Novel 5-dehydro-2-deoxygluconate kinase variants and method for producing 5 '-inosinic acid using same

Abstract

The present invention relates to a novel 5-dehydro-2-deoxygluconate kinase variant and a method for producing 5 '-inosinic acid using the same. According to the present invention, the 5-dehydro-2-deoxygluconate kinase variant has a modified protein activity due to the substitution of one or more amino acids in the amino acid sequence constituting a 5-dehydro-2-deoxygluconate kinase, and thus a recombinant microorganism comprising the 5-dehydro-2-deoxygluconate kinase variant can efficiently produce 5 '-inosinic acid.

Description

Technical Field

[0001] This invention relates to novel 5-dehydro-2-deoxygluconic acid kinase variants and methods for producing 5'-inosine using them. Background Technology

[0002] 5'-Inosinic acid (or inosine monophosphate (IMP)) is an intermediate in the nucleic acid biosynthesis metabolic system, playing important physiological roles not only in plants and animals but also in various applications, including food, pharmaceuticals, and multiple medical applications. In particular, 5'-Inosinic acid is a nucleic acid-based flavoring agent, attracting considerable attention as a savory flavoring agent due to its significant synergistic effect with monosodium glutamate (MSG) in taste.

[0003] Methods for producing 5'-inosine include enzymatic degradation of ribonucleic acid extracted from yeast cells and chemical phosphorylation of inosine produced through fermentation. Recently, the primary method has been to cultivate microorganisms that produce 5'-inosine and recover the 5'-inosine accumulated in the culture medium.

[0004] To improve the efficiency of 5'-inosine production using microorganisms, various recombinant or mutant strains with excellent 5'-inosine productivity have been developed by applying genetic recombination technology to microorganisms widely used to produce useful substances such as nucleic acids or L-amino acids (e.g., *Escherichia coli* and *Corynebacterium*), along with methods for producing 5'-inosine using them. Specifically, attempts have been made to enhance 5'-inosine production by targeting genes involved in the 5'-inosine biosynthesis pathway, such as enzymes, transcription factors, and transporters, or by inducing promoter mutations that regulate the expression of these genes. However, dozens to hundreds of types of proteins (e.g., enzymes, transcription factors, and transporters) are directly or indirectly involved in 5'-inosine production, and therefore, significant research is still needed to improve 5'-inosine productivity by altering the activity of these proteins.

[0005] [Existing Technical Documents]

[0006] [Patent Literature]

[0007] Korean Patent No. 10-116602 Summary of the Invention

[0008] Technical issues

[0009] It is an object of the present invention to provide new 5-dehydro-2-deoxygluconate kinase variants.

[0010] It is another object of the present invention to provide polynucleotides encoding said variants.

[0011] It is yet another object of the present invention to provide transformants comprising said variants or polynucleotides.

[0012] It is yet another object of the present invention to provide methods of producing 5'-inosinate using said transformants.

[0013] Technical solution

[0014] One aspect of the present invention provides 5-dehydro-2-deoxygluconate kinase variants consisting of the amino acid sequence of SEQ ID NO: 2, wherein the serine at position 146 of the amino acid sequence of SEQ ID NO: 4 is replaced by an asparagine.

[0015] The term "inventive dehydro-2-deoxygluconate kinase" used in the present invention catalyzes the reaction producing ADP and 6-phospho-5-dehydro-2-deoxy-D-gluconate using ATP and 5-dehydro-2-deoxy-D-gluconate as substrates, and it can be a polypeptide or protein consisting of the amino acid sequence of SEQ ID NO: 4 and having 5-dehydro-2-deoxygluconate kinase activity.

[0016] Information on nucleic acid and protein sequences of 5-dehydro-2-deoxygluconate kinases can be obtained from known sequence databases (e.g. GenBank, UniProt).

[0017] According to one embodiment of the present invention, the 5-dehydro-2-deoxygluconate kinase can be encoded by the nucleotide sequence of SEQ ID NO: 3.

[0018] The amino acid sequence of the 5-dehydro-2-deoxygluconate kinase according to the present invention or the nucleotide sequence encoding the same can include an amino acid sequence or a nucleotide 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. The term "homology" or "identity" used herein means the percentage rate of identity between two sequences, which is determined by aligning the reference nucleotide sequence or amino acid sequence with any other nucleotide sequence or amino acid sequence to correspond to each other as much as possible and analyzing the aligned sequences.

[0019] According to one embodiment of the present application, the 5-dehydro-2-deoxygluconokinase can be derived from wild-type Corynebacterium stationis.

[0020] The term "variant" used in the present application means a protein having an amino acid sequence different from that before mutation, but retaining the function or property of the protein before mutation, which is caused by a mutation in the nucleotide sequence of a gene encoding the protein by conservative substitution and / or modification of one or more amino acids at the N-terminus, C-terminus and / or inside of the amino acid sequence. The term "conservative substitution" used herein means substitution of one amino acid with another amino acid having similar structure and / or chemical property. The conservative substitution can have little or no effect on the activity of the protein or polypeptide. In addition, the term "modification" means substitution, insertion, deletion, etc. of one or more 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).

[0021] In addition, some variants include those in which one or more portions (e.g., N-terminal leader sequence or transmembrane region) have been removed, or those in which a portion has been removed from the N-terminus and / or C-terminus of the mature protein.

[0022] The variant can have an improved (enhanced), unaltered, or reduced (diminished) ability compared to the protein before mutation. Here, the term "improved or enhanced" includes: a case in which the activity of the protein itself is improved compared to that of the protein before mutation; a case in which the overall activity of the protein in the cell is higher than those in a wild-type strain or a strain expressing the protein before mutation due to an increase in the expression or translation of the gene encoding the protein; and combinations of these. In addition, the term "reduced or diminished" includes: a case in which the activity of the protein itself is reduced compared to that of the protein before mutation; a case in which the overall activity of the protein in the cell is lower than those in a wild-type strain or a strain expressing the protein before mutation due to a decrease in the expression or translation of the gene encoding the protein; and combinations of these. In the present application, the term "variant" can be used interchangeably with terms such as variant type, modification, variant polypeptide, mutated protein, mutant, etc.

[0023] The 5-dehydro-2-deoxygluconate kinase variant according to the present application can comprise an 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: 2.

[0024] Another aspect of the present application provides a polynucleotide encoding the 5-dehydro-2-deoxygluconate kinase variant.

[0025] The term "polynucleotide" used in the present application refers to a DNA or RNA chain having a certain length or more, which is a long chain polymer of nucleotides formed by covalently linking nucleotide monomers. More specifically, the term "polynucleotide" refers to a polynucleotide fragment encoding the variant.

[0026] According to one embodiment, the polynucleotide can comprise a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2.

[0027] More specifically, the polynucleotide can comprise the nucleotide sequence of SEQ ID NO: 1, wherein the nucleotide "G" at position 437 of the nucleotide sequence of SEQ ID NO: 3 encoding the 5-dehydro-2-deoxygluconate kinase is replaced with the nucleotide "A".

[0028] Still another aspect of the present application provides a vector comprising a polynucleotide encoding the 5-dehydro-2-deoxygluconate kinase variant.

[0029] Still another aspect of the present application provides a transformant comprising the 5-dehydro-2-deoxygluconate kinase variant or the polynucleotide.

[0030] The term "vector" used in the present application refers to any type of nucleic acid sequence transfer structure used as a means for transferring and expressing a gene of interest in a host cell. Unless otherwise specified, the term "vector" can mean a vector allowing a nucleic acid sequence contained therein to be expressed after being inserted into the genome of a host cell and / or a vector allowing a nucleic acid sequence to be independently expressed. The vector comprises necessary regulatory elements operably linked so that the inserted gene can be expressed. The term "operably linked" used herein means that a gene of interest and its regulatory sequences are functionally linked together in a manner that enables the gene to be expressed, and the "regulatory element" includes a promoter for initiating transcription, any operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence for regulating termination of transcription and translation.

[0031] The vector in the present application is not particularly limited as long as it can replicate in a host cell, and any vector known in the art can be used. Some examples of the vector include natural or recombinant plasmids, cosmids, viruses, and bacteriophages. Some examples of the bacteriophage vector or cosmid vector include, but are not limited to, pWE15, M13, λMBL3, λMBL4, λIXII, λASHII, λAPII, λt10, λt11, Charon 4A, and Charon 21A, and some examples of the plasmid vector include, but are not limited to, pBR series, pUC series, pBluescript II series, pGEM series, pTZ series, pCL series, and pET series.

[0032] The vector can be generally constructed as a vector for cloning or a vector for expression. The vector for expression can be a conventional vector used in the art for expressing foreign genes or proteins in plants, animals, or microorganisms, and can be constructed by various methods known in the art.

[0033] The term "recombinant vector" used in the present application can be transformed into a suitable host cell, and can then replicate without the influence of the host cell genome, or can be integrated into the genome itself. In this case, the "suitable host cell" can contain an origin of replication, which is a specific nucleotide sequence that enables the vector to replicate in a suitable host cell and initiates replication therefrom. For example, when the vector used is an expression vector and a prokaryotic cell is used as a host, the vector generally contains a strong promoter capable of promoting transcription (e.g., pLλ promoter, CMV promoter, trp promoter, lac promoter, tac promoter, T7 promoter, etc.); a ribosome binding site for initiating translation; and a transcription / translation termination sequence. When a eukaryotic cell is used as a host, the vector contains an origin of replication manipulated in a eukaryotic cell, and some examples of the origin of replication include, but are 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, the recombinant vector can contain a promoter derived from a mammalian cell genome (e.g., metallothionein promoter) or a promoter derived from a mammalian virus (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, HSV-tk promoter, etc.), and generally has a polyadenylation sequence as a transcription termination sequence.

[0034] The recombinant vector can contain a selection marker. The selection marker is used to select transformants (host cells) transformed with the vector, and since only cells expressing the selection marker can survive in a medium treated with the selection marker, the transformed cells can be selected. Some representative examples of the selection marker include, but are not limited to, kanamycin, streptomycin, and chloramphenicol.

[0035] The transformant can be produced by inserting the recombinant vector into a host cell, and the transformant can 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 an expression vector, and any host cell known in the art can be used.

[0036] In the case where the vector is transformed into a prokaryotic cell to produce a recombinant microorganism, some examples of the host cell that can be used include, but are not limited to, E. coli sp. strains 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, and E. coli XL1-Blue; Bacillus sp. strains such as Bacillus subtilis and Bacillus thuringiensis; Corynebacterium sp. strains such as Corynebacterium glutamicum and Corynebacterium ambiguum; and various Enterobacteriaceae strains such as Salmonella typhimurium, Serratia marcescens, and Pseudomonas sp.

[0037] In the case where the vector is transformed into a eukaryotic cell to produce a recombinant microorganism, some examples of the host cell that can be used include, but are 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.

[0038] The term "transformation" used in the present application refers to a phenomenon in which external DNA is introduced into a host cell, thereby artificially causing a genetic change, and the term "transformant" refers to a host cell into which external DNA is introduced and in which expression of a gene of interest is stably maintained.

[0039] The transformation can be performed using a suitable vector introduction technique according to selection of a host cell, so that the gene of interest or a recombinant vector containing the same can be expressed in the host cell. For example, the introduction of the vector can be performed by electroporation, heat shock, calcium phosphate (CaP04) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, lithium acetate-DMSO method, or any combination thereof, but is not limited thereto. As long as the transformed gene can be expressed in the host cell, it can be inserted into the chromosome of the host cell, or can exist outside the chromosome, but is not limited thereto.

[0040] The transformant can include a cell transfected, transformed, or infected in vivo or in vitro with the recombinant vector of the present application, and can be used in the same meaning as the recombinant host cell, recombinant cell, or recombinant microorganism.

[0041] The gene inserted into the recombinant vector of the present application can be introduced into a host cell, for example, a Corynebacterium strain, by crossing through homologous recombination.

[0042] According to one embodiment of the present application, the transformant can be a Corynebacterium microorganism.

[0043] The microorganism of the genus Corynebacterium can be, but is not limited to, Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium callunae, Corynebacterium suranareeae, Corynebacterium lubricantis, Corynebacterium doosanense, Corynebacterium efficiens, Corynebacterium uterequi, Corynebacterium stagnense, Corynebacterium pacaense, Corynebacterium singulare, Corynebacterium humireducens, Corynebacterium marinum, Corynebacterium halotolerans, Corynebacterium spheniscorum, Corynebacterium freiburgense, Corynebacterium striatum, Corynebacterium canis, Corynebacterium ammoniagenes, Corynebacterium renale, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium caspium, Corynebacterium testudinoris, Corynebacterium pseudopelargi, or Corynebacterium flavescens.

[0044] The transformant in the present application can be a strain comprising the above-mentioned 5-dehydro-2-deoxygluconate kinase variant or a polynucleotide encoding 5-dehydro-2-deoxygluconate kinase, or a vector comprising the polynucleotide; a strain expressing the 5-dehydro-2-deoxygluconate kinase variant or the polynucleotide; or a strain having the activity of the 5-dehydro-2-deoxygluconate kinase variant, but is not limited thereto.

[0045] In addition to the 5-dehydro-2-deoxygluconokinase variant, the transformant of the present application can comprise other protein variants or genetic mutants.

[0046] According to one embodiment of the present application, the transformant can have the ability to produce inosinic acid.

[0047] 5'-inosinic acid is a nucleic acid-based compound that imparts a flavor, especially umami (salty) taste, to food, and it is used in the same meaning as inosine monophosphate (IMP).

[0048] The transformant can naturally have the ability to produce 5'-inosinic acid, or can be a transformant artificially imparted with the ability to produce 5'-inosinic acid.

[0049] According to one embodiment of the present application, the transformant can have an improved ability to produce 5'-inosinic acid due to the change in 5-dehydro-2-deoxygluconokinase activity.

[0050] The term "improved production ability" used in the present application means that 5'-inosinic acid productivity is improved compared to that of the parent strain. The term "parent strain" used herein refers to a wild-type strain or a mutant strain to be mutated, and includes a strain to be directly mutated or transformed with a recombinant vector or the like. In the present application, the parent strain can be a wild-type Corynebacterium strain or a Corynebacterium strain mutated from a wild-type strain.

[0051] The transformant according to the present application exhibits an improved ability to produce 5'-inosinic acid due to the change in 5-dehydro-2-deoxygluconokinase activity caused by the introduction of the 5-dehydro-2-deoxygluconokinase variant therein compared to the parent strain. More specifically, the amount of 5'-inosinic acid produced by the transformant can be at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% higher than the amount of 5'-inosinic acid produced by the parent strain, or can be 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 higher than the amount of 5'-inosinic acid produced by the parent strain, but is not limited thereto. For example, the amount of 5'-inosinic acid produced by the transformant comprising the 5-dehydro-2-deoxygluconokinase variant can be at least 5% higher, specifically 5% to 50% (preferably 10% to 40%) than the amount of 5'-inosinic acid produced by the parent strain.

[0052] Yet another aspect of the present application provides a method for producing 5'-inosinic acid, comprising the steps of culturing the transformant in a culture medium; and recovering 5'-inosinic acid from the transformant or from the culture medium in which the transformant is cultured.

[0053] The culturing can be performed using a suitable culture medium and culture conditions known in the art, and the culture medium and culture conditions can be easily adjusted and used by one skilled in the art. Specifically, the culture medium can be a liquid medium, but is not limited thereto. Some examples of the culturing method include, but are not limited to, batch culture, continuous culture, fed-batch culture, or a combination thereof.

[0054] According to one embodiment of the present application, the culture medium should satisfy the requirements of a specific strain in an appropriate manner, and can be appropriately modified by one skilled in the art. For the culture medium of Escherichia sp. strains, reference can be made to known literature (Manual of Methods for General Bacteriology, American Society for Bacteriology, Washington D.C., USA, 1981), but is not limited thereto.

[0055] According to one embodiment of the present application, the culture medium can comprise various carbon sources, nitrogen sources, and trace element components. Some examples of the carbon sources that can be used 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 alone or as a mixture, but are not limited thereto. Some examples of the nitrogen sources that can be used include peptone, yeast extract, meat extract, malt extract, corn steep liquor, soybean powder, urea, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate. The nitrogen sources can also be used alone or as a mixture, but are not limited thereto. Some examples of the phosphorus sources that can be used include, but are not limited to, potassium dihydrogen phosphate or dipotassium hydrogen phosphate, or a corresponding sodium-containing salt. In addition, the culture medium can comprise, but is not limited to, metal salts required for growth, such as magnesium sulfate or ferric sulfate. In addition, the culture medium can comprise essential growth substances such as amino acids and vitamins. Furthermore, suitable precursors can be used in the culture medium. The culture medium or individual components can be added to the culture medium in a batch or continuous manner by a suitable method during the culturing, but are not limited thereto.

[0056] According to one embodiment of the 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. Additionally, antifoaming agents such as fatty acid polyethylene glycol esters can be used to suppress foaming during cultivation. Furthermore, to maintain the culture medium under aerobic conditions, oxygen or an oxygen-containing gas (e.g., air) can be injected into the culture medium. The temperature of the culture medium can typically be 20°C to 45°C, for example, 25°C to 40°C. Cultivation can continue until the desired amount of useful substances is produced. For example, the cultivation time can be 10 hours to 160 hours.

[0057] According to one embodiment of the invention, in the step of recovering 5'-inosinic acid from the cultured transformant or from the culture medium in which the transformant was cultured, the generated 5'-inosinic acid may be collected or recovered from the culture medium using suitable methods known in the art, according to the culture method. Some examples of methods that can be used to recover the generated 5'-inosinic acid include, but are not limited to, 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), etc.

[0058] According to one embodiment of the present invention, the step of recovering 5'-inosinic acid can be carried out by centrifuging the culture medium at low speed to remove biomass and separating the obtained supernatant by ion exchange chromatography.

[0059] According to one embodiment of the present invention, the step of recovering 5'-inosine acid may include a process for purifying 5'-inosine acid.

[0060] Beneficial effects

[0061] The 5-dehydro-2-deoxygluconic acid kinase variant according to the present invention is obtained by replacing one or more amino acids constituting the amino acid sequence of 5-dehydro-2-deoxygluconic acid kinase to alter the activity of the protein, and the recombinant microorganism containing the 5-dehydro-2-deoxygluconic acid kinase variant is able to efficiently produce 5'-inosine. Attached Figure Description

[0062] Figure 1 The structure of the pK19msb plasmid according to one embodiment of the present invention is shown. Detailed Implementation

[0063] The invention will be described in more detail below. However, this description is presented by way of example only to facilitate understanding of the invention, and the scope of the invention is not limited to this exemplary description.

[0064] Example 1. Construction of a strain expressing a 5-dehydro-2-deoxyglucose kinase variant

[0065] To evaluate the effect of a variant (SEQ ID NO: 2) in which serine (S) at position 146 of the amino acid sequence of 5-dehydro-2-deoxygluconate kinase (SEQ ID NO: 4) is replaced with asparagine (N) on the production of 5'-inosinic acid, the present inventors constructed a vector for expressing the 5-dehydro-2-deoxygluconate kinase variant and a strain into which the vector was introduced.

[0066] 1-1. Construction of a vector for expressing a 5-dehydro-2-deoxygluconate kinase variant

[0067] Using genomic DNA of wild-type C. glutamicum ATCC6872 as a template, PCR reactions were performed using primer pairs of primers 1 and 2 and primers 3 and 4, respectively. Thereafter, using the two PCR products as templates, overlap PCR was performed using a primer pair of primers 1 and 4 to obtain a single fragment. The PCR fragment and pK19msb plasmid (SEQ ID NO: 5) were treated with a restriction enzyme smal (NEB) and ligated together using T4 ligase. The resulting plasmid was named pK_DD.

[0068] PCR amplification was performed using Pfu PreMix (Bioneer) under the following conditions: denaturation at 95°C for 5 minutes, and then 30 cycles each consisting of 95°C for 30 seconds, 58°C for 30 seconds, and 72°C for 1 minute and 30 seconds, followed by reaction at 72°C for 5 minutes.

[0069] Primer sequences used for plasmid construction are shown in Table 1 below.

[0070] [Table 1]

[0071]

[0072] 1-2. Construction of a mutant strain into which a 5-dehydro-2-deoxygluconate kinase variant is introduced

[0073] The electroporation method (a modified form of the method of van der Rest et al.) was used as a method for transforming C. glutamicum KCCM13339P.

[0074] First, the stationary rod-shaped bacterium KCCM13339P was primarily 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. Hydrazine isonicotinic acid was added to 100 ml of 2YT medium without glucose at a concentration of 1 mg / ml, and 2.5% glycine was added thereto. Then, the seed culture was inoculated into the 2YT medium to make the OD 610 value reach 0.3, and then cultured for 5 to 8 hours at 30°C and 180 rpm so that the OD 610 value reached 0.6 to 0.7. The culture was kept on ice for 30 minutes, and then centrifuged at 3,500 rpm for 10 minutes at 4°C. Thereafter, the supernatant was discarded, and the precipitated stationary rod-shaped bacterium KCCM13339P was washed with a 10% glycerol solution 4 times and finally resuspended in 0.5 ml of a 10% glycerol solution to prepare competent cells. Electroporation was performed using a Bio-Rad electroporator. The prepared competent cells and the constructed pK_DD vector were placed in an electroporation cup (0.2 mm), and then electroporation was performed under the conditions of 2.5 kV, 200 Ω, and 12.5 μF. Immediately after the completion of electroporation, 1 ml of a recovery (RG) medium (containing 18.5 g / l of brain heart infusion and 0.5 M of sorbitol) was added to the cells, and then the cells were heat-treated at 46°C for 6 minutes. Then, the cells were cooled at room temperature, transferred to a 15 ml cap tube, incubated at 30°C for 2 hours, and plated 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 cells were cultured at 30°C for 72 hours, and the produced colonies were cultured in the medium until the stationary phase to induce secondary recombination. Then, the cells were diluted to 10 -5 to 10 -7 , and plated on an antibiotic-free plate medium (containing 10% sucrose), and a strain having no kanamycin resistance and capable of growing on the medium containing 10% sucrose was selected and named IDD-1.

[0075] Experimental Example 1. Evaluation of 5'-inosinic acid productivity of a strain expressing a 5-dehydro-2-deoxygluconate kinase variant

[0076] 5'-inosinic acid productivity was compared between the parent strain KCCM13339P and the mutant strain IDD-1 in which a 5-dehydro-2-deoxygluconate kinase variant was introduced.

[0077] Each strain (parent strain or mutant strain) was inoculated at 1% by volume into a 100 mL flask containing 10 mL of the medium shown in Table 2 below for 5'-inosinic acid production, and was cultured with shaking at 200 rpm for 45 hours. After completion of the culture, the concentration of 5'-inosinic acid in the culture medium was measured using HPLC (Agilent), and the results are shown in Table 3 below.

[0078] [Table 2]

[0079] Components Amount Glucose 70 g / L (NH4)2SO4 2 g / L MgSO4 1 g / L Urea 2 g / L Yeast extract 20 g / L KH2PO4 2 g / L FeSO4 10 mg / L MnSO4 10 mg / L Thiamine HCl 5 mg / L Biotin 20 ug / L Cysteine 20 mg / L Beta-alanine 20 mg / L Adenine 30 mg / L

[0080] [Table 3]

[0081] Strain 5'-inosinic acid production (g / L) KCCM13339P 19.8 IDD-1 23.0

[0082] As shown in Table 3 above, it was confirmed that the amount of 5'-inosinic acid produced by the mutant strain in which the 5-dehydro-2-deoxygluconokinase variant was introduced was increased by about 16% compared to the amount of 5'-inosinic acid produced by the parent strain, due to the substitution of serine at position 146 with asparagine. These results indicate that the introduction of a point mutation in the 5-dehydro-2-deoxygluconokinase provides a significant effect on 5'-inosinic acid productivity.

[0083] The present application has been described with reference to some preferred embodiments. Ordinary skill in the art to which the present application pertains will understand that the present application can be implemented in modified forms without departing from the essential characteristics of the present application. Therefore, the disclosed embodiments should be considered illustrative rather than restrictive. The scope of the present application is defined by the claims rather than the above description, and all differences within the equivalent range should be interpreted as included in the present application.

[0084] [Accession No.]

[0085] Depositing Agency: Korean Culture Center of Microorganisms (KCCM)

[0086] Accession No.: KCCM13339P

[0087] Date of Deposit: March 29, 2023

[0088] Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure

[0089] International Form

[0090] Original Certificate of Deposit

[0091]

[0092]

[0093] 1 In the case where clause 6.4(d) applies, the date is the date of obtaining the international depositary authority status.

[0094] BP / 4 table (one page).

Claims

1,5-Dehydro-2-deoxygluconic acid kinase variant, which consists of the amino acid sequence of SEQ ID NO: 2, wherein the serine at position 146 in the amino acid sequence of SEQ ID NO: 4 is replaced by asparagine.

2. A polynucleotide encoding the variant of claim 1.

3. A transformant comprising the variant of claim 1 or the polynucleotide of claim 2.

4. The transformant of claim 3 is a Corynebacterium microorganism.

5. The transformant of claim 3, which has the ability to produce 5'-inosine.

6. A method for producing 5'-inosine, comprising the following steps: The transformant of claim 3 is cultured in a culture medium; as well as 5'-inosinic acid is recovered from the transformant or from the culture medium in which the transformant is cultured.

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