Purine nucleotide-producing microorganisms and method for producing purine nucleotides using the same

Abstract

The present application relates to a microorganism in which the protein activity of formate dehydrogenase is weakened compared to its endogenous activity; a method for producing purine nucleotides, which includes culturing the microorganism in a medium; a composition for producing purine nucleotides, which includes the microorganism, a culture of the microorganism, a fermentation product of the microorganism, or a combination of two or more of these; and use of the microorganism for producing purine nucleotides.

Description

[Technical Field]

[0001] The present application relates to a microorganism in which the protein activity of formate dehydrogenase is weakened compared to its endogenous activity; a method for producing purine nucleotides, which includes culturing the microorganism in a medium; a composition for producing purine nucleotides, which includes the microorganism, a culture of the microorganism, a fermentation product of the microorganism, or a combination of two or more of these; and use of the microorganism for producing purine nucleotides. [Background technology]

[0002] Purine nucleotides, such as 5'-inosine monophosphate (IMP), 5'-xanthosine monophosphate (XMP), and 5'-guanosine monophosphate (GMP), are intermediates in nucleic acid biosynthesis and play important physiological roles in the body. They are widely used in foods and pharmaceuticals. Specifically, IMP imparts a beefy flavor, while GMP, derived from XMP, imparts a mushroom flavor. Both substances are known to enhance the flavor of monosodium glutamate (MSG), making them attractive nucleotide seasonings.

[0003] Methods for producing purine nucleotides include (1) enzymatic degradation of ribonucleic acid (RNA) extracted from yeast cells, (2) fermentation, in which a microorganism that produces the purine nucleotides is cultured and the purine nucleotides are directly recovered from the culture medium, (3) chemical phosphorylation of nucleosides produced by fermentation, and (4) enzymatic phosphorylation of nucleosides produced by fermentation (Korean Patent Registration No. 10-1049023, Japanese Patent Publication No. 4363042, Korean Patent Registration No. 10-1210704, Agri. Biol. Chem., 36(9), 1511-1522). Among these, method (1) has problems with raw material supply and demand and economic feasibility, while method (2) is economically and environmentally advantageous and is widely used. On the other hand, the production of GMP, a purine nucleotide, has the disadvantage of low yield due to cell membrane permeability issues, so a method of enzymatically converting XMP produced by microbial fermentation to produce GMP is also being used.

[0004] However, during the fermentative production of purine nucleotides using microorganisms, the microorganisms are subjected to stresses such as temperature, pH, osmotic pressure, nutrient deficiency, and oxidative factors. Among these, oxidative stress, particularly that caused by reactive oxygen species (ROS), which are unavoidable elements generated during fermentation production, can lead to abnormal growth of the microorganisms.

[0005] Therefore, research into the efficient production of purine nucleotides is still needed. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Korean Patent Registration No. 10-1049023 [Patent Document 2] Patent No. 4363042 [Patent Document 3] Korean Patent Registration No. 10-1210704 [License 4] U.S. Patent No. 7662943 [Patent Document 5] U.S. Patent No. 10584338 [License 6] U.S. Patent No. 10273491 [License 7] Korean Patent Publication No. 10-2020-0136813 [License 8] Korean Patent Publication No. 10-2011-0105662 [License 9] Korean application number 10-2004-0107215 [License 10] Korean Registration Patent No. 10-1950141 [Non-licensed literature]

[0007] [Non-licensed Document 1] Agri.Biol.Chem., 36(9), 1511-1522 [Non-licensed Document 2] Pearson et al (1988)[Proc.Natl.Acad.Sci.USA 85]:2444 [Non-licensed Document 3] Rice et al., 2000, Trends Genet.16:276-277 [Non-licensed Document 4] Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453 [Non-licensed Document 5] Devereux,J.,et al,Nucleic Acids Research 12:387 (1984) [Non-licensed Document 6] Atschul,[S.] [F.,] [ET AL,J MOLEC BIOL 215]:403 (1990) [Non-licensed Document 7] Guide to Huge Computers,Martin J.Bishop,[ED.,] Academic Press,San Diego, 1994 [Non-licensed document 8] [CARILLO et al.](1988) SIAM J Applied Math 48:1073

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

Non-licensed Document 12

Non-licensed Document 13

Non-licensed Document 14

Non-licensed Document 15

Non-licensed Document 16

[0008] The present inventors have developed a microorganism in which the protein activity of formate dehydrogenase is weakened compared to its endogenous activity, a method for producing purine nucleotides which includes culturing said microorganism in a medium, a composition for producing purine nucleotides which includes said microorganism, a culture of said microorganism, a fermentation product of said microorganism or a combination of two or more of these, and use of said microorganism for producing purine nucleotides, and have completed the present application. [Means for solving the problem]

[0009] One aspect of the present application provides a microorganism capable of producing purine nucleotides, in which the protein activity of formate dehydrogenase is weakened compared to the endogenous activity.

[0010] In one specific example, the formate dehydrogenase may consist of any one or more amino acid sequences selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, and combinations thereof.

[0011] As a microorganism according to any one of the above-mentioned specific examples, the microorganism may further be one in which the activity of one or more proteins selected from the group consisting of the following (a) to (c) is regulated: (a) Enhancement of glycine degrading enzyme protein activity compared to endogenous activity; (b) the protein activity of serine hydroxymethyltransferase is enhanced compared to the endogenous activity; and (c) Enhancement of formate-dependent phosphoribosylglycine amidoformyltransferase protein activity compared to endogenous activity.

[0012] As a microorganism according to any one of the above-mentioned embodiments, the microorganism may be a Corynebacterium microorganism.

[0013] In any one of the above-mentioned embodiments, the Corynebacterium microorganism may be Corynebacterium stachyonis.

[0014] In a microorganism according to any one of the above-mentioned embodiments, the microorganism may have an increased ability to produce purine nucleotides compared to an unmodified microorganism.

[0015] Another aspect of the present application provides a method for producing purine nucleotides, comprising culturing in a medium a microorganism in which the protein activity of formate dehydrogenase is weakened compared to the endogenous activity.

[0016] In one embodiment, the method may further comprise the step of recovering a target substance from the cultured microorganism, the culture of the microorganism, the fermentation product of the microorganism, or the culture medium.

[0017] Another aspect of the present application provides a composition for producing purine nucleotides, comprising a microorganism in which the protein activity of formate dehydrogenase is weakened compared to the endogenous activity, a culture of the microorganism, a fermentation product of the microorganism, or a combination of two or more of these.

[0018] Another aspect of the present application provides use of a microorganism in which the protein activity of formate dehydrogenase is weakened compared to the endogenous activity, for producing purine nucleotides. [Effects of the Invention]

[0019] The microorganism of the present invention in which the protein activity of formate dehydrogenase is weakened compared to the endogenous activity can produce purine nucleotides at a high yield and can be usefully utilized for the industrial production of purine nucleotides. DETAILED DESCRIPTION OF THE INVENTION

[0020] 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.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] In this application, terms such as "first, second, third," "i), ii), iii)...," or "(a), (b), (c), (d)..." are used to distinguish between similar components and do not imply sequential or orderly performance. For example, when the terms are used in connection with steps of a method, use, or analysis, these steps may be performed without any time interval between them, simultaneously, or separated by a distance of a few seconds, minutes, hours, days, or months.

[0025] In this application, the term "consisting essentially of" means that unspecified components may be present if the characteristics of the subject matter claimed in this application are not substantially affected by the presence of the unspecified components.

[0026] In this application, the term "consisting of" means that the percentages of the specified component(s) total 100%. The components or features following the term "consisting of" may be essential or mandatory. In some embodiments, other optional or non-essential components may be excluded, other than the components or features following "consisting of."

[0027] 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.

[0028] One aspect of the present application provides a microorganism capable of producing purine nucleotides, in which the protein activity of formate dehydrogenase is weakened compared to the endogenous activity.

[0029] The attenuated formate dehydrogenase protein activity may be defined as, but is not limited to, an increased purine nucleotide productivity of the microorganism of the present application compared to the productivity of a natural wild-type microorganism or an unmodified microorganism (e.g., a microorganism expressing a polypeptide having the protein activity of a wild-type formate dehydrogenase (e.g., any one or more polypeptides selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, and combinations thereof), or a strain in which the protein activity of the formate dehydrogenase of the present application is not attenuated compared to its endogenous activity or has not been attenuated).

[0030] For example, the protein activity of the formate dehydrogenase can be measured by measuring the purine nucleotide productivity or yield, but is not limited thereto.

[0031] In the present application, the term "formate dehydrogenase" refers to an enzyme that is composed of formate dehydrogenase subunit 1 (Fdh subunit 1), formate dehydrogenase subunit 2 (Fdh subunit 2), and formate dehydrogenase subunit 3 (Fdh subunit 3), and catalyzes the oxidation reaction of formate as a substrate to produce NAD +to produce NADH and CO2. Specifically, the formate dehydrogenase of the present application may be, but is not limited to, one or more proteins selected from the group consisting of formate dehydrogenase subunit 1 (Fdh subunit 1), formate dehydrogenase subunit 2 (Fdh subunit 2), and formate dehydrogenase subunit 3 (Fdh subunit 3). The formate dehydrogenase of the present application may also refer to a complex of formate dehydrogenase subunit 1 (Fdh subunit 1), formate dehydrogenase subunit 2 (Fdh subunit 2), and formate dehydrogenase subunit 3 (Fdh subunit 3), and may be used interchangeably with Fdh.

[0032] The term "fdh gene" used herein refers to a gene encoding the formate dehydrogenase of the present application, which is composed of the fdh 1, fdh 2, and fdh 3 genes. Specifically, the term "fdh gene" used herein refers to a gene encoding a complex of formate dehydrogenase subunit 1, formate dehydrogenase subunit 2, and formate dehydrogenase subunit 3 of the present application.

[0033] Specifically, the formate dehydrogenase of the present application may be a protein having the activity of formate dehydrogenase subunit 1 (Fdh subunit 1), formate dehydrogenase subunit 2 (Fdh subunit 2), and formate dehydrogenase subunit 3 (Fdh subunit 3), which are encoded by the fdh 1, fdh 2, and fdh 3 genes, respectively, but is not particularly limited to a specific type as long as it has activity corresponding to formate dehydrogenase. Formate dehydrogenase subunit 1 (Fdh subunit 1), formate dehydrogenase subunit 2 (Fdh subunit 2), and formate dehydrogenase subunit 3 (Fdh subunit 3), encoded by the fdh 1, fdh 2, and fdh 3 genes, respectively, are known in the art, and the amino acid and polynucleotide sequences of formate dehydrogenase subunit 1 (Fdh subunit 1), formate dehydrogenase subunit 2 (Fdh subunit 2), and formate dehydrogenase subunit 3 (Fdh subunit 3), respectively, can be obtained from publicly known databases, such as, but not limited to, NCBI's GenBank.

[0034] For example, the formate dehydrogenase subunit 1 (Fdh subunit 1), formate dehydrogenase subunit 2 (Fdh subunit 2), and formate dehydrogenase subunit 3 (Fdh subunit 3) may comprise the amino acid sequences of SEQ ID NO: 39, SEQ ID NO: 41, and SEQ ID NO: 43, respectively, or amino acid sequences having 60% or more homology or identity thereto, but are not limited thereto as long as they have the protein activity of formate dehydrogenase. Specifically, the polypeptides having the activity of formate dehydrogenase subunit 1 (Fdh subunit 1), formate dehydrogenase subunit 2 (Fdh subunit 2), and formate dehydrogenase subunit 3 (Fdh subunit 3) may have, comprise, consist of, or essentially consist of an amino acid sequence set forth in SEQ ID NO: 39, SEQ ID NO: 41, and SEQ ID NO: 43, respectively, or an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity to SEQ ID NO: 39, SEQ ID NO: 41, and SEQ ID NO: 43, respectively. For example, the formate dehydrogenase subunit 1 (Fdh subunit 1), formate dehydrogenase subunit 2 (Fdh subunit 2), and formate dehydrogenase subunit 3 (Fdh subunit 3) proteins refer to proteins endogenously present in Corynebacterium microorganisms or Corynebacterium stathionis, but are not limited thereto. Specifically, the formate dehydrogenase subunit 1 (Fdh subunit 1) protein having the amino acid sequence of SEQ ID NO: 39, the formate dehydrogenase subunit 2 (Fdh subunit 2) protein having the amino acid sequence of SEQ ID NO: 41, and the formate dehydrogenase subunit 3 (Fdh subunit 3) protein having the amino acid sequence of SEQ ID NO: 43, which are endogenously present in Corynebacterium microorganisms or Corynebacterium stathionis, are not limited thereto.

[0035] In the present application, in relation to an amino acid sequence, a polypeptide "comprising" an amino acid sequence set forth in a particular SEQ ID NO, a polypeptide "consisting of" an amino acid sequence set forth in a particular SEQ ID NO, or a polypeptide or protein "having" an amino acid sequence set forth in a particular SEQ ID NO, is clearly within the scope of the present application, provided that it has the same or corresponding activity as a protein consisting of the amino acid sequence of the SEQ ID NO. For example, addition of sequences before or after the amino acid sequence that do not change the function of the protein, naturally occurring mutations, silent mutations, or conservative substitutions thereof is not excluded, and it is clearly within the scope of the present application, even if such additions or mutations of sequences are present, provided that they have the same or corresponding activity as the mutant protein.

[0036] For example, the variant polypeptide of the present application may have additional sequences, naturally occurring mutations, silent mutations, or conservative substitutions at the N-terminus, C-terminus, and / or internally of the amino acid sequence that do not alter the function of the variant polypeptide. For example, the polypeptide may be conjugated to an N-terminal signal (or leader) sequence of a protein involved in co- or post-translational protein transfer. The polypeptide may also be conjugated to other sequences or linkers that allow the polypeptide to be identified, purified, or synthesized.

[0037] As used herein, the term "conservative substitution" refers to the substitution of one amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions may generally be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. For example, positively charged (basic) amino acids include arginine, lysine, and histidine; negatively charged (acidic) amino acids include glutamic acid and aspartic acid; amino acids with nonpolar side chains (nonpolar amino acids) include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline; and amino acids with polar or hydrophilic side chains (polar amino acids) include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. As another example, amino acids can be classified into electrically charged amino acids (arginine, lysine, histidine, glutamic acid, and aspartic acid) and uncharged amino acids (glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, and glutamine). Phenylalanine, tryptophan, and tyrosine can be classified as aromatic amino acids. Valine, leucine, and isoleucine can be classified as branched-chain amino acids.As another example, the 20 amino acids can be classified by size and divided into five groups, starting with the amino acid group with the smallest volume: glycine, alanine, serine; cysteine, proline, threonine, aspartic acid, asparagine; valine, histidine, glutamic acid, glutamine; isoleucine, leucine, methionine, lysine, arginine; and phenylalanine, tryptophan, and tyrosine. However, conservative substitutions are not necessarily limited to these. Typically, conservative substitutions have little or no effect on the activity of a polypeptide.

[0038] The base sequence encoding the formate dehydrogenase may be a base sequence encoding a protein that exhibits the activity of formate dehydrogenase.

[0039] For example, the nucleotide sequences encoding formate dehydrogenase subunit 1 (Fdh subunit 1), formate dehydrogenase subunit 2 (Fdh subunit 2), and formate dehydrogenase subunit 3 (Fdh subunit 3) may be nucleotide sequences encoding proteins exhibiting formate dehydrogenase activity.

[0040] For example, the formate dehydrogenase subunit 1 (Fdh subunit 1), formate dehydrogenase subunit 2 (Fdh subunit 2), and formate dehydrogenase subunit 3 (Fdh subunit 3), having the amino acid sequences of SEQ ID NOs: 39, 41, and 43, respectively, may be encoded by a polynucleotide having, comprising, consisting of, or essentially consisting of the sequences of SEQ ID NOs: 40, 42, and 44, respectively, or having 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homology or identity to the sequences of SEQ ID NOs: 40, 42, and 44, respectively, but are not limited thereto. The nucleotide sequences of SEQ ID NOs: 40, 42, and 44, respectively, can be obtained from publicly known databases, such as, but not limited to, GenBank of NCBI.

[0041] In the present application, for example, a gene comprising the base sequence of SEQ ID NO: 40 may be used interchangeably with a polynucleotide comprising the base sequence of SEQ ID NO: 40, a gene or polynucleotide having the base sequence of SEQ ID NO: 40, or a gene or polynucleotide consisting of the base sequence of SEQ ID NO: 40.

[0042] The polynucleotides of the present application may be modified in various ways in their coding regions without altering the amino acid sequence of the formate dehydrogenase of the present application, taking into account codon degeneracy or preferred codons in the organism in which the formate dehydrogenase of the present application is to be expressed. Therefore, it is clear that the polynucleotides of the present application include polynucleotides that, due to codon degeneracy, are translated into a polypeptide consisting of the amino acid sequence of the formate dehydrogenase of the present application or a polypeptide having homology or identity thereto. For example, the polynucleotides of the present application may be SEQ ID NO: 40, SEQ ID NO: 42, and / or SEQ ID NO: 44, or degenerated sequences thereof.

[0043] As another example, the polynucleotide of the present application may have or comprise a nucleotide sequence that is 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homologous or identical to SEQ ID NO: 40, SEQ ID NO: 42, and / or SEQ ID NO: 44, or may consist of or essentially consist of a nucleotide sequence that is 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homologous or identical to SEQ ID NO: 40, SEQ ID NO: 42, and / or SEQ ID NO: 44, but is not limited thereto.

[0044] Furthermore, the polynucleotide of the present application may include, without limitation, a probe prepared from a known gene sequence, for example, a sequence that hybridizes under stringent conditions with a complementary sequence to all or part of the polynucleotide sequence of the present application and encodes the formate dehydrogenase of the present application.

[0045] As used herein, the terms "homology" or "identity" refer to the degree of relatedness between two given amino acid or nucleotide sequences, and can be expressed as a percentage. The terms homology and identity are often used interchangeably.

[0046] Sequence homology or identity of conserved polynucleotides or polypeptides can be determined by standard sequence algorithms, optionally with default gap penalties established by the program used. Substantially homologous or identical sequences are generally hybridizable under moderately or highly stringent conditions over at least about 50%, 60%, 70%, 80%, or 90% of the entire sequence or its entire length. Hybridization obviously also includes polynucleotides containing common codons in polynucleotides or codons that take codon degeneracy into account.

[0047] Whether any two polynucleotide or polypeptide sequences have homology, similarity, or identity can be determined using known computer algorithms such as the "FASTA" program using default parameters, for example, as in Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]:2444. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), as implemented in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) (version 5.0.0 or later), as implemented in the GCG program package (Devereux, J., et al., Nucleic Acids Research 12:387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.] [ET AL, J MOLEC BIOL 215]:403 (1990); Guide to Huge Computers, Martin J. Bishop, [Ed.] Academic Press, San Diego, 1994, and CARILLO et al. (1988) SIAM J Applied Math 48:1073. For example, BLAST or ClustalW from the National Center for Biotechnology Information can be used to determine homology, similarity, or identity.

[0048] Homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using a GAP computer program, such as that of Needleman et al. (1970), J Mol Biol. 48:443, as known, for example, in Smith and Waterman, Adv. Appl. Math (1981) 2:482. Briefly, the GAP program defines a match as the total number of symbols in the shorter of the two sequences divided by the number of similar aligned symbols (i.e., nucleotides or amino acids). Default parameters for the GAP program include: (1) unitary matrices (containing values ​​of 1 for identity and 0 for non-identity), the PAM Matrix (see the disclosure by Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation (1978)), the weighted comparison matrix of Gribskov et al. (1986) Nucl. Acids Res. 14:6745 (or the EDNAFULL (the EMBOSS version of NCBI NUC4.4) substitution matrix); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap (or a gap open penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for end gaps.

[0049] Furthermore, whether any two polynucleotide or polypeptide sequences have homology, similarity, or identity can be confirmed by comparing the sequences in a Southern hybridization experiment under defined stringent conditions. Suitable defined hybridization conditions are within the skill of the art and can be determined by methods well known to those skilled in the art (e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual; FM Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York), but are not limited thereto.

[0050] In the present application, the term "stringent conditions" refers to conditions that allow specific hybridization between polynucleotides. Such conditions are specifically described in the literature (see Sambrook et al., supra, 9.50-9.51, 11.7-11.8). For example, conditions include conditions under which polynucleotides with high homology or identity, such as polynucleotides with a homology or identity of 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, hybridize with each other, but polynucleotides with lower homology or identity do not hybridize with each other; or conditions under which washing is performed once, specifically 2 to 3 times, at a salt concentration and temperature equivalent to the washing conditions for conventional Southern hybridization, such as 60°C, 1×SSC, 0.1% SDS, specifically 60°C, 0.1×SSC, 0.1% SDS, more specifically 68°C, 0.1×SSC, 0.1% SDS.

[0051] Such hybridization requires that the two nucleotides have complementary sequences, even though mismatches between bases are possible depending on the stringency of the hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that can hybridize to each other. For example, with respect to DNA, adenosine is complementary to thymine, and cytosine is complementary to guanine. Thus, the polynucleotides of the present application can also include isolated nucleic acid fragments that are complementary to the entire sequence, as well as substantially similar base sequences.

[0052] For example, polynucleotides having homology or identity to the polynucleotides of the present application can be detected using the above-mentioned hybridization conditions, including a hybridization step at a Tm value of 55° C. The Tm value may be, but is not limited to, 60° C., 63° C., or 65° C., and can be appropriately adjusted by those skilled in the art depending on the purpose.

[0053] The appropriate stringency for hybridizing such polynucleotides depends on the length and degree of complementation of the polynucleotides, variables well known in the art (eg, J. Sambrook et al., supra).

[0054] In this application, the term "purine nucleotide" specifically refers to one or more nucleotides selected from the group consisting of 5'-inosine monophosphate (IMP), 5'-xanthosine monophosphate (XMP), and 5'-guanosine monophosphate (GMP). IMP is a compound formed by deamination of adenine, and refers to a nucleotide composed of one molecule of hypoxanthine ribose and one molecule of phosphate. It is biosynthesized from 5'-phosphoribosyl-1-pyrophosphate (PRPP). Specifically, the pyrophosphate group attached to the first carbon atom of PRPP is replaced with a nitrogen atom, and an imidazole ring and a pyrimidine ring are formed in nine steps. XMP refers to a nucleotide dehydrogenated from IMP. GMP can be synthesized from XMP by 5'-inosine-5'-monophosphate dehydrogenase. GMP refers to a nucleotide having a structure in which a phosphate group is ester-bonded to the ribose moiety of a guanosine molecule. GMP can be synthesized by adding an ammonia molecule to XMP by 5'-guanine acid biosynthesis enzyme (GMP synthase). The method for producing GMP from XMP and / or the means used for the method may be selected from known techniques.

[0055] In this application, the term "microorganism (or strain)" includes all wild-type microorganisms and naturally or artificially genetically modified microorganisms, and may be a microorganism in which a specific mechanism is weakened or enhanced by inserting an exogenous gene or by increasing or inactivating the activity of an endogenous gene, and may also be a microorganism that contains a genetic modification for producing a desired polypeptide, protein, or product. In this application, the terms "microorganism" and "strain" may be used interchangeably without limitation.

[0056] For example, the microorganism of the present application may be, but is not limited to, a microorganism (e.g., a recombinant strain) in which the protein activity of formate dehydrogenase is weakened compared to the endogenous activity.

[0057] In this application, the term "microorganism capable of producing purine nucleotides" refers to a prokaryotic or eukaryotic microbial strain capable of producing purine nucleotides within the organism, and includes both microorganisms in which the ability to produce purine nucleotides has been imparted to a parent strain that does not have the ability to produce purine nucleotides, and microorganisms that have the ability to produce purine nucleotides endogenously. The ability to produce purine nucleotides can be imparted or enhanced by species improvement.

[0058] As used herein, the term "unmodified microorganism" does not exclude strains containing mutations that may occur naturally in microorganisms, but refers to a wild-type or naturally occurring strain itself, or a strain before its characteristics are altered by genetic mutations due to natural or artificial factors. The term "unmodified microorganism" may be used interchangeably with "pre-modified strain," "pre-modified microorganism," "non-mutated strain," "non-mutated microorganism," "parent strain before mutation," "wild-type microorganism," "reference microorganism," or "reference microorganism." As used herein, "unmodified microorganism" refers to a strain in which the formate dehydrogenase protein activity of the present application is not attenuated compared to its endogenous activity, or before it is attenuated, but is not limited thereto. Furthermore, as used herein, "unmodified microorganism" may be a microorganism containing the amino acid sequence of SEQ ID NO: 39, SEQ ID NO: 41, and / or SEQ ID NO: 43, or a polynucleotide sequence of SEQ ID NO: 40, SEQ ID NO: 42, and / or SEQ ID NO: 44, but is not limited thereto.

[0059] For purposes of this application, the microorganism of this application includes all microorganisms capable of producing a desired purine nucleotide by attenuating the formate dehydrogenase protein activity relative to its endogenous activity. For example, the microorganism of this application may be, but is not limited to, a genetically modified or recombinant microorganism characterized by an increased ability to produce purine nucleotides due to attenuation of the formate dehydrogenase protein activity relative to its endogenous activity. Specifically, the recombinant strain with increased ability to produce purine nucleotides may be, but is not limited to, a microorganism with increased ability to produce purine nucleotides relative to a native wild-type microorganism or an unmodified microorganism that has endogenous activity of the formate dehydrogenase protein.

[0060] For example, a microorganism capable of producing purine nucleotides is a prokaryotic or eukaryotic microbial strain capable of producing purine nucleotides within the organism, and may include any microorganism that inherently has the ability to produce purine nucleotides or a microorganism in which the ability to produce purine nucleotides has been imparted to a parent strain that does not have the ability to produce purine nucleotides by the weakened formate dehydrogenase protein activity of the present application. The ability to produce purine nucleotides can be imparted or enhanced by species improvement.

[0061] The term "intrinsic activity" refers to the activity of a specific polypeptide that a parent strain or an unaltered microorganism originally possessed before the trait change, when the trait has been changed due to genetic mutation caused by natural or artificial factors. This term may be used interchangeably with "activity before the change."

[0062] In the present application, the term "weakening" of a polypeptide activity is a concept that encompasses weakening of activity compared to the endogenous activity or the absence of activity. The term "weakening" may be used interchangeably with terms such as deficiency, inactivation, deletion, disruption, down-regulation, decrease, attenuation, repression, and reduction.

[0063] For example, the attenuation means a state in which the protein exhibits activity but is not completely inactivated by deletion, and the activity of the protein is weakened compared to that of a non-transformed microorganism, a wild-type strain, or a parent strain, but is not limited thereto.

[0064] For example, the attenuation may be, but is not limited to, inactivation, which means that the expression of a protein comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, and combinations thereof is not expressed at all, or even if expressed, its activity is absent or attenuated, compared to that of a parent strain or an unmodified strain.

[0065] The weakening may include cases where the activity of the polypeptide itself is weakened or eliminated compared to the activity of the polypeptide originally possessed by the microorganism due to a mutation in the polynucleotide encoding the polypeptide, cases where the overall polypeptide activity in the cell is lower than that of a wild-type strain due to inhibition of expression of the gene encoding it or inhibition of translation into the polypeptide, cases where the gene is not expressed at all, and cases where the gene is expressed but the polypeptide has no activity.

[0066] "Polypeptide activity is weakened compared to its intrinsic activity" means that the activity of a particular polypeptide is lower than that originally possessed by the parent strain or non-transformed microorganism before transformation. Whether or not the activity of the polypeptide is weakened can be confirmed by examining the activity, expression level, or amount of a product secreted from the polypeptide.

[0067] For example, the attenuation may be such that the activity of the formate dehydrogenase protein is less than about 100%, about 90% or less, about 80% or less, about 70% or less, about 60% or less, about 50% or less, about 40% or less, about 30% or less, about 20% or less, about 10% or less, about 5% or less, or 0% of the activity of the formate dehydrogenase protein of the parent strain or untransformed microorganism before transformation, but is not limited thereto.

[0068] For example, the inactivation means that the formate dehydrogenase protein is not expressed at all, or is expressed but has no or weakened activity compared to an untransformed microorganism.

[0069] The activity of such a polypeptide can be attenuated by any method known in the art, but is not limited to this, and may be achieved by applying various methods well known in the art (e.g., Nakashima N et al., Bacterial cellular engineering by genome editing and gene silencing. Int J Mol Sci. 2014; 15(2): 2773-2793, Sambrook et al. Molecular Cloning 2012, etc.).

[0070] Specifically, the attenuation of the activity of the polypeptide of the present application is 1) Deletion of all or part of the gene encoding the polypeptide; 2) Modifying the expression control region (or expression control sequence) so that expression of the gene encoding the polypeptide is attenuated; 3) modification of the amino acid sequence constituting the polypeptide (e.g., deletion / substitution / addition of one or more amino acids in the amino acid sequence) so as to eliminate or attenuate the activity of the polypeptide; 4) modification of a polynucleotide sequence encoding a polypeptide so as to eliminate or attenuate the activity of the polypeptide (e.g., deletion / substitution / addition of one or more nucleic acid bases in the nucleic acid base sequence of the polypeptide gene so as to encode a polypeptide that has been altered so as to eliminate or attenuate the activity of the polypeptide); 5) a modification of the initiation codon or 5'-UTR region nucleotide sequence of a gene encoding a polypeptide; 6) introduction of an antisense oligonucleotide (e.g., antisense RNA) that binds complementary to the transcript of the gene encoding the polypeptide; 7) Addition of a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of a gene encoding a polypeptide to form a secondary structure that prevents ribosome attachment; 8) Addition of a promoter transcribed in the opposite direction to the 3' end of the ORF (open reading frame) of the polynucleotide sequence encoding the polypeptide (reverse transcription engineering, RTE); or 9) It may be a combination of two or more selected from the above 1) to 8), but is not particularly limited thereto.

[0071] for example, The deletion of a part or all of the gene encoding the polypeptide (1) may be removal of the entire polynucleotide encoding the endogenous target polypeptide in the chromosome, replacement with a polynucleotide lacking a partial nucleotide sequence, or replacement with a marker gene.

[0072] Methods for deleting a portion or all of such polynucleotides include, but are not limited to, deleting the polynucleotide through homologous recombination using a vector for chromosomal insertion in a microorganism, or inducing mutations using light such as ultraviolet light or chemicals, and then selecting a strain in which the target gene is deleted from the resulting mutants. Methods for deleting a portion or all of the gene include DNA recombination techniques. For example, a nucleotide sequence or vector containing a nucleotide sequence homologous to the target gene may be injected into the microorganism to cause homologous recombination, thereby deleting a portion or all of the gene. The injected nucleotide sequence or vector may contain, but is not limited to, a dominant selectable marker.

[0073] Furthermore, the modification of the expression regulatory sequence (2) may involve the generation of a mutation in the expression regulatory region (or expression regulatory sequence) by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, or replacement with a sequence having a weaker activity. The expression regulatory region may include, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating the termination of transcription and translation.

[0074] Furthermore, the modifications of the amino acid sequence or polynucleotide sequence in 3) and 4) above may include, but are not limited to, the generation of sequence mutations by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide, so as to attenuate the activity of the polypeptide, or the replacement with an amino acid sequence or polynucleotide sequence that has been improved to have weaker activity or an amino acid sequence or polynucleotide sequence that has been improved to have no activity. For example, but not limited to, the introduction of a mutation in the polynucleotide sequence to form a stop codon may inhibit or attenuate gene expression.

[0075] Furthermore, the nucleotide sequence modification of the start codon or 5'-UTR region of the gene encoding the polypeptide may be, for example, but is not limited to, substituting another start codon that has a lower polypeptide expression rate than the endogenous start codon.

[0076] 6) The introduction of an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the transcript of the gene encoding the polypeptide can be carried out by referring to, for example, the literature [Weintraub, H. et al., Antisense-RNA as a molecular tool for genetic analysis, Reviews - Trends in Genetics, Vol. 1(1) 1986].

[0077] 7) Addition of a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of a gene encoding a polypeptide to form a secondary structure that prevents ribosome attachment may disable or slow down mRNA translation.

[0078] 8) Addition of a promoter transcribed in the opposite direction to the 3' end of the ORF (open reading frame) of a polynucleotide sequence encoding a polypeptide (reverse transcription engineering, RTE) may be performed to attenuate activity by creating an antisense nucleotide complementary to the transcript of the gene encoding the polypeptide.

[0079] Modification of a portion or all of a polynucleotide in the microorganism of the present application may be induced by, but is not limited to, (a) homologous recombination using a vector for chromosomal insertion in the microorganism or genome editing using engineered nucleases (e.g., CRISPR-Cas9) and / or (b) light and / or chemical treatment, such as ultraviolet light and radiation. Methods for modifying a portion or all of the gene include DNA recombination techniques. For example, a nucleotide sequence or vector containing a nucleotide sequence homologous to a target gene may be injected into the microorganism to cause homologous recombination, thereby deleting a portion or all of the gene. The injected nucleotide sequence or vector may contain, but is not limited to, a dominant selectable marker.

[0080] For example, the recombinant microorganism capable of producing purine nucleotides of the present application may include any microorganism that is transformed with a vector and has attenuated formate dehydrogenase protein activity of the present application, thereby being capable of producing purine nucleotides.

[0081] The vector of the present application may comprise a DNA construct comprising a base sequence of a polynucleotide encoding a polypeptide of interest operably linked to a suitable expression control region (or expression control sequence) so as to enable expression of the polypeptide of interest in a suitable host. The expression control region may comprise a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosomal binding site, and a sequence regulating the termination of transcription and translation. After being transformed into a suitable host cell, the vector can replicate or function independently of the host genome, or it may be integrated into the genome itself.

[0082] The vectors used in this application are not particularly limited, and any vector known in the art can be used. Examples of commonly used vectors include naturally occurring or recombinant plasmids, cosmids, viruses, and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage or cosmid vectors, and pDZ, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, pSK, pSKH, and pET can be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pSK, pSKH130, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC vectors can be used.

[0083] For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome via a vector for chromosomal integration in a cell. The polynucleotide can be inserted into a chromosome by any method known in the art, including, but not limited to, homologous recombination. A selection marker for confirming the presence or absence of insertion into the chromosome may be further included. The selection marker is used to select cells transformed with the vector, i.e., to confirm the presence or absence of insertion of the target nucleic acid molecule. A marker that confers a selectable phenotype, such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of a surface polypeptide, may be used. In an environment treated with a selective agent, only cells expressing the selection marker will survive or exhibit other phenotypes, allowing the selection of transformed cells.

[0084] The term "transformation" as used herein refers to introducing a vector containing a polynucleotide encoding a target polypeptide into a host cell or microorganism, thereby enabling the expression of the polypeptide encoded by the polynucleotide in the host cell. A transformed polynucleotide may include any polynucleotide, whether it is located intrachromosomally or extrachromosomally, as long as it is expressible in the host cell. The polynucleotide may also include DNA and / or RNA encoding the target polypeptide. The polynucleotide may be introduced in any form that can be introduced and expressed in the host cell. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a genetic construct containing all elements necessary for its own expression. The expression cassette typically includes a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the polynucleotide. The expression cassette may be in the form of an autonomously replicating expression vector. The polynucleotide may also be introduced into the host cell in its own form, operably linked to sequences necessary for expression in the host cell, but is not limited thereto.

[0085] As used herein, the term "operably linked" refers to a configuration in which a regulatory sequence is positioned appropriately so that it directs the expression of a coding sequence. Thus, "operably linked" includes known regulatory regions, such as promoters, terminators, signal sequences, or enhancer regions, or functional domains having a desired activity, attached or linked to a target (gene or polypeptide) so that the expression, secretion, or function of the target can be regulated by the known or desired activity. For example, "operably linked" refers to a polynucleotide sequence being operably linked to a promoter sequence that initiates and mediates transcription of the polynucleotide encoding the target mutant polypeptide of the present application.

[0086] In this application, the term "expression" includes any step involved in the production of a polypeptide, such as, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0087] As used herein, the term "expression vector" refers to a linear or circular nucleic acid molecule that contains a coding sequence and regulatory sequences operably linked thereto for the expression thereof.

[0088] In this application, the term "regulatory sequence" refers to a polynucleotide sequence required for the expression of a coding sequence. Each regulatory sequence may be native to the coding sequence (having the same origin) or foreign (derived from another gene). Examples of the regulatory sequence include a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal peptide sequence, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating transcription and translation termination. The minimum unit of the regulatory sequence may include a promoter, and a transcription and translation termination sequence.

[0089] As used herein, the term "recombinant" in reference to a cell, polynucleotide, polypeptide, or vector means that the cell, polynucleotide, polypeptide, or vector has been transformed by the introduction of a heterologous nucleic acid or polypeptide or the alteration of a naturally occurring polynucleotide or polypeptide, or that the cell is derived from a cell so transformed. Thus, for example, a recombinant cell can express genes that are not found within the native (non-recombinant) form of the cell, or express native genes that are not expressed or not expressed at all or are aberrantly expressed.

[0090] For example, the microorganism that produces the purine nucleotide may be a microorganism that endogenously contains a protein consisting of the amino acid sequence of SEQ ID NO:39, SEQ ID NO:41, and / or SEQ ID NO:43, or a protein consisting of an amino acid sequence having at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% or more homology or identity to SEQ ID NO:39, SEQ ID NO:41, and / or SEQ ID NO:43.

[0091] For example, the microorganism that produces the purine nucleotide may be a microorganism that endogenously contains a polynucleotide sequence capable of encoding a protein comprising an amino acid sequence having at least 80% homology to SEQ ID NO:39, SEQ ID NO:41, and / or SEQ ID NO:43, a nucleotide sequence of SEQ ID NO:40, SEQ ID NO:42, and / or SEQ ID NO:44, or a nucleotide sequence that has 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homology or identity to the nucleotide sequence of SEQ ID NO:40, SEQ ID NO:42, and / or SEQ ID NO:44.

[0092] The microorganisms of the present application may include all microorganisms in which the protein activity of formate dehydrogenase has been weakened compared to the endogenous activity by various known methods.

[0093] As a specific example, the microorganism of the present application in which the formate dehydrogenase protein activity is weakened compared to the endogenous activity may be, but is not limited to, a microorganism in which the polynucleotide encoding the formate dehydrogenase protein is deleted or modified, specifically, a microorganism in which the nucleotide sequences of SEQ ID NO: 40, SEQ ID NO: 42, and SEQ ID NO: 44 are deleted and the microorganism has no formate dehydrogenase protein activity, but is not limited to this.

[0094] For example, the microorganism of the present application having increased purine nucleotide-producing ability may be, but is not limited to, a microorganism having increased purine nucleotide-producing ability compared to a non-modified microorganism. For example, the non-modified microorganism, which is the subject strain for comparing the increase in purine nucleotide-producing ability, may be, but is not limited to, the CJX1664 strain or the KCCM12151P strain.

[0095] For example, the microorganism having increased purine nucleotide productivity may have an increase of about 1% or more, specifically about 1% or more, about 2.5% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, or about 35% or more (there is no particular upper limit, and it may be, for example, about 200% or less, about 150% or less, about 100% or less, about 50% or less, about 45% or less, or about 40% or less), compared to the purine nucleotide productivity of the parent strain or unmodified microorganism before mutation, but is not limited thereto as long as there is an increase in the + value compared to the productivity of the parent strain or unmodified microorganism before mutation. In other examples, the recombinant strain with increased purine nucleotide production ability may have an increased purine nucleotide production ability of about 1.1 times or more, about 1.15 times or more, about 1.2 times or more, about 1.25 times or more, about 1.3 times or more, or about 1.35 times or more (there is no particular upper limit, and the increase may be, for example, about 10 times or less, about 5 times or less, about 3 times or less, about 2 times or less, about 1.5 times or less, or about 1.4 times or less) compared to the parent strain or untransformed microorganism before mutation, but is not limited thereto.

[0096] For example, the microorganism capable of producing purine nucleotides may be either a prokaryotic cell or a eukaryotic cell, specifically a prokaryotic cell. Examples of the prokaryotic cell include microbial strains belonging to the genera Escherichia, Erwinia, Serratia, Providencia, Corynebacteria, Pseudomonas, Leptospira, Salmonella, Brevibacteria, Hypomonas, Chromobacterium, and Norcardia, or fungi or yeast. Specifically, the microorganisms include strains of the genera Escherichia, Corynebacterium, and Leptospira, and yeasts. More specifically, the microorganisms may be strains of the genus Corynebacterium.

[0097] As a microorganism according to any one of the above-mentioned embodiments, the microorganism of the present application may be a Corynebacterium microorganism.

[0098] As an example of the present application, the microorganism of the present application is Corynebacterium stationis, Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris, or Corynebacterium flavescens. Specifically, the microorganism of the present application may be a Corynebacterium microorganism, more specifically, Corynebacterium stationis or Corynebacterium glutamicum, but is not limited thereto.

[0099] The microorganism capable of producing purine nucleotides of the present application may further be a microorganism in which the activity of one or more proteins selected from the group consisting of the following (a) to (c) is regulated, thereby improving the ability to produce purine nucleotides, but is not limited thereto: (a) Enhancement of glycine degrading enzyme protein activity compared to endogenous activity; (b) the protein activity of serine hydroxymethyltransferase is enhanced compared to the endogenous activity; and (c) Enhancement of formate-dependent phosphoribosylglycine amidoformyltransferase protein activity compared to endogenous activity.

[0100] As a microorganism according to any one of the above-mentioned specific examples, the microorganism capable of producing purine nucleotides of the present application may further be a microorganism in which the activity of one or more proteins selected from the group consisting of the following (a) to (d) is regulated, thereby improving the ability to produce purine nucleotides, but is not limited thereto: (a) Enhancement of glycine degrading enzyme protein activity compared to endogenous activity; (b) enhanced serine hydroxymethyltransferase protein activity compared to endogenous activity; (c) enhanced formate-dependent phosphoribosylglycine amidoformyltransferase protein activity compared to endogenous activity; and (d) The protein activity of serine dehydratase is weakened compared to the endogenous activity.

[0101] As used herein, the term "enhancement" of a polypeptide activity means that the activity of the polypeptide is enhanced compared to its endogenous activity. The term "enhancement" may be interchangeably used with terms such as activation, up-regulation, overexpression, and enhancement.

[0102] The enhancement can include exhibiting an activity that the substance did not originally have, or exhibiting an activity that is improved compared to the intrinsic activity or the activity before the modification.

[0103] For example, the "exhibition of an activity not originally possessed" may refer to, but is not limited to, "introduction of a protein." The introduction of a protein refers to the expression in the microorganism of a gene not originally possessed by the microorganism, thereby exhibiting the activity of a specific protein, or the activity of the protein being enhanced or improved compared to the endogenous activity or activity before modification. For example, the introduction may refer to the introduction of a polynucleotide encoding a specific protein into a chromosome in the microorganism, or the introduction of a vector containing a polynucleotide encoding a specific protein into the microorganism, resulting in the exhibiting of the activity.

[0104] The term "intrinsic activity" refers to the activity of a specific polypeptide that a parent strain or an unaltered microorganism originally possessed before the trait change, when the trait has been changed due to genetic mutation caused by natural or artificial factors. This term may be used interchangeably with "activity before the change."

[0105] The activity of a polypeptide being enhanced compared to its endogenous activity means that the activity and / or concentration (expression level) of a particular polypeptide is improved compared to that originally possessed by the parent strain or non-transformed microorganism before transformation.

[0106] By way of example, the enhancement may be, but is not limited to, an enhancement of the corresponding protein activity, or an enhancement of its activity or concentration, generally by about 1%, about 10%, about 25%, about 50%, about 75%, about 100%, about 150%, about 200%, about 300%, about 400%, or about 500%, up to about 1000% or about 2000% or more, based on the activity or concentration of the wild-type protein or the initial microbial strain.

[0107] The activity of the polypeptide can be enhanced by introducing an exogenous polypeptide or by enhancing the activity of an endogenous polypeptide. Whether the activity of the polypeptide is enhanced can be confirmed by enhancing the activity, expression level, or amount of a product secreted from the polypeptide.

[0108] The activity of the polypeptide can be enhanced by various methods well known in the art, and is not limited thereto, as long as the activity of the polypeptide of interest can be enhanced compared to that of the microorganism before transformation. Specifically, the enhancement may be achieved by using genetic engineering and / or protein engineering, which are routine methods in molecular biology and well known to those skilled in the art, but is not limited thereto (e.g., Sitnicka et al., Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol. 2, 1-16; Sambrook et al., Molecular Cloning 2012, etc.).

[0109] Specifically, the activity of the polypeptide of the present application is enhanced by: 1) enhancing the intracellular copy number of a polynucleotide encoding a polypeptide; 2) Modification of the expression regulatory region of the gene on the chromosome encoding the polypeptide (e.g., mutation within the expression regulatory region, replacement with a sequence having stronger activity, or insertion of a sequence having stronger activity); 3) a modification of the nucleotide sequence encoding the initiation codon or 5'-UTR region of a gene transcript encoding a polypeptide; 4) modifying the amino acid sequence of the polypeptide so that the polypeptide activity is enhanced; 5) modifying a polynucleotide sequence encoding the polypeptide so as to enhance the polypeptide's activity (e.g., modifying the polynucleotide sequence of the polypeptide gene so as to encode a polypeptide modified so as to enhance the polypeptide's activity); 6) introduction of a foreign polypeptide exhibiting the activity of the polypeptide or a foreign polynucleotide encoding the same; 7) codon optimization of the polynucleotide encoding the polypeptide; 8) analyzing the tertiary structure of the polypeptide and selectively altering or chemically modifying exposed sites; or 9) It may be a combination of two or more selected from the above 1) to 8), but is not particularly limited thereto.

[0110] for example, The 1) intracellular copy number of a polynucleotide encoding a polypeptide may be increased by introducing into a host cell a vector operably linked to the polynucleotide encoding the polypeptide, which can replicate and function independently of the host. Alternatively, one or more copies of the polynucleotide encoding the polypeptide may be introduced into a chromosome in the host cell. The introduction into a chromosome can be achieved by, but is not limited to, introducing into the host cell a vector capable of inserting the polynucleotide into a chromosome in the host cell. The vector is as described above.

[0111] The 2) replacement of the expression regulatory region (or expression regulatory sequence) of a gene on a chromosome encoding a polypeptide with a sequence with stronger activity may be, for example, by introducing a mutation in the sequence through deletion, insertion, non-conservative or conservative substitution, or a combination thereof, to further enhance the activity of the expression regulatory region, or by replacing the sequence with a sequence with stronger activity. The expression regulatory region may include, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating the termination of transcription and translation. For example, the original promoter may be replaced with a strong promoter, but this is not limited thereto.

[0112] Examples of known strong promoters include, but are not limited to, the cj1 to cj7 promoters (U.S. Patent No. 7,662,943 B2), the lac promoter, the trp promoter, the trc promoter, the tac promoter, the lambda phage PR promoter, the PL promoter, the tet promoter, the gapA promoter, the SPL7 promoter, the SPL13 (sm3) promoter (U.S. Patent No. 10,584,338 B2), the O2 promoter (U.S. Patent No. 10,273,491 B2), the tkt promoter, and the yccA promoter.

[0113] The nucleotide sequence modification of the start codon or 5'-UTR region of the gene encoding the polypeptide may be, for example, but is not limited to, substituting another start codon that results in a higher polypeptide expression rate than the endogenous start codon.

[0114] The modification of the amino acid sequence or polynucleotide sequence in 4) and 5) above may be, but is not limited to, a sequence mutation such as deletion, insertion, non-conservative or conservative substitution, or a combination thereof, in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide, or an amino acid sequence or polynucleotide sequence modified to have stronger activity or to have enhanced activity, so as to enhance the activity of the polypeptide. Specifically, the modification can be performed by, but is not limited to, inserting the polynucleotide into a chromosome by homologous recombination. The vector used in this case may further contain a selection marker for detecting the presence or absence of insertion into the chromosome. The selection marker is as described above.

[0115] The introduction of an exogenous polynucleotide that exhibits the activity of a polypeptide (6) may be the introduction of an exogenous polynucleotide that encodes a polypeptide that exhibits the same or similar activity as the polypeptide into a host cell. The exogenous polynucleotide is not limited in its origin or sequence, as long as it exhibits the same or similar activity as the polypeptide. The method used for the introduction can be any known transformation method appropriately selected by those skilled in the art. The introduced polynucleotide is expressed in a host cell to produce a polypeptide, and its activity can be enhanced.

[0116] The codon optimization of the polynucleotide encoding the polypeptide (7) may be that of an endogenous polynucleotide that has been codon-optimized to enhance transcription or translation in a host cell, or that of an exogenous polynucleotide that has been codon-optimized to optimize transcription and translation in a host cell.

[0117] 8) Analyzing the tertiary structure of a polypeptide and selecting and modifying or chemically modifying exposed sites may involve, for example, comparing the sequence information of the polypeptide to be analyzed with a database storing sequence information of known proteins, determining candidate template proteins according to the degree of sequence similarity, confirming the structure based on the candidate template proteins, and selecting and modifying exposed sites to be modified or chemically modified.

[0118] Such enhancement of polypeptide activity may be, but is not limited to, an enhancement of the activity or concentration of the corresponding polypeptide relative to the activity or concentration of the polypeptide expressed in a wild-type or unaltered microbial strain, or an increase in the amount of product produced from the polypeptide.

[0119] As an example of the present application, the microorganism capable of producing purine nucleotides of the present application may be a microorganism in which the activity of a glycine degrading enzyme protein is further enhanced compared to the endogenous activity, thereby improving the ability to produce purine nucleotides, but is not limited thereto.

[0120] In the present application, the term "glycine degrading enzyme protein" refers to a protein directly or indirectly involved in the glycine degradation pathway, and may refer to each protein constituting the glycine degradation system, a complex of such proteins, or the glycine degradation system itself. Specifically, the glycine degrading enzyme protein may be, but is not limited to, one or more proteins selected from the group consisting of T-protein (GcvT), P-protein (GcvP), L-protein (GcvL), and H-protein (GcvH) constituting the glycine degradation system, and coenzymes LipB and LipA of the glycine degradation system (John E. Cronan, Microbiology and Molecular Biology Reviews, April 13, 2016). The glycine degrading enzyme protein of the present application may be used in combination with GcvPTH or the glycine cleavage system.

[0121] In this application, the term "glycine cleavage system (GCV)" refers to a system that is composed of two or more subunits, namely, the subunits GcvP, GcvT, and GcvH, and cleaves carbon dioxide, ammonium ion, and N from glycine. 5-10 -methylenetetrahydrofolate. The glycine cleavage system of the present application can be used in conjunction with a glycine degradation system.

[0122] Specifically, the glycine degrading enzyme protein of the present application may be a protein having the activity of the GcvP, GcvT, and GcvH proteins encoded by the GcvP, GcvT, and gcvH genes, respectively, but is not particularly limited to its type as long as it has the activity corresponding to the glycine degrading enzyme protein. The GcvP, GcvT, and GcvH proteins encoded by the GcvP, GcvT, and gcvH genes, respectively, are known in the art, and the amino acid and polynucleotide sequences of the GcvP, GcvT, and GcvH proteins can be obtained from publicly known databases, such as, but not limited to, NCBI's GenBank.

[0123] For example, the GcvP, GcvT, and GcvH proteins may comprise the amino acid sequences of SEQ ID NOs: 47, 49, and 51, respectively, or amino acid sequences having 60% or more homology or identity thereto, but are not limited thereto as long as they have glycine decomposition enzyme protein activity. Specifically, the polypeptides having GcvP, GcvT, and GcvH protein activity may have, comprise, consist of, or essentially consist of the amino acid sequences of SEQ ID NOs: 47, 49, and 51, respectively, or amino acid sequences having at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity thereto. For example, the GcvP, GcvT, and GcvH proteins refer to proteins endogenously present in Corynebacterium microorganisms or Corynebacterium stathionis, but are not limited thereto. Specifically, the GcvP protein having the amino acid sequence of SEQ ID NO: 47, the GcvT protein having the amino acid sequence of SEQ ID NO: 49, and the GcvH protein having the amino acid sequence of SEQ ID NO: 51, which are endogenously present in Corynebacterium microorganisms or Corynebacterium stathionis, are not limited thereto.

[0124] Furthermore, the nucleotide sequences encoding the GcvP, GcvT, and GcvH proteins may be nucleotide sequences encoding proteins that exhibit the activity of glycine decomposition enzyme proteins.

[0125] For example, the GcvP, GcvT, and GcvH proteins having the amino acid sequences of SEQ ID NOs: 47, 49, and 51, respectively, may be encoded by a polynucleotide having, comprising, consisting of, or essentially consisting of the sequences of SEQ ID NOs: 48, 50, and 52, respectively, or having 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homology or identity to the sequences of SEQ ID NOs: 48, 50, and 52, respectively, but are not limited thereto. The nucleotide sequences of SEQ ID NOs: 48, 50, and 52, respectively, can be obtained from publicly known databases, such as, but not limited to, NCBI's GenBank.

[0126] As an example of the present application, the microorganism capable of producing purine nucleotides of the present application may be a microorganism in which the protein activity of serine hydroxymethyltransferase is further enhanced compared to the endogenous activity, thereby improving the ability to produce purine nucleotides, but is not limited thereto.

[0127] As used herein, the term "serine hydroxymethyltransferase" refers to an enzyme that catalyzes the conversion of serine to glycine. The term "serine hydroxymethyltransferase" as used herein may be interchangeable with "GlyA." Specifically, the serine hydroxymethyltransferase of the present application may be a protein having serine hydroxymethyltransferase activity encoded by the glyA gene, but is not particularly limited to its type as long as it has activity corresponding to that of serine hydroxymethyltransferase. The serine hydroxymethyltransferase encoded by the glyA gene is known in the art, and the amino acid and polynucleotide sequences of the serine hydroxymethyltransferase can be obtained from publicly known databases, such as, but not limited to, GenBank at NCBI.

[0128] For example, the serine hydroxymethyltransferase protein may include the amino acid sequence of SEQ ID NO: 53 or an amino acid sequence having 60% or more homology or identity thereto, but is not limited thereto as long as it has the protein activity of serine hydroxymethyltransferase. Specifically, the polypeptide having the protein activity of serine hydroxymethyltransferase may have, comprise, consist of, or essentially consist of the amino acid sequence of SEQ ID NO: 53 or an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity thereto. For example, the serine hydroxymethyltransferase protein refers to a protein endogenously present in Corynebacterium microorganisms or Corynebacterium stathionis, but is not limited thereto. Specifically, the serine hydroxymethyltransferase protein may be endogenously present in Corynebacterium microorganisms or Corynebacterium stathionis and have the amino acid sequence of SEQ ID NO: 53, but is not limited thereto.

[0129] The base sequence encoding the serine hydroxymethyltransferase may also be a base sequence encoding a protein that exhibits the activity of serine hydroxymethyltransferase.

[0130] For example, the serine hydroxymethyltransferase protein having the amino acid sequence of SEQ ID NO: 53 may be encoded by a polynucleotide having, comprising, consisting of, or essentially consisting of the sequence of SEQ ID NO: 54 or a nucleotide sequence having 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or less than 100% homology or identity to the sequence of SEQ ID NO: 54, but is not limited thereto. The nucleotide sequence of SEQ ID NO: 54 can also be obtained from publicly known databases, such as, but not limited to, GenBank of NCBI.

[0131] As an example of the present application, the microorganism capable of producing purine nucleotides of the present application may be a microorganism in which the protein activity of formate-dependent phosphoribosylglycine amidoformyltransferase is further enhanced compared to the endogenous activity, thereby improving the ability to produce purine nucleotides, but is not limited thereto.

[0132] In the present application, the term "formate-dependent phosphoribosylglycine amidoformyltransferase" refers to an enzyme that catalyzes the chemical reaction 10-formyltetrahydrofolate + N1-(5-phospho-D-ribosyl)glycinamide ⇔ tetrahydrofolate + N2-formyl-N1-(5-phospho-D-ribosyl)glycinamide. The term "formate-dependent phosphoribosylglycine amidoformyltransferase" in the present application may be used interchangeably with "PurT." Specifically, the formate-dependent phosphoribosylglycine amidoformyltransferase in the present application may be a protein encoded by the purT gene and having formate-dependent phosphoribosylglycine amidoformyltransferase activity. However, the type of the formate-dependent phosphoribosylglycine amidoformyltransferase is not particularly limited as long as it has activity corresponding to that of a formate-dependent phosphoribosylglycine amidoformyltransferase. The formate-dependent phosphoribosylglycine amidoformyltransferase encoded by the purT gene is known in the art, and the amino acid and polynucleotide sequences of the formate-dependent phosphoribosylglycine amidoformyltransferase can be obtained from publicly known databases, such as, but not limited to, GenBank at NCBI.

[0133] For example, the formate-dependent phosphoribosylglycine amidoformyltransferase protein may include the amino acid sequence of SEQ ID NO: 55 or an amino acid sequence having 60% or more homology or identity thereto, but is not limited thereto as long as it has the protein activity of a formate-dependent phosphoribosylglycine amidoformyltransferase. Specifically, the polypeptide having the protein activity of a formate-dependent phosphoribosylglycine amidoformyltransferase may have, comprise, consist of, or essentially consist of the amino acid sequence of SEQ ID NO: 55 or an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity thereto. For example, the formate-dependent phosphoribosylglycine amidoformyltransferase protein refers to a protein endogenously present in Corynebacterium microorganisms or Corynebacterium stathionis, but is not limited thereto. Specifically, the formate-dependent phosphoribosylglycine amidoformyltransferase protein may be endogenously present in Corynebacterium microorganisms or Corynebacterium stathionis and have the amino acid sequence of SEQ ID NO: 55, but is not limited thereto.

[0134] The base sequence encoding the formate-dependent phosphoribosylglycine amidoformyltransferase may also be a base sequence encoding a protein that exhibits the activity of formate-dependent phosphoribosylglycine amidoformyltransferase.

[0135] For example, the formate-dependent phosphoribosylglycine amidoformyltransferase protein having the amino acid sequence of SEQ ID NO: 55 may be encoded by a polynucleotide having, comprising, consisting of, or essentially consisting of the sequence of SEQ ID NO: 56 or a nucleotide sequence having 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or less than 100% homology or identity to the sequence of SEQ ID NO: 56, but is not limited thereto. The nucleotide sequence of SEQ ID NO: 56 can also be obtained from publicly known databases, such as, but not limited to, NCBI's GenBank.

[0136] As an example of the present application, the microorganism capable of producing purine nucleotides of the present application may be a microorganism in which the protein activity of serine dehydratase is weakened compared to the endogenous activity, thereby improving the ability to produce purine nucleotides, but is not limited thereto.

[0137] The term "serine dehydratase (L-serine dehydratase)" used herein refers to an enzyme that catalyzes the chemical reaction L-serine = pyruvate + NH3. The term "serine dehydratase" used herein may be interchangeable with "SdaA" or "L-serine ammonialytic enzyme." Specifically, the serine dehydratase used herein may be a protein having the activity of a serine dehydratase encoded by the sdaA gene, but is not particularly limited to the type of protein as long as it has the activity corresponding to a serine dehydratase. The serine dehydratase encoded by the sdaA gene is known in the art, and the amino acid and polynucleotide sequences of the serine dehydratase can be obtained from publicly known databases, such as, but not limited to, NCBI's GenBank.

[0138] For example, the serine dehydratase protein may include the amino acid sequence of SEQ ID NO: 45 or an amino acid sequence having 60% or more homology or identity thereto, but is not limited thereto as long as it has the protein activity of serine dehydratase. Specifically, the polypeptide having the protein activity of serine dehydratase may have, comprise, consist of, or essentially consist of the amino acid sequence of SEQ ID NO: 45 or an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity thereto. For example, the serine dehydratase protein refers to, but is not limited to, a protein endogenously present in Corynebacterium microorganisms or Corynebacterium stathionis. Specifically, the serine dehydratase protein may be, but is not limited to, a serine dehydratase protein having the amino acid sequence of SEQ ID NO: 45 endogenously present in Corynebacterium microorganisms or Corynebacterium stathionis.

[0139] The base sequence encoding the serine dehydratase may be a base sequence encoding a protein that exhibits serine dehydratase activity.

[0140] For example, the serine dehydratase protein having the amino acid sequence of SEQ ID NO: 45 may be encoded by a polynucleotide having, comprising, consisting of, or essentially consisting of the sequence of SEQ ID NO: 46 or a nucleotide sequence having 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or less than 100% homology or identity to the sequence of SEQ ID NO: 46, but is not limited thereto. The nucleotide sequence of SEQ ID NO: 46 can be obtained from publicly known databases, such as, but not limited to, GenBank of NCBI.

[0141] As a microorganism according to any one of the above-mentioned specific examples, the microorganism having the ability to produce purine nucleotides of the present application may be a microorganism in which the protein activity of formate dehydrogenase is weakened compared to the endogenous activity and the protein activity of formate-dependent phosphoribosylglycine amidoformyltransferase is enhanced compared to the endogenous activity, thereby improving the ability to produce purine nucleotides, but is not limited thereto.

[0142] As a microorganism according to any one of the above-mentioned specific examples, the microorganism having the ability to produce purine nucleotides of the present application may be a microorganism in which the protein activity of formate dehydrogenase is weakened compared to the endogenous activity, the protein activity of formate-dependent phosphoribosylglycine amidoformyltransferase is enhanced compared to the endogenous activity, and the protein activity of glycine degrading enzyme is enhanced compared to the endogenous activity, thereby improving the ability to produce purine nucleotides, but is not limited thereto.

[0143] As a microorganism according to any one of the above-mentioned specific examples, the microorganism having the ability to produce purine nucleotides of the present application may be a microorganism having improved ability to produce purine nucleotides, in which the protein activity of serine dehydratase is weakened compared to its endogenous activity, the protein activity of serine hydroxymethyltransferase is enhanced compared to its endogenous activity, the protein activity of glycine dehydrogenase is enhanced compared to its endogenous activity, the protein activity of formate dehydrogenase is weakened compared to its endogenous activity, and the protein activity of formate-dependent phosphoribosylglycine amidoformyltransferase is enhanced compared to its endogenous activity, but is not limited thereto.

[0144] Another aspect of the present application provides a method for producing purine nucleotides, comprising culturing in a medium a microorganism in which the protein activity of the formate dehydrogenase of the present application is weakened compared to its endogenous activity.

[0145] 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.

[0146] As used herein, the term "culture medium" refers to a mixture of nutrients, primarily those required for culturing the microorganisms 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 may be any medium commonly used for culturing microorganisms, without any particular limitations. The microorganisms of the present application may be cultured under aerobic conditions in a conventional medium containing appropriate carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids, and / or vitamins, with temperature, pH, and other parameters adjusted. 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).

[0147] 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 into reducing sugars) can be used. A variety of other carbon sources can also be used in appropriate amounts without limitation. These carbon sources can be used alone or in combination of two or more, and are not limited thereto.

[0148] 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 thereto.

[0149] The phosphorus source may include monopotassium phosphate, dipotassium phosphate, or a corresponding sodium-containing salt thereof. 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 thereto.

[0150] During the cultivation of the microorganism of the present application, the pH of the medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid to the medium in an appropriate manner. Furthermore, during cultivation, foam formation can be suppressed using an antifoaming agent such as a fatty acid polyglycol ester. Furthermore, to maintain an aerobic state in the medium, oxygen or an oxygen-containing gas can be injected into the medium, and to maintain an anaerobic or microaerobic state, no gas can be injected, or nitrogen, hydrogen, or carbon dioxide gas can be injected, but this is not limiting.

[0151] In the culture of the present invention, the culture temperature can be maintained at 27 to 37°C, specifically 30 to 33°C, and the culture can be carried out for about 20 to 120 hours, but is not limited thereto.

[0152] 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 microorganism in a culture medium, where the culture solution contains the specific microorganism, and the culture filtrate is substantially free of the specific microorganism (here, "substantially" means that the specific microorganism is removed by filtration or the like, but does not mean that the microorganism is completely removed 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 purine nucleotides.

[0153] In this application, the term "fermentation" refers to the process in which microorganisms use their own enzymes to decompose organic matter, 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.

[0154] 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.

[0155] 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.

[0156] In the method of the present application, the microorganism 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.

[0157] The purine nucleotides produced by the culture of the present application are either secreted into the medium or remain intracellularly.

[0158] In one embodiment, the method for producing purine nucleotides of the present application may further include a step of preparing the microorganism 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.

[0159] The method for producing purine nucleotides of the present application may further include a step of recovering a target substance, specifically, a purine nucleotide, from the cultured microorganism, the culture of the microorganism, the fermentation product of the microorganism, or the culture medium. The recovery step may be further included after the culturing step.

[0160] The recovery may involve collecting the target purine nucleotides using a suitable method known in the art based on the microbial culture method 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), 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, the purine nucleotides can be recovered from the medium or the microorganisms using a suitable method known in the art.

[0161] Furthermore, the method for producing purine nucleotides of the present application may further include a purification step. The purification can be performed using any suitable method known in the art. In one example, when the method for producing purine nucleotides 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 in any order, or can be performed simultaneously or integrated into one step, but are not limited thereto.

[0162] In the method of the present application, the attenuation of the formate dehydrogenase protein activity and the purine nucleotides are as described above in other embodiments.

[0163] Another aspect of the present application provides a composition for producing purine nucleotides, comprising a microorganism in which the protein activity of the formate dehydrogenase of the present application is weakened compared to its endogenous activity, a culture of the microorganism, a fermentation product of the microorganism, or a combination of two or more of these.

[0164] The compositions of the present application may further contain any suitable excipient commonly used in compositions for producing purine nucleotides, and such excipients may be, for example, but are not limited to, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers, or isotonicity agents.

[0165] In one embodiment, each component present in the compositions of the present application may be included in a microbiologically effective amount, or in an amount that may suitably be present in a production composition.

[0166] In the composition of the present application, the attenuation of the formate dehydrogenase protein activity and the purine nucleotides are as described above in other aspects.

[0167] Another aspect of the present application provides use of a microorganism in which the formate dehydrogenase protein activity of the present application is weakened compared to its endogenous activity, for producing purine nucleotides.

[0168] In the present application, the attenuation of the formate dehydrogenase protein activity and the purine nucleotides are as described above in other aspects.

[0169] 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.

[0170] Example 1. Construction of a formate dehydrogenase-deficient strain, construction of a formate-dependent phosphoribosylglycine amidoformyltransferase-enhanced strain, and evaluation of 5'-inosinic acid production ability Example 1-1. Construction of formate dehydrogenase-deficient recombinant vector and 5'-inosinic acid-producing strain The formate dehydrogenase derived from Corynebacterium stathionis may consist of formate dehydrogenase subunit 1 (Fdh subunit 1), formate dehydrogenase subunit 2 (Fdh subunit 2), and formate dehydrogenase subunit 3 (Fdh subunit 3).

[0171] To confirm whether the increase in formate production due to the deficiency of formate dehydrogenase subunit 1, formate dehydrogenase subunit 2, and formate dehydrogenase subunit 3 (hereafter referred to as formate dehydrogenase) leads to an increase in IMP production, we conducted an experiment to create an IMP-producing strain, Corynebacterium stathionis KCCM12151P, lacking endogenous formate dehydrogenase.

[0172] Specifically, experiments were conducted to create strains lacking the fdh 1, fdh 2, and fdh 3 genes (hereinafter referred to as fdh genes), which encode formate dehydrogenase subunit 1, formate dehydrogenase subunit 2, and formate dehydrogenase subunit 3, respectively.

[0173] The chromosomal DNA of the wild-type Corynebacterium stachyonis strain ATCC 6872 was isolated using Intron's G-spin Total DNA extraction mini kit (Cat. No. 17045) according to the protocol provided with the kit. A pair of gene fragments (fdh-A and fdh-B) was isolated by polymerase chain reaction using the primer pair SEQ ID NO: 1 and SEQ ID NO: 2 and the primer pair SEQ ID NO: 3 and SEQ ID NO: 4, respectively. The PCR conditions were denaturation at 94°C for 5 minutes, followed by 20 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 1 minute, followed by polymerization at 72°C for 7 minutes. As a result, polynucleotides of 1091 bp and 1105 bp, respectively, were obtained.

[0174] A second PCR was performed using these two fragments as templates to obtain a 2.2 kbp polynucleotide template. The resulting gene fragment was digested with the restriction enzyme XbaI (New England Biolabs, Beverly, MA). Using T4 ligase (New England Biolabs, Beverly, MA), the gene fragment was ligated to linearized pDCM2 (Korea Publication No. 10-2020-0136813) digested with XbaI restriction enzyme. The resulting vector was designated pDCM2-del_fdh.

[0175] The sequences of the primers used to construct the vector are as follows:

[0176] [Table 1]

[0177] The pDCM2-del_fdh vector was transformed into Corynebacterium stachyonis KCCM12151P (an IMP-producing strain), a strain capable of producing 5'-inosinic acid, by electroporation. Strains in which both the mutated gene and the vector were integrated into the chromosome were selected as primary candidates on a selection medium containing 25 mg / L kanamycin. Subsequently, a secondary crossover process utilizing the homology between the existing gene on the chromosome and the gene inserted via the vector was performed to obtain the final strain in which the endogenous formate dehydrogenase gene on the chromosome was deleted and the vector containing the kanamycin resistance gene was removed. The deleted strains were initially confirmed by PCR using primers set forth in SEQ ID NOs: 5 and 6, and then finally confirmed by gene sequence analysis. The formate dehydrogenase-deficient strain obtained by the above method was named CJI-3162 (KCCM12151P_fdh_deletion) and deposited with the Korea Microorganism Collection, a depository under the Budapest Treaty, on November 24, 2022, and was assigned the accession number KCCM13275P.

[0178] [Table 2]

[0179] Example 1-2. Construction of an integrated strain lacking formate dehydrogenase and enriched for formate-dependent phosphoribosylglycine amidoformyltransferase, and construction of a 5'-inosinic acid-producing strain A strain integrating formate dehydrogenase deficiency and formate-dependent phosphoribosylglycine amidotransferase enhancement was constructed and evaluated.

[0180] To create a strain in which the endogenous purT gene activity was further enhanced in the CJI-3162 strain constructed in Example 1-1, an experiment was conducted to enhance purT in the form of the Pcj7 promoter (Korea Patent Application No. 10-2004-0107215).

[0181] The chromosomal DNA of wild-type Corynebacterium stathionis strain ATCC 6872 was isolated using Intron's G-spin Total DNA extraction mini kit (Cat. No. 17045) according to the protocol provided with the kit. Pairs of gene fragments (Pcj7 / purT-A, Pcj7 / purT-B, and Pcj7 / purT-C) were isolated by polymerase chain reaction using primer pairs SEQ ID NOs: 7 and 8, SEQ ID NOs: 9 and 10, and SEQ ID NOs: 11 and 12. PCR conditions included denaturation at 94°C for 5 minutes, followed by 20 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 1 minute, followed by polymerization at 72°C for 7 minutes. Polynucleotides of 851 bp, 318 bp, and 882 bp were obtained, respectively.

[0182] A second PCR was performed using the three fragments as templates to obtain a 2-kbp polynucleotide template. The resulting gene fragment was digested with the restriction enzyme XbaI (New England Biolabs, Beverly, MA). Using T4 ligase (New England Biolabs, Beverly, MA), the gene fragment was ligated to a linearized pDCM2 vector that had been digested with XbaI. The resulting vector was designated pDCM2-Pcj7 / purT.

[0183] The sequences of the primers used to construct the vector are as follows:

[0184] [Table 3]

[0185] After transforming the pDCM2-Pcj7 / purT vector into CJI-3162 (KCCM12151P_fdh_deletion) prepared in Example 1-1 by electroporation, strains in which both the mutant gene and the vector were inserted onto the chromosome were selected as the primary candidate group from a selection medium containing 25 mg / L of kanamycin. Then, through a secondary crossover process utilizing the homology of the gene inserted through the existing gene and vector on the chromosome, the promoter of the endogenous purT on the chromosome was enhanced to Pcj7, and the final strain in which the vector containing the kanamycin resistance gene was removed was obtained. The strain with enhanced purT was initially confirmed by PCR using the primers of SEQ ID NO: 13 and SEQ ID NO: 14, and finally confirmed by gene sequence analysis. The Pcj7 / purT strain obtained by the method as described above was named CJI-3163 (CJI3162_Pcj7 / purT).

[0186]

Table 4

[0187] Example 1-3. Evaluation of the 5'-inosinic acid production ability of two strains, a formate dehydrogenase-deficient strain and a strain with enhanced formate-dependent phosphoribosylglycinamide formyltransferase To measure the IMP production ability of the prepared strains, flask evaluation was performed. Corynebacterium stationis KCCM12151P, CJI-3162, and CJI-3163 were inoculated into a 14 ml tube containing 2.5 ml of the following seed medium and cultured with shaking at 170 rpm at 30°C for 24 hours. 2 ml of the seed culture solution was inoculated into a 250 ml corner baffle flask containing 29 ml of the following production medium (24 ml of the main medium + 5 ml of the separate killing medium) and cultured with shaking at 170 rp at 30°C for 72 hours. After the culture was completed, the production amount of 5'-inosinic acid was measured by the method using HPLC.

[0188] The compositions of the above seed medium and fermentation medium are as follows.

[0189] <IMP seed medium> 1% glucose, 1% peptone, 1% gravy, 1% yeast extract, 0.25% sodium chloride, 100 mg / L adenine, 100 mg / L guanine, pH 7.2

[0190] <IMP flask fermentation medium> 0.1% sodium glutamate, 1% ammonium chloride, 1.2% magnesium sulfate, 0.01% calcium chloride, 20 mg / L iron sulfate, 20 mg / L manganese sulfate, 20 mg / L zinc sulfate, 5 mg / L copper sulfate, 23 mg / L L-cysteine, 24 mg / L beta-alanine, 8 mg / L nicotinic acid, 45 μg / L biotin, 5 mg / L thiamine hydrochloride, 30 mg / L adenine, 1.9% phosphoric acid (85%), 4.2% glucose, 2.4% fructose added

[0191] The culture results of formate dehydrogenase deficiency and enhancement of formate-dependent phosphoribosylglycinamide formyltransferase in Corynebacterium stationis are shown in Table 5 below.

[0192]

Table 5

[0193] The IMP-producing strain with fdh deficiency showed a 7.4% increase in IMP production ability compared to the control group. It was confirmed that when purT‘ was enhanced in the fdh-deficient strain, the 5’-inosinic acid production ability was further improved.

[0194] Example 2. Production and evaluation of enhanced gcvPTH, sda deficiency, and glyA-enhanced strains based on formate dehydrogenase deficiency and enhanced formate-dependent phosphoribosylglycinamide formyl enzyme Example 2-1. Production of an enhanced recombinant vector with gcvPTH gene addition Copy addition of gcvPTH was performed.

[0195] A vector was constructed to insert a copy of the endogenous gcvPTH gene of Corynebacterium stachyonis in the form of a native promoter. It was constructed as follows using the plasmid pDCM2 (Korea Publication No. 10-2020-0136813), which is used for inserting and exchanging genes into the Corynebacterium chromosome.

[0196] The chromosomal DNA of wild-type Corynebacterium stathionis ATCC6872 strain was isolated using Intron's G-spin Total DNA extraction mini kit (Cat. No. 17045) according to the protocol provided with the kit. Gene fragments (Pn / gcvPTH-A, Pn / gcvPTH-B, Pn / gcvPTH-C, and Pn / gcvPTH-D) were isolated by polymerase chain reaction using primer pairs SEQ ID NOs: 15 and 16, 17 and 18, 19 and 20, and 21 and 22. PCR conditions included denaturation at 94°C for 5 minutes, followed by 20 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 1 minute, followed by polymerization at 72°C for 7 minutes.

[0197] The resulting gene fragment was digested with the restriction enzyme XbaI (New England Biolabs, Beverly, MA). Using T4 ligase (New England Biolabs, Beverly, MA), the gene fragment was ligated to linearized pDCM2 (Korea Publication No. 10-2020-0136813) digested with XbaI. The resulting vector was designated pDCM2-Pn / gcvPTH.

[0198] The sequences of the primers used to construct the vector are as follows:

[0199] [Table 6]

[0200] Example 2-2. Construction of a 5'-inosinic acid producing strain with enhanced gcvPTH gene An experiment was conducted to further enhance gcvPTH expression in CJI-3163 (CJI3162_Pcj7 / purT) constructed in Example 1-2. The pDCM2-Pn / gcvPTH vector constructed in Example 2-1 was transformed into CJI-3163 (CJI3162_Pcj7 / purT) constructed in Example 1-2 by electroporation. Strains in which both the mutant gene and the vector were integrated into the chromosome were selected as primary candidates using a selection medium containing 25 mg / L kanamycin. Strains in which homologous recombination had occurred were then initially confirmed by PCR using the primer pair of SEQ ID NO: 23 and SEQ ID NO: 24, and then finally confirmed by gene sequence analysis. The selected strains were designated CJI-3188 (CJI-3163_Pn / gcvPTH).

[0201] [Table 7]

[0202] Example 2-3. Construction of sda-deficient and glyA-added recombinant vector and construction of 5'-inosinic acid-producing strain A vector was constructed in which the endogenous gene sdaA of Corynebacterium stathionis was deleted and multiple copies of the glyA gene were added to the location of the deleted gene.

[0203] The chromosomal genes of the wild-type Corynebacterium stachyonis strain ATCC6872 were isolated using Intron's G-spin Total DNA extraction mini kit (Cat. No. 17045) according to the protocol provided with the kit. Gene fragments (del_sda::Pcj7 / glyA-A, del_sda::Pcj7 / glyA-B, del_sda::Pcj7 / glyA-C, del_sda::Pcj7 / glyA-D) were obtained by polymerase chain reaction using the primer pairs of SEQ ID NOs: 25 and 26, 27 and 28, 29 and 30, and 31 and 32, respectively. The PCR conditions were as follows: denaturation at 94°C for 5 minutes, followed by denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 1 minute, repeated 20 times, followed by polymerization at 72°C for 7 minutes.

[0204] A second PCR was performed using the four fragments obtained above as templates, and the resulting gene fragment was digested with the restriction enzyme XbaI (New England Biolabs, Beverly, MA). Using T4 ligase (New England Biolabs, Beverly, MA), the gene fragment was ligated to linearized pDCM2 (Korea Publication No. 10-2020-0136813) digested with XbaI restriction enzyme. The resulting plasmid was designated pDCM2-del_sdaA-Pn / glyA.

[0205] The sequences of the primers used to construct the vector are as follows:

[0206] [Table 8]

[0207] The pDCM2-del_sdaA-Pcj7 / glyA vector was transformed into the CJI-3188 strain (CJI-3163_Pn / gcvPTH) constructed in Example 2-2 by electroporation, and strains in which both the mutant gene and the vector were integrated into the chromosome were selected as a primary candidate group using a selection medium containing 25 mg / L kanamycin. Subsequently, from the strains in which homologous recombination had occurred, strains in which the sdaA gene had been deleted and the glyA gene linked to the Pcj7 promoter had been inserted at that position were selected using the primer pair of SEQ ID NO: 33 and SEQ ID NO: 34. The strain obtained in this manner was designated CJI-3189 (CJI-3188_del_sdaA-Pn / glyA).

[0208] The sequences of the primers used to construct the strains are as follows:

[0209] [Table 9]

[0210] Example 2-4 Evaluation of 5'-inosinic acid production ability of a strain based on a formate dehydrogenase-deficient and formate-dependent phosphoribosylglycine amidoformylase-enhanced strain, which is a combination of gcvPTH-enhanced, sda-deficient, and glyA-enhanced strains Flask titer was evaluated to measure the 5'-inosinic acid producing ability of the strains and parent strains prepared in Examples 2-3 and 2-4. Corynebacterium stathionis KCCM12151P, CJI-3188, and CJI-3189 were inoculated into 14 ml tubes containing 2.5 ml of the following seed medium and cultured at 30°C for 24 hours with shaking at 170 rpm. 2 ml of the seed culture was inoculated into a 250 ml corner baffle flask containing 29 ml of the following production medium (24 ml of main medium + 5 ml of sterilization medium) and cultured at 30°C for 72 hours with shaking at 170 rpm. After the culture was completed, the amount of 5'-inosinic acid produced was measured using HPLC.

[0211] The compositions of the seed medium and fermentation medium were the same as those in Examples 1-3.

[0212] The results of culturing Corynebacterium stathionis with formate dehydrogenase deficiency and formate-dependent phosphoribosylglycine amidoformyltransferase enhancement, combined with gcvPTH enhancement, glyA enhancement, and sda deficiency, are shown in Table 10 below.

[0213] [Table 10]

[0214] When fdh deficiency, purT enhancement, and gcvPTH enhancement were combined in an IMP-producing strain, it led to an increase in IMP production, and furthermore, when sdaA deficiency and glyA enhancement were combined, the IMP concentration was confirmed to further increase.

[0215] Example 3. Construction of XMP (5'-xanthylic acid)-producing strains deficient in formate dehydrogenase and enhanced in formate-dependent phosphoribosylglycine amidoformyltransferase and evaluation of XMP production ability Example 3-1. Construction of formate dehydrogenase-deficient recombinant vector and XMP-producing strain To confirm whether the increase in formate concentration due to the deficiency of formate dehydrogenase leads to an increase in XMP production, we conducted an experiment to create an XMP-producing Corynebacterium stachyonis strain lacking endogenous formate dehydrogenase.To this end, we constructed a vector deleting the gene encoding formate dehydrogenase using the plasmid pDCM2 (Korea Publication No. 10-2020-0136813), which is used for the deletion, insertion, and exchange of genes within the Corynebacterium chromosome, as follows.

[0216] Using wild-type Corynebacterium stathionis ATCC 6872 gDNA (genomic DNA) as a template, PCR was performed using the primer pair of SEQ ID NOs: 35 and 36 and the primer pair of SEQ ID NOs: 37 and 38. PCR was performed at 94°C for 5 minutes, followed by 30 cycles of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute and 30 seconds, followed by 5 minutes at 72°C. The pDCM2 vector was digested with XbaI, and the resulting PCR product was subjected to fusion cloning. Fusion cloning was performed using the In-Fusion® HD Cloning Kit (Clontech). The resulting plasmid was designated pDCM2-del fdh.

[0217] The sequences of the primers used to construct the vector are as follows:

[0218] [Table 11]

[0219] The constructed vector was transformed into Corynebacterium stachyonis CJX1664 (KR 10-1950141 B1) by electroporation, and strains in which the vector had been integrated into the chromosome were selected as the primary candidate group on a selection medium containing 25 mg / L kanamycin. Subsequently, strains in which homologous recombination had occurred were selected using the primer pair of SEQ ID NO: 35 and SEQ ID NO: 38 to identify strains in which the formate dehydrogenase gene had been deleted. The selected strain was designated CJX1664_del fdh.

[0220] Example 3-2. Construction of a recombinant vector for enhanced expression of formate-dependent phosphoribosylglycine amidoformyltransferase and an XMP-producing strain To confirm whether enhancing formate-dependent phosphoribosylglycine amidotransferase would lead to improved XMP production, we conducted an experiment to create an XMP-producing Corynebacterium stathionis strain with enhanced expression of endogenous formate-dependent phosphoribosylglycine amidotransferase.

[0221] At the same time, to confirm whether the increase in formate due to formate dehydrogenase deficiency leads to an additional increase in XMP production through formate-dependent phosphoribosylglycine amidotransferase, we also enhanced the expression of formate-dependent phosphoribosylglycine amidotransferase in the formate dehydrogenase-deficient strain.

[0222] The vector pDCM2-Pcj7 / purT, which enhances the promoter of the purT gene encoding formate-dependent phosphoribosylglycine amidotransferase constructed in Example 1-2, was transformed into Corynebacterium stathionis CJX1664 (KR 10-1950141 B1) and the formate dehydrogenase-deficient strain CJX1664_del fdh constructed in Example 3-1 by electroporation. Strains with chromosomal integration of the vector were selected as primary candidates on a selection medium containing 25 mg / L kanamycin. Subsequently, strains in which homologous recombination had occurred were selected using the primer pair of SEQ ID NOs: 9 and 12 to select strains in which the purT gene was linked to the Pcj7 promoter. The selected strains were designated CJX1664_Pcj7 / purT and CJX1664_del fdh_Pcj7 / purT, respectively.

[0223] Example 3-3. Evaluation of XMP production ability of two strains: one lacking formate dehydrogenase and one enriched for formate-dependent phosphoribosylglycine amidoformyltransferase The XMP productivity of the three strains prepared in Examples 3-1 and 3-2 and the parent strain was evaluated by evaluating the flask fermentation titer.

[0224] First, each strain was inoculated into a test tube with a diameter of 18 mm containing 2 ml of the seed medium and cultured with shaking at 30°C for 24 hours to be used as the seed culture solution. 0.7 ml of the seed culture solution was inoculated into a 250 ml conical baffle flask containing 32 ml of the following production medium (24 ml of this medium + 8 ml of separately sterilized medium), and cultured at 30°C for 75 hours at 170 rpm. After the culture was completed, the production ability of XMP was measured by HPLC. The XMP concentration and the concentration increase rate in the culture solution for each strain tested are as shown in Table 3 below.

[0225] <Seed medium (pH 7.5)> Glucose 1%, peptone 1%, gravy 1%, yeast extract 1%, sodium chloride 0.25%, adenine 100 mg / l, guanine 100 mg / l (based on 1 liter of distilled water)

[0226] <XMP flask production medium (this medium)> Glucose 40 g / L, magnesium sulfate 10 g / L, calcium chloride 100 mg / L, iron sulfate 20 mg / L, manganese sulfate 10 mg / L, zinc sulfate 10 mg / L, copper sulfate 0.8 mg / L, histidine 20 mg / L, cystine 15 mg / L, beta-alanine 15 mg / L, biotin 100 ug / L, thiamine 5 mg / L, adenine 50 mg / L, guanine 25 mg / L, niacin 15 mg / L, pH 7.0

[0227] <XMP flask production medium (separately sterilized medium)> Potassium dihydrogen phosphate 18 g / L, dipotassium hydrogen phosphate 42 g / L, urea 7 g / L, ammonium sulfate 5 g / L (based on 1 liter of distilled water)

[0228] The above experiment was repeated 3 times, and the average value of the analysis results is shown in Table 12 below.

[0229]

Table 12

[0230] As shown in Table 12, the CJX1664_del fdh and CJX1664_Pcj7 / purT strains showed 3.04% and 2.81% increases in XMP production compared to the control CJX1664, respectively, and the CJX1664_del fdh-Pcj7 / purT strain showed a 5.62% increase in XMP production. This confirms that increasing formate through the deletion of formate dehydrogenase leads to increased XMP production, and that XMP production is further improved when formate-dependent phosphoribosylglycine amidoformyltransferase, which uses formate as a substrate, is also enhanced.

[0231] Example 3-4. Confirmation of GMP production ability of formate dehydrogenase-deficient strain and formate-dependent phosphoribosylglycine amidoformyltransferase-enhanced strain The GMP production ability was confirmed using the XMP culture medium of the strains prepared in Examples 3-1 and 3-2 and the control parent strain as follows (see Korean Patent Publication No. 10-2011-0105662 A).

[0232] Specifically, the strain was cultured using the method for evaluating the fermentation titer in Example 3-3. After the culture was completed, the amount of XMP (5'-xanthylic acid) produced was measured by a method using HPLC.

[0233] To convert the produced XMP to GMP, the following conversion reaction additives and E. coli XMP aminase were added to the flask fermentation broth, and the conversion reaction was carried out at 40°C for 2.5 hours. The conversion rate, which indicates the amount of GMP produced relative to the amount of XMP consumed, is shown in Table 13.

[0234] [Table 13]

[0235] As shown in Table 13, it was confirmed that GMP was produced through a conversion reaction from XMP produced by each strain.

[0236] <Conversion reaction additives> 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, glutamine 2g / L, glucose 46g / L

[0237] Example 4. Construction of a gcvPTH-enhanced, sdaA-deficient, and glyA-enhanced strain based on a formate dehydrogenase-deficient and formate-dependent phosphoribosylglycine amidoformyltransferase-enhanced strain, and evaluation of XMP (5'-xanthylic acid) production ability Example 4-1. Construction of XMP-producing strain with enhanced gcvPTH gene A strain was constructed by adding a copy of the gcvPTH gene in the form of a native promoter to a strain that lacks fdh and has an enhanced purT promoter.

[0238] The pDCM2-Pn / gcvPTH vector prepared in Example 2-1 was transformed into the CJX1664_del fdh-Pcj7 / purT strain prepared in Example 3-2 by electroporation, and strains in which the vector had been integrated into the chromosome were selected as a primary candidate group using a selection medium containing 25 mg / L kanamycin. Subsequently, strains in which homologous recombination had occurred were selected for an additional copy of the gcvPTH gene using the primer pair of SEQ ID NO: 23 and SEQ ID NO: 24. The selected strain was designated CJX1664_del fdh-Pcj7 / purT-Pn / gcvPTH.

[0239] Example 4-2. Construction of an XMP-producing strain with Sda deletion and glyA addition Based on an XMP-producing strain in which purT and gcvPTH were introduced, we constructed a strain in which the endogenous gene sdaA of Corynebacterium stathionis that encodes serine, encoding glycine, was deleted and multiple copies of the glyA gene were added to the position of the gene in order to secure a serine pool that leads to glycine, and we confirmed the effect on XMP production when the glycine pool was further enhanced.

[0240] The pDCM2-del_sdaA-Pn / glyA vector prepared in Example 2-3 was transformed into the CJX1664_del fdh-Pcj7 / purT-Pn / gcvPTH prepared in Example 4-1 by electroporation, and strains in which the vector had been integrated into the chromosome were selected as a primary candidate group using a selection medium containing 25 mg / L kanamycin. From the strains in which homologous recombination had occurred, strains in which the sdaA gene had been deleted and the glyA gene linked to the Pcj7 promoter had been inserted at that position were selected using the primer pair of SEQ ID NO: 33 and SEQ ID NO: 34. The strain obtained in this manner was designated CJX1664_del fdh-Pcj7 / purT-Pn / gcvPTH_del sdaA-Pn / glyA.

[0241] Example 4-3. Evaluation of XMP production ability of gcvPTH-enhanced, sdaA-deficient, and glyA-enhanced strains based on formate dehydrogenase-deficient and formate-dependent phosphoribosylglycine amidoformyltransferase-enhanced strains The XMP productivity of the strains and parent strains prepared in Examples 4-1 and 4-2 was evaluated by flask fermentation titer evaluation in the same manner as in Example 3-3. The experiment was repeated three times, and the average values ​​of the analytical results are shown in Table 14 below.

[0242] [Table 14]

[0243] As shown in Table 14, XMP production was further improved in CJX1664_del fdh_Pcj7 / purT_Pn / gcvPTH, which was enriched with gcvPTH, compared to the CJX1664_del fdh_Pcj7 / purT strain, and XMP production was also further increased in CJX1664_del fdh_Pcj7 / purT_Pn / gcvPTH_del sdaA_Pn / glyA, a strain in which sdaA was deleted and glyA was enriched. This confirmed that XMP production was further improved when sdaA deletion, glyA enrichment, and gcvPTH enrichment were combined with fdh deletion and purT enhancement, and sdaA deletion was combined with gcvPTH enhancement to enrich both gcvPTH and glyA.

[0244] Example 4-4. Confirmation of GMP production ability of gcvPTH-enhanced, sdaA-deficient, and glyA-enhanced strains based on formate dehydrogenase-deficient and formate-dependent phosphoribosylglycine amidoformyltransferase-enhanced strains GMP production was confirmed using the XMP culture medium of the strains prepared in Examples 4-1 and 4-2 in the same manner as in Examples 3-4, and the results are shown in Table 15.

[0245] [Table 15]

[0246] As shown in Table 15, it was confirmed that GMP was produced through a conversion reaction from XMP produced by each strain.

[0247] 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.

[0248] Table 16

Claims

1. A microorganism capable of producing purine nucleotides, in which the protein activity of formate dehydrogenase is weakened compared to its endogenous activity.

2. The microorganism capable of producing purine nucleotides according to claim 1, wherein the formate dehydrogenase consists of one or more amino acid sequences selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43 and combinations thereof.

3. The microorganism capable of producing purine nucleotides according to claim 1, wherein the activity of one or more proteins selected from the group consisting of the following (a) to (c) is further regulated: (a) enhanced glycine degrading enzyme protein activity compared to endogenous activity; (b) the protein activity of serine hydroxymethyltransferase is enhanced compared to the endogenous activity; and (c) Enhancement of formate-dependent phosphoribosylglycine amidoformyltransferase protein activity compared to endogenous activity.

4. The microorganism capable of producing purine nucleotides according to claim 1 , wherein the microorganism is a Corynebacterium microorganism.

5. The microorganism capable of producing purine nucleotides according to claim 4, wherein the Corynebacterium microorganism is Corynebacterium stachyonis.

6. 6. The microorganism capable of producing purine nucleotides according to claim 1, wherein the microorganism has an increased ability to produce purine nucleotides compared to a non-transformed microorganism.

7. A method for producing purine nucleotides, comprising the step of culturing in a medium a microorganism in which the protein activity of formate dehydrogenase is weakened compared to the endogenous activity.

8. The method according to claim 7, further comprising recovering a target substance from the cultured microorganism, the culture of the microorganism, the fermentation product of the microorganism, or the culture medium.

9. A composition for producing purine nucleotides, comprising a microorganism in which the protein activity of formate dehydrogenase is weakened compared to its endogenous activity, a culture of said microorganism, a fermentation product of said microorganism, or a combination of two or more of these.

10. Use of a microorganism in which the protein activity of formate dehydrogenase is weakened compared to the endogenous activity for the production of purine nucleotides.

Images