5'-Guanine Acid Producing Strain and Method for Producing 5'-Guanine Acid Using the Same

Abstract

The present application relates to the Corynebacterium ammoniagenes CJG0497 strain deposited under accession number KCCM13320P; a method for producing 5'-guanic acid, the method comprising culturing the strain in a medium; a composition for producing 5'-guanic acid, the composition comprising the strain, a culture of the strain, a fermentation product of the strain, or a combination of two or more thereof; and use of the strain for producing 5'-guanic acid.

Description

[Technical Field]

[0001] The present application relates to the Corynebacterium ammoniagenes CJG0497 strain deposited under accession number KCCM13320P; a method for producing 5'-guanic acid, the method comprising culturing the strain in a medium; a composition for producing 5'-guanic acid, the composition comprising the strain, a culture of the strain, a fermentation product of the strain, or a combination of two or more thereof; and use of the strain for producing 5'-guanic acid. [Background technology]

[0002] 5'-guanosine monophosphate (GMP) is an intermediate in the nucleic acid biosynthesis metabolic pathway, and is not only physiologically important in animals and plants, but is also used in a wide range of fields, including food, pharmaceuticals, and various medical applications. In particular, when used with monosodium glutamate (MSG), it has a strong synergistic flavor effect, making it one of the nucleic acid seasonings that has been gaining attention as a flavor enhancer.

[0003] There are several methods for producing GMP: (1) using microbial enzymes or chemically decomposing yeast RNA, (2) directly producing nucleotides using microorganisms in a medium containing sugars, nitrogen sources, and phosphate sources, and (3) chemically or enzymatically converting intermediates in nucleotide synthesis. Currently, a combined production method combining fermentation, chemical synthesis, and enzymatic conversion is widely used industrially.

[0004] This complex production method consists of a fermentation process using microorganisms to produce 5'-xanthosine monophosphate (XMP), an intermediate product in the metabolic pathway of purine nucleotide biosynthesis, and an enzymatic reaction process to convert the fermentation product into GMP. Both XMP-producing microorganisms and microorganisms that convert XMP to GMP are used. Therefore, for efficient GMP production, GMP degradation by the microorganisms involved in GMP production must be suppressed, and in particular, the microorganisms added during the enzymatic reaction process must have a high combined ability to convert XMP to GMP.

[0005] Therefore, various studies are being conducted to develop highly efficient production microorganisms and fermentation process technologies. For example, target substance-specific approaches such as increasing the expression of genes encoding enzymes involved in XMP or GMP biosynthesis or deleting genes unnecessary for biosynthesis are mainly used (EP 3722430 A1, US 2020-0347346 A1).

[0006] However, in order to effectively increase the production of 5'-guanine acid, research into new strains with high GMP conversion ability and low GMP degradation rate is still needed. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] European Patent Application Publication No. 3722430 [Patent Document 2] US Patent Application Publication No. 2020 / 0347346 [Non-patent literature]

[0008] [Non-Patent Document 1] “Manual of Methods for General Bacteriology” by the American Society for Bacteriology (Washington DC, USA, 1981) Summary of the Invention [Problem to be solved by the invention]

[0009] The present inventors have confirmed that the Corynebacterium ammoniagenes CJG0497 strain deposited under accession number KCCM13320P has a high GMP conversion ability and a low GMP degradation rate, and is therefore capable of producing 5'-guanine acid in high yields, and have completed the present application. [Means for solving the problem]

[0010] One aspect of the present application provides Corynebacterium ammoniagenes strain CJG0497, deposited under accession number KCCM13320P.

[0011] In one embodiment, the strain may be capable of producing 5'-guanine acid.

[0012] In another embodiment, the strain may be capable of converting 5'-xanthylic acid to 5'-guanic acid.

[0013] Another aspect of the present application provides a method for producing 5'-guanine acid, comprising culturing the Corynebacterium ammoniagenes CJG0497 strain deposited under accession number KCCM13320P in a medium.

[0014] In one embodiment, the method may further comprise recovering 5'-guanine acid from the cultured strain, a culture of the strain, a fermentation product of the strain, or the culture medium.

[0015] Another aspect of the present application provides a composition for producing 5'-guanine acid, comprising the Corynebacterium ammoniagenes CJG0497 strain deposited under accession number KCCM13320P, a culture of the strain, a fermentation product of the strain, or a combination of two or more thereof.

[0016] Another aspect of the present application provides use of the Corynebacterium ammoniagenes strain CJG0497, deposited under accession number KCCM13320P, for producing 5'-guanine acid. [Effects of the Invention]

[0017] The Corynebacterium ammoniagenes CJG0497 strain deposited in the present application under accession number KCCM13320P has a high GMP conversion ability and a low GMP degradation rate, and is therefore capable of producing 5'-guanine acid in high yields, making it useful for the industrial production of 5'-guanine acid. DETAILED DESCRIPTION OF THE INVENTION

[0018] This will be explained in more detail as follows: Meanwhile, each description and embodiment disclosed in this application also applies to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the specific descriptions described below are not considered to limit the category of this application.

[0019] Additionally, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein and such equivalents are intended to be encompassed by this application.

[0020] As used in this specification and the appended claims, the singular articles "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Unless the context dictates otherwise, singular terms include plurals and plural terms include the singular. In this specification and the appended claims, the use of "or" is intended to include "and / or" unless specifically stated otherwise.

[0021] In this application, the term "about" is used before a specific numerical value. As used in this application, the term "about" includes not only the exact number listed after the term, but also approximately that number or a range close to that number. Whether a number is close to or approximately the specific number mentioned can be determined by considering the context in which the number is presented. As an example, the term "about" can refer to a range of 10% to +10% of the numerical value. As another example, the term "about" can refer to a range of -5% to +5% of the given numerical value. However, the present invention is not limited to this.

[0022] In this application, the term "comprising" means the presence of the feature, step, or component described below that term, and does not exclude the presence or addition of one or more features, steps, or components. In this application, the components or features described below as "comprising" may be essential or mandatory, but some embodiments may further include other optional or non-essential components or features.

[0023] One aspect of the present application provides Corynebacterium ammoniagenes strain CJG0497, deposited under accession number KCCM13320P.

[0024] The term "strain" as used herein refers to a mutant strain obtained by mutating a parent strain, specifically, any strain capable of producing the desired 5'-guanine acid obtained by mutating the Corynebacterium ammoniagenes ATCC 6872 strain (currently referred to as the Corynebacterium stathionis ATCC 6872 strain).

[0025] In this regard, mutagenesis of microorganisms can be carried out by a variety of means well known in the art, including physical and chemical mutagenesis methods. For example, gamma radiation and ultraviolet light can be used as physical mutagen suitable for the present invention, but are not limited thereto. Chemical mutagen suitable for the present invention include, but are not limited to, N-methyl-N'-nitro-N-nitrosoguanidine (NTG), diepoxybutane, ethyl methanesulfonate, mustard compounds, hydrazine, and nitrous acid.

[0026] During mutation induction, the parent strain is affected by a mutagen under conditions that leave a specific size of surviving individual bacteria. These conditions vary depending on the type of mutagen and depend on the amount of mutations the mutagen induces in surviving individual bacteria at a certain kill rate. For example, in the case of gamma radiation, the kill rate is approximately 0.00001% to 20% of the starting population. In the case of NTG, the kill rate is approximately 10% to 50% of the starting population, and in the case of mutations induced by nitrite, the kill rate is approximately 0.01% to 0.1% of the starting population, but these are not limiting.

[0027] In one embodiment, the strain may be capable of producing 5'-guanine acid.

[0028] In another embodiment, the strain may be capable of converting 5'-xanthylic acid to 5'-guanic acid.

[0029] In this application, the term "5'-guanosine monophosphate (GMP)" refers to a nucleotide having a structure in which a phosphate group is ester-linked to the ribose moiety of a guanosine molecule. The 5'-guanosine monophosphate can be used in combination with GMP. GMP can be synthesized by adding an ammonia molecule to 5'-xanthosine monophosphate (XMP) using 5'-guanosine synthase (GMP synthase). The method for producing GMP from XMP and / or the means used in the method can be selected from known techniques.

[0030] In this application, the term "5'-xanthosine monophosphate (XMP)" refers to a nucleotide dehydrogenated with IMP. 5'-XMP may be used interchangeably with XMP. XMP can be synthesized from IMP by 5'-inosine-5'-monophosphate dehydrogenase.

[0031] The Corynebacterium ammoniagenes CJG0497 strain deposited in the present application under accession number KCCM13320P has a high GMP conversion ability and a low GMP degradation rate, and is therefore capable of producing 5'-guanine acid in high yields, making it useful for industrial production of 5'-guanine acid.

[0032] In one specific example, the strain of the present application may be a strain having increased 5'-guanine acid production ability compared to a parent strain, but is not limited thereto. For example, the parent strain, which is the subject strain for comparing the increase in 5'-guanine acid production ability, may be, but is not limited to, Corynebacterium ammoniagenes ATCC 6872.

[0033] In another specific example, the strain of the present application may be a strain that has an increased ability to convert 5'-xanthylic acid to 5'-guanic acid compared to the parent strain, but is not limited thereto.

[0034] In another specific example, the strain of the present application may be a strain that has a reduced decomposition rate of 5'-guanine acid compared to the parent strain, but is not limited thereto.

[0035] Meanwhile, the Corynebacterium ammoniagenes CJG0497 strain of the present application, which is capable of producing 5'-guanine acid and deposited under accession number KCCM13320P, may include the strain itself, a strain that has improved 5'-guanine acid production ability by increasing or decreasing the activity of a gene related to the 5'-guanine acid production mechanism, and a strain that has improved 5'-guanine acid production ability by introducing or increasing the activity of an exogenous gene.

[0036] Another aspect of the present application provides a method for producing 5'-guanine acid, comprising culturing the Corynebacterium ammoniagenes CJG0497 strain deposited under accession number KCCM13320P of the present application in a medium.

[0037] In the present application, the term "culturing" refers to growing the strain of the present application under appropriately controlled environmental conditions. The culturing process of the present application can be carried out using appropriate media and culture conditions known in the art. Such a culturing process can be easily adjusted and used by those skilled in the art depending on the selected strain. Specifically, the culturing may be, but is not limited to, a batch, continuous, and / or fed-batch culture.

[0038] As used herein, the term "culture medium" refers to a mixture of nutrients, primarily those required for culturing the strains of the present application, and provides nutrients, including water, and growth factors essential for survival and growth. Specifically, the culture medium and other culture conditions used to culture the strains of the present application can be any medium commonly used for culturing strains, without any particular limitations. The strains of the present application can be cultured under aerobic conditions in a conventional medium containing appropriate carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids, and / or vitamins, while adjusting the temperature, pH, and other parameters. For example, culture media for Corynebacterium strains can be found in the literature, "Manual of Methods for General Bacteriology" by the American Society for Bacteriology (Washington, DC, USA, 1981).

[0039] In the present application, examples of the carbon source include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, and maltose; sugar alcohols such as mannitol and sorbitol; organic acids such as pyruvic acid, lactic acid, and citric acid; and amino acids such as glutamic acid, methionine, and lysine. Natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn steeping liquid can also be used. Specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted to reducing sugars) can be used. Various other carbon sources can also be used in appropriate amounts without limitation. These carbon sources can be used alone or in combination, and are not limited thereto.

[0040] Examples of the nitrogen source include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate, and organic nitrogen sources such as amino acids such as glutamic acid, methionine, and glutamine, peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steeping liquid, casein hydrolysate, fish or its degradation products, and defatted soybean cake or its degradation products. These nitrogen sources may be used alone or in combination of two or more, and are not limited to these.

[0041] The phosphorus source may include monopotassium phosphate, dipotassium phosphate, or the corresponding sodium-containing salts. Inorganic compounds may include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, and other compounds, including amino acids, vitamins, and / or appropriate precursors. These components or precursors may be added to the culture medium in a batch or continuous manner. However, the present invention is not limited to these compounds.

[0042] During the cultivation of the strain of the present application, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid can be added to the medium in an appropriate manner to adjust the pH of the medium. During cultivation, foam formation can be suppressed using an antifoaming agent such as a fatty acid polyglycol ester. To maintain an aerobic state in the medium, oxygen or an oxygen-containing gas can be injected into the medium, and to maintain anaerobic and microaerobic states, no gas can be injected or nitrogen, hydrogen, or carbon dioxide can be injected, but this is not a limitation.

[0043] In the culture of the present application, the culture temperature can be maintained at 27 to 37°C, specifically 30 to 33°C, and the culture can be performed for about 20 to 120 hours, but is not limited thereto.

[0044] In this application, the term "culture" refers to a culture solution, concentrated culture solution, dried culture solution, culture filtrate, concentrated culture filtrate, or dried culture filtrate obtained by culturing a specific strain in a culture medium, and the culture solution contains the specific strain, while the culture filtrate does not substantially contain the specific strain (here, "substantially" means that the specific strain separated by filtration or the like is excluded, but does not mean that the strain is completely excluded in the filtrate). The form of the culture is not limited, and may be, for example, a liquid, emulsion, or solid. Specifically, for purposes of this application, the culture may contain 5'-guanine acid.

[0045] In this application, the term "fermentation" refers to the process of decomposing organic matter using the enzymes that bacteria possess, but is not a putrefaction reaction. Fermentation and putrefaction proceed through similar processes, but if useful substances are produced as a result of decomposition, it is called fermentation, and if foul odors or harmful substances are produced, it is called putrefaction.

[0046] In the present application, the method for obtaining a fermented product from the strain is not particularly limited, and the product can be obtained by a method commonly used in the technical field or a similar field.

[0047] In the present application, the term "fermented product" refers not only to the fermented substance itself but also to any kind of substance containing the fermented product generated from the strain, such as a culture medium of the strain in which the strain and the culture coexist, a fermented product obtained by filtering the strain from the culture medium, a fermented product obtained by sterilizing the strain from the culture medium and filtering it, an extract obtained by extracting the fermented product or a culture medium containing the fermented product, a diluted solution or concentrate obtained by diluting the fermented product or an extract thereof, a dried product obtained by drying the fermented product or an extract thereof, and a lysate obtained by collecting and disrupting the cells of the strain.

[0048] In the method of the present application, the strain can be cultured under any culture conditions and by any culture method known in the art, and such a culture process can be easily adjusted by a person skilled in the art depending on the selected strain.

[0049] The 5'-guanine acid produced by the culture of the present application is either secreted into the medium or remains intracellularly.

[0050] In one embodiment, the method for producing 5'-guanine acid of the present application may further include a step of preparing the strain of the present application, a step of preparing a medium for culturing the strain, or a combination thereof (in any order), for example, before the culturing step.

[0051] The method for producing 5'-guanine acid of the present application may further include a step of recovering the target substance, specifically 5'-guanine acid, from the cultured strain, the culture of the strain, the fermentation product of the strain, or the culture medium. The recovery step may be further included after the culturing step.

[0052] The recovery may involve collecting the target 5'-guanine acid using an appropriate method known in the art based on the culture method of the strain of the present application, such as a batch, continuous, or fed-batch culture method. For example, centrifugation, filtration, treatment with a crystallized protein precipitant (salting-out method), extraction, ultrasonic disruption, ultrafiltration, dialysis, various types of chromatography such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, and affinity chromatography, HPLC, or a combination of these methods can be used. The target substance, specifically, 5'-guanine acid, can be recovered from the medium or the strain using an appropriate method known in the art.

[0053] Furthermore, the method for producing 5'-guanine acid of the present application may further include a purification step. The purification can be performed using an appropriate method known in the art. In one example, when the method for producing 5'-guanine acid of the present application includes both a recovery step and a purification step, the recovery step and the purification step can be performed continuously or discontinuously, simultaneously, or integrated into one step, regardless of the order, but are not limited thereto.

[0054] In the method of the present application, the strain, 5'-guanine acid, etc. are as described above in other aspects.

[0055] Another aspect of the present application provides a composition for producing 5'-guanine acid, comprising: the Corynebacterium ammoniagenes CJG0497 strain deposited under accession number KCCM13320P of the present application; a culture of the strain; a fermentation product of the strain; or a combination of two or more thereof.

[0056] The composition of the present application may further contain any suitable excipient commonly used in compositions for producing 5′-guanine acid, and such excipients may be, for example, but are not limited to, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers, or isotonic agents.

[0057] In one embodiment, each component present in the compositions of the present application may be included in a mycologically effective amount, or in an amount that may be suitably present in a production composition.

[0058] In the composition of the present application, the strain and 5'-guanine acid are as described above in other aspects.

[0059] Another aspect of the present application provides use of the Corynebacterium ammoniagenes CJG0497 strain deposited in the present application under accession number KCCM13320P for producing 5'-guanine acid.

[0060] Another aspect of the present application provides use of the Corynebacterium ammoniagenes CJG0497 strain deposited under accession number KCCM13320P; a culture of the strain, a fermentation product of the strain, or a composition comprising a combination of two or more thereof, for producing 5'-guanine acid.

[0061] For purposes of this application, the strain and 5'-guanine acid etc. are as described above in other aspects.

[0062] Example The present application will be described in more detail below with reference to experimental examples. However, the following examples are merely preferred embodiments for illustrating the present application, and are not intended to limit the scope of the present application. Meanwhile, technical matters not described in this specification can be fully understood and easily performed by those of ordinary skill in the technical field of the present application or a similar technical field.

[0063] Example 1: Selection of mutant strains through artificial mutation To obtain a microbial mutant capable of producing GMP, mutations were induced in Corynebacterium ammoniagenes ATCC6872.

[0064] The Corynebacterium ammoniagenes ATCC6872 strain is now called Corynebacterium stathionis ATCC6872 strain, and Corynebacterium ammoniagenes ATCC6872 is also used.

[0065] Specifically, we applied gamma-ray irradiation, a physical method, to induce mutations. High-energy gamma rays emitted from a Co-60 source travel along chromosomes, randomly inducing changes in base sequences, such as base substitutions, deletions, and insertions. Because the level of base sequence changes is proportional to the gamma-ray irradiation intensity, high-level gamma rays induce a high rate of mutations and also increase the kill rate of the strain. To ensure a sufficient, high-quality mutant library with high mutation rates and high diversity, we first established the gamma-ray irradiation conditions for the Corynebacterium strains to be irradiated. Using a Nordion high-level gamma-ray irradiation device at the Advanced Radiation Research Institute of the Korea Atomic Energy Research Institute, 30 mL of seed medium culture with an Absorbance 562 nm value of 6.09 was irradiated for 1 hour with gamma rays at intensities of 0, 0.5, 1, 2, 3, 4, 5, 7.5, and 10 kGy / hr. Then, the seed medium was resuspended in 100 ml of gamma rays. 0 , 10 -1 , 10 -2 , 10 -3 , 10 -4The gamma-irradiated solution diluted at the dilution ratio of 0, 0.5, 1, 2, 3, 4, 5, 7.5, and 10 kGy / hr was smeared in 100 μl of each solution, and the plate medium on which the gamma-irradiated solution was smeared was cultured in a static incubator at 30°C for 48 hours. The number of individual colonies formed was counted, and the killing rate for each irradiation condition was calculated. The CFU / mL for gamma-irradiation conditions of 0, 0.5, 1, 2, 3, 4, 5, 7.5, and 10 kGy / hr was 3.6 x 10 8 , 6.9X10 7 , 9.8X10 6 , 2.7X10 5 , 2.1X10 3 , 3.0X10 1 , 0, 0, 0, which correspond to the killing rates of 0, 81.00467, 97.31540, 99.92589, 99.99942, 99.99999, 100, 100, and 100%, respectively. Based on the above irradiation conditions, it was confirmed that the gamma ray irradiation conditions for obtaining a high-quality mutation library with high mutation diversity by gamma ray irradiation of Corynebacterium strains are 4 kGy / hr.

[0066] To identify a microorganism with high GMP conversion potential, we constructed a gamma-irradiated mutation library for the ATCC 6872 strain and screened for mutants with high GMP conversion potential. For gamma irradiation of the ATCC 6872 strain, a 500-mL flask containing 50 mL of seed medium was cultured in a shaking incubator at 30°C for 24 hours to obtain a culture with an Absorbance 562nm value of 7.84. The culture was then diluted with the seed medium to an Absorbance 562nm value of 6.10. Using a high-level gamma irradiator at the Advanced Radiation Research Institute of the Korea Atomic Energy Research Institute, 30 mL of the diluted solution was irradiated with 4 kGy / hr of gamma rays for 1 hour, and 100 μl of the irradiated stock solution was then spread onto 100 seed medium-containing 90 x 15 mm Petri dishes. The Petri dishes were cultured in a static incubator at 30°C for 48 hours to obtain a total of 40,000 individual colonies. To ensure the stability of the mutants, all individual colonies generated in all Petri dishes were collected and suspended in a 20% glycerol solution, then aliquoted into 1.5-mL microtubes in 1-mL portions and stored in a deep freezer at -80°C. During the screening of GMP-highly converting mutants, the 20% glycerol suspension was removed from the deep freezer and thawed at room temperature. The 20% glycerol suspension was diluted with saline for 10 min. -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5After serial dilutions, 100 μl of each dilution was smeared onto seed medium and incubated in a static incubator at 30°C for 24 hours to reactivate individual colonies. The reactivated individual colonies were inoculated into 96 deep-well plates containing 350 μl of seed medium (17% fill rate) per well using a Molecular Devices Qpix420 colony picker. The same plates were inoculated with ATCC 6872 strains that had not been gamma-irradiated (four wells per plate) to serve as controls for GMP high-converting mutant screening. The 96 deep-well plates containing the control strain and mutants were sealed with an Azenta Gas Permeable Seal Mark2 and incubated in an Infors-HT Multitron shaking incubator at 30°C and 1,000 rpm for 48 hours. The cultured strains and mutants were incubated in 1% xylene for 1 hour, followed by 150 μl of titer evaluation solution containing XMP. The cultures were then incubated for 12 hours at 30°C and 1,000 rpm in an Infors-HT Multitron shaking incubator. The 96-deep-well plates were then centrifuged for 20 minutes at 15°C and 4,000 rpm in an Eppendorf Centrifuge 5810R. 100 μl of the culture supernatant was transferred to a Corning 96-well Black Polystyrene Microplate using a Beckman Coulter Biomek i5 liquid handler for NIR spectrometry analysis. Next, the NIR spectrometry developed by the Analysis & Quality Department of CJ CheilJedang Bio Technology Research Institute was applied to obtain individual analysis spectra for each well. The GMP concentration was determined through a preliminary HPLC analysis based on the culture sample, and a regression analysis prediction model with a crystallization coefficient of 0.96 for the GMP concentration range of 0-20g / L was applied. Selection logic was applied to improve GMP concentration by 15% or more compared to the control group, and 64 strains were initially selected from 11,451 mutants.The 64 selected strains were cultured in the same manner as above, and the top 5 strains with the highest GMP concentrations were finally selected.

[0067] The mutant strains obtained by the above method were named Corynebacterium ammoniagenes CJG0497, Corynebacterium ammoniagenes CJG0498, Corynebacterium ammoniagenes CJG0499, Corynebacterium ammoniagenes CJG0500, and Corynebacterium ammoniagenes CJG0501, respectively. Among them, Corynebacterium ammoniagenes CJG0497 strain was deposited with the Korea Microorganism Collection, an international depository under the Budapest Treaty, on January 10, 2023, and was assigned the accession number KCCM13320P.

[0068] The compositions of the media used in Examples 1 and 2 are as follows:

[0069] <Seed medium> Glucose 30g / L, peptone 15g / L, yeast extract 15g / L, sodium chloride 2.5g / L, urea 3g / L, adenine 150mg / L, guanine 150mg / L, agar 20g / L, pH 7.0 (based on 1 liter of distilled water)

[0070] <Titer evaluation solution> Trizma-base 24.20g / L, ATP 30g / L, XMP·2Na·7H2O 30g / L, magnesium sulfate 15g / L, ammonium sulfate 20g / L

[0071] Example 2: Investigation of the ability of GMP-producing mutant strains to convert XMP to GMP To confirm the GMP conversion ability of Corynebacterium ammoniagenes CJG0497, CJG0498, CJG0499, CJG0500, and CJG0501 obtained in Example 1, they were cultured as follows.

[0072] The parent strain, Corynebacterium ammoniagenes ATCC 6872, and the five mutant strains were each inoculated into a 250 ml corner-baffled flask containing 25 ml of seed medium and cultured at 30°C for 20 hours with shaking at 200 rpm. 800 μl of titer evaluation solution was added to 200 μl of culture medium, and the mixture was incubated at 42°C for 30 minutes to produce GMP. After the culture was completed, the amount of GMP produced was measured using high-performance liquid chromatography, and the GMP concentration in the culture medium for each strain tested is shown in Table 1 below.

[0073] [Table 1]

[0074] As a result, as shown in Table 1, the parent strain Corynebacterium ammoniagenes ATCC6872 produced (converted) GMP from XMP at a concentration of 0.58 g / L, while the mutants Corynebacterium ammoniagenes CJG0497, CJG0498, CJG0499, CJG0500, and CJG0501 of the present application were confirmed to convert XMP to GMP at concentrations of 3.45 g / L, 1.69 g / L, 2.84 g / L, 2.58 g / L, and 2.81 g / L, respectively.

[0075] That is, it was confirmed that the mutant strain Corynebacterium ammoniagenes CJG0497 of the present application has excellent GMP conversion ability and GMP production ability.

[0076] Example 3: Investigation of GMP degradation rate of mutant strains for GMP production To confirm the GMP degradation rate of Corynebacterium ammoniagenes CJG0497, CJG0498, CJG0499, CJG0500, and CJG0501 obtained in Example 1, they were cultured as follows.

[0077] 250 ml corner baffle flasks containing 25 ml of the culture medium were each inoculated with the parent strain Corynebacterium ammoniagenes ATCC6872 and the five mutant strains, and then cultured with shaking at 30 °C for 20 hours at 200 rpm. 500 μl of the GMP degradation degree evaluation medium was added to 500 μl of the culture solution, and after reacting at 30 °C for 12 hours, the GMP concentration was measured using liquid high performance chromatography. The GMP concentrations in the culture solutions for each of the tested strains are shown in Table 2 below.

[0078]

Table 2

[0079] As a result, as shown in Table 2 above, the parent strain Corynebacterium ammoniagenes ATCC6872 decomposed 3.56 g / l of GMP, while the mutant strains Corynebacterium ammoniagenes mutant strains CJG0497, CJG0498, CJG0499, CJG0500, and CJG0501 according to the present invention decomposed 0.50 g / l, 3.38 g / l, 2.67 g / l, 2.50 g / l, and 2.81 g / l of GMP, respectively.

[0080] That is, it was confirmed that the mutant strain Corynebacterium ammoniagenes CJG0497 strain of the present application can produce GMP with high efficiency and high yield by reducing the GMP degradation rate compared to the parent strain.

[0081] The composition of the medium used in Example 3 is as follows.

[0082] <GMP degradation degree evaluation medium> GMP at 20 g / L, phytic acid at l.8 g / L, magnesium sulfate at 4.8 g / L, nymeen at 3 ml / L, xylene at 2%, adenine at 100 mg / L, sodium hydrogen phosphate (Na2HPO4) at 7.7 g / L

[0083] Example 4: Investigation of the XMP degradation degree of the mutant strain for GMP production To confirm the XMP degradation degrees of Corynebacterium ammoniagenes CJG0497, CJG0498, CJG0499, CJG0500, and CJG0501 obtained from Example 1 above, they were cultured by the following method.

[0084] 250 ml Erlenmeyer flasks containing 25 ml of the seed medium were each inoculated with Corynebacterium ammoniagenes ATCC6872, the parental strain, and the five mutant strains, and then cultured with shaking at 30 °C for 20 hours at 200 rpm. After adding 500 μl of the XMP degradation degree evaluation medium to 500 μl of the culture broth and reacting at 30 °C for 12 hours, the XMP concentration was measured using liquid high performance chromatography, and the XMP concentrations in the culture broth for each of the strains tested are shown in Table 3 below.

[0085]

Table 3

[0086] As a result, as shown in Table 3 above, Corynebacterium ammoniagenes ATCC6872, the parental strain, degraded 2.53 g / l of XMP, while the mutant strains of Corynebacterium ammoniagenes mutant strains CJG0497, CJG0498, CJG0499, CJG0500, and CJG0501 according to the present invention degraded 1.59 g / l, 2.3 g / l, 2.07 g / l, 1.88 g / l, and 2.2 g / l of XMP, respectively, were confirmed.

[0087] That is, it was confirmed that the mutant strain Corynebacterium ammoniagenes CJG0497 strain of the present application can produce GMP with high efficiency and high yield by reducing the XMP degradation rate compared to the parental strain.

[0088] The composition of the medium used in Example 4 is as follows.

[0089] <XMP degradation degree evaluation medium> XMP 20g / L, phytic acid 1.8g / L, magnesium sulfate 4.8g / L, nymeen 3ml / L, xylene 2%, adenine 100mg / L, sodium hydrogen phosphate (Na2HPO4) 7.7g / L

[0090] Example 5: Genetic analysis of mutant strains for GMP production In order to analyze the genetic differences of the Corynebacterium ammoniagenes CJG0497 strain obtained in Example 1, a genome comparison analysis experiment was carried out with the wild-type Corynebacterium ammoniagenes strain ATCC6872.

[0091] Specifically, genomic DNA (gDNA) extraction for full-length genome sequence analysis (next generation sequencing; NGS) was performed using Corynebacterium ammoniagenes CJG0497 and ATCC6872 strains, which had been plated on optimal culture medium and grown for at least 24 hours. The resulting cells were suspended in 300 μL of PBS using a 10-μL loop (SPL Inc., Cat. No. 90010). After stirring, 175 μL of the suspension was mixed with Lysis / Binding Solution (232 μL Lysis / Binding Solution Concentrate and 3 μL Carrier NA; components of the MagMax™ Total Nucleic Acid Isolation Kit) and lysed twice in a bead tube at 6500 rpm for 1 minute. The subsequent process was performed using the MagMax™ Total Nucleic Acid Isolation Kit (Thermo Fisher Scientific Inc., Cat. No. AM1840) according to the manufacturer's recommendations, and the resulting gDNA was quantitatively analyzed using a Qubit Fluorometer (Thermo Fisher Scientific Inc.).

[0092] The gDNA from the strains reserved for long-read sequencing was diluted to 1500 ng gDNA / 48 μL DW and sequencing libraries were prepared using a Ligation Sequencing Kit (Oxford Nanopore Technologies Inc., Cat. No. SQK-LSK109) and a Native Barcoding Expansion 1-12 (PCR-free) Kit (Oxford Nanopore Technologies Inc., Cat. No. EXP-NBD104) following the manufacturer's recommended procedures. Nick and gap filling, dA tailing, barcoding, and adapter ligation were performed according to the manufacturer's instructions. The prepared library samples were sequenced for approximately 24 hours using a MinION Mk1C instrument (Oxford Nanopore Technologies Inc.).

[0093] The gDNA from the strains reserved for short-read sequencing was diluted to 100 ng gDNA / 50 μL DW and then subjected to DNA shearing and barcoding using the Ion Plus Fragment Library Kit for Library Builder™ (Thermo Fisher Scientific Inc., Cat. No. 4477597), Ion Xpress™ Plus Fragment Library (Thermo Fisher Scientific Inc., Cat. No. 4477597), and AB Library Builder™ equipment (Thermo Fisher Scientific Inc.) according to the manufacturer's recommended procedures. Similarly, adapter ligation, nick-repair, and library amplification were also performed according to the manufacturer's recommended procedures. The resulting final sequencing library was diluted to a final concentration of 50 pM with Ion 510. TM & Ion 520 TM& Ion 530 TM Emulsion PCR and enrichment were performed using the Ion Chef System (Thermo Fisher Scientific Inc., Cat. No. A34461) and the Ion Chef System (Thermo Fisher Scientific Inc.). TM Chip(Ion 530 TM The fragments were loaded onto a Chip Kit (Thermo Fisher Scientific Inc., Cat. No. A27764) and sequence analysis was performed. After sequence analysis, adapter sequences were removed and quality checked via a torrent server, and the raw sequence data (raw fastq data) was used for full-length genome assembly and polishing.

[0094] [Table 4]

[0095] [Table 5]

[0096] As a result, as shown in Table 4, it was confirmed that non-synonymous mutations in two genes (hrpB (P327S) and sraP (S88F)) were observed in the mutant strain Corynebacterium ammoniagenes CJG0497 of the present application compared to the parent strain Corynebacterium ammoniagenes ATCC6872.

[0097] In addition, as shown in Table 5, it was confirmed that nucleotide mutations were observed at two rRNA ORF positions in the mutant strain Corynebacterium ammoniagenes CJG0497 of the present application compared to the parent strain Corynebacterium ammoniagenes ATCC6872.

[0098] That is, it was confirmed that the Corynebacterium ammoniagenes mutant strain of the present application has genetic differences from the parent strain.

[0099] From the above description, those skilled in the art to which the present application pertains will understand that the present application may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are merely illustrative and not limiting. The scope of the present application should be interpreted as including within the meaning and scope of the claims below, and any modifications or variations derived from the equivalent concepts thereof, rather than the above detailed description.

Claims

1. Corynebacterium ammoniagenes strain CJG0497, deposited under accession number KCCM13320P.

2. The strain according to claim 1, which is capable of producing 5'-guanine acid.

3. The strain according to claim 1, which has the ability to convert 5'-xanthylic acid to 5'-guanic acid.

4. A method for producing 5'-guanine acid, comprising culturing the Corynebacterium ammoniagenes CJG0497 strain deposited under accession number KCCM13320P in a medium.

5. 5. The method of claim 4, further comprising recovering 5'-guanine acid from the cultured strain, a culture of the strain, a fermentation product of the strain, or the culture medium.

6. A composition for producing 5'-guanine acid, comprising the Corynebacterium ammoniagenes CJG0497 strain deposited under accession number KCCM13320P, a culture of said strain, a fermentation product of said strain, or a combination of two or more thereof.

7. Use of Corynebacterium ammoniagenes strain CJG0497, deposited under accession number KCCM13320P, for the production of 5'-guanine acid.

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