Novel mutant of ABC transporter permease and method for producing 5'-inosinic acid using the same

Abstract

The present invention relates to a novel mutant of an ABC transporter permease and a method for producing 5'-inosinic acid using the same. In the ABC transporter permease mutant, one or more amino acids in the amino acid sequence constituting the ABC transporter permease 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 ABC transporter permease and a method for producing 5'-inosinic acid using the same. [Background technology]

[0002] 5'-inosinic acid (also known as inosine monophosphate, IMP) is an intermediate in the nucleic acid biosynthesis metabolic pathway. It not only plays an important physiological role in animals and plants, but is also used in a wide range of applications, including food, medicine, and various medical uses. In particular, when used together with monosodium glutamate (MSG), IMP is one of the nucleic acid seasonings that has been attracting attention as a flavor seasoning due to its significant flavor synergy.

[0003] Methods for producing 5'-inosinic acid include enzymatic decomposition of ribonucleic acid extracted from yeast cells and chemical phosphorylation of inosinic acid produced by fermentation. Recently, the most widely used method involves culturing microorganisms that produce 5'-inosinic acid and recovering the 5'-inosinic acid that accumulates in the culture medium.

[0004] To improve the efficiency of 5'-inosinic acid production using microorganisms, genetic engineering has been applied to microorganisms such as Escherichia coli and Corynebacterium, which are often used to produce useful substances such as nucleic acids and L-amino acids, to develop various recombinant strains or mutants with excellent 5'-inosinic acid production capabilities and methods for producing 5'-inosinic acid using these strains. In particular, attempts have been made to increase 5'-inosinic acid production by targeting genes for enzymes, transcription factors, transport proteins, and other proteins involved in the 5'-inosinic acid biosynthetic pathway or by inducing mutations in promoters that regulate their expression. However, because there are dozens to hundreds of proteins, including enzymes, transcription factors, and transport proteins, directly or indirectly involved in 5'-inosinic acid production, much research remains to be done to determine whether altering the activity of these proteins increases 5'-inosinic acid production. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Korean Patent No. 10-116602 Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide a novel ABC transporter permease mutant.

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

[0008] A further object of the present invention is to provide a transformant comprising 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 problem]

[0010] One aspect of the present invention provides an ABC transporter permease mutant in which one or more of the amino acids at positions 10, 49 and 169 in the amino acid sequence of SEQ ID NO: 4 have been substituted with other amino acids.

[0011] The "ABC transporter permease" used in the present invention is involved in the translocation of an unknown substrate across a membrane, contains an ATP-binding domain for energy generation, and may be a polypeptide or protein having the amino acid sequence of SEQ ID NO: 4 and having the activity of an ABC transporter permease.

[0012] The nucleic acid and protein sequence information of the ABC transporter permease can be obtained through publicly known sequence databases (eg, GenBank, UniProt).

[0013] According to one embodiment of the present invention, the ABC transporter permease may be encoded by the nucleotide sequence of SEQ ID NO:3.

[0014] The amino acid sequence of an ABC transporter permease according to the present invention or the nucleotide sequence encoding the same may comprise a nucleotide sequence or amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% homology or identity to the amino acid sequence of SEQ ID NO: 4 or the nucleotide sequence of SEQ ID NO: 3. Here, "homology" or "identity" refers to the percentage match between a reference nucleotide sequence or amino acid sequence and any other nucleotide sequence or amino acid sequence when the two sequences are aligned and analyzed for maximum correspondence.

[0015] According to one embodiment of the present invention, the ABC transporter permease may be derived from wild-type Corynebacterium stationis.

[0016] As used herein, the term "mutant" refers to a mutation in the base sequence of a gene encoding a protein, resulting in conservative substitution and / or modification of one or more amino acids at the N-terminus, C-terminus, and / or internally, resulting in a difference from the amino acid sequence prior to the mutation, while maintaining the functions or properties of the original amino acid. Here, "conservative substitution" refers to the replacement of one amino acid with another amino acid with similar structural and / or chemical properties, which may have little or no effect on the activity of the protein or polypeptide. Furthermore, "modification" refers to amino acid substitution, insertion, deletion, etc. 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] Variants also include those in which one or more portions have been removed, such as the N-terminal leader sequence or transmembrane domain, or portions have been removed from the N- and / or C-termini of the mature protein.

[0018] The ability of such a mutant may be increased (strengthened), unchanged, or decreased (weakened) compared to the protein before mutation. Here, "increased or enhanced" includes cases where the activity of the protein itself is increased compared to the protein before mutation, cases where the overall activity of the protein in cells is higher than that of a wild-type strain or a strain expressing the protein before mutation due to increased expression or translation of the gene encoding the protein, or a combination thereof. Furthermore, "decreased or weakened" includes cases where the activity of the protein itself is decreased compared to the protein before mutation, cases where the overall activity of the protein in cells is lower than that of a wild-type strain or a strain expressing the protein before mutation due to inhibited expression or translation of the gene encoding the protein, or a combination thereof. In the present invention, "mutant" may be interchangeably referred to as "mutated type," "transformed," "mutated polypeptide," "mutated protein," "mutation," etc.

[0019] According to one embodiment of the present invention, the mutant may consist of the amino acid sequence of SEQ ID NO: 2, in which the proline at position 10 in the amino acid sequence of SEQ ID NO: 4 is replaced with serine, the arginine at position 49 is replaced with cysteine, and the threonine at position 169 is replaced with isoleucine.

[0020] More specifically, the ABC transporter permease mutant may comprise 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 invention provides polynucleotides encoding the ABC transporter permease mutants.

[0022] The term "polynucleotide" as used herein refers to a nucleotide polymer in which nucleotide units are linked in a long chain by covalent bonds, and is a DNA or RNA chain of a certain length or more, and more specifically, refers to a polynucleotide fragment encoding the above variant.

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

[0024] More specifically, the polynucleotide may comprise the base sequence of SEQ ID NO: 1, in which the 28th base c is replaced with t, the 145th base c is replaced with t, and the 506th base c is replaced with t in the base sequence of SEQ ID NO: 3, which encodes an ABC transporter permease.

[0025] Another aspect of the present invention provides a vector comprising a polynucleotide encoding the ABC transporter permease mutant.

[0026] Another aspect of the present invention provides a transformant comprising the ABC transporter permease mutant or polynucleotide.

[0027] As used herein, the term "vector" refers to any type of nucleic acid sequence delivery structure used to transfer and express a gene of interest in a host cell. Unless otherwise specified, the term "vector" can refer to a vector that inserts a nucleic acid sequence into a host cell and expresses it therein, and / or a vector that expresses it independently. Such vectors contain essential regulatory elements operably linked to allow expression of the gene insert. "Operably linked" means that the gene of interest and its regulatory sequences are functionally linked to each other to enable gene expression. "Regulatory elements" include a promoter for transcription, an optional operator sequence for transcription regulation, a sequence encoding a suitable mRNA ribosomal binding site, and sequences regulating the termination of transcription and translation.

[0028] The vector used in the present invention is not particularly limited as long as it is replicable in host cells, and any vector known in the art can be used. Examples of such vectors include naturally occurring or recombinant plasmids, cosmids, viruses, and bacteriophages. Examples of phage or cosmid vectors include pWE15, M13, λMBL3, λMBL4, λIXII, λASHII, λAPII, λt10, λt11, Charon4A, and Charon21A. Examples of plasmid vectors include, but are not limited to, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, and pET.

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

[0030] The "recombinant vector" used in the present invention may be replicable independently of the genome of a suitable host cell after transformation, or may be integrated into the genome itself. In this regard, the "suitable host cell" refers to a cell in which the vector is replicable and may contain a replication origin, which is a specific base sequence that initiates replication. For example, when the vector used is an expression vector and a prokaryotic cell is used as the host, it typically contains a strong promoter (e.g., pLλ promoter, CMV promoter, trp promoter, lac promoter, tac promoter, T7 promoter) that can drive transcription, a ribosome binding site for the initiation of transcription, and a transcription / transcription termination sequence. When a eukaryotic cell is used as the host, replication origins that function in eukaryotic cells and are contained in the vector include, but are not limited to, the f1 origin, SV40 origin, pMB1 origin, adenovirus origin, AAV origin, and BBV origin. Alternatively, promoters derived from the genome of mammalian cells (e.g., metallothionine promoter) or promoters derived from mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, HSV tk promoter) may be used, and generally have a polyadenylation sequence as a transcription termination sequence.

[0031] The recombinant vector may contain a selection marker, which is used to select transformants (host cells) transformed with the vector. Only cells expressing the selection marker can survive in a medium treated with the selection marker, allowing for the selection of transformed cells. Representative examples of the selection marker include, but are not limited to, kanamycin, streptomycin, and chloramphenicol.

[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 a suitable host cell. The host cell is a cell that can stably and continuously clone or express the expression vector, and any host cell known in the art can be used.

[0033] When transforming prokaryotic cells to produce recombinant microorganisms, host cells that can be used include, but are not limited to, Escherichia coli strains 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; Bacillus strains such as Bacillus subtilis and Bacillus thuringiensis; Corynebacterium strains such as Corynebacterium glutamicum and Corynebacterium stachyonis; and various Enterobacteriaceae strains such as Salmonella typhimurium, Serratia marcescens, and Pseudomonas species.

[0034] When transforming eukaryotic cells to produce recombinant microorganisms, host cells may be used, including, but not limited to, yeast (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, and MDCK cell lines.

[0035] As used herein, "transformation" refers to the phenomenon of artificially causing genetic changes by introducing exogenous DNA into a host cell, and "transformant" refers to a host cell into which exogenous DNA has been introduced and which stably maintains expression of a target gene.

[0036] For the transformation, a vector introduction technique appropriate for the host cell can be selected to express the target gene or a recombinant vector containing the target gene in the host cell. For example, vector introduction may be performed by, but is not limited to, electroporation, heat shock, calcium phosphate (CaPO) precipitation, calcium chloride (CaCl) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, lithium acetate-DMSO method, or a combination thereof. The transformed gene may be inserted into the host cell's chromosome or located extrachromosomally, as long as it can be expressed in the host cell.

[0037] The transformant includes cells transfected, transformed, or infected with a recombinant vector according to the present invention in vivo or in vitro, and may be used interchangeably with recombinant host cells, recombinant cells, or recombinant microorganisms.

[0038] Genes inserted into the recombinant vectors of the present invention may be introduced into host cells, such as Corynebacterium strains, by homologous recombination crossing over.

[0039] According to one embodiment of the present invention, the transformant may be a microorganism of the genus Corynebacterium.

[0040] Examples of the Corynebacterium microorganism include Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium callunae, Corynebacterium suranareeae, Corynebacterium lubricantis, Corynebacterium doosanense, Corynebacterium efficiens, Corynebacterium uterequii, and the like. Corynebacterium uterequi, Corynebacterium stationis, Corynebacterium pacaense, Corynebacterium singulare, Corynebacterium humireducens, Corynebacterium marinum, Corynebacterium halotolerans, Corynebacterium spheniscorum, Corynebacterium freiburgense, Corynebacterium striatum striatum, Corynebacterium canis, Corynebacterium ammoniagenes, Corynebacterium renale, Corynebacterium portisoriiThe bacterium may be, but is not limited to, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium caspium, Corynebacterium testudinoris, Corynebacterium pseudopelargi, or Corynebacterium flavescens.

[0041] The transformant of the present invention may be, but is not limited to, a strain containing the above-mentioned ABC transporter permease mutant or a polynucleotide encoding it, or a strain containing a vector containing it, a strain expressing the ABC transporter permease mutant or polynucleotide, or a strain having activity against the ABC transporter permease mutant.

[0042] The transformant of the present invention may contain other protein mutants or gene mutations in addition to the ABC transporter permease mutant.

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

[0044] 5'-inosinic acid is a nucleic acid compound that imparts flavor, especially umami, to food, and is used interchangeably with inosine monophosphate (IMP).

[0045] The transformant may naturally have the ability to produce 5'-inosinic acid, or may be one to which the ability to produce 5'-inosinic acid has been artificially imparted.

[0046] According to one embodiment of the present invention, the transformant may have an improved ability to produce 5'-inosinic acid by altering the activity of an ABC transporter permease.

[0047] As used herein, "improved productivity" means increased productivity of 5'-inosinic acid compared to the parent strain. The parent strain refers to a wild-type or mutant strain that can be mutated, and includes strains that can be directly mutated or transformed with a recombinant vector, etc. 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 of the present invention exhibits increased 5'-inosinic acid production compared to the parent strain due to altered ABC transporter permease activity resulting from the introduction of an ABC transporter permease mutant. More specifically, the transformant may exhibit at least a 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% increase in 5'-inosinic acid production compared to the parent strain, or a 1.1-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, or 10-fold increase in 5'-inosinic acid production compared to the parent strain, but is not limited thereto. For example, the transformant containing the ABC transporter permease mutant may have an increased production of 5'-inosinic acid of 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, comprising 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 culture may be performed using an appropriate medium and culture conditions known in the art, and a person skilled in the art can easily adjust the medium and culture conditions. Specifically, the medium may be, but is not limited to, a liquid medium. The culture method may include, but is not limited to, batch culture, continuous culture, fed-batch culture, or a combination thereof.

[0051] According to one embodiment of the present invention, the medium should be appropriately adapted to meet the requirements of a specific strain and can be modified by a person skilled in the art. Culture media for Escherichia strains can be found in the well-known literature (Manual of Methods for General Bacteriology, American Society for Bacteriology, Washington DC, USA, 1981), but are not limited thereto.

[0052] According to one embodiment of the present invention, the medium can contain a variety of carbon sources, nitrogen sources, and trace element components. Usable carbon sources include sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch, and cellulose; oils and fats such as soybean oil, sunflower oil, castor oil, and coconut oil; fatty acids such as palmitic acid, stearic acid, and linoleic acid; alcohols such as glycerol and ethanol; and organic acids such as acetic acid. These substances can be used individually or in mixtures, but are not limited to these. Usable nitrogen sources include peptone, yeast extract, broth, 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. Nitrogen sources can also be used individually or in mixtures, but are not limited to these. Usable phosphorus sources include, but are not limited to, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts. The culture medium may also contain, but is not limited to, metal salts necessary for growth, such as magnesium sulfate or iron sulfate. Other essential growth substances, such as amino acids and vitamins, may also be included. Appropriate precursors may also be used for the culture medium. The medium or individual components may be added to the culture solution in a suitable manner during the culture process, such as batchwise or continuous, but not limited to these.

[0053] According to one embodiment of the present invention, the pH of the culture medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid to the microbial culture medium in an appropriate manner during cultivation. Furthermore, foam formation can be suppressed during cultivation using an antifoaming agent such as a fatty acid polyglycol ester. Additionally, oxygen or an oxygen-containing gas (e.g., air) can be injected into the culture medium to maintain an aerobic state in the culture medium. The temperature of the culture medium can generally be 20 to 45°C, for example, 25 to 40°C. The cultivation period can be continued until the desired amount of useful substance is obtained, and can be, for example, 10 to 160 hours.

[0054] According to one embodiment 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 be carried out by collecting or recovering the produced 5'-inosinic acid from the medium using a suitable method known in the art, depending on the culture method, including, but not limited to, centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, differential dissolution (e.g., ammonium sulfate precipitation), and chromatography (e.g., ion exchange, affinity, hydrophobic, and size exclusion).

[0055] According to one embodiment of the present invention, the step of recovering 5'-inosinic acid can be performed by subjecting the culture medium to low-speed centrifugation to remove biomass, and then separating the resulting supernatant by ion exchange chromatography.

[0056] According to one embodiment of the present invention, the step of recovering 5'-inosinic acid may include a step of purifying 5'-inosinic acid. [Effects of the Invention]

[0057] The ABC transporter permease mutant of the present invention has altered protein activity due to substitution of one or more amino acids in the amino acid sequence constituting the ABC transporter permease, and a recombinant microorganism containing the mutant can efficiently produce 5'-inosinic acid. [Brief explanation of the drawings]

[0058] [Figure 1] 1 shows the structure of pK19msb plasmid according to one embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0059] The present invention will be described in more detail below. However, such description is merely provided as an example for understanding the present invention, and the scope of the present invention is not limited by such exemplary description.

[0060] Example 1. Construction of strains expressing ABC transporter permease mutants To confirm the effect of a mutant (SEQ ID NO: 2) in which proline (P) at position 10 in the amino acid sequence of an ABC transporter permease (SEQ ID NO: 4) is replaced with serine (S), arginine (R) at position 49 is replaced with cysteine ​​(C), and threonine (T) at position 169 is replaced with isoleucine (I) on the production of 5'-inosinic acid, a vector expressing the mutant ABC transporter permease and a bacterial strain into which the vector was introduced were constructed.

[0061] 1-1. Construction of vectors for expression of ABC transporter permease mutants Using wild-type Corynebacterium stachyonis ATCC6872 genomic DNA as a template, PCR was performed using primer pairs 1 and 2 and primer pairs 3 and 4. Subsequently, overlapping PCR was performed using the two PCR products as templates with primer pairs 1 and 4 to ligate them into a single fragment. The PCR fragment and pK19msb plasmid (SEQ ID NO: 5) were treated with the restriction enzyme smaI (NEB) and ligated using T4 ligase. The constructed plasmid was designated pK_AT.

[0062] For PCR amplification, pfu premix (Bioneer) was used, and after denaturation at 95°C for 5 minutes, the mixture was subjected to 30 cycles of 95°C for 30 seconds, 58°C for 30 seconds, and 72°C for 1 minute 30 seconds, followed by a reaction at 72°C for 5 minutes.

[0063] The primer sequences used to prepare the plasmids are shown in Table 1 below.

[0064] [Table 1]

[0065] 1-2. Construction of mutant strains containing ABC transporter permease mutants For the transformation of Corynebacterium stathionis KCCM13339P, a modified electrocompetent cell preparation method based on the method of van der Rest et al. was used.

[0066] First, Corynebacterium stachyonis KCCM13339P was cultured in 10 ml of 2YT medium (containing 16 g / L tryptone, 10 g / L yeast extract, and 5 g / L sodium chloride) supplemented with 2% glucose to prepare a seed culture. 1 mg / ml of isonicotinic acid hydrazine and 2.5% glycine were added to 100 ml of glucose-free 2YT medium. The OD 610 After inoculating the seed culture so that the OD value was 0.3, the culture was incubated at 30°C and 180 rpm for 5 to 8 hours, and the OD 610The pH was adjusted to a value between 0.6 and 0.7. The culture was incubated on ice for 30 minutes and then centrifuged at 3500 rpm at 4°C for 10 minutes. The supernatant was discarded, and the precipitated Corynebacterium stachyonis KCCM13339P was washed four times with 10% glycerol solution and finally resuspended in 0.5 ml of 10% glycerol solution to prepare competent cells. Electroporation was performed using a Bio-Rad electroporator. The prepared competent cells and the prepared pK_AT vector were placed in an electroporation cuvette (0.2 mm), and an electric shock was applied under 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 Brain Heart Infusion and 0.5 M sorbitol) was added, and the mixture was heated at 46°C for 6 minutes. After cooling at room temperature, the mixture was transferred to a 15 ml cap tube and cultured at 30°C for 2 hours, and then smeared on a selective medium (containing tryptone 5 g / L, NaCl 5 g / L, yeast extract 2.5 g / L, Brain Heart infusion powder 18.5 g / L, agar 15 g / L, sorbitol 91 g / L, and kanamycin 20 μg / L). The colonies formed after 72 hours of culture at 30°C were cultured in the medium until stationary phase to induce secondary recombination, and then 10 -5 ~10 -7 The resulting mixture was diluted to 100°C and smeared on an antibiotic-free plate medium (containing 10% sucrose). A strain that had no kanamycin resistance and could grow on the medium containing 10% sucrose was selected and named IAT-1.

[0067] Experimental Example 1. Evaluation of 5'-inosinic acid production ability of strains expressing ABC transporter permease mutants The 5'-inosinic acid production ability of the parent strain KCCM13339P was compared with that of the mutant strain IAT-1, which had an ABC transporter permease mutant introduced therein.

[0068] Each strain (parent strain or mutant strain) was inoculated at 1% volume into a 100 mL flask containing 10 mL of the 5'-inosinic acid production medium shown in Table 2 below, and cultured with shaking at 34°C and 200 rpm for 45 hours. After the culture 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.

[0069] [Table 2]

[0070] [Table 3]

[0071] As shown in Table 3, the mutant strain into which the ABC transporter permease mutant was introduced showed an approximately 18% increase in 5'-inosinic acid production compared to the parent strain due to the substitution of amino acids at positions 10, 49, and 169. These results suggest that the introduction of point mutations in the ABC transporter permease has a beneficial effect on 5'-inosinic acid productivity.

[0072] The present invention has been described above with reference to its preferred embodiments. Those skilled in the art will understand that the present invention can be embodied in various modified forms without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a restrictive perspective. The scope of the present invention is defined by the claims, not the above description, and all variations within the scope of the claims should be construed as being within the scope of the present invention.

[0073] [Accession number] Depository institution: Korea Center for Microorganisms (KCCM) Accession number: KCCM13339P Date of acceptance: 20230329

Claims

1. An ABC transporter permease mutant consisting of the amino acid sequence of SEQ ID NO: 4 in which the 10th proline is replaced with serine, the 49th arginine is replaced with cysteine, and the 169th threonine is replaced with isoleucine.

2. The ABC transporter permease mutant of claim 1, which consists of the amino acid sequence of SEQ ID NO:

2.

3. A polynucleotide encoding the variant of claim 1.

4. A transformant comprising the mutant of claim 1 or the polynucleotide of claim 3.

5. The transformant according to claim 4, which is a microorganism of the genus Corynebacterium.

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

7. Cultivating the transformant according to claim 4 in a medium; and recovering 5'-inosinic acid from the transformant or from the medium in which the transformant has been cultured.

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