Novel glutamine-hydrolyzing GMP synthase variant and method for producing purine nucleotides using the same

Abstract

The present invention relates to a glutamine hydrolyzing GMP synthetase mutant and a method for producing purine nucleotides using the same.

Description

Technical Field

[0001] The present invention relates to a glutamine-hydrolyzing GMP synthase variant and a method for producing purine nucleotides using the same.

Background Art

[0002] Purine nucleotides, which are one of nucleic acid substances, are widely used substances for food flavor additives or foods, and have been in the limelight as flavor nucleic acid seasonings.

[0003] In order to produce purine nucleotides, various studies have been conducted for the development of highly efficient production microorganisms and fermentation process technologies. For example, a target substance-specific approach such as increasing the expression of a gene encoding an enzyme involved in the biosynthesis of purine nucleotides or removing genes unnecessary for biosynthesis is mainly used (EP 3722430 A1, US 2020-0347346 A1).

[0004] As the demand for purine nucleotides increases, research for effective production of purine nucleotides is still necessary at present.

Prior Art Documents

Patent Documents

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[0007] The present invention provides a glutamine-hydrolyzing GMP synthase variant. [Means for Solving the Problems]

[0008] An object of the present invention is to provide a glutamine-hydrolyzing GMP synthase variant.

[0009] Another object of the present invention is to provide a polynucleotide encoding the variant.

[0010] Another object of the present invention is to provide a microorganism containing the variant or the polynucleotide encoding it.

[0011] Another object of the present invention is to provide a method for producing purine nucleotides including the step of culturing the microorganism. [Effects of the Invention]

[0012] High-yield production of purine nucleotides is possible using the variant of the present invention. [Modes for Carrying Out the Invention]

[0013] The present invention will be specifically described as follows. On the other hand, each of the descriptions and embodiments disclosed in this specification can also be applied to different descriptions and embodiments. That is, all combinations of various elements disclosed in this specification belong to the scope of the present invention. Also, the scope of the present invention is not limited by the specific descriptions described below. Also, a number of papers and patent documents are referred to throughout this specification, and their citations are indicated. The disclosure content of the cited papers and patent documents is incorporated herein by reference in its entirety, and the level of the technical field to which the present invention belongs and the content of the present invention are more clearly explained.

[0014] One aspect of the present invention provides a glutamine-hydrolyzing GMP synthase variant in which the amino acid corresponding to the 29th position of SEQ ID NO: 3 is substituted with another amino acid.

[0015] The glutamine-hydrolyzing GMP synthase variant means a polypeptide having the activity of glutamine-hydrolyzing GMP synthase or a variant containing a substitution of the amino acid corresponding to the 29th position from the N-terminus of SEQ ID NO: 3 with another amino acid in glutamine-hydrolyzing GMP synthase.

[0016] In the present invention, "glutamine-hydrolyzing GMP synthase" is an enzyme involved in converting 5'-xanthosine monophosphate (XMP) to 5'-guanosine monophosphate (hereinafter, GMP).

[0017] The glutamine-hydrolyzing GMP synthase of the present invention may be a glutamine-hydrolyzing GMP synthase or a polypeptide having glutamine-hydrolyzing GMP synthase activity that is modified to produce the glutamine-hydrolyzing GMP synthase variant provided by the present invention.

[0018] Specifically, it may be a naturally occurring polypeptide or a wild-type polypeptide, which may be its mature polypeptide, or may contain its variant or functional fragment, but is included without limitation as long as it can be a parent of the glutaminase GMP synthase variant of the present invention.

[0019] As an embodiment, the glutaminase GMP synthase of the present invention may be derived from the genus Corynebacterium, and more specifically may be derived from Corynebacterium stationis, but is not limited thereto.

[0020] As an embodiment, the glutaminase GMP synthase of the present invention may be the polypeptide of SEQ ID NO: 3. As an embodiment, the glutaminase GMP synthase of the present invention may be a polypeptide having about 60%, 70%, 75%, 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98% or 99% or more sequence identity with the polypeptide of SEQ ID NO: 3, and is included in the scope of the glutaminase GMP synthase without limitation as long as it has the same or corresponding activity as the polypeptide consisting of the amino acid sequence of SEQ ID NO: 3. Specifically, the glutaminase GMP synthase may have, contain, or be essentially consisting of the amino acid sequence described in SEQ ID NO: 3, but is not limited thereto.

[0021] In the present invention, the glutamine-hydrolyzing GMP synthase to be mutated may be a glutamine-hydrolyzing GMP synthase derived from Corynebacterium sp., specifically, a polypeptide / protein containing the amino acid sequence set forth in SEQ ID NO: 3, but polypeptides / proteins having the same activity may be included without limitation. As an example, it may include the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence having 80% or more homology or identity therewith, but is not limited thereto. Specifically, it may include an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity with SEQ ID NO: 3. Also, a polypeptide / protein having an amino acid sequence in which some sequences are deleted, modified, substituted, or added, as long as it has such homology or identity and exhibits glutamine-hydrolyzing GMP synthase activity, is obviously included within the scope of the polypeptide / protein to be mutated in the present application.

[0022] The glutamine-hydrolyzing GMP synthase of the present invention can obtain its sequence from GenBank of NCBI, a known database. As an example, the glutamine-hydrolyzing GMP synthase of the present invention may be a polypeptide encoded by the guaA gene. As an example, the glutamine-hydrolyzing GMP synthase of the present application may be derived from Corynebacterium stationis, Corynebacterium casei, or Corynebacterium ammoniagenes, and as an example, it may be an enzyme represented by NCBI Reference sequence WP_194285183.1, WP_006823296.1, or WP_040355890.1, but is not limited thereto.

[0023] In the present invention, the terms "homology" or "identity" refer to the degree of similarity between two given amino acid sequences or nucleotide sequences, and can be expressed as a percentage. The terms homology and identity can often be used interchangeably.

[0024] The sequence homology or identity of a conserved polynucleotide or polypeptide is determined by standard sequence algorithms, and the default gap penalties established by the programs used may be used together. Substantially, homologous or identical sequences can generally hybridize with all or part of the sequence under moderately or highly stringent conditions. Hybridization is also understood to include hybridization with polynucleotides containing codons that take into account the general codons or codon degeneracy in polynucleotides.

[0025] Whether any two polynucleotide sequences or polypeptide sequences have homology, similarity or identity can be determined using known computer algorithms such as the "FASTA" program with default parameters as, for example, 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 performed by 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 versions) (including 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, homology, similarity or identity can be determined using BLAST or ClustalW of the National Center for Biotechnology Information Database Center.

[0026] The homology, similarity or identity of polynucleotides or polypeptides can be determined by, for example, comparing sequence information using a GAP computer program such as that described in Needleman et al. (1970), J Mol Biol. 48:443, as is known, for example, in Smith and Waterman, Adv. Appl. Math (1981) 2:482. Briefly, the GAP program can be defined as the total number of symbols in the shorter of two sequences, minus the number of similar aligned symbols (i.e., nucleotides or amino acids). The default parameters for the GAP program are: (1) a binary comparison matrix (with a value of 1 for identity and 0 for non-identity), and a weighted comparison matrix as disclosed by Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979), Gribskov et al(1986) Nucl. Acids Res. 14: 6745 (or the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or a gap opening penalty of 10 and a gap extension penalty of 0.5); (3) no penalty for terminal gaps.

[0027] In the present invention, the term "variant" refers to a polypeptide in which one or more amino acids are conservatively substituted and / or modified, which is different from the amino acid sequence before the mutation of the variant, but maintains functions or properties. Such variants may generally be identified by modifying one or more amino acids in the amino acid sequence of the polypeptide and evaluating the properties of the modified polypeptide. That is, the ability of the variant can be increased, unchanged, or decreased compared to the polypeptide before the mutation. In addition, some variants may include variants in which one or more parts such as an N-terminal leader sequence or a transmembrane domain are removed. Other variants may include variants in which a part is removed from the N- and / or C-terminus of the mature protein. The term "variant" can be used interchangeably with terms such as modification, modified polypeptide, modified protein, mutant, mutein, divergent, etc. (in English expressions), and is not limited to this as long as it is used in a mutated meaning.

[0028] In addition, the variant may include deletions or additions of amino acids that have a minimal impact on the properties and secondary structure of the polypeptide. For example, a signal (or leader) sequence involved in the translocation of the protein may be conjugated to the N-terminus of the variant co-translationally or post-translationally. In addition, the variant may be conjugated to other sequences or linkers for confirmation, purification, or synthesis.

[0029] The glutaminase GMP synthase variant provided by the present invention may be a polypeptide comprising an amino acid sequence in which the amino acid corresponding to the 29th position of SEQ ID NO: 3 is substituted with another amino acid. Specifically, the other amino acid may be selected from glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine and histidine.

[0030] The "other amino acid" is not limited as long as it is different from the amino acid before substitution. On the other hand, when expressing "a specific amino acid is substituted" in the present invention, even if it is not particularly stated that it is substituted with another amino acid, it is obvious that it is substituted with an amino acid different from the amino acid before substitution.

[0031] As an embodiment, the variant of the present invention may be a variant in which the amino acid corresponding to the 29th position in the amino acid sequence of SEQ ID NO: 3, which is a comparative (reference) protein, is an amino acid other than arginine. Specifically, the amino acid corresponding to the 29th position of SEQ ID NO: 3 of the variant may be selected from glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine and histidine.

[0032] As an embodiment, the glutaminase GMP synthase variant of the present invention has an amino acid at the 29th position corresponding to the amino acid sequence set forth in SEQ ID NO: 3 that is an amino acid other than arginine, and has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.8% or more homology or identity with the amino acid sequence set forth in SEQ ID NO: 3.

[0033] As an example, the glutaminase GMP synthase variant of the present invention may consist of the amino acid sequence of SEQ ID NO: 1, or may include an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.8% or more identity thereto, but is not limited thereto.

[0034] Moreover, as long as it has such homology or identity and exhibits the efficacy corresponding to the variant of the present invention, a variant having an amino acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted or added is also clearly included within the scope of the present invention. For example, it is a case where there is an addition or deletion of a sequence that does not modify the function of the variant of the present invention at the N-terminus, C-terminus and / or inside of the amino acid sequence, a naturally occurring mutation, a silent mutation or a conservative substitution.

[0035] The "conservative substitution" means substituting an amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions can generally occur based on the similarity in the polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphipathic nature of the residues. Usually, conservative substitutions have little or no effect on the activity of a protein or polypeptide.

[0036] In the present invention, the term "corresponding to" refers to an amino acid residue at the position listed in the polypeptide, or an amino acid residue similar or identical or homologous to the residue listed in the polypeptide. Determining the amino acid at the corresponding position may be determining a specific amino acid of a sequence with reference to a specific sequence. The "corresponding region" used in the present invention generally refers to a similar or corresponding position in a related protein or a reference protein.

[0037] For example, any amino acid sequence can be aligned with SEQ ID NO: 3, and based on this, each amino acid residue of the amino acid sequence can be numbered by referring to the numerical position of the corresponding amino acid residue in SEQ ID NO: 3. For example, sequence alignment algorithms such as those described in the present invention can identify the positions of amino acids or the positions where modifications such as substitutions, insertions, or deletions occur, compared to a query sequence (also referred to as an "inquiry sequence").

[0038] For such alignment, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), etc. can be used, but it is not limited thereto, and sequence alignment programs, pairwise sequence comparison algorithms, etc. known in the art can be appropriately used.

[0039] Another aspect of the present invention provides a polynucleotide encoding a variant of the present invention.

[0040] In the present invention, the term "polynucleotide" refers to a polymer of nucleotides in which nucleotide monomers are covalently linked in a long chain, and means a DNA or RNA strand having a certain length or more.

[0041] As an example, the polynucleotide encoding the glutamine-hydrolyzing GMP synthase of the present invention can have or contain a sequence encoding the nucleotide sequence of SEQ ID NO: 4. For example, the polynucleotide encoding the glutamine-hydrolyzing GMP synthase may consist of or be essentially composed of the sequence of SEQ ID NO: 4.

[0042] The polynucleotide of the present invention may be subjected to various modifications in the coding region within the range that does not change the amino acid sequence of the variant of the present invention, taking into account the degeneracy of codons or the codons preferred in the organism in which the variant of the present invention is to be expressed.

[0043] Specifically, the polynucleotide encoding the glutaminase GMP synthase variant of the present invention has a base sequence having 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 with the sequence of SEQ ID NO: 4, or contains, or consists of, or may be essentially composed of, but is not limited to, a base sequence having 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 with the sequence of SEQ ID NO: 4. At this time, in the sequence having the homology or identity, the codon encoding the amino acid corresponding to the 29th position of SEQ ID NO: 3 may be one of the codons encoding amino acids other than arginine.

[0044] In addition, the polynucleotide of the present invention includes, without limitation, a probe that can be produced from a known gene sequence, for example, a sequence that can hybridize under stringent conditions with a complementary sequence to the whole or a part of the polynucleotide sequence of the present invention. The "stringent condition" means a condition that enables specific hybridization between polynucleotides. Such conditions are specifically described in the literature (see J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; F.M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50-9.51, 11.7-11.8). For example, conditions under which polynucleotides with high homology or identity, such as polynucleotides having 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 homology or identity hybridize with each other, and polynucleotides with lower homology or identity do not hybridize with each other, or the washing conditions for ordinary Southern hybridization, i.e., 60°C, 1×SSC, 0.1% SDS, specifically, 60°C, 0.1×SSC, 0.1% SDS, more specifically 68°C, 1×SSC, 0.1% SDS, and washing once, specifically 2 to 3 times, at the salt concentration and temperature corresponding thereto can be listed.

[0045] Hybridization requires that two nucleic acids have complementary sequences, even if base mismatches 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, adenine is complementary to thymine, and cytosine is complementary to guanine. Thus, the polynucleotides of the present invention may also include not only substantially similar nucleic acid sequences but also isolated nucleic acid fragments complementary to the entire sequence.

[0046] Specifically, polynucleotides having homology or identity with the polynucleotides of the present invention can be detected using hybridization conditions including a hybridization step at a Tm value of 55°C, using the above-described conditions. Also, the Tm value may be 60°C, 63°C or 65°C, but is not limited thereto and can be appropriately adjusted by those skilled in the art according to the purpose.

[0047] The appropriate stringency for hybridizing the polynucleotide depends on the length and degree of complementarity of the polynucleotide, and the variables are well known in the art (e.g., J. Sambrook et al., ibid.).

[0048] Another aspect of the present invention provides a vector comprising a polynucleotide encoding a variant of the present invention. The vector may be, but is not limited to, an expression vector for expressing the polynucleotide in a host cell.

[0049] The "vector" of the present invention may include a DNA product containing the nucleotide sequence of a polynucleotide encoding the target polypeptide operably linked to an appropriate expression control region (or expression control sequence) so that the target polypeptide can be expressed in an appropriate host. The expression control region may include a promoter capable of initiating transcription, any operator sequence for regulating such transcription, a sequence encoding an appropriate mRNA ribosome binding site, and a sequence for controlling the termination of transcription and translation. After being transformed into an appropriate host cell, the vector can replicate or function independently of the host genome and may be integrated into the genome itself.

[0050] The vectors used in the present invention are not particularly limited, and any vector known in the art can be utilized. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages in their natural or recombinant states. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage vectors or cosmid vectors, and pDZ series, pBR series, pUC series, pBluescriptII series, pGEM series, pTZ series, pCL series, and pET series can be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vector, etc. can be used.

[0051] As an example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome through a vector for chromosomal insertion within a cell. The insertion of the polynucleotide into the chromosome may be carried out by any method known in the art, such as, but not limited to, homologous recombination. A selection marker for confirming the presence or absence of the chromosomal insertion may further be included. The selection marker is for selecting cells transformed with the vector, that is, for confirming the presence or absence of the insertion of the target nucleic acid molecule, and markers that confer selectable phenotypes such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of surface polypeptides can be used. In an environment treated with a selective agent, only the cells expressing the selection marker survive or exhibit other expressed traits, so that the transformed cells can be selected.

[0052] As used herein, the term "transformation" means introducing a vector containing a polynucleotide encoding a target polypeptide into a host cell or microorganism so that the polypeptide encoded by the polynucleotide can be expressed in the host cell. The transformed polynucleotides can include all of them, whether inserted and located within the host cell's chromosome or located outside the chromosome, as long as they can be expressed within the host cell. Further, the polynucleotide includes DNA and / or RNA encoding the polypeptide of interest. The polynucleotide can be introduced in any form as long as it can be introduced into the host cell and expressed. For example, the polynucleotide can be introduced into the host cell in the form of an expression cassette, which is a gene construct containing all the elements necessary for its own expression. The expression cassette usually can include a promoter operably linked to the polynucleotide, a transcription termination signal, a ribosome binding site, and a translation termination signal. The expression cassette can also be in the form of a self-replicable expression vector. Further, the polynucleotide can be introduced into the host cell in its own form and be operably linked to the sequences necessary for expression in the host cell, and is not limited thereto.

[0053] Also, as used herein, the term "operably linked" means that the promoter sequence that initiates and mediates the transcription of the polynucleotide encoding the variant of the present invention is functionally linked to the polynucleotide sequence.

[0054] Another aspect of the present invention provides a microorganism comprising the variant of the present invention or a polynucleotide encoding the variant thereof.

[0055] The microorganism of the present invention may include the variant of the present invention, the polynucleotide encoding it, or a vector containing the polynucleotide.

[0056] In the present invention, the "microorganism (or strain)" includes all wild-type microorganisms and microorganisms that have undergone natural or artificial genetic modifications, such as those in which foreign genes are inserted, or the activities of endogenous genes are enhanced or inactivated, resulting in the weakening or strengthening of specific mechanisms, and may be a microorganism that includes genetic modifications for the production of a target polypeptide, protein, or product.

[0057] The microorganism of the present invention may be a microorganism containing any one or more of the variant of the present invention, a polynucleotide encoding the variant of the present invention, and a vector containing a polynucleotide encoding the variant of the present application; a microorganism modified to express the variant of the present invention or a polynucleotide encoding the variant of the present invention; a microorganism expressing the variant of the present invention or a polynucleotide encoding the variant of the present invention (for example, a recombinant strain); or a microorganism having the variant activity of the present invention (for example, a recombinant strain), but is not limited thereto.

[0058] The microorganism of the present invention may have the ability to produce purine nucleotides.

[0059] The microorganism of the present invention may be a microorganism that naturally has glutamine-hydrolyzing GMP synthase or the ability to produce purine nucleotides; or a parent strain that does not have glutamine-hydrolyzing GMP synthase or the ability to produce purine nucleotides, into which the variant of the present invention or a polynucleotide encoding the same (or a vector containing the polynucleotide) is introduced, and / or a microorganism given the ability to produce purine nucleotides, but is not limited thereto. As an example, the microorganism of the present invention may be a microorganism into which the glutamine-hydrolyzing GMP synthase variant of the present invention is introduced and given the ability to produce purine nucleotides. As an example, the microorganism of the present invention may be a microorganism into which the glutamine-hydrolyzing GMP synthase variant of the present application is introduced and the ability to produce purine nucleotides is increased. However, it is not limited thereto.

[0060] As an example, the strain of the present invention is a cell or microorganism transformed with a vector containing the polynucleotide of the present invention or a polynucleotide encoding a variant of the present application, and expressing the variant of the present invention. For the purpose of the present invention, the strain of the present invention may include all microorganisms capable of producing purine nucleotides, including the variant of the present invention. For example, the strain of the present invention may be a natural wild-type microorganism, or a recombinant strain in which a polynucleotide encoding the variant of the present invention is introduced into a microorganism producing purine nucleotides, thereby expressing a glutamine-hydrolyzing GMP synthase variant and increasing the purine nucleotide production ability. The recombinant strain with increased purine nucleotide production ability may be a microorganism with increased purine nucleotide production ability compared to a natural wild-type microorganism or a non-transformed microorganism of glutamine-hydrolyzing GMP synthase (i.e., a microorganism expressing wild-type glutamine-hydrolyzing GMP synthase (SEQ ID NO: 3); or a microorganism not expressing a mutant protein), but is not limited thereto.

[0061] In the present invention, the term "non-transformed microorganism" does not exclude a strain containing mutations that can occur naturally in the microorganism, but may mean a wild-type strain or a natural strain itself, or a strain before being transformed by genetic mutations due to natural or artificial factors. For example, the non-transformed microorganism may mean a strain before the introduction of the glutamine-hydrolyzing GMP synthase variant described in the present specification or before the introduction. "Non-transformed microorganism" may be used interchangeably with "strain before transformation", "microorganism before mutation", "non-mutant strain", "non-transformed strain", "non-mutant microorganism" or "reference microorganism".

[0062] As an embodiment, the microorganism of the present invention may be a microorganism belonging to the genus Corynebacterium. For example, the microorganism of the present invention may be Corynebacterium stationis, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium glutamicum, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris or Corynebacterium flavescens. Specifically, it may be Corynebacterium stationis, but is not limited thereto.

[0063] In the present invention, "purine nucleotide" means a nucleotide containing a purine structure. The purine nucleotide may be XMP, IMP, GMP or AMP, and specifically may be IMP, XMP or GMP. More specifically, it may be XMP or GMP, but is not limited thereto.

[0064] "IMP (inosine 5'-monophosphate, 5'-inosinic acid)" and "GMP (guanosine-5'-monophosphate, 5'-Guanylic acid)" of the present invention are intermediates in the nucleic acid biosynthesis metabolic system, play physiologically important roles in the body, and are widely used in foods, pharmaceuticals, etc. Specifically, IMP is known to impart the flavor of beef by itself, and GMP derived from XMP is known to impart the flavor of mushrooms. Both substances are known to enhance the flavor of monosodium glutamate (MSG), and have been in the spotlight as flavor nucleic acid seasonings.

[0065] As an embodiment, GMP may be produced by conversion from XMP, but is not limited thereto. For example, GMP can also be converted from XMP with increased production volume, and it is obvious that the said GMP is also included in the scope of the present invention where its production volume can be increased.

[0066] "XMP" of the present invention is also called xanthosine-5’-monophosphate, 5’-Xanthylic acid, and can be formed from IMP through the action of IMP dehydrogenase, or can be converted to GMP through the action of GMP synthase.

[0067] In the present invention, the "microorganism that produces purine nucleotides" includes all wild-type microorganisms and microorganisms that have undergone natural or artificial genetic modifications. Due to reasons such as insertion of foreign genes, enhancement or inactivation of the activity of endogenous genes, it is a microorganism in which a specific mechanism has been weakened or strengthened, and may be a microorganism in which a genetic mutation has occurred or the activity has been enhanced for the production of the target purine nucleotide.

[0068] The microorganism that produces the purine nucleotide of the present invention can be characterized by including a glutamine-hydrolyzing GMP synthase variant and having an increased production ability of the target purine nucleotide.

[0069] As an embodiment, in the present invention, a microorganism that produces purine nucleotides or a microorganism having the ability to produce purine nucleotides may be a microorganism in which some of the genes in the purine nucleotide biosynthesis pathway are enhanced or weakened, or some of the genes in the purine nucleotide degradation pathway are enhanced or weakened.

[0070] The term "weakening" of polypeptide activity in the present invention is a concept that includes all cases where the activity is decreased or absent compared to the intrinsic activity. The weakening can be used interchangeably with terms such as inactivation, deficiency, down-regulation, decrease, reduce, attenuation, etc.

[0071] The weakening includes cases where the activity of the polypeptide itself is decreased or removed compared to the activity of the polypeptide originally possessed by the microorganism due to a mutation in the polynucleotide encoding the polypeptide, inhibition of gene expression of the polynucleotide encoding it, or inhibition of translation into the polypeptide, etc., resulting in the overall polypeptide activity and / or concentration (expression level) in the cell being lower than that of the wild-type strain, cases where the expression of the polynucleotide is not carried out at all, and / or cases where the polypeptide has no activity despite the expression of the polynucleotide. The "intrinsic activity" means the activity of a specific polypeptide originally possessed by the parental strain, wild-type, or non-transformed microorganism before transformation when the trait is changed due to genetic mutations caused by natural or artificial factors. This can be used interchangeably with "activity before transformation". The fact that the activity of the polypeptide is "inactivated, deficient, decreased, down-regulated, reduced, attenuated" compared to the intrinsic activity means that it is lower than the activity of the specific polypeptide originally possessed by the parental strain or non-transformed microorganism before transformation.

[0072] The attenuation of the activity of such a polypeptide can be carried out by any method known in the art, but is not limited thereto, and can be achieved by applying various methods well known in the art (for example, 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.).

[0073] Specifically, the attenuation of the polypeptide of the present invention is 1) deletion of all or part of the gene encoding the polypeptide; 2) modification of the expression regulatory region (or expression regulatory sequence) so that the expression of the gene encoding the polypeptide decreases; 3) modification of the amino acid sequence constituting the polypeptide so that the activity of the polypeptide is removed or weakened (for example, deletion / substitution / addition of one or more amino acids on the amino acid sequence); 4) modification of the gene sequence encoding the polypeptide so that the activity of the polypeptide is removed or weakened (for example, deletion / substitution / addition of one or more nucleobases on the nucleobase sequence of the polypeptide gene so as to encode a polypeptide modified so that the activity of the polypeptide is removed or weakened); 5) modification of the base sequence encoding the start codon or 5'-UTR region of the gene transcript encoding the polypeptide encoding the polypeptide; 6) introduction of an antisense oligonucleotide (for example, antisense RNA) that binds complementarily to the transcript of the gene encoding the polypeptide; 7) addition of a sequence complementary to the Shine-Dalgarno sequence in front of the Shine-Dalgarno sequence of the gene encoding the polypeptide in order 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 open reading frame (ORF) of the gene 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.

[0074] For example, The deletion of part or all of the gene encoding the polypeptide may be the removal of the entire polynucleotide encoding the endogenous target polypeptide in the chromosome, the replacement with a polynucleotide with some nucleotides deleted, or the replacement with a marker gene.

[0075] In addition, the modification of the expression regulatory region (or expression regulatory sequence) may be a deletion, insertion, non-conservative or conservative substitution, or a combination thereof, resulting in a mutation in the expression regulatory region (or expression regulatory sequence), or a replacement with a sequence having a weaker activity. The expression regulatory region includes, 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.

[0076] In addition, the modification of the amino acid sequence or polynucleotide sequence in 3) and 4) may be a deletion, insertion, non-conservative or conservative substitution, or a combination thereof, of the amino acid sequence of the polypeptide or the sequence of the polynucleotide encoding the polypeptide so as to weaken the activity of the polypeptide, resulting in a mutation in the sequence, or a replacement with an amino acid sequence or polynucleotide sequence improved to have a weaker activity or an amino acid sequence or polynucleotide sequence improved to have no activity, but is not limited thereto. For example, the expression of the gene can be inhibited or weakened by introducing a mutation in the polynucleotide sequence to form a stop codon, but is not limited to this.

[0077] In addition, the modification of the start codon of the gene transcript encoding the polypeptide or the nucleotide sequence encoding the 5'-UTR region may, for example, be substitution with a nucleotide sequence encoding another start codon with a lower polypeptide expression rate compared to the endogenous start codon, but is not limited thereto.

[0078] The introduction of an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the transcript of the gene encoding the polypeptide (6) above can be referred to, for example, in the literature [Weintraub, H. et al., Antisense-RNA as a molecular tool for genetic analysis, Reviews - Trends in Genetics, Vol. 1(1) 1986].

[0079] The addition of a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of the gene encoding the polypeptide to form a secondary structure that prevents ribosome attachment may render mRNA translation impossible or may reduce the rate.

[0080] The addition of a promoter transcribed in the opposite direction to the 3' end of the open reading frame (ORF) of the gene sequence encoding the polypeptide (8) above (Reverse transcription engineering, RTE) may create an antisense nucleotide complementary to the transcript of the gene encoding the polypeptide and weaken the activity.

[0081] In the present invention, the term "enhancement" of polypeptide activity means that the activity of the polypeptide increases as compared to the intrinsic activity. The said enhancement may be used interchangeably with terms such as activation, up-regulation, overexpression, increase, etc. Here, activation, enhancement, up-regulation, overexpression, increase can all include showing an activity that was not originally present, or showing an activity improved as compared to the intrinsic activity or the activity before modification. The said "intrinsic activity" means the activity of a specific polypeptide that the parental strain before transformation or the unmodified microorganism originally had when the trait changed due to genetic mutation by natural or artificial factors. This may be used interchangeably with "activity before modification". That the activity of a polypeptide "enhances", "is up-regulated", "is overexpressed" or "increases" as compared to the intrinsic activity means that it is improved as compared to the activity and / or concentration (expression level) of the specific polypeptide that the parental strain before transformation or the unmodified microorganism originally had.

[0082] The said enhancement can be achieved by introducing a foreign polypeptide or through enhancing the activity and / or concentration (expression level) of an intrinsic polypeptide. Whether the activity of the said polypeptide is enhanced can be confirmed from an increase in the activity level of the said polypeptide, the expression level or the amount of the product excreted from the said polypeptide.

[0083] The enhancement of the activity of the said polypeptide can be achieved by applying various methods well known in the art, and is not limited as long as the activity of the polypeptide of interest can be enhanced as compared to the microorganism before modification. Specifically, it may be, but is not limited to, those using genetic engineering and / or protein engineering well known to an ordinary technician in the art, which are routine methods in molecular biology (for example, Sitnicka et al. Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol. 2. 1-16, Sambrook et al. Molecular Cloning 2012, etc.).

[0084] Specifically, the enhancement of the polypeptide of the present application is 1) an increase in the intracellular copy number of the polynucleotide encoding the polypeptide; 2) replacing the gene expression regulatory region on the chromosome encoding the polypeptide with a sequence having strong activity; 3) modification of the base sequence encoding the start codon or 5'-UTR region of the gene transcript encoding the polypeptide; 4) modification of the amino acid sequence of the polypeptide so as to enhance the polypeptide activity; 5) modification of the polynucleotide sequence encoding the polypeptide so as to enhance the polypeptide activity (for example, modification of the polynucleotide sequence of the polypeptide gene so as to encode a polypeptide modified so as to enhance the activity of the polypeptide); 6) introduction of a foreign polypeptide showing 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 to select and modify the exposed site or chemically modify it; or 9) It may be a combination of two or more selected from the above 1) to 8), but is not particularly limited thereto.

[0085] More specifically, The increase in the intracellular copy number of the polynucleotide encoding the polypeptide in 1) above may be achieved by introducing into the host cell a vector that can replicate and function independently of the host and to which the polynucleotide encoding the polypeptide is operably linked. Alternatively, it may be achieved by introducing one copy or two or more copies of the polynucleotide encoding the polypeptide into the chromosome in the host cell. The introduction into the chromosome may be carried out by introducing into the host cell a vector capable of inserting the polynucleotide into the chromosome in the host cell, but is not limited thereto. The vector is as described above.

[0086] Exchanging the gene expression regulatory region (or expression regulatory sequence) on the chromosome encoding the polypeptide in 2) above with a sequence having strong activity may, for example, generate mutations in the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof so as to further enhance the activity of the expression regulatory region, or may be an exchange with a sequence having stronger activity. The expression regulatory region may include, but is not particularly limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating the termination of transcription and translation. As an example, it may be to exchange the original promoter with a strong promoter, but is not limited thereto.

[0087] Examples of known strong promoters include, but are not limited to, the CJ1-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 SPL 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, the yccA promoter, etc.

[0088] Modification of the start codon of the gene transcript encoding the polypeptide in 3) above or the nucleotide sequence encoding the 5'-UTR region may, for example, be to substitute the nucleotide sequence encoding another start codon with a higher polypeptide expression rate compared to the endogenous start codon, but is not limited thereto.

[0089] The modifications of the amino acid sequences or polynucleotide sequences in the above 4) and 5) may be deletions, insertions, non-conservative or conservative substitutions, or the occurrence of sequence mutations by combinations thereof in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide so as to enhance the activity of the polypeptide, or the amino acid sequence or polynucleotide sequence improved to have stronger activity or the exchange to an amino acid sequence or polynucleotide sequence improved to increase the activity, but are not limited thereto. Specifically, the above exchange can be carried out by inserting a polynucleotide into a chromosome by homologous recombination, but is not limited thereto. The vector used at this time may further contain a selection marker for confirming the presence or absence of chromosomal insertion. The selection marker is as described above.

[0090] The introduction of a foreign polynucleotide showing the activity of the above 6) polypeptide may be the introduction of a foreign polynucleotide encoding a polypeptide showing the same / similar activity as the above polypeptide into a host cell. The origin and sequence of the foreign polynucleotide are not limited as long as it shows the same / similar activity as the above polypeptide. The method used for the above introduction can be appropriately selected by those skilled in the art from known transformation methods, and the introduced polynucleotide is expressed in the host cell to generate a polypeptide, and its activity may be increased.

[0091] The codon optimization of the polynucleotide encoding the above 7) polypeptide is such that the endogenous polynucleotide is codon-optimized so that transcription or translation increases in the host cell, or the codon of the foreign polynucleotide is optimized so that optimized transcription and translation are carried out in the host cell.

[0092] Analyzing the tertiary structure of the polypeptide described in 8) to select an exposed site for modification or chemical modification may involve, for example, comparing the sequence information of the polypeptide to be analyzed with a database storing the sequence information of known proteins. Based on the sequence similarity, candidate template proteins are determined, and based on this, the structure is confirmed, and an exposed site for modification or chemical modification is selected for modification or chemical modification.

[0093] Such enhancement of polypeptide activity may be that the activity or concentration expression level of the corresponding polypeptide increases based on the activity or concentration of the polypeptide expressed in the wild-type or unmodified microbial strain, or the amount of the product produced from the polypeptide increases, but is not limited thereto.

[0094] In the microorganism of the present invention, partial or total modification of the polynucleotide can be induced by (a) homologous recombination using a chromosomal insertion vector in the microorganism or genome editing using an engineered nuclease (e.g., CRISPR-Cas9) and / or (b) treatment with light such as ultraviolet rays and radiation and / or chemicals, but is not limited thereto. The method for partial or total modification of the gene can include a method using DNA recombination technology. For example, a nucleotide sequence or vector containing a nucleotide sequence homologous to the target gene can be injected into the microorganism to cause homologous recombination, and partial or total deletion of the gene may be achieved. The injected nucleotide sequence or vector may include a dominant selection marker, but is not limited thereto.

[0095] Another aspect of the present invention provides a method for producing purine nucleotides, which includes culturing a microorganism containing the mutant of the present invention or a polynucleotide encoding the same.

[0096] The method for producing purine nucleotides of the present invention may include the step of culturing a microorganism containing the variant of the present invention, or a polynucleotide encoding the same, or a vector containing the polynucleotide in a medium.

[0097] In the present invention, the term "culturing" means growing the microorganism of the present invention under appropriately controlled environmental conditions. The culturing process of the present invention can be carried out according to appropriate media and culturing conditions known in the art. Such a culturing process can be easily adjusted and used by those skilled in the art according to the selected strain. Specifically, the culturing may be batch, continuous, and / or fed-batch, but is not limited thereto.

[0098] In the present invention, the term "medium" means a substance mainly composed of nutrients required for culturing the microorganism of the present application, and supplies nutrients and growth factors including water essential for survival and growth. Specifically, the medium and other culturing conditions used for culturing the microorganism of the present invention can be any medium used for culturing ordinary microorganisms without particular limitation, but the microorganism of the present invention can be cultured under aerobic conditions while adjusting temperature, pH, etc. in an ordinary medium containing an appropriate carbon source, nitrogen source, phosphorus source, inorganic compound, amino acid, and / or vitamin.

[0099] For example, the culture medium for Corynebacterium strains can be found in the literature ["Manual of Methods for General Bacteriology" by the American Society for Bacteriology (Washington D.C., USA, 1981)].

[0100] In the present invention, the carbon source may include carbohydrates such as glucose, sucrose, lactose, fructose, sucrose, maltose; sugar alcohols such as mannitol, sorbitol; organic acids such as pyruvic acid, lactic acid, citric acid; amino acids such as glutamic acid, methionine, lysine, etc. In addition, natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn steep liquor can be used. Specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted to reducing sugar) can be used, and other appropriate carbon sources can be used in various ways without limitation. These carbon sources may be used alone or in combination of two or more, and are not limited thereto.

[0101] As the nitrogen source, inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate; amino acids such as glutamic acid, methionine, glutamine; peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolysate, fish or its decomposition products, defatted soybean cake or its decomposition products and other organic nitrogen sources can be used. These nitrogen sources may be used alone or in combination of two or more, and are not limited thereto.

[0102] The phosphorus source may include monopotassium phosphate, dipotassium phosphate, or their corresponding sodium-containing salts. As inorganic compounds, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc. may be used, and in addition, amino acids, vitamins and / or appropriate precursors, etc. may be included. These components or precursors can be added to the medium in a batch or continuous manner. However, it is not limited thereto.

[0103] During the cultivation of the microorganism of the present invention, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. can be added to the medium in an appropriate manner to adjust the pH of the medium. Also, during cultivation, foaming can be suppressed using an antifoaming agent such as a fatty acid polyglycol ester. Further, in order to maintain the aerobic state of the medium, oxygen or an oxygen-containing gas can be injected into the medium, or in order to maintain anaerobic and microaerobic states, without gas injection, or nitrogen, hydrogen or carbon dioxide gas can be injected, but is not limited thereto.

[0104] In the cultivation of the present invention, the cultivation temperature can be maintained at 20 to 45 °C, specifically 25 to 40 °C, and can be cultivated for about 10 to 160 hours, but is not limited thereto.

[0105] The purine nucleotide produced by the cultivation of the present invention may be secreted into the medium or remain intracellularly.

[0106] The method for producing a purine nucleotide of the present invention can further include, for example, before the cultivation step, the step of preparing the microorganism of the present invention, the step of preparing a medium for cultivating the microorganism, or a combination thereof (the order is irrelevant, in any order).

[0107] The method for producing a purine nucleotide of the present invention can further include the step of recovering the purine nucleotide from the medium (cultivated medium) by the cultivation or from the microorganism. The step of recovering can be further included after the step of cultivating.

[0108] The recovery may be to collect the target purine nucleotide by using an appropriate method known in the art, such as a batch, continuous, or fed-batch culture method, etc., of the method for culturing the microorganism of the present invention. For example, centrifugation, filtration, treatment with a crystallization protein precipitant (salting-out method), extraction, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, and other various chromatographies, HPLC, or a combination of these methods can be used, and the target purine nucleotide can be recovered from the culture medium or the microorganism by using an appropriate method known in the art.

[0109] Furthermore, the method for producing a purine nucleotide of the present invention may further include a purification step. The purification can be carried out by using an appropriate method known in the art. As an example, when the method for producing a purine nucleotide of the present invention includes both a recovery step and a purification step, the recovery step and the purification step can be carried out continuously or discontinuously regardless of the order, or simultaneously or integrated into one step, but it is not limited thereto.

[0110] As an example, the GMP production method in the present invention may further include a step of converting XMP to GMP. In the GMP production method of the present invention, the converting step can be further included after the culturing step or the recovering step. The converting step can be carried out by using an appropriate method known in the art. For example, the conversion can be carried out by using a coryneform microorganism, Escherichia coli, or 5'-xanthylic acid aminase (such as KR 10-0655902 B1, US 2013-0095529 A1, etc.), but it is not limited thereto.

[0111] In the method of the present invention, the mutant, polynucleotide, vector, strain, purine nucleotide, etc. are as described in the above other aspects.

[0112] Another aspect of the present invention provides a composition for producing purine nucleotides, which comprises a variant of the present application, a polynucleotide encoding the variant, a vector containing the polynucleotide, or a microorganism containing the polynucleotide of the present invention; a medium obtained by culturing the same; or a combination thereof.

[0113] The composition of the present invention may further contain any suitable excipient commonly used in compositions for producing purine nucleotides, and such excipients may include, for example, preservatives, wetting agents, dispersants, suspending agents, buffering agents, stabilizers or isotonic agents, etc., but are not limited thereto.

[0114] In the composition of the present invention, the variant, polynucleotide, vector, microorganism, medium, purine nucleotide, etc. are as described in the other aspects.

[0115] Another aspect of the present invention provides a method for increasing the purine nucleotide production ability of a microorganism, which comprises the step of modifying the microorganism to express the variant of the present invention.

[0116] In the method of the present invention, the variant, polynucleotide, vector, microorganism, medium, purine nucleotide, etc. are as described in the other aspects.

[0117] Another aspect of the present invention provides the use of the variant of the present invention in the production of purine nucleotides.

[0118] Another aspect of the present invention provides the use of the variant of the present invention, a polynucleotide encoding the same, or a microorganism containing the vector containing the same in the production of purine nucleotides.

[0119] The variant, polynucleotide, vector, microorganism, etc. are as described in the other aspects.

[0120] Hereinafter, the present invention will be described in more detail through examples and experimental examples. However, these examples and experimental examples are for illustrative purposes of this application, and the scope of the present invention is not limited to these examples and experimental examples.

[0121] Example 1: Production of guaA synthase-attenuated mutants To discover guaA mutants for the production of XMP, a mutant library of guaA, the gene encoding it, was prepared.

[0122] Example 1-1. Preparation of a vector containing guaA To prepare a guaA mutant library, first, a recombinant vector containing guaA was prepared. Using the chromosome of Corynebacterium stationis ATCC6872 as a template, PCR was performed using the primers of SEQ ID NO: 5 and SEQ ID NO: 6, and the amplification product was cloned into the pCR2.1 vector using the TOPO Cloning Kit (Invitrogen), and this was named pCR-guaA.

[0123] [Table 1]

[0124] Example 1-2: Preparation of a guaA mutant library Using the pCR-guaA vector prepared in Example 1-1 as a template, a guaA mutant library was prepared. The library was prepared using an error-prone PCR kit (clontech DiversifyR PCR Random Mutagenesis Kit), and random mutations were induced according to the manufacturer's manual using the primers of SEQ ID NO: 7 and SEQ ID NO: 8 to obtain guaA mutant PCR products with different sequences. After cloning the amplified products using the TOPO Cloning Kit (Invitrogen), they were transformed into Escherichia coli DH5α and spread on LB solid medium containing kanamycin (25 mg / L). The transformed E. coli colonies were collected and plasmids were extracted, which were named pCR-guaA-library.

[0125]

Table 2

[0126] Example 1-3: Evaluation of the prepared library and selection of strains After transforming the pCR-guaA-library prepared in Example 1-2 into the wild-type strain Corynebacterium stationis ATCC6872 by electroporation, it was spread on a nutrient medium containing 25 mg / L of kanamycin to secure 5,000 colonies of strains into which the mutant gene was inserted, and each colony was named from C.st ATCC6872_pCR_guaA(mt)1 to C.st ATCC6872_pCR_guaA(mt)5000.

[0127] - Nutrient medium: 1% peptone, 1% gravy, 0.25% sodium chloride, 1% yeast extract, 150 mg / L adenine, 150 mg / L guanine, 2% agar, pH 7.2 (based on 1 liter of distilled water)

[0128] After subjecting 5,000 colonies that had been ensured to be sterilized under pressure to autoclaving, each was inoculated with 100 μl of a seed medium containing kanamycin (25 mg / L), and cultured with shaking at a temperature of 30°C and 1,200 rpm for 24 hours using a microplate shaker (TAITEC) in a 96-deep well plate, and used as a seed culture solution. After dispensing 320 μl of the autoclaved fermentation medium (240 μl of the main medium + 80 μl of separately sterilized medium) into a 96-deep well plate, 15 μl of the seed culture solution was inoculated into each well, and cultured with shaking for 72 hours under the same conditions as above.

[0129] To analyze the production amount of XMP produced in the culture solution, after the completion of the culture, 640 μl of sterilized water was dispensed into the culture solution, and centrifuged at 4,000 rpm for 15 minutes. Then, 3 μl of the supernatant was transferred to a 96-well UV-plate into which 297 μl of distilled water had been dispensed. Next, using a microplate reader, it was shaken for 30 seconds, and the absorbance was measured with a spectrophotometer at 25°C and a wavelength of 260 nm. Ten colonies of mutants with an increased specific absorbance compared to the wild-type strain were selected. The other colonies showed similar or decreased absorbance compared to the control group.

[0130] The selected 10 strains were repeatedly subjected to confirmation of the XMP production amount by measuring the absorbance in the same manner as described above, and one strain (C.st ATCC6872_pCR_guaA(mt)726) with a significantly improved XMP production ability compared to the wild-type strain was selected.

[0131] To verify the effectiveness of the finally selected strain, the fermentation titer was evaluated.

[0132] 14 ml of a tube containing 2.5 ml of the following seed medium was inoculated with Corynebacterium stationis ATCC6872 wild-type strain and the selected C. st ATCC6872_pCR_guaA(mt)726 strain, and cultured with shaking at 170 rpm for 24 hours at 30°C. 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 with shaking at 170 rpm for 75 hours at 30°C.

[0133] The compositions of the above-mentioned seed medium, this medium, and the separately sterilized medium are as follows.

[0134] <Seed medium for XMP flask> Glucose 30 g / L, peptone 15 g / L, yeast extract 15 g / L, sodium chloride 2.5 g / L, urea 3 g / L, adenine 150 mg / L, guanine 150 mg / L, pH 7.0 (based on 1 liter of distilled water)

[0135] <XMP flask production medium (this medium)> Glucose 50 g / L, magnesium sulfate 10 g / L, yeast extract 3 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, histidine 20 mg / L, cysteine 15 mg / L, β-alanine 15 mg / L, biotin 100 μg / L, thiamine 5 mg / L, adenine 50 mg / L, guanine 25 mg / L, niacin 15 mg / L, pH 7.0 (based on 1 liter of distilled water)

[0136] <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)

[0137] After the cultivation was completed, the results of measuring the production amount of XMP by the method using HPLC are as shown in Table 3 below.

[0138]

Table 3

[0139] As shown in Table 3 above, it was confirmed that an XMP concentration of 4.83 g / L was produced in C.st ATCC6872_pCR_guaA(mt)726 compared to the wild-type strain.

[0140] Examples 1-4: Confirmation of mutations through gene sequencing To confirm the guaA mutant sequence of the C.st ATCC6872_pCR_guaA(mt)726 strain, PCR and nucleotide sequence analysis were performed on the C.st ATCC6872_pCR_guaA(mt)726 strain using the primers of SEQ ID NO: 8 and SEQ ID NO: 9. As a result of comparison with the wild-type strain guaA gene sequence, it was confirmed that the 29th amino acid of the guaA gene of the C.st ATCC6872_pCR_guaA(mt)726 strain was substituted from arginine to cysteine (substitution of the 85th nucleotide from c to t: SEQ ID NO: 2).

[0141] The confirmed mutant sequence is as shown in Table 4 below.

[0142] [Table 4]

[0143] Therefore, in the following examples, an attempt was made to confirm that the guaA mutation affects the production of XMP.

[0144] Example 2: Confirmation of the effects of guaA mutants Example 2-1: Preparation of a vector for the expression of the guaA mutant To confirm the effect of the mutant in which arginine at the 29th position of the guaA enzyme amino acid sequence is substituted with cysteine (R29C; SEQ ID NO: 1) on XMP production, a vector for preparing its expression strain was prepared as follows using plasmid pDCM2 (Korean Patent Publication No. 10-2020-0136813) for insertion and exchange of genes in the Corynebacterium chromosome.

[0145] Using the chromosome of Corynebacterium stationis ATCC6872 as a template, PCR was performed using the primer pairs of SEQ ID NO: 10 and SEQ ID NO: 11, and SEQ ID NO: 12 and SEQ ID NO: 13. The polymerase used was SolgTM Pfu-X DNA polymerase, and the PCR amplification conditions were denaturation at 95°C for 5 minutes, followed by 30 cycles of denaturation at 95°C for 30 seconds, annealing at 58°C for 30 seconds, polymerization at 72°C for 60 seconds, and finally a polymerization reaction at 72°C for 5 minutes to obtain each PCR product. The amplified product was mixed with the pDCM2 vector prepared by pre-cutting with SmaI restriction enzyme and cloned using the Gibson Assembly (DG Gibson et al., NATURE METHODS, VOL.6 NO.5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix) method to obtain a recombinant plasmid, which was named pDCM2-guaA(R29C). Cloning was performed by mixing the Gibson Assembly reagent and the calculated molar amounts of each gene fragment and then storing at 50°C for 1 hour.

[0146] Example 2-2: Preparation of guaA Mutant Expression Strain The pDCM2-guaA(R29C) vector prepared in the above example was transformed into Corynebacterium stationis ATCC6872 strain by electroporation. The strain in which the vector was inserted onto the chromosome by recombination of homologous sequences was selected on a medium containing 25 mg / L of kanamycin. The selected primary strain was again subjected to a secondary cross-over to select a strain into which a mutation of the target gene was introduced. The presence or absence of gene mutation introduction in the finally transformed strain was confirmed through PCR using the primer pair of SEQ ID NO: 14 and SEQ ID NO: 15 and nucleotide sequence analysis, and this was named C.st ATCC6872::guaA(R29C).

[0147] Example 2-3: Comparison of XMP production ability of guaA mutant-expressing strains The XMP production abilities of the C.st ATCC6872::guaA(R29C) strain and the wild-type strain prepared in Example 2-2 above were confirmed through the fermentation titer evaluation method of Example 1-3. After the completion of culture, the production amount of XMP was measured using HPLC, and the culture results are as shown in Table 5 below.

[0148] The above experiment was repeated three times, and the average value of the analysis results is shown in Table 5 below.

[0149]

Table 5

[0150] Similar to Table 5 above, it was confirmed that the XMP concentration of 5.21 g / L was produced in the C.st ATCC6872::guaA(R29C) strain.

[0151] The C.st ATCC6872::guaA(R29C) strain was named CN02-2299 and deposited with the Korean Culture Center of Microorganisms, a depository institution under the Budapest Treaty, on August 9, 2021, and was assigned the deposit number KCCM13029P.

[0152] Example 3: Substitution of the amino acids of guaA mutations with other amino acids Example 3-1: Preparation of Vector for guaA Mutant Amino Acid Substitution and Insertion According to the above example, it was confirmed that XMP can be produced by the guaA(R29C) mutation. To confirm the positional importance of the guaA mutation, a vector was prepared to substitute the 29th amino acid with other amino acids, and it was confirmed whether it affects the XMP production ability. Site-directed mutagenesis was performed using the pDCM2-guaA(R29C) vector prepared in Example 2-1 as a template.

[0153] Specifically, PCR was performed using the primer pairs of SEQ ID NO: 10 and SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 13, SEQ ID NO: 10 and SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 13, SEQ ID NO: 10 and SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 13, SEQ ID NO: 10 and SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 13, SEQ ID NO: 10 and SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 13, SEQ ID NO: 10 and SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 13, SEQ ID NO: 10 and SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 13, SEQ ID NO: 10 and SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 13, SEQ ID NO: 10 and SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 13, SEQ ID NO: 10 and SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 13, SEQ ID NO: 10 and SEQ ID NO: 36, SEQ ID NO: 37 and SEQ ID NO: 13, SEQ ID NO: 10 and SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 13, SEQ ID NO: 10 and SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO: 13, SEQ ID NO: 10 and SEQ ID NO: 42, SEQ ID NO: 43 and SEQ ID NO: 13, SEQ ID NO: 10 and SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 13, SEQ ID NO: 10 and SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 13, SEQ ID NO: 10 and SEQ ID NO: 48, SEQ ID NO: 49 and SEQ ID NO: 13, SEQ ID NO: 10 and SEQ ID NO: 50, SEQ ID NO: 51 and SEQ ID NO: 13 respectively. At this time, SolgTM Pfu-X DNA polymerase was used as the polymerase, and the PCR amplification conditions were denaturation at 95°C for 5 minutes, followed by 30 cycles of denaturation at 95°C for 30 seconds, annealing at 58°C for 30 seconds, and polymerization at 72°C for 60 seconds, and then polymerization reaction at 72°C for 5 minutes to obtain each PCR product. Each of the amplified products was previously digested with SmaI restriction enzyme, mixed with the prepared pDCM2 vector, and cloned by using the Gibson assembly method to obtain a recombinant plasmid. The plasmid information obtained in this way is as shown in Table 6. Cloning was performed by mixing the Gibson assembly reagent and the calculated molar amount of each gene fragment, and then storing at 50°C for 1 hour.

[0154]

Table 6

[0155] Example 3-2: Preparation of Strains in Which the Mutation of the guaA Variant is Substituted with Other Amino Acids and Comparison of XMP Production Ability Each of the 18 mutagenesis vectors prepared in Example 3-1 was transformed into the wild-type strain of Corynebacterium stationis ATCC6872 by electroporation, and the strain in which the vector was inserted into the chromosome by homologous sequence recombination was selected on a medium containing 25 mg / L of kanamycin. The selected primary strains were again subjected to secondary cross-over to select strains in which mutations were introduced into the target gene. The presence or absence of gene mutagenesis in the finally transformed strains was confirmed through PCR using the primer pair of SEQ ID NO: 14 and SEQ ID NO: 15 and nucleotide sequence analysis, and the strain names due to the inserted mutations are as shown in Table 7 below.

[0156]

Table 7

[0157] The prepared strains, the C. st ATCC6872::guaA(R29C) strain, and the wild-type strain were cultured by the fermentation titer evaluation method of Example 1-3 to analyze the concentration of XMP. The experiment was repeated 3 times, and the average value of the analysis results is shown in Table 8 below.

[0158]

Table 8

[0159] Referring to Table 8 above, in the case of a strain containing guaA in which the 29th amino acid of the amino acid sequence encoded by the guaA gene is substituted with an amino acid other than arginine, it was confirmed that XMP was produced as compared to the wild-type strain. That is, the 29th amino acid of the amino acid sequence encoded by the guaA gene is a major mutation site in the production of XMP. More specifically, when the 29th amino acid of the amino acid sequence encoded by the guaA gene is not arginine but is substituted with another amino acid (cysteine, glycine, phenylalanine, serine, tyrosine, tryptophan, isoleucine, valine, proline, threonine, alanine, aspartic acid, asparagine, histidine, leucine, methionine, glutamine, lysine, and glutamic acid), it was confirmed that the XMP production amount of the microorganism containing this can be significantly increased.

[0160] From this, it can be seen that a mutant in which the 29th amino acid of guaA is substituted with another amino acid can be usefully used for the production of purine nucleotides.

[0161] Example 4: Confirmation of the GMP production ability of guaA mutant-expressing strains Using the XMP culture solution of C.st ATCC6872::guaA(R29C), which is an XMP-producing strain, GMP production was confirmed by the following method (see Korean Patent KR 10-0655902 B1 and US Patent Publication No. US 2013-0095529 A1).

[0162] The C.st ATCC6872::guaA(R29C) strain was cultured by the fermentation titer evaluation method of Examples 1-3. After the culture was completed, the production amount of XMP was measured by a method using HPLC. In order to perform the conversion reaction of the generated XMP to GMP, the following conversion reaction additives and XMP aminase of Escherichia coli were added to the Erlenmeyer flask fermentation broth, and the conversion reaction was carried out at 40 °C for 2.5 hours.

[0163] <Conversion reaction additives> Phytic acid 1.8 g / L, magnesium sulfate 4.8 g / L, nymeen 3 ml / L, xylene 2%, adenine 100 mg / L, sodium hydrogen phosphate (Na2HPO4) 7.7 g / L, glutamine 2 g / L, glucose 46 g / L

[0164] As a result of conducting the above experiment, Table 9 shows the results of the conversion rate, which represents the production amount of GMP with respect to the XMP consumption amount.

[0165]

Table 9

[0166] As shown in Table 9 above, it was confirmed that 3.54 g / L of GMP was produced through the conversion reaction from XMP produced by the C.st ATCC6872::guaA(R29C) strain.

[0167] As a result, when it was confirmed that a mutant in which the 29th amino acid of guaA was substituted with another amino acid increased GMP production, it was found that the mutant provided in the present invention can be usefully used for the production of purine nucleotides.

[0168] From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present invention can be implemented in other specific forms without changing its technical idea and essential features. In this regard, it should be understood that the embodiments described above are merely exemplary and not restrictive. The scope of the present invention should be interpreted as including the meaning and scope of the following claims, and all changes or modified forms derived from the equivalent concept thereof within the scope of the present invention.

Claims

1. The amino acid corresponding to the 29th position of SEQ ID NO: 3 is substituted with another amino acid, and the other amino acid is selected from glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, and histidine, and has at least 90% sequence identity with the polypeptide of SEQ ID NO: 3, a glutamine-hydrolyzing GMP synthase variant.

2. A polynucleotide encoding the variant according to Claim 1.

3. A microorganism, Corynebacterium stationis, comprising the glutamine-hydrolyzing GMP synthase variant according to Claim 1 or the polynucleotide encoding the variant.

4. A method for producing a purine nucleotide, comprising culturing a microorganism, Corynebacterium stationis, comprising the variant according to Claim 1 or the polynucleotide encoding the variant.

5. The method for producing a purine nucleotide according to Claim 4, further comprising recovering the purine nucleotide from the culture medium or the microorganism.

6. A composition for producing a purine nucleotide, comprising at least one of the glutamine-hydrolyzing GMP synthase variant according to Claim 1 and the microorganism, Corynebacterium stationis, comprising the variant.

7. A method for increasing the purine nucleotide-producing ability of a microorganism, Corynebacterium stationis, comprising introducing the glutamine-hydrolyzing GMP synthase variant according to Claim 1 or a polynucleotide encoding the same into the microorganism.

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