5 apos is produced; -guanylic acid strains and the use thereof to produce 5apos; method for producing guanylic acid

Abstract

The present disclosure relates to a Corynebacterium ammoniacum CJG0497 strain preserved at a preservation number of KCCM13320P, a strain of Corynebacterium ammoniacum CJG0497, a A method of producing 5 '-guanylate comprising culturing the strain in a culture medium; a composition for producing 5 '-guanylate comprising the strain, a culture product of the strain, a fermentation product of the strain, or a combination of two or more thereof; and the use of the strain for the production of 5 '-guanylate (GMP).

Description

Technical Field

[0001] The present disclosure relates to the Corynebacterium ammoniagenes CJG0497 strain deposited under the accession number KCCM13320P; a method for producing 5′-guanosine monophosphate (GMP), comprising culturing the strain in a culture medium; a composition for producing 5′-guanosine monophosphate (GMP), comprising the strain, a culture product 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′-guanosine monophosphate (GMP). Background Art

[0002] 5′-Guanosine monophosphate (GMP) is an intermediate in the nucleic acid biosynthetic metabolic system. It is physiologically important in animals and plants and is used in a wide range of fields, including the food and pharmaceutical industries, as well as for various medical purposes. In particular, GMP is a nucleic acid-based seasoning that is attractive as a flavoring agent due to its high flavor-enhancing effect on foods when combined with monosodium glutamate (MSG).

[0003] Examples of methods for producing GMPs may include: (1) methods for degrading yeast RNA using enzymes derived from microorganisms or chemically degrading yeast RNA; (2) methods for directly producing nucleotides by culturing microorganisms in a culture medium containing sugars, nitrogen sources, and phosphate sources; and (3) methods for chemically or enzymatically converting intermediates in nucleotide synthesis into nucleotides. Currently, combined methods of fermentation and chemical synthesis and enzymatic conversion are widely used for industrial purposes.

[0004] The combined method comprises a fermentation process for producing 5'-xanthosine monophosphate (XMP), an intermediate in the nucleic acid biosynthesis metabolic system, using a microorganism, and an enzymatic reaction process for converting the fermentation product into GMP. The method uses both XMP-producing microorganisms and microorganisms capable of converting XMP into GMP. Therefore, in order to efficiently produce GMP, it is necessary to inhibit the degradation of GMP by the microorganisms involved in GMP production. In particular, the microorganisms used in the enzymatic reaction process must have a high degree of complexation ability to convert XMP into GMP.

[0005] Therefore, various studies are underway to develop efficient production microorganisms and fermentation process technologies. For example, target-specific approaches are mainly used, such as increasing the expression of genes encoding enzymes related to XMP or GMP biosynthesis, or deleting genes that are unnecessary for their biosynthesis (EP 3722430 A1, US2020-0347346A1).

[0006] However, in order to effectively improve GMP production capacity, new strains with high GMP conversion ability and low GMP degradation rate are still needed to be studied. Summary of the Invention

[0007]

Technical Issues

[0008] The present inventors confirmed that the Corynebacterium ammoniagenes CJG0497 strain deposited under the accession number KCCM13320P has a high GMP conversion ability and a low GMP degradation rate, and thus can produce 5′-guanosine monophosphate (GMP) in high yield, thereby completing the present disclosure.

[0009]

Technical solution

[0010] One object of the present disclosure is to provide the Corynebacterium ammoniagenes CJG0497 strain deposited under the accession number KCCM13320P.

[0011] In one embodiment, the strain may have the ability to produce 5'-guanosine monophosphate (GMP).

[0012] In another embodiment, the strain may have the ability to convert 5'-xanthosine monophosphate (XMP) into 5'-guanosine monophosphate (GMP).

[0013] Another object of the present disclosure is to provide a method for producing 5′-guanosine monophosphate (GMP), comprising culturing the Corynebacterium ammoniagenes CJG0497 strain deposited under the accession number KCCM13320P in a culture medium.

[0014] In one embodiment, the method may further comprise recovering 5'-guanosine monophosphate (GMP) from the cultured strain, the culture product of the strain, the fermentation product of the strain, or the culture medium.

[0015] Another object of the present disclosure is to provide a composition for producing 5′-guanosine monophosphate (GMP), comprising the Corynebacterium ammoniagenes CJG0497 strain deposited under the accession number KCCM13320P, a culture product of the strain, a fermentation product of the strain, or a combination of two or more thereof.

[0016] Another object of the present disclosure is to provide use of the Corynebacterium ammoniagenes CJG0497 strain deposited under the accession number KCCM13320P for producing 5′-guanosine monophosphate (GMP).

[0017] Beneficial effects

[0018] The disclosed Corynebacterium ammoniagenes CJG0497 strain, deposited under the accession number KCCM13320P, has high GMP conversion capacity and low GMP degradation rate, and can therefore produce 5′-guanosine monophosphate (GMP) in high yield. Therefore, it can be effectively used in the industrial production of GMP. DETAILED DESCRIPTION

[0019] The present disclosure will be described in detail below. Meanwhile, each description and embodiment disclosed herein can be applied to other descriptions and embodiments respectively. That is to say, all combinations of the various elements disclosed herein fall within the scope of the present disclosure. In addition, the scope of the present disclosure is not limited by the specific description described below.

[0020] Furthermore, those skilled in the art will be able to recognize or ascertain using no more than routine experimentation many equivalents to the specific aspects of the invention described herein, and it is intended that such equivalents be encompassed within this disclosure.

[0021] As used in the specification and the appended claims, the singular forms ("a," "an," and "the") include plural referents unless the context clearly dictates otherwise. Singular terms shall include pluralities and plural terms shall include the singular unless the context clearly dictates otherwise. As used in the specification and the appended claims, the use of "or" may be intended to include "and / or" unless the context clearly dictates otherwise.

[0022] As used herein, the term "about" may appear before a particular numerical value. As used herein, the term "about" includes not only the exact numerical value listed after the term, but also a range that is close to or approximately that numerical value. Whether any number is close to or approximately the particular numerical value presented can be determined by taking into account the context of the numerical value presented. In one example, the term "about" may refer to a range of -10% to +10% of a numerical value. In another example, the term "about" may refer to a range of -5% to +5% of a given numerical value, but is not limited thereto.

[0023] As used herein, the term "comprises / includes" means the presence of the features, steps, or components listed after the term, and does not exclude the presence or addition of one or more features, steps, or components. The components or features listed after the term "comprises / includes" herein may be essential or mandatory. However, in some embodiments, the term may also include any other or non-essential components or features.

[0024] One aspect of the present disclosure provides a Corynebacterium ammoniagenes CJG0497 strain deposited under the accession number KCCM13320P.

[0025] The bacterial strain of the present disclosure may refer to a mutant strain obtained by mutation of its parent strain. Specifically, the bacterial strain of the present disclosure may include all bacterial strains capable of producing the desired 5′-guanosine monophosphate (GMP) by mutating the Corynebacterium ammoniagenes ATCC6872 strain (currently known as the Corynebacterium stationis ATCC6872 strain).

[0026] In this regard, the mutagenesis of microorganisms can be carried out by various methods well known in the art, and physical mutagenesis or chemical mutagenesis can be used. For example, examples of physical mutagens suitable for the present disclosure may include gamma radiation and ultraviolet radiation, but physical mutagens are not limited thereto. In addition, examples of chemical mutagens may include N-methyl-N'-nitro-N-nitrosoguanidine (NTG), diepoxybutane, ethyl methanesulfonate, mustard compounds, hydrazine and nitrite, but chemical mutagens are not limited thereto.

[0027] When inducing mutagenesis, the parent strain is exposed to the mutagen under conditions that leave a specific size of the parent strain surviving. These conditions vary depending on the type of mutagen and depend on the amount of mutations induced in the surviving population at a constant kill rate. For example, with ultraviolet radiation, the kill rate can be approximately 0.00001% to 20% of the initial population. With NTG, the kill rate can be approximately 10% to 50% of the initial population, and with nitrite mutagenesis, approximately 0.01% to 0.1% of the initial population can survive, but mutagens are not limited to these.

[0028] In one embodiment, the strain may have the ability to produce 5'-guanosine monophosphate (GMP).

[0029] In another embodiment, the strain may have the ability to convert 5'-xanthosine monophosphate (XMP) into 5'-guanosine monophosphate (GMP).

[0030] As used herein, the term "5'-guanosine monophosphate (GMP)" refers to a nucleotide having a structure in which a phosphate group forms an ester bond with the ribose sugar of a guanosine molecule. 5'-Guanosine monophosphate can be used interchangeably with GMP. GMP can be synthesized by adding an ammonia molecule to 5'-xanthosine monophosphate (XMP) by a 5'-guanylate biosynthetic enzyme (GMP synthase). The method for preparing GMP from XMP and / or the means used in the method can be selected from known techniques.

[0031] As used herein, the term "5'-xanthosine monophosphate (XMP)" refers to a nucleotide produced by dehydrogenation of IMP and can be used interchangeably with XMP. XMP can be synthesized from IMP by inosine-5'-monophosphate dehydrogenase.

[0032] The disclosed Corynebacterium ammoniagenes CJG0497 strain, deposited under the accession number KCCM13320P, has high GMP conversion capacity and low GMP degradation rate, and can therefore produce 5′-guanosine monophosphate (GMP) in high yield. Therefore, it can be effectively used in the industrial production of GMP.

[0033] In one embodiment, the strain of the present disclosure may be a strain having enhanced 5′-guanosine monophosphate (GMP) production capacity compared to a parent strain, but is not limited thereto. In one example, the parent strain (which is the target strain for comparison of enhanced 5′-guanosine monophosphate (GMP) production capacity) may be Corynebacterium ammoniagenes ATCC6872 strain, but is not limited thereto.

[0034] In another embodiment, the strain of the present disclosure may be a strain having enhanced conversion ability of 5′-xanthosine monophosphate (XMP) to 5′-guanosine monophosphate (GMP) compared to the parent strain, but is not limited thereto.

[0035] In another embodiment, the strain of the present disclosure may be a strain having a reduced 5′-guanosine monophosphate (GMP) degradation rate compared to a parent strain, but is not limited thereto.

[0036] At the same time, the ammonia-producing bacterium CJG0497 strain with the ability to produce 5'-guanosine monophosphate (GMP) deposited under the accession number KCCM13320P may include the strain itself, as well as all strains with enhanced 5'-guanosine monophosphate (GMP) production ability by increasing or decreasing the activity of genes related to the GMP production mechanism, or strains with enhanced 5'-guanosine monophosphate (GMP) production ability by introducing or increasing the activity of exogenous genes.

[0037] Another aspect of the present disclosure provides a method for producing 5′-guanosine monophosphate (GMP), comprising culturing the Corynebacterium ammoniagenes CJG0497 strain deposited under the accession number KCCM13320P in a culture medium.

[0038] As used herein, the term "cultivation" refers to growing the strains of the present invention under appropriately controlled environmental conditions. The cultivation process of the present invention can be carried out under suitable culture media and culture conditions known in the art. Depending on the strain to be selected, those skilled in the art can easily adjust this cultivation process for use. Specifically, the cultivation can be batch culture, continuous culture and / or fed-batch culture, but is not limited thereto.

[0039] As used herein, the term "culture medium" refers to a mixture of substances containing the nutrients required for cultivating bacterial strains of the present invention as a main component, which provides nutrients and growth factors, as well as water necessary for survival and growth. Specifically, the culture medium and other culture conditions for cultivating bacterial strains of the present invention can be any culture medium for conventional bacterial strain culture, without any particular restrictions. However, bacterial strains of the present invention can be cultivated under aerobic conditions in a conventional culture medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compound, amino acid and / or vitamin, while regulating temperature, pH etc. For example, the culture medium for Corynebacterium genus bacterial strains can be found in document ["Manual of Methods for General Bacteriology" by the American Society for Bacteriology (Washington DC, USA, 1981)].

[0040] In the present disclosure, the carbon source can include carbohydrates such as glucose, sucrose, lactose, fructose, sucrose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvic acid, lactic acid, citric acid, etc.; amino acids such as glutamic acid, methionine, lysine, etc. In addition, the carbon source can include natural organic nutrients such as starch hydrolysate, molasses, blackstrap molasses, rice bran, cassava, sugarcane molasses, and corn steep liquor, etc. Specifically, carbohydrates such as glucose and sterile pretreated molasses (i.e., molasses converted into reducing sugars) can be used, and in addition, various other carbon sources can be used in appropriate amounts without restriction. These carbon sources can be used alone or in combination of two or more, but are not limited thereto.

[0041] The nitrogen source may include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc.; amino acids such as glutamic acid, methionine, glutamine, etc.; and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or its decomposition product, defatted soybean cake or its decomposition product, etc. These nitrogen sources may be used alone or in combination of two or more, but are not limited thereto.

[0042] The phosphorus source may include potassium dihydrogen phosphate, dipotassium hydrogen phosphate or corresponding sodium salts. Examples of inorganic compounds may include sodium chloride, calcium chloride, ferric chloride, magnesium sulfate, ferric sulfate, manganese sulfate, calcium carbonate, etc. In addition, amino acids, vitamins and / or suitable precursors may be included. These components or precursors may be added to the culture medium in a batch or continuous manner, but these phosphorus sources are not limited thereto.

[0043] In addition, during the process of culturing the strain of the present invention in an appropriate manner, the pH of the culture medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid. In addition, defoamers such as fatty acid polyethylene glycol esters can be used to prevent bubble formation during the culture process. In addition, oxygen or oxygen-containing gas can be injected into the culture medium to maintain aerobic conditions of the culture medium; or nitrogen, hydrogen or carbon dioxide can be injected or no gas can be injected to maintain anaerobic or microaerobic conditions, but the gas is not limited thereto.

[0044] The temperature during the culture of the present disclosure may be in the range of 27° C. to 37° C., specifically 30° C. to 33° C., and the culture may last for 20 to 120 hours, but is not limited thereto.

[0045] As used herein, the term "culture product" refers to a culture solution, a concentrated culture solution, a dried product of the culture solution, a culture filtrate, a concentrated culture filtrate, or a dried product of the culture filtrate obtained by culturing a specific strain in a culture medium, and means that the culture solution may contain a specific strain, while the culture filtrate does not substantially contain a specific strain (particularly, it substantially means excluding a specific strain separated by filtration, etc., but does not mean that the strain is completely excluded from the filtrate). The dosage form of the culture product is not limited and can be, for example, a liquid, an emulsion, or a solid. Specifically, for the purposes of the present disclosure, the culture product can include 5'-guanosine monophosphate (GMP).

[0046] As used herein, the term "fermentation" refers to the process by which a strain decomposes organic matter using its own enzymes without becoming corrupt. Fermentation and decay reactions proceed through similar processes, but when decomposition produces useful substances, it is called fermentation, and when decomposition produces odorous or harmful substances, it is called decay.

[0047] In the present disclosure, the method of obtaining the fermentation product from the strain is not particularly limited, and can be obtained according to a method commonly used in this art or similar fields.

[0048] As used herein, the term "fermentation product" may include not only the fermentation material itself, but also all kinds of materials, including the fermentation product produced by the strain, such as the strain culture medium in which the strain and the culture coexist, the fermentation product produced by the culture medium, the fermentation product obtained by filtering the strain from the culture medium, the fermentation product obtained by sterilizing the strain from the culture medium and filtering the strain, the extract obtained by extracting the fermentation product or the culture medium containing the fermentation product, the diluted solution obtained by diluting the fermentation product or its extract, the concentrated solution, the dried product obtained by drying the fermentation product or its extract, and the lysate obtained by collecting and lysing the strain cells, etc.

[0049] In the method disclosed herein, the strain can be cultured using any culture conditions and methods known in the art. Those skilled in the art can easily adjust and use this culture process according to the selected strain.

[0050] 5'-Guanosine monophosphate (GMP) produced by the culture of the present disclosure may be released into the culture medium or retained in the cells.

[0051] In one embodiment, the method for producing 5′-guanosine monophosphate (GMP) of the present disclosure may further include, for example, before the culturing step, a step of preparing the strain of the present disclosure, a step of preparing a culture medium for culturing the strain, or a combination thereof (regardless of the order, in any order).

[0052] The method for producing 5′-guanosine monophosphate (GMP) disclosed herein may further include recovering the desired substance, specifically 5′-guanosine monophosphate (GMP), from the cultured strain, the culture product of the strain, the fermentation product of the strain, or the culture medium. A recovery step may also be included after the culture step.

[0053] In the recovery step, the method for culturing the strain of the present invention can be used, for example, according to a batch culture, continuous culture or fed-batch culture method, using a suitable method known in the art to collect the desired 5'-guanosine monophosphate (GMP). For example, methods such as centrifugation, filtration, treatment with a protein crystallization precipitant (salting out method), extraction, ultrasonication, ultrafiltration, dialysis, various types of chromatography (such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, etc.), HPLC or a combination thereof can be used, and the desired substance, specifically 5'-guanosine monophosphate (GMP), can be recovered from the culture medium or strain using a suitable method known in the art.

[0054] In addition, the method for producing 5'-guanosine monophosphate (GMP) disclosed herein may further include a purification step, which may be performed using a suitable method known in the art. In one example, when the method for producing 5'-guanosine monophosphate (GMP) disclosed herein includes a recovery step and a purification step, the recovery step and the purification step may be performed continuously or intermittently without regard to the order, or may be performed simultaneously, or may be integrated into one step, but the method is not limited thereto.

[0055] In the methods disclosed herein, the strains and 5′-guanosine monophosphate (GMP) are as described above in other aspects.

[0056] Another aspect of the present disclosure provides a composition for producing 5′-guanosine monophosphate (GMP), comprising the Corynebacterium ammoniagenes CJG0497 strain deposited under the accession number KCCM13320P, a culture product of the strain, a fermentation product of the strain, or a combination of two or more thereof.

[0057] The composition of the present disclosure may also include any suitable excipients commonly used in compositions for producing 5′-guanosine monophosphate (GMP), and such excipients may include, for example, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, or isotonic agents, but are not limited thereto.

[0058] In one embodiment, the various components present in the compositions of the present disclosure may be included in a mycologically effective amount, or in an amount that may be suitably present in the composition for manufacture.

[0059] In the composition of the present disclosure, the strain and 5′-guanosine monophosphate (GMP) etc. are as described in other aspects above.

[0060] Another aspect of the present disclosure provides use of the Corynebacterium ammoniagenes CJG0497 strain deposited under the accession number KCCM13320P for producing 5′-guanosine monophosphate (GMP).

[0061] Another aspect of the present disclosure provides use of a combination comprising the Corynebacterium ammoniagenes CJG0497 strain deposited under accession number KCCM13320P, a culture product of the strain, a fermentation product of the strain, or a combination of two or more thereof for producing 5′-guanosine monophosphate (GMP).

[0062] In the use disclosed herein, the strain and 5′-guanosine monophosphate (GMP) are as described in other aspects above.

[0063] [Mode for Carrying Out the Invention]

[0064] The present disclosure will be described in detail below by way of examples. However, these examples are preferred embodiments provided for illustrative purposes only, and therefore, the scope of the present disclosure is not intended to be limited by these examples. Furthermore, those skilled in the art of the present disclosure or similar technical fields can fully understand and easily implement technical features not described herein.

[0065] Example 1: Screening of mutant strains by artificial mutagenesis

[0066] In order to obtain a microbial mutant strain having GMP production capability, mutation was induced in Corynebacterium ammoniagenes ATCC6872.

[0067] The Corynebacterium ammoniagenes ATCC6872 strain is currently referred to as the Corynebacterium stagnantum ATCC6872 strain and is also used as Corynebacterium ammoniagenes ATCC6872.

[0068] Specifically, for mutagenesis, a physical method of gamma ray radiation was applied. High-energy gamma rays emitted from the radiation source Co-60 are transmitted along the chromosomes and randomly trigger changes in the nucleotide sequence, such as nucleotide substitutions, deletions, insertions, and the like. Since the degree of nucleotide sequence change is proportional to the intensity of gamma ray irradiation, high levels of gamma rays induce mutations at a high ratio while increasing the killing rate of the strain. In order to fully ensure a high-quality mutant library with high diversity and high mutation rates, gamma irradiation conditions for gamma-irradiated coryneform bacteria strains were established in advance. Using a high-level gamma ray irradiation device from Nordion Inc. at the Advanced Radiation Technology Research Institute of the Korea Atomic Energy Research Institute, 30 mL of seed culture solution (562 nm absorbance 6.09) was subjected to gamma rays with intensities of 0, 0.5, 1, 2, 3, 4, 5, 7.5, and 10 kGy / h for 1 hour, and 100 μl was irradiated at 10 0 , 10 -1 , 10 -2 , 10 -3 and 10 -4 The γ-ray irradiation solutions diluted at the dilution rate of 0.5 were spread on the seed culture medium. The plate culture medium coated with the γ-ray irradiation solution was incubated in a static incubator at 30°C for 48 hours. The number of single colonies produced was then counted, and the killing rate under each irradiation condition was calculated. The CFU / mL for the 0, 0.5, 1, 2, 3, 4, 5, 7.5, and 10-kGy / hr γ-irradiation conditions was 3.6×10 8 , 6.9×10 7 , 9.8×10 6 , 2.7×10 5 , 2.1×10 3 , 3.0×10 1 , 0, 0 and 0, and the killing rates calculated therefrom were 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 γ irradiation conditions for obtaining a high-quality mutant library with high mutation diversity by γ irradiation based on Corynebacterium strains were 4 kGy / hr.

[0069] To obtain microorganisms with high GMP conversion capacity, a γ-irradiation-based mutant library was constructed based on the ATCC6872 strain, and mutants with high GMP conversion capacity were screened. For γ-irradiation of the ATCC6872 strain, a 500 mL flask containing 50 mL of seed culture medium was cultured in a shaking incubator at 30°C for 24 hours to obtain a culture solution with an absorbance of 7.84 at 562 nm. The culture solution was then diluted with the seed culture medium to an absorbance of 6.10 at 562 nm. After irradiating 30 mL of the diluted solution with 4 kGy / hr of γ-rays for 1 hour using a high-level γ-ray irradiation device at the Advanced Radiation Technology Research Institute of the Korea Atomic Energy Research Institute, 100 μl of the irradiated stock solution was spread on the seed culture medium in 100 Petri dishes (90×15 mm). Then, a total of approximately 40,000 single colonies were obtained by culturing the Petri dish in a 30°C static incubator for 48 hours, and to ensure the stability of the mutants, all single colonies generated in the Petri dish were recovered and suspended in a 20% glycerol solution, and then subdivided into 1 mL in 1.5 mL microtubes and stored in a -80°C low-temperature freezer. When screening mutants with high GMP conversion ability, the 20% glycerol suspension stored in the low-temperature freezer was thawed at room temperature and serially diluted to 10 with saline. -1 , 10 -2 , 10 -3 , 10 -4 and 10 -5. Then, 100 μl of the diluted solution corresponding to each dilution factor was spread on the seed culture medium and reactivated in the form of a single colony by culturing in a static incubator at 30°C for 24 hours. The reactivated single colony was inoculated into a 96-deep-well plate containing 350 μl of seed culture medium using Qpix420 (a colony picker from Molecular Devices) with a filling rate of 17% per well. The ATCC6872 strain that was not irradiated with gamma rays was inoculated into 4 wells per plate on the same plate and used as a control for screening mutants with high GMP conversion ability. The 96-deep-well plate inoculated with the control strain and the mutant strain was sealed with an Azenta breathable sealing film 2 and then cultured at 30°C and 1,000 rpm for 48 hours in a culture shaker Multitron manufactured by Infors-HT. The strains and mutants were then cultured for an additional hour by adding 1% xylene, and 150 μl of a titer solution containing XMP was added, followed by further 12-hour culture in an Infors-HT Multitron shaker at 30°C and 1,000 rpm. The 96-deep-well plate, cultured for 12 hours, was centrifuged for 20 minutes at 15°C and 4,000 rpm using an Eppendorf 5810R centrifuge, and 100 μl of the culture supernatant from which the cells had been separated was transferred to a 96-well black polystyrene microplate manufactured by Corning using a Beckman Coulter Biomek i5 liquid handler for NIR spectroscopy analysis. Afterwards, the strains were subjected to an in-house NIR spectrometry method developed at the CJ Cheil Jedang Institute of Biotechnology's Analytical and Quality Department to ensure individual analytical spectra for each well. Sixty-four of the 11,451 mutants were initially selected using a regression analysis prediction model with a coefficient of determination of 0.96 for a GMP concentration range of 0 to 20 g / L, established based on culture samples previously quantified for GMP concentration by HPLC analysis, and a screening logic where GMP concentrations were increased by 15% or more compared to controls. The 64 selected strains were cultured in the same manner as above, and the top five strains with high GMP concentrations were ultimately selected.

[0070] 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 was deposited in the Korean Center for Culture of Microorganisms (KCCM), an international depository institution, on January 10, 2023 in accordance with the Budapest Treaty, with the deposit number KCCM13320P.

[0071] The culture medium components used in Examples 1 and 2 are as follows:

[0072] <Seed culture medium>

[0073] 30 g / L glucose, 15 g / L peptone, 15 g / L yeast extract, 2.5 g / L sodium chloride, 3 g / L urea, 150 mg / L adenine, 150 mg / L guanine, 20 g / L agar, pH 7.0 (based on 1 liter of distilled water)

[0074] <Potency Evaluation Solution>

[0075] 24.20g / L tris(hydroxymethyl)aminomethane, 30g / L ATP, 30g / L XMP·2Na·7H2O, 15g / L magnesium sulfate, 20g / L ammonium sulfate

[0076] Example 2: Study on the conversion ability of GMP-producing mutant strains to convert XMP to GMP

[0077] In order to confirm the conversion ability of Corynebacterium ammoniagenes CJG0497, CJG0498, CJG0499, CJG0500 and CJG0501 obtained in Example 1 to convert XMP into GMP, the strains were cultured in the following manner.

[0078] Corynebacterium ammoniagenes ATCC6872 (parent strain) and five mutant strains were each inoculated into a 250 ml baffled flask containing 25 ml of seed culture medium and then cultured with shaking at 30°C and 200 rpm for 20 hours. GMP was produced by adding 800 μl of the titer evaluation solution to 200 μl of the culture medium and reacting at 42°C for 30 minutes. After the culture was completed, GMP production was measured using high-performance liquid chromatography, and the GMP concentrations in the culture medium of each test strain are shown in Table 1 below.

[0079] [Table 1]

[0080] Comparison of GMP Conversion Abilities among Corynebacterium ammoniagenes ATCC6872, CJG0497, CJG0498, CJG0499, CJG0500, and CJG0501

[0081]

[0082] As a result, as shown in Table 1, it was confirmed that Corynebacterium ammoniagenes ATCC6872 (parent strain) produced (converted) GMP at a concentration of 0.58 g / l from XMP, but Corynebacterium ammoniagenes CJG0497, CJG0498, CJG0499, CJG0500 and CJG0501 (mutant strains disclosed in the present invention) converted 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 from XMP, respectively.

[0083] That is, it was confirmed that the Corynebacterium ammoniagenes CJG0497 strain of the present disclosure exhibited excellent GMP conversion ability and GMP production ability.

[0084] Example 3: Study on GMP degradation by GMP-producing mutant strains

[0085] In order to confirm the GMP degradation of Corynebacterium ammoniagenes CJG0497, CJG0498, CJG0499, CJG0500 and CJG0501 obtained in Example 1, the strains were cultured in the following manner.

[0086] Corynebacterium ammoniagenes ATCC6872 (parent strain) and five mutant strains were each inoculated into a 250 ml baffled flask containing 25 ml of seed culture medium and then cultured with shaking at 30°C and 200 rpm for 20 hours. 500 μl of GMP degradation evaluation medium was added to 500 μl of the culture solution and reacted at 30°C for 12 hours to produce GMP. After the culture was completed, the GMP concentration was measured using high-performance liquid chromatography, and the GMP concentration in the culture solution of each test strain is shown in Table 2 below.

[0087] [Table 2]

[0088] Comparison of GMP degradation by Corynebacterium ammoniagenes ATCC6872, CJG0497, CJG0498, CJG0499, CJG0500, and CJG0501

[0089]

[0090] The results, as shown in Table 2, confirmed that Corynebacterium ammoniagenes ATCC6872 (parent strain) degraded 3.56 g / l of GMP, but Corynebacterium ammoniagenes CJG0497, CJG0498, CJG0499, CJG0500 and CJG0501 (mutant strains disclosed in the present invention) degraded 0.50 g / l, 3.38 g / l, 2.67 g / l, 2.50 g / l and 2.81 g / l of GMP, respectively.

[0091] That is, it was confirmed that compared with the parental strain, the Corynebacterium ammoniagenes CJG0497 strain of the present disclosure has a reduced GMP degradation rate and can produce GMP with high efficiency and high yield.

[0092] The components of the culture medium used in Example 3 are as follows.

[0093] <GMP Degradation Evaluation Medium>

[0094] 20 g / L GMP, 1.8 g / L phytic acid, 4.8 g / L magnesium sulfate, 3 ml / L nymeen, 2% xylene, 100 mg / l adenine, 7.7 g / l Na2HPO4

[0095] Example 4: Study on XMP Degradation of Mutant Strains for Producing GMP

[0096] To confirm the XMP degradation of Corynebacterium ammoniagenes CJG0497, CJG0498, CJG0499, CJG0500 and CJG0501 obtained in Example 1, the strains were cultured in the following manner.

[0097] Corynebacterium ammoniagenes ATCC6872 (parental strain) and five mutant strains were each inoculated into a 250 ml Erlenmeyer flask containing 25 ml of seed medium, and then cultured with shaking at 30 °C and 200 rpm for 20 hours. 500 μl of XMP degradation evaluation medium was added to 500 μl of the culture solution, and the reaction was carried out at 30 °C for 12 hours to prepare XMP. After the culture was completed, the XMP concentration was measured using high performance liquid chromatography, and the XMP concentrations in the culture solutions of each test strain are shown in Table 3 below.

[0098] [Table 3]

[0099] Comparison of XMP Degradation Amounts of Corynebacterium ammoniagenes ATCC6872, CJG0497, CJG0498, CJG0499, CJG0500 and CJG0501

[0100]

[0101] As a result, as shown in Table 3, it was confirmed that Corynebacterium ammoniagenes ATCC6872 (parental strain) degraded 2.53 g / l of XMP, but Corynebacterium ammoniagenes CJG0497, CJG0498, CJG0499, CJG0500 and CJG0501 (mutant strains of the present disclosure) degraded 1.59 g / l, 2.30 g / l, 2.07 g / l, 1.88 g / l and 2.20 g / l of XMP, respectively.

[0102] That is, it was confirmed that, compared with the parental strain, the Corynebacterium ammoniagenes CJG0497 strain of the present disclosure has a reduced XMP degradation rate and is capable of producing GMP with high efficiency and high yield.

[0103] The components of the culture medium used in Example 4 are as follows.

[0104] <XMP Degradation Evaluation Medium>

[0105] 20 g / L XMP, 1.8 g / L phytic acid, 4.8 g / L magnesium sulfate, 3 ml / L nymeen, 2% xylene, 100 mg / l adenine, 7.7 g / l Na2HPO4

[0106] Example 5: Genetic Analysis of Mutant Strains for Producing GMP

[0107] To analyze the genetic differences of the Corynebacterium ammoniagenes CJG0497 strain obtained in Example 1, a comparative genomic analysis experiment was performed using the wild-type strain ATCC6872 of Corynebacterium ammoniagenes.

[0108] Specifically, the Corynebacterium ammoniagenes CJG0497 and ATCC6872 strains spread on the optimal medium and cultured for at least 24 hours were used to extract genomic DNA (gDNA) for next-generation sequencing (NGS), and were suspended in 300 μL of PBS using 10 μL loops (SPL Inc., Cat. No: 90010). After stirring, 175 μL of the strain suspension was taken and mixed with the lysis / binding solution (a mixture of 232 μL of lysis / binding solution concentrate and 3 μL of carrier NA; components in the MagMaxTM Total Nucleic Acid Isolation Kit), and lysed twice at 6500 rpm for 1 minute each in a bead tube. The subsequent process was carried out using the MagMaxTM Total Nucleic Acid Isolation Kit (Thermo Fisher Scientific Inc., Cat. No. AM184). Quantitative analysis of the obtained gDNA was performed using a Qubit fluorometer (Thermo Fisher Scientific Inc.).

[0109] The gDNA of the strain obtained for long read sequencing was diluted to 1500-ng gDNA / 48-μL DW and used for sequencing using a ligation sequencing kit (Oxford Nanopore Technologies Inc., Cat.No: SQK-LSK109) and a native barcode extension 1-12 (no PCR) kit (Oxford Nanopore Technologies Inc., Cat.No: EXP-NBD104). In addition, sequencing libraries were prepared by performing nicking and gap filling, dA tailing and barcode labeling, and adapter ligation according to the manufacturer's recommended method. Sequence analysis of the prepared library samples was performed for approximately 24 hours using a MinION Mk1C (Oxford Nanopore Technologies Inc.).

[0110] The gDNA of the strain obtained for short-read sequencing was diluted to 100-ng gDNA / 50-μL DW, and DNA fragmentation and barcode labeling were performed 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™ (Thermo Fisher Scientific Inc.) according to the manufacturer's recommended methods. Similarly, adapter ligation, nick repair and library amplification processes were performed according to the manufacturer's recommended methods. The final sequencing library was diluted to a final concentration of 50-pM and amplified by PC Ion 510 TM &Ion 520 TM &Ion 530 TM Emulsion PCR enrichment was performed using Kit-Chef (Thermo Fisher Scientific Inc., Cat. No: A34461) and Ion Chef system (Thermo Fisher Scientific Inc). Finally, the samples were loaded onto the Ion 530 TM On-chip (Ion530 TMSequence analysis was performed using a Chip Kit, Thermo Fisher Scientific Inc., Cat. No: A27764. After sequence analysis, adapter sequences were removed and quality checked using a Torrent Server, and the resulting raw sequence data (raw fastq data) was used for full-length genome assembly and polishing.

[0111] [Table 4]

[0112] Comparative Analysis of Gene Sequence Differences between Corynebacterium ammoniagenes ATCC6872 and CJG0497

[0113] [Table 5]

[0114] Comparative Analysis of Sequence Differences in the Non-coding Regions of Corynebacterium ammoniagenes ATCC6872 and CJG0497

[0115]

[0116] As a result, as shown in Table 4, it was confirmed that non-synonymous mutations of two genes (hrpB (P327S) and sraP (S88F)) were observed in the mutant strain Corynebacterium ammoniagenes CJG0497 of the present disclosure compared with the parent strain Corynebacterium ammoniagenes ATCC6872.

[0117] Furthermore, as shown in Table 5, it was confirmed that nucleotide mutations at two rRNA ORF positions were observed in the mutant strain Corynebacterium ammoniagenes CJG0497 of the present disclosure compared to the parent strain Corynebacterium ammoniagenes ATCC6872.

[0118] That is, it was confirmed that the mutant strain of Corynebacterium ammoniagenes disclosed in the present invention had genetic differences from the parent strain.

[0119] Based on the foregoing, those skilled in the art to which the present disclosure pertains will appreciate that the present disclosure may be implemented in other specific forms without modifying the technical concepts or essential features of the present disclosure. In this regard, the exemplary embodiments disclosed herein are for illustrative purposes only and should not be construed as limiting the scope of the present disclosure. On the contrary, the present disclosure is intended to cover not only the exemplary embodiments, but also various alternatives, modifications, equivalent replacements, and other embodiments that may be included within the spirit and scope of the present disclosure as defined by the appended claims.

[0120] International Form of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure

[0121] To CJ CheilJedang Co., Ltd., The international depositary organizations designated at the bottom of this page are published in accordance with Article 7.1.

[0122] CJ CheilJedang Center, original preservation certificate

[0123] 330, Dongho-ro, Jung-gu, Seoul, 100-400,

[0124] South Korea

[0125]

[0126] 1 Where Article 6.4(d) applies, this date is the date on which the international depositary authority status was obtained.

[0127] Form BP / 4 (single page)

[0128] Korea Microbial Culture Center Korea Culture Collection Association

[0129] Yurim Bldg, 45, Hongjenae-2ga-gil, Seodaemun-gu, Seoul, 03641, South Korea Tel: 82-2-391-0950 Fax: 82-2-392-2859

Claims

1. A Corynebacterium ammoniagenes CJG0497 strain deposited under the accession number KCCM13320P. The strain according to claim 1 , wherein the strain has 5′-guanosine monophosphate (GMP)-producing ability. 3 . The strain according to claim 1 , wherein the strain has the ability to convert 5′-xanthosine monophosphate (XMP) into 5′-guanosine monophosphate (GMP).

4. A method for producing 5'-guanosine monophosphate (GMP), comprising culturing the Corynebacterium ammoniagenes CJG0497 strain deposited under the accession number KCCM13320P in a culture medium. 5 . The method according to claim 4 , further comprising recovering 5′-guanosine monophosphate (GMP) from the cultured strain, a culture product of the strain, a fermentation product of the strain, or a culture medium.

6. A composition for producing 5'-guanosine monophosphate (GMP), comprising the Corynebacterium ammoniagenes CJG0497 strain deposited under the accession number KCCM13320P, a culture product of the strain, a fermentation product of the strain, or a combination of two or more thereof.

7. Use of the Corynebacterium ammoniagenes CJG0497 strain deposited under the accession number KCCM13320P for producing 5'-guanosine monophosphate (GMP).

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