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

Abstract

The present invention relates to a Corynebacterium stachyonis microorganism that produces purine nucleotides by enhancing the activity of the phosphate influx system (Pit system), and a method for producing purine nucleotides using the same.

Description

[Technical Field]

[0001] The present invention relates to a Corynebacterium stachyonis microorganism having enhanced activity of the phosphate influx system (Pit system) and the ability to produce purine nucleotides, and a method for producing purine nucleotides using the microorganism. [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] Phosphate is a component of purine nucleotides, provides the energy necessary for microbial growth, is essential for the biosynthesis of phospholipids in cell membranes, nucleic acids, and proteins, and also plays a key role in cell signaling processes.

[0004] Phosphate is primarily absorbed into cells in the form of inorganic orthophosphate (Pi). The entry of Pi into microorganisms depends on the function of importers present in the cell membrane. Known Pi importers include the Pst system, a high-affinity Pi entry system whose expression is regulated by sensing the extracellular Pi concentration; the Pit system, which is expressed regardless of Pi concentration and imports Pi in a mixed form with metal ions; and the antiporter transport system, which imports the organic phosphate forms glycerol-3-phosphate and glucose-6-phosphate.

[0005] Among the phosphate influx systems, several methods for regulating the Pit system to produce amino acids have been reported, but no reports have been published on its effects on nucleic acid production. Furthermore, it has been confirmed that weakening or strengthening the expression of the Pit system leads to different results depending on the microorganism and the amino acid, confirming that in the case of amino acid production, regulating the Pi influx system in a specific direction does not have a uniform effect on increasing or decreasing production (US9506094 B2, US9873898 B2). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] US9506094 B2 [Patent Document 2] US9873898 B2 [Patent Document 3] US Patent US7662943 B2 [Patent Document 4] US Patent US10584338 B2 [Patent Document 5] US Patent US10273491 B2 [Patent Document 6] KR10-1950141 B1 [Patent Document 7] US2020-0392478 A1

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[0008] The present invention provides a Corynebacterium stachyonis microorganism capable of producing purine nucleotides by enhancing the activity of the phosphate influx system (Pit system), and a method for producing purine nucleotides using the microorganism. [Means for solving the problem]

[0009] One aspect of the present invention is to provide a Corynebacterium stathionis microorganism having enhanced activity of the phosphate influx system (Pit system) and the ability to produce nucleotides.

[0010] Another aspect of the present invention is to provide a method for producing purine nucleotides, which comprises culturing the Corynebacterium stachyonis microorganism capable of producing purine nucleotides in a medium.

[0011] Another aspect of the present invention is to provide a composition for producing purine nucleotides, comprising the Corynebacterium stachyonis microorganism capable of producing purine nucleotides; a medium in which the microorganism has been cultured; or a combination thereof.

[0012] Another aspect of the present invention is to provide use of the Corynebacterium stachyonis microorganism having the ability to produce purine nucleotides for producing purine nucleotides. [Effects of the Invention]

[0013] By enhancing the activity of the phosphate importer according to the present invention, it is possible to facilitate the inflow of phosphate, an important component for the production of purine nucleotides, into microorganisms. As a result, microorganisms capable of producing purine nucleotides can efficiently produce purine nucleotides, which can significantly contribute to cost reduction when producing purine nucleotides on an industrial scale. DETAILED DESCRIPTION OF THE INVENTION

[0014] This will be explained in more detail as follows: Each description and embodiment disclosed in the present invention can also be applied to each other description and embodiment. In other words, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Furthermore, the specific descriptions set forth below are not considered to limit the category of the present invention. Furthermore, numerous papers and patent documents are referenced throughout this specification, and citations thereof are provided. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to more clearly explain the state of the art to which the present invention pertains and the content of the present invention.

[0015] One aspect of the present invention is to provide a Corynebacterium stachyonis microorganism capable of producing purine nucleotides by enhancing the activity of the phosphate influx system (Pit system) encoded by the pit gene.

[0016] As used herein, the term "phosphate influx system" refers to a polypeptide or a system comprising such a polypeptide that functions to influx phosphate into cells. The phosphate influx system may comprise a phosphate importer. For purposes of this application, the phosphate influx system may also be the Pit system.

[0017] As used herein, the term "phosphate importer" refers to a polypeptide capable of absorbing phosphate into cells. Phosphate is absorbed into cells primarily in the form of inorganic orthophosphate (hereinafter referred to as Pi), and this absorption depends on the function of importers present in the cell membrane. Specifically, known Pi importers include, but are not limited to, the Pst system, the Pit system, and antiporter transport systems that transport the organic phosphate forms glycerol-3-phosphate and glucose-6-phosphate.

[0018] In the present invention, the term "Pit system" refers to a phosphate transporter (low-affinity; tellurite importer; hereafter referred to as "pit") that exhibits low affinity for Pi. Expression of the Pit system is independent of the extracellular Pi concentration, and Pi is known to enter the cell in the form of a metal cation complex. (JOURNAL OF BACTERIOLOGY, September 2001, pp. 5008-5014, Vol. 183, No. 17)

[0019] The Pit system may include a polypeptide encoded by one or more genes selected from pit, pit1, pit2, pit3, pitA, pitB, and pitC, or one or more of Pit, Pit1, Pit2, Pit3, PitA, PitB, and PitC polypeptides. For purposes of the present invention, the Pit system may be, but is not limited to, a polypeptide encoded by the pitA (inorganic phosphate transporter) gene or a PitA polypeptide.

[0020] The Pit system may include a polypeptide having the amino acid sequence set forth in SEQ ID NO: 1, but is not limited thereto, as there may be differences in the amino acid sequence of the enzyme exhibiting the activity depending on the species or strain of the microorganism.

[0021] The PitA polypeptide or the polypeptide encoded by the pitA gene may consist of the polypeptide set forth in the amino acid sequence of SEQ ID NO: 1, but is not limited thereto.

[0022] The PitA polypeptide or the polypeptide encoded by the pitA gene may have, comprise, consist of, or essentially consist of the amino acid sequence set forth in SEQ ID NO: 1, but is not limited thereto. More specifically, SEQ ID NO: 1 may be the sequence of a polypeptide having phosphate importer activity encoded by the pitA gene.

[0023] In one embodiment, the polypeptide encoded by the pitA gene may include, but is not limited to, a polypeptide having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity to the amino acid sequence of SEQ ID NO: 1.

[0024] The amino acid sequence of SEQ ID NO: 1 can be obtained from various publicly known databases, such as NCBI's GenBank, but is not limited thereto. For example, the amino acid sequence may be derived from Escherichia coli, but is not limited thereto, and sequences having the same activity as the amino acid sequence are included without limitation. Furthermore, although the polypeptide having the activity of a phosphate importer, Pit system, or PitA of the present invention is described as a polypeptide containing the amino acid sequence of SEQ ID NO: 1, it will be obvious to those skilled in the art that this does not exclude meaningless additions of sequences before or after the amino acid sequence of SEQ ID NO: 1, naturally occurring mutations, or silent mutations thereof. Any polypeptide having the same or corresponding activity as a polypeptide containing the amino acid sequence of SEQ ID NO: 1 is also considered to be a polypeptide having the activity of a phosphate importer, Pit system, or PitA of the present invention.

[0025] Specifically, for example, a polypeptide having the activity of a phosphate importer or Pit system of the present invention may be a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity thereto.

[0026] A polypeptide having the activity of the PitA polypeptide of the present application or the polypeptide encoded by the pitA gene may be a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity thereto.

[0027] It is also clear that polypeptides having an amino acid sequence in which a portion of the sequence has been deleted, modified, substituted, or added are also included within the scope of the polypeptides of the present application, as long as the amino acid sequence has such homology or identity and exhibits the efficacy corresponding to the above polypeptide.

[0028] In the present invention, even if a polypeptide is described as "a polypeptide comprising an amino acid sequence set forth in a specific SEQ ID NO," "a polypeptide consisting of an amino acid sequence set forth in a specific SEQ ID NO," or "a polypeptide having an amino acid sequence set forth in a specific SEQ ID NO," it is obvious that a polypeptide having an amino acid sequence in which a portion of the sequence has been deleted, modified, substituted, conservatively substituted, or added is also used in the present application, so long as it has the same or equivalent activity as a polypeptide consisting of the amino acid sequence of the SEQ ID NO. For example, a polypeptide having an addition of a sequence at the N-terminus and / or C-terminus of the amino acid sequence that does not change the function of the protein, a naturally occurring mutation, a silent mutation, or a conservative substitution thereof.

[0029] The term "conservative substitution" refers to the substitution of an amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions may generally be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. Typically, conservative substitutions have little or no effect on the activity of a polypeptide.

[0030] The Pit system may be encoded by one or more genes selected from pit, pit1, pit2, pit3, pitA, pitB, and pitC, but is not limited thereto. Specifically, the Pit system may be encoded by a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 2, but is not limited thereto due to codon degeneracy. The Pit system may be encoded by a polynucleotide comprising a nucleotide sequence having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity to the nucleotide sequence of SEQ ID NO: 2, but is not limited thereto.

[0031] The PitA polypeptide or the polypeptide encoded by the pitA gene may be, but is not limited to, one encoded by a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 2. The PitA polypeptide or the polypeptide encoded by the pitA gene may be, but is not limited to, one encoded by a polynucleotide comprising a nucleotide sequence having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity to the nucleotide sequence of SEQ ID NO: 2.

[0032] In the present invention, the amino acid sequence of SEQ ID NO: 1 may be encoded by a polynucleotide containing the nucleic acid sequence of SEQ ID NO: 2, for example.

[0033] In the present invention, the term "polynucleotide" refers to a nucleotide polymer in which nucleotide units are linked together in a long chain by covalent bonds, and refers to a DNA or RNA chain of a certain length or greater. In the present invention, the polynucleotide may encode a polypeptide having the amino acid sequence of SEQ ID NO: 1 or exhibiting activity of a phosphate importer or Pit system, but is not limited thereto.

[0034] The polynucleotide may include, without limitation, a polynucleotide encoding a polypeptide having the activity of the phosphate importer, Pit system, or PitA according to the present invention. In the present application, the gene encoding the amino acid sequence of the phosphate importer or Pit system is one or more genes selected from pit, pit1, pit2, pit3, pitA, pitB, and pitC, and the gene may be derived from Escherichia coli, but is not limited thereto.

[0035] Specifically, the polynucleotide encoding the polypeptide exhibiting the activity of the phosphate importer, the Pit system, or PitA can comprise a base sequence encoding the amino acid set forth in SEQ ID NO:1.

[0036] The polynucleotide may be modified in various ways in the coding region without changing the amino acid sequence of the polypeptide, taking into consideration codon degeneracy or preferred codons in the organism in which the polypeptide is to be expressed. The polynucleotide may, for example, comprise the nucleotide sequence of SEQ ID NO: 2, or may consist of a nucleotide sequence having 80% homology or identity thereto, specifically 90% or more, more specifically 95% or more, 96% or more, 97% or more, 98% or more, or even more specifically 99% or more, but is not limited thereto.

[0037] Furthermore, the polynucleotide of the present invention includes, without limitation, a sequence that hybridizes under stringent conditions with a probe that can be prepared from a known gene sequence, for example, a sequence complementary to all or part of the base sequence, and encodes the amino acid sequence of SEQ ID NO: 1. The term "stringent conditions" refers to conditions that allow specific hybridization between polynucleotides. Such conditions are specifically described in the literature (e.g., J. Sambrook et al., supra). For example, conditions include conditions under which polynucleotides with high homology or identity, such as 40% or more, specifically 90% or more, more specifically 95% or more, 96% or more, 97% or more, 98% or more, and even more specifically 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.

[0038] Hybridization requires that two nucleic acids 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 one another. For example, with respect to DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Thus, polynucleotides of the present invention can include isolated nucleic acid fragments that are complementary to entire sequences, as well as substantially similar nucleic acid sequences.

[0039] Specifically, polynucleotides having homology or identity can be detected using the hybridization conditions described above, 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. Those skilled in the art can adjust the Tm value as appropriate depending on the purpose.

[0040] The appropriate stringency for hybridizing polynucleotides depends on the length of the polynucleotides and the degree of complementation, variables well known in the art (see Sambrook et al., supra, 9.50-9.51, 11.7-11.8).

[0041] As used herein, the term "homology" or "identity" refers 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.

[0042] Sequence homology or identity of conserved polynucleotides or polypeptides can be determined by standard sequence algorithms, with default gap penalties established by the program used. Substantially homologous or identical sequences generally hybridize under moderately or highly stringent conditions over at least about 50%, 60%, 70%, 80%, or 90% of the entire sequence or over the entire length of the sequence. Hybridization obviously also includes polynucleotides containing common codons in polynucleotides or codons that take codon degeneracy into account.

[0043] 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), 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]). ETA / .](1988) SIAM J Applied Math 48:1073). For example, BLAST or ClustalW from the National Database Center for Biotechnology Information can be used to determine homology, similarity or identity.

[0044] Homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using a GAP computer program, such as that described in Smith and Waterman, Adv. Appl. Math (1981) 2:482, or in Needleman et al. (1970), J. Mol. Biol. 48:443. 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) a binary comparison matrix (containing a value of 1 for identity and 0 for non-identity) and 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) as disclosed by Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979); (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.

[0045] 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. Defining appropriate hybridization conditions is 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, 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).

[0046] In one embodiment, the enhancement of the activity of the phosphate influx system may be, but is not limited to, enhancement of the activity of a polypeptide encoded by the pitA gene.

[0047] In one embodiment, the pitA gene of the present invention may be a foreign pitA gene. In another embodiment, the microorganism of the present invention may be, but is not limited to, a microorganism into which a foreign pitA gene or foreign pit polypeptide has been introduced. The microorganism may also include a microorganism in which the introduced foreign gene or polypeptide has been enhanced. Specifically, the foreign pitA gene may be derived from Escherichia sp. or Escherichia coli, but is not limited thereto.

[0048] As used herein, the term "enhancement" of polypeptide activity means that the polypeptide activity is increased compared to its endogenous activity. The term "enhancement" may be used interchangeably with terms such as "activation," "up-regulation," "overexpression," and "increase." Here, activation, enhancement, up-regulation, overexpression, and increase may all encompass the display of an activity not inherently possessed, or an activity that is improved compared to the endogenous activity or the activity before modification. The term "endogenous activity" refers to the activity of a specific polypeptide inherently possessed by a parent strain or an unaltered microorganism before the trait has been altered due to genetic mutation caused by natural or artificial factors. This term may be used interchangeably with "activity before modification." The terms "enhancement," "up-regulation," "overexpression," or "increase" of a polypeptide activity compared to its endogenous activity mean that the activity is improved compared to the activity of a specific polypeptide inherently possessed by a parent strain or an unaltered microorganism before the trait has been altered.

[0049] The enhancement can be achieved by introducing an exogenous polypeptide or by enhancing the activity and / or concentration (expression level) of an endogenous polypeptide. The enhancement of the activity of the polypeptide can be confirmed by an increase in the activity, expression level, or amount of a product secreted from the polypeptide.

[0050] 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 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.).

[0051] Specifically, the activity of the polypeptide of the present invention is enhanced by: 1) an increase in the intracellular copy number of the polynucleotide encoding the polypeptide; 2) replacing the expression regulatory region of a gene on a chromosome that encodes a polypeptide with a sequence with strong 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 a polypeptide and selectively modifying 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.

[0052] More specifically, The 1) increase in the intracellular copy number of a polynucleotide encoding a polypeptide may be achieved by introducing into a host cell a vector operably linked to the polynucleotide encoding the polypeptide, which vector is capable of replicating and functioning independently of the host. Alternatively, the increase may be achieved by introducing one or more copies of the polynucleotide encoding the polypeptide 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.

[0053] The 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 involve, for example, mutation of the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, to further enhance the activity of the expression regulatory region, or replacement 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.

[0054] Examples of known strong promoters include, but are not limited to, the CJ1 to CJ7 promoters (U.S. Patent US7662943 B2), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13 (sm3) promoter (U.S. Patent US10584338 B2), O2 promoter (U.S. Patent US10273491 B2), tkt promoter, and yccA promoter.

[0055] 3) the initiation codon or 5'-UTR of the gene transcript encoding the polypeptide Modification of the base sequence encoding the region may be, for example, but is not limited to, substitution with a base sequence encoding another start codon that results in a higher polypeptide expression rate than the endogenous start codon.

[0056] 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 to enhance the activity of the polypeptide, or an amino acid sequence or polynucleotide sequence modified to have stronger activity or to have increased activity. Specifically, the modification can be achieved by, but is not limited to, inserting the polynucleotide into a chromosome via 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.

[0057] 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 the host cell to produce a polypeptide, and its activity can be increased.

[0058] The codon optimization of the polynucleotide encoding the polypeptide (7) may be that of an endogenous polynucleotide that has been codon-optimized to increase transcription or translation in a host cell, or that of an exogenous polynucleotide that has been codon-optimized to optimize transcription or translation in a host cell.

[0059] 8) Analyzing the tertiary structure of a polypeptide and selecting and deforming 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 deforming or chemically modifying exposed sites.

[0060] Such enhanced polypeptide activity may refer to, but is not limited to, an increase in the activity or expression level of the corresponding polypeptide relative to the activity or concentration of the polypeptide expressed in a wild-type or untransformed microbial strain, or an increase in the amount of a product produced from the polypeptide.

[0061] Modification of a portion or the entire polynucleotide in the microorganism of the present invention 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) treatment with light and / or chemicals, such as ultraviolet light and radiation. Methods for modifying a portion or the entire 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 induce homologous recombination, thereby causing a partial or entire deletion of the gene. The injected nucleotide sequence or vector may contain, but is not limited to, a dominant selectable marker.

[0062] In the present invention, the polypeptide whose activity is to be enhanced may be a phosphate importer, specifically, a Pit system, a polypeptide encoded by the pitA gene, or a PitA polypeptide. For purposes of the present invention, the enhancement of activity may be achieved by, but is not limited to, introducing an exogenous Pit system, an exogenous polypeptide encoded by the pitA gene, or an exogenous PitA polypeptide.

[0063] For the purposes of this application, enhancing the activity of the phosphate influx system may be, but is not limited to, introducing a foreign pitA gene or a polypeptide encoded by a foreign pitA gene.

[0064] In the present invention, "introduction of a polypeptide" means exhibiting the activity of a specific polypeptide that a microorganism does not originally possess, or exhibiting improved activity compared to the endogenous activity of the polypeptide or the activity of the polypeptide before modification. For example, this may mean introducing a specific polypeptide, introducing a polynucleotide encoding a specific polypeptide into a chromosome of a microorganism, or introducing a vector containing a polynucleotide encoding a specific polypeptide into a microorganism, and then exhibiting the activity.

[0065] In the present invention, the term "foreign gene" refers to a gene that is non-natively (non-naturally) contained in a microorganism. Examples include, but are not limited to, (a) an exogenous gene that cannot be found naturally in a microorganism, (b) an endogenous gene that can be found naturally in a microorganism, but whose transcription or translation results in an unnatural amount (larger or smaller than the amount found in nature), (c) a polypeptide sequence that is endogenously present in a microorganism, but whose encoding gene sequence is different, and / or (d) a combination of two or more of the above-mentioned features in a microorganism. For purposes of the present invention, since the Pit system is not present in the Corynebacterium stachyonis microorganism, the foreign gene may be, but is not limited to, a foreign pitA gene, a pitA gene derived from Escherichia sp., or a pitA gene derived from Escherichia coli.

[0066] In the present invention, a vector may comprise a DNA construct containing 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 include 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 can be integrated into the genome itself.

[0067] The vectors used in the present invention are not particularly limited, and any vector known in the art can be used. Examples of commonly used vectors include natural 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 pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, and pET can be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC vectors can be used.

[0068] For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome of a cell using a vector for chromosomal insertion. The insertion of the polynucleotide into a chromosome can be performed by any method known in the art, including, but not limited to, homologous recombination. A selection marker for confirming the presence or absence of the insertion into the chromosome may also be included. The selection marker is used to select cells transformed with the vector, i.e., to confirm the presence or absence of the insertion of the target nucleic acid molecule. 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 cells expressing the selection marker survive or exhibit other phenotypes, allowing the transformed cells to be selected.

[0069] As used herein, the term "transformation" 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. The transformed polynucleotide may be any polynucleotide, regardless of 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 may typically include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the polynucleotide. The expression cassette may also be in the form of a self-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.

[0070] In addition, the term "operably linked" means that the polynucleotide sequence is functionally linked to a promoter sequence that initiates and mediates transcription of the polynucleotide encoding the target mutant of the present invention.

[0071] In the present invention, the term "strain (or microorganism)" includes all wild-type microorganisms and microorganisms that have undergone natural or artificial genetic modification, and may be a microorganism in which a specific mechanism has been weakened or enhanced by inserting an exogenous gene or by enhancing or inactivating the activity of an endogenous gene, and may also be a microorganism that contains genetic modifications for the production of a desired polypeptide, protein, or product.

[0072] As used herein, the term "Corynebacterium stathionis microorganism capable of producing purine nucleotides" refers to a microorganism that, when cultured in a medium, produces and accumulates purine nucleotides as a target product within the organism, or secretes and accumulates them in the medium. In one embodiment, the term "capable of producing purine nucleotides" may be used interchangeably with "producing purine nucleotides." The ability to produce purine nucleotides may be inherent in wild-type strains of Corynebacterium stathionis microorganisms, or may be conferred or enhanced by genetic modification.

[0073] The microorganism capable of producing purine nucleotides of the present invention may be a recombinant strain having increased purine nucleotide production ability due to enhanced activity of the phosphate influx system (Pit system) encoded by the pit gene. The recombinant strain having increased purine nucleotide production ability may be, but is not limited to, a strain having enhanced purine nucleotide production compared to a natural wild-type microorganism or a microorganism without a modified phosphate influx system (i.e., a microorganism expressing a wild-type phosphate influx system, a microorganism without an enhanced phosphate influx system, or a microorganism not expressing a phosphate influx system). For example, the phosphate influx system unaltered microorganism, which is the subject strain for comparing the increase in purine nucleotide production ability, may be, but is not limited to, CJX1664 (KCCM12285P, KR10-1950141 B1, US2020-0392478 A1), CJX1665 (KCCM12286P, KR10-1950141 B1, US2020-0392478 A1), CJI2332 (KCCM12277P, KR10-1950141 B1, US2020-0392478 A1), or CJI2335 (KCCM12278P, KR10-1956510 B1, EP3705572 A1).

[0074] For example, the recombinant strain with increased production capacity may have an increase in purine nucleotide production capacity 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 11% or more, about 12% or more, or about 13% 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 40% or less, or about 30% or less), compared to 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 parent strain or unmodified microorganism. In another example, the recombinant strain having increased productivity may have an increased purine nucleotide productivity of about 1.01-fold or more, about 1.02-fold or more, about 1.03-fold or more, about 1.05-fold or more, about 1.06-fold or more, about 1.07-fold or more, about 1.08-fold or more, about 1.09-fold or more, about 1.10-fold or more, about 1.11-fold or more, about 1.12-fold or more, or about 1.13-fold or more (there is no particular upper limit, and the increase may be, for example, about 10-fold or less, about 5-fold or less, about 3-fold or less, or about 2-fold or less) compared to the parent strain or unmodified microorganism before mutation, but is not limited thereto. The term "about" refers to a range that includes ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all numerical values ​​in a range that is equal to or similar to the value following the term "about," but is not limited thereto.

[0075] The microorganism capable of producing purine nucleotides of the present invention may be, but is not limited to, a microorganism that contains one or more of the Pit system, the PitA polypeptide, a polynucleotide encoding the PitA polypeptide, and a vector containing the polynucleotide, and is capable of producing a target polypeptide or a target product involved in the production of the target polypeptide.The microorganism may be, but is not limited to, a microorganism that naturally has the ability to produce the target polypeptide or target product, or a microorganism in which the ability to produce the target polypeptide or target product has been imparted to a parent strain that does not have the ability to produce the target polypeptide or target product.

[0076] As used herein, the term "non-modified 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 traits are changed due to genetic mutations caused by natural or artificial factors. For example, the non-modified microorganism refers to a strain before the phosphate flux system described herein has been enhanced or introduced, or before it has been enhanced or introduced. The term "non-modified microorganism" may be used interchangeably with "pre-modified strain," "pre-modified microorganism," "non-mutated strain," "non-modified strain," "non-mutated microorganism," or "reference microorganism."

[0077] In the present invention, the term "purine nucleotide" may refer to one or more nucleotides selected from the group consisting of 5'-inosinic acid (IMP), 5'-xanthosine monophosphate (XMP), and 5'-guanosine monophosphate (GMP). 5'-inosinic acid 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. 5'-inosinic acid can be biosynthesized from 5'-phosphoribosyl-1-pyrophosphate (PRPP). Specifically, the pyrophosphate group attached to carbon number 1 of PRPP is replaced with a nitrogen atom, and it can be formed by forming an imidazole ring and a pyrimidine ring in nine steps, but this is not limiting. 5'-Xanthosine acid refers to a nucleotide dehydrogenated from 5'-inosinic acid. 5'-XMP can be synthesized from 5'-inosinic acid by, but is not limited to, 5'-inosinic acid dehydrogenase. 5'-Guanic acid has a structure in which a phosphate group is ester-bonded to the ribose moiety of a guanosine molecule, and can be synthesized by, but is not limited to, adding an ammonia molecule to 5'-XMP by 5'-guanic acid biosynthetic enzyme (GMP synthase).

[0078] Another aspect of the present invention provides a method for producing purine nucleotides, comprising culturing in a medium a Corynebacterium stachyonis microorganism capable of producing purine nucleotides and having enhanced activity of the phosphate influx system (Pit system).

[0079] The phosphate influx system, Pit system, enrichment, purine nucleotides, and Corynebacterium stachyonis microorganism are as described elsewhere.

[0080] In the present invention, the term "culturing" refers to growing the microorganism 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 performed in a batch, continuous, or fed-batch manner, but is not limited thereto.

[0081] In the present invention, the term "culture medium" refers to a mixture of nutrients required for culturing the microorganism as the main components, and provides nutrients such as water and growth factors essential for survival and growth. Specifically, the culture medium and other culture conditions used for culturing the microorganism of the present application are not particularly limited as long as they are media used for culturing conventional microorganisms. The microorganism of the present invention can be cultured in a conventional culture medium containing an appropriate carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids, and / or vitamins under aerobic conditions by adjusting the temperature, pH, etc.

[0082] In the present invention, the carbon source may 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 hydrolysate, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn steeping liquid may also be used. Specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted into reducing sugars) may be used. A variety of other carbon sources may also be used in appropriate amounts without limitation. These carbon sources may be used alone or in combination of two or more, and are not limited thereto.

[0083] 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 hydrolyzed products, defatted soybean cake or its hydrolyzed products, etc. These nitrogen sources may be used alone or in combination of two or more, and are not limited thereto.

[0084] The phosphorus source may include monopotassium phosphate, dipotassium phosphate, or the corresponding sodium-containing salts. Inorganic compounds may include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, and other compounds, including amino acids, vitamins, and / or appropriate precursors. These components or precursors may be added to the culture medium in a batch or continuous manner. However, the present invention is not limited to these.

[0085] During the cultivation of the microorganism, the pH of the medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. to the medium in an appropriate manner. Also, during cultivation, foam formation can be suppressed using an antifoaming agent such as a fatty acid polyglycol ester. Oxygen or an oxygen-containing gas can be injected into the medium to maintain an aerobic state, or nitrogen, hydrogen, or carbon dioxide gas can be injected without gas injection or to maintain an anaerobic or microaerobic state, but the methods are not limited thereto.

[0086] The temperature of the medium may be, but is not limited to, 27° C. to 37° C., specifically 30° C. to 33° C. The culture period may be, but is not limited to, 20 hours to 120 hours, and can be continued until a desired amount of useful substance is produced.

[0087] Specifically, 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)].

[0088] The purine nucleotides produced by the culture of the present invention are either secreted into the medium or remain intracellularly.

[0089] The method for producing purine nucleotides of the present invention may further include a step of preparing the Corynebacterium stachyonis microorganism of the present invention, a step of preparing a medium for culturing the microorganism, or a combination thereof (in any order), for example, before the culturing step.

[0090] The method for producing purine nucleotides of the present invention may further include a step of recovering purine nucleotides from the culture medium (the culture medium) or the microorganism (the cultured microorganism). The recovery step may be performed after the culturing step.

[0091] The recovery may involve collecting the desired purine nucleotides using a suitable method known in the art based on the culture method of the microorganism of the present invention, 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 to recover the desired purine nucleotides from the medium or the microorganism using a suitable method known in the art.

[0092] The production method of the present invention may further include a purification step. The purification can be performed using any suitable method known in the art. For example, when the purine nucleotide production method 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 this is not limiting.

[0093] Specifically, in the method for producing 5'-guanine acid (5'-guanosine monophosphate, GMP) of the present invention, the culturing step may further include a step of converting 5'-xanthosine monophosphate (XMP) to 5'-guanine acid. In the method for producing GMP of the present invention, the conversion step may be performed after the culturing step or the recovering step. The conversion step may be performed using an appropriate method known in the art. For example, the conversion may be performed using a coryneform microorganism, Escherichia coli, or 5'-xanthosine monophosphate aminase (KR10-0655902 B1), but is not limited thereto.

[0094] Another aspect of the present invention is to provide a composition for producing purine nucleotides, comprising the Corynebacterium stachyonis microorganism of the present invention; a medium in which the microorganism has been cultured; or a combination thereof.

[0095] The composition of the present invention may further contain any suitable excipient commonly used in compositions for producing purine nucleotides, such as, but not limited to, a preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer, or isotonicity agent.

[0096] In the composition of the present invention, the microorganism, medium, purine nucleotides, etc. are the same as those described in the other embodiments above.

[0097] Another aspect of the present invention is to provide use of the Corynebacterium stachyonis microorganism of the present application for producing purine nucleotides.

[0098] In the use of the present invention for producing purine nucleotides, the microorganisms, purine nucleotides, etc. are as described above in other aspects.

[0099] The present invention will be described in more detail with reference to the following examples. However, these examples are merely for illustrative purposes and are not intended to limit the scope of the present invention, and are obvious to those skilled in the art.

[0100] Example 1: Construction of strains incorporating exogenous pitA and evaluation of IMP production ability 1-1: Construction of a recombinant vector for introducing the foreign pitA gene and construction of an IMP-producing strain The wild-type strain of Corynebacterium stathionis does not contain a Pit system. Therefore, a vector was constructed to introduce a foreign Pit system into Corynebacterium stathionis. The foreign pitA gene was then fused to the CJ1 promoter (Korea Patent Registration No. 10-0620092, SEQ ID NO: 3), which is known as an enhanced promoter among Corynebacterium microorganisms. A vector was then constructed to replace this with the fepB gene locus, a periplasmic binding protein of Corynebacterium microorganisms.

[0101] Specifically, to construct an insertion vector for deleting the fepB gene of Corynebacterium stathionis, the chromosomal gene of the wild-type Corynebacterium stathionis strain ATCC 6872 was isolated using the Intron G-spin Total DNA extraction mini kit (Cat. No. 17045) according to the protocol provided with the kit, and PCR was performed using this as a template. The polymerase used was Maxime PCR PreMix (i-pfu) high-fidelity DNA polymerase (Intron). PCR consisted of denaturation at 95°C for 5 minutes, followed by 24 cycles of denaturation at 95°C for 30 seconds, annealing at 54°C for 30 seconds, and polymerization at 72°C for 1 minute and 30 seconds. As a result, two distinct PCR products (fepBdel-A and fepBdel-B) were obtained. fepB-A was 1,038 bp in size and was amplified using primers sequenced as SEQ ID NO: 4 and SEQ ID NO: 5. The fepB-B fragment was 1111 bp in size and was amplified using SEQ ID NO: 6 and SEQ ID NO: 7 as primers. A second PCR was performed using the two amplification products as templates using the sewing PCR technique. PCR was performed under the same conditions as the first PCR, resulting in a 2131 bp PCR product lacking the fepB gene and containing SpeI and NotI restriction enzyme sites in the center. The amplification product was then excised using the restriction enzyme sites located at both ends (fepBdel-A: HindIII, fepBdel-B: HindIII) and cloned into the pDZ vector using T4 ligase, thereby obtaining the pDZ-fepBdel vector for the fepB gene deletion. The sequences of the primers used are as follows:

[0102] [Table 1]

[0103] A vector in which the nucleic acid portion of the fepB gene deleted from the pDZ-fepBdel vector was replaced with a foreign pitA gene was constructed as follows. Specifically, chromosomes of wild-type Corynebacterium stachyonis ATCC 6782 and Escherichia coli MG1655 were isolated as templates. Using Maxime PCR PreMix (i-pfu) high-fidelity DNA polymerase (Intron) as the polymerization enzyme, PCR was performed by denaturing at 95°C for 5 minutes, followed by denaturing at 95°C for 30 seconds, annealing at 54°C for 30 seconds, and polymerizing at 72°C for 2 minutes and 30 seconds, 24 cycles. As a result of PCR, two PCR products (pCJ1-pitA-A and pCJ1-pitA-B) containing the pitA gene linked to the CJ1 promoter were obtained. pCJ1-pitA-A was a 341-bp promoter PCR product amplified using primers SEQ ID NO: 8 and SEQ ID NO: 9. pCJ1-pitA-B is a 1537-bp PCR product of the pitA gene, amplified using primers SEQ ID NO: 10 and SEQ ID NO: 11. A second PCR was performed using the two amplification products as templates using the sewing PCR technique. PCR was performed under the same conditions as above, resulting in the pitA gene linked to the pCJ1 promoter, which contains nucleotide sequences at both ends that share homology with the pDZ-fepBdel vector. This gene was cloned into the pDZ-fepBdel vector, which had been treated with SpeI and NotI restriction enzymes, using a homologous recombinase method to obtain the pDZ-ΔfepB::pCJ1 / pitA(Eco) vector. The sequences of the primers used are as follows:

[0104] [Table 2]

[0105] Then, the obtained vector pDZ-△fepB::pCJ1 / pitA(Eco) was transformed into IMP-producing strains CJI2332 (an IMP-producing strain derived from Corynebacterium stathionis ATCC6872, KCCM12277P, Korean Patent No. 10-1950141, US2020-0392478 A1) and CJI2335 (an IMP-producing strain derived from Corynebacterium stathionis ATCC6872, KCCM12278P, Korean Patent No. 10-1956510, EP3705572 A1) by electroporation, and a strain containing the CJ1 promoter-pitA gene in the endogenous fepB gene on the chromosome was obtained through a second crossover process. The newly inserted CJ1 promoter-pitA gene was confirmed using primers SEQ ID NO: 12 and SEQ ID NO: 13, which can amplify the region where both ends of the inserted gene and the fepB gene meet. In this way, we obtained two strains, CJI2332_pCJ1 / pitA and CJI2335_pCJ1 / pitA, which consistently overexpress the exogenously introduced pitA gene via the CJ1 promoter.

[0106] [Table 3]

[0107] 1-2. Evaluation of IMP production ability of the strain into which the exogenous pitA gene was introduced The following experiment was carried out to confirm the improvement of IMP production ability for the CJI2332_pCJ1 / pitA and CJI2335_pCJ1 / pitA strains constructed in Example 1-1.

[0108] Specifically, CJI2332_pCJ1 / pitA, CJI2332, CJI2335_pCJ1 / pitA, and CJI2335 were each inoculated into 5 ml of autoclaved seed medium in an 18 mm diameter test tube and cultured with shaking at 30°C for 24 hours to prepare seed cultures. 29 ml of fermentation medium was dispensed into a 250 ml shaker Erlenmeyer flask, autoclaved at 121°C for 15 minutes, and then inoculated with 2 ml of seed culture medium and cultured for 3 days. Culture conditions were adjusted to a rotation speed of 170 rpm, a temperature of 30°C, and a pH of 7.5. The compositions of the seed medium and fermentation medium were as follows:

[0109] IMP seed medium Glucose 1%, peptone 1%, meat juice 1%, yeast extract 1%, sodium chloride 0.25%, adenine 100 mg / L, guanine 100 mg / L, pH 7.2 (based on 1 L of medium)

[0110] IMP flask fermentation medium Monosodium glutamate 0.1%, ammonium chloride 1%, magnesium sulfate 1.2%, calcium chloride 0.01%, iron sulfate 20mg / L, manganese sulfate 20mg / L, zinc sulfate 20mg / L, copper sulfate 5mg / L, L-cysteine ​​23mg / L, alanine 24mg / L, nicotinic acid 8mg / L, biotin 45μg / L, thiamine hydrochloride 5mg / L, adenine 30mg / L, phosphoric acid (85%) 1.9%, glucose 4.2%, raw sugar 2.4% (based on 1L of medium)

[0111] After the cultivation was completed, the amount of IMP produced was measured by a method using HPLC, and the results are shown in Table 4 below.

[0112] [Table 4]

[0113] As shown in Table 4, under the same conditions, the yield of IMP accumulation produced by the CJI2332_pCJ1 / pitA and CJI2335_pCJ1 / pitA strains was confirmed to be increased by approximately 13% and approximately 12.5%, respectively, compared to the parent strains CJI2332 and CJI2335. CJI2332_pCJ1 / pitA was designated CJI2630 and deposited with the Korea Microorganism Collection, a depository under the Budapest Treaty, on April 13, 2021, and was assigned accession number KCCM12974P.

[0114] The CJI2335_pCJ1 / pitA was named CJI2631 and deposited with the Korea Microorganism Collection, a depository under the Budapest Treaty, on April 13, 2021, and was assigned the accession number KCCM12975P.

[0115] Example 2: Construction of strains into which exogenous pitA has been introduced and evaluation of XMP production ability 2-1: Construction of strains incorporating exogenous pitA The XMP-producing strain CJX1664 (an XMP-producing strain derived from Corynebacterium stathionis, KCCM12285P, Korean Patent No. 10-1950141, US2020-0392478 A1) and the purA (G85S) mutant strain of CJX1664, CJX1665 (KCCM12286P, Korean Patent No. 10-1950141, US2020-0392478 A1), were transformed with pDZ-△fepB::pCJ1 / pitAA(Eco) by electroporation, and then, through a second crossover process, strains CJX1664_pCJ1 / pitA and CJX1665_pCJ1 / pitA, which contain the pCJ1-pitA gene in the endogenous fepB gene on the chromosome, were obtained. The newly inserted pCJ1-pitA gene was confirmed using primers SEQ ID NO: 12 and SEQ ID NO: 13, which can amplify the region where both ends of the inserted gene meet the fepB gene.

[0116] 2-2. Evaluation of XMP production ability of the strains into which the exogenous pitA gene was introduced To evaluate the XMP production ability of the CJX1664_pCJ1 / pitA and CJX1665_pCJ1 / pitA strains prepared in Example 2-1, flask evaluation was performed. CJX1664, CJX1664_pCJ1 / pitA, CJX1665, and CJX1665_pCJ1 / pitA were inoculated into 14 ml tubes containing 2.5 ml of the following seed medium, respectively, and cultured at 30°C for 24 hours with shaking at 170 rpm. 0.7 ml of the seed culture was inoculated into a 250 ml corner-baffled flask containing 32 ml of the following production medium (24 ml of main medium + 8 ml of separate sterilized medium) and cultured at 30°C for 75 hours with shaking at 170 rpm. The compositions of the seed medium, main medium, and separate sterilized medium are as follows:

[0117] XMP flask seed medium Glucose 30g / L, peptone 15g / L, yeast extract 15g / L, sodium chloride 2.5g / L, urea 3g / L, adenine 150mg / L, guanine 150mg / L, pH 7.0 (based on 1L of medium)

[0118] XMP flask production medium (main medium) Glucose 50g / L, magnesium sulfate 10g / L, calcium chloride 100mg / L, ferrous sulfate 20mg / L, manganese sulfate 10mg / L, zinc sulfate 10mg / L, copper sulfate 0.8mg / L, histidine 20mg / L, cystine 15mg / L, beta-alanine 15mg / L, biotin 100ug / L, thiamine 5mg / L, adenine 50mg / L, guanine 25mg / L, niacin 15mg / L, pH 7.0 (based on 1L of medium)

[0119] XMP flask production medium (separate sterilized medium) Potassium phosphate monobasic 18g / L, potassium phosphate dibasic 42g / L, urea 7g / L, ammonium sulfate 5g / L (based on 1L of medium)

[0120] After the cultivation was completed, the amount of XMP produced was measured using HPLC, and the results are shown in Table 5 below.

[0121] [Table 5]

[0122] As shown in Table 5, the XMP concentrations produced by CJX1664_pCJ1 / pitA and CJX1665_pCJ1 / pitA, strains that constantly express E. coli pitA, increased by 6.7% and 6.4%, respectively, compared to the parent strains CJX1664 and CJX1665.

[0123] From the above description, those skilled in the art to which the present invention pertains will understand that the present invention 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 invention should be interpreted as including within the meaning and scope of the claims below, and all modifications and variations derived from the equivalent concepts thereof, rather than the above detailed description.

[0124] JPEG0007827748000006.jpg228170

[0125] JPEG0007827748000007.jpg224170

Claims

1. A Corynebacterium stachyonis microorganism, in which the activity of the phosphate influx system encoded by the pitA gene is enhanced compared to its endogenous activity, the phosphate influx system comprises a polypeptide having 97% or more identity to the amino acid sequence of SEQ ID NO: 1, and the microorganism is capable of producing purine nucleotides.

2. The microorganism according to claim 1, wherein the polypeptide encoded by the pitA gene comprises a polypeptide having 99% or more identity to the amino acid sequence of SEQ ID NO:

1.

3. 2. The microorganism according to claim 1, wherein the purine nucleotide is at least one selected from the group consisting of 5'-inosinic acid, 5'-xanthylic acid, and 5'-guanic acid.

4. A method for producing purine nucleotides, comprising culturing the microorganism according to any one of claims 1 to 3 in a medium.

5. 5. The method for producing purine nucleotides according to claim 4, further comprising recovering purine nucleotides from the cultured microorganism or medium.

6. 5. The method for producing a purine nucleotide according to claim 4, wherein the purine nucleotide is at least one selected from the group consisting of 5'-inosinic acid, 5'-xanthylic acid, and 5'-guanic acid.

7. A composition for producing purine nucleotides, comprising: the Corynebacterium stathionis microorganism according to any one of claims 1 to 3; a medium in which the Corynebacterium stathionis microorganism has been cultured, the medium containing the Corynebacterium stathionis microorganism according to any one of claims 1 to 3; or a combination thereof.

8. Use of the Corynebacterium stachyonis microorganism according to any one of claims 1 to 3 for producing purine nucleotides.

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