mutants Introduced As a method for the transformation of Corynebacterium stationis KCCM13339P, a method for producing electrocompetent cells modified based on the method of van der Rest et al. was used.

[0066] First, Corynebacterium stationis KCCM13339P was precultured 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 to 8 hours, and the OD 610The value was set to be 0.6 - 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. Thereafter, the supernatant was discarded, and the precipitated Corynebacterium stationis KCCM13339P was washed 4 times with a 10% glycerol solution, and finally 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_PC vector to an electroporation cuvette (0.2 mm), an electric shock was applied under the conditions of 2.5 kV, 200 Ω, and 12.5 μF. Immediately after the electric 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. Thereafter, after cooling at room temperature, it was transferred to a 15 ml cap 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 by culturing at 30°C for 72 hours were cultured in the medium until the stationary phase to induce secondary recombination, and diluted 10 -5 ~10 -7 times and spread on a plate medium without antibiotics (containing 10% sucrose), and strains that were not kanamycin-resistant and were viable on the medium containing 10% sucrose were selected and named IPC-1.

[0067] Experimental Example 1. Evaluation of 5'-inosinic acid production ability of a strain expressing a phosphoenolpyruvate carboxylase mutant The 5'-inosinic acid production abilities of the parent strain KCCM13339P and the mutant strain IPC-1 into which a phosphoenolpyruvate carboxylase mutant was introduced were compared.

[0068] 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% by volume, and shake-cultured at 34 °C and 200 rpm for 45 hours. After completion of the culture, the concentration of 5'-inosinic acid in the medium was measured using HPLC (Agilent), and the results are shown in Table 3 below.

[0069]

Table 2

[0070]

Table 3

[0071] As shown in Table 3 above, in the mutant strain into which the phosphoenolpyruvate carboxylase mutant was introduced, the 472nd and 673rd amino acids were substituted with other amino acids, and it was confirmed that the production amount of 5'-inosinic acid was improved by about 16% compared to the parent strain. Such results suggest that the introduction of point mutations in phosphoenolpyruvate carboxylase provides an effective effect on the productivity of 5'-inosinic acid.

[0072] 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 thereof should be construed as being included in the present invention.

[0073] [Deposit number] Name of depository institution: Korea Culture Center of Microorganisms (KCCM) Deposit number: KCCM13339P Date of deposit: 20230329

Claims

1. A phosphoenolpyruvate carboxylase variant in which one or more of the 472nd and 673rd amino acids in the amino acid sequence of SEQ ID NO: 4 are substituted with other amino acids.

2. The phosphoenolpyruvate carboxylase variant according to claim 1, wherein the variant consists of the amino acid sequence of SEQ ID NO: 2, in which the 472nd proline in the amino acid sequence of SEQ ID NO: 4 is substituted with serine and the 673rd glycine is substituted with aspartic acid.

3. A polynucleotide encoding the variant according to claim 1.

4. A transformant comprising the variant according to claim 1 or the polynucleotide according to claim 3.

5. The transformant according to claim 4, wherein the transformant is a microorganism belonging to the genus Corynebacterium.

6. The transformant according to claim 4, wherein the transformant has the ability to produce 5'-inosinic acid.

7. A method for producing 5'-inosinic acid, comprising culturing the transformant according to claim 4 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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