RU2843581C2 - Purine nucleotides producing microorganism, and method for producing purine nucleotides using same - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology and is a microorganism Corynebacterium stationis, having the ability to produce purine nucleotides, in which the activity of the phosphate importer system coded by the pit gene is enhanced, and wherein the amplification of the activity of the phosphate importer system is an amplification of the activity of a polypeptide encoded by the pitA gene. Such a microorganism is meant for producing a purine nucleotide.

EFFECT: invention enables to obtain purine nucleotides with high efficiency.

10 cl, 5 tbl, 2 ex

Description

Field of technology

The present invention relates to a microorganism Corynebacterium stationis, which has the ability to produce purine nucleotides, in which the activity of the phosphate transport system is enhanced, and to a method for producing purine nucleotides using this microorganism.

Prior art

Purine nucleotides, i.e., 5'-inosine monophosphate (hereinafter referred to as IMP), 5'-xanthosine monophosphate (hereinafter referred to as XMP) and 5'-guanosine monophosphate (hereinafter referred to as GMP), which are intermediates in the metabolic system of nucleic acid biosynthesis, play an important physiological role in the body and are widely used in food, pharmaceuticals, etc. In particular, IMP itself is known to impart a beef flavor, and GMP, which is derived from XMB, is known to impart a mushroom flavor. Both substances are known to enhance the flavor intensity of monosodium glutamate (MSG), and thus have attracted much attention as a nucleic acid-based flavor-enriching seasoning.

Phosphate, which is a component of purine nucleotides, provides the energy needed for the growth of microorganisms and is essential for the biosynthesis of phospholipids of cell membranes, nucleic acids and proteins. It also plays an important role in the process of signaling between cells.

Phosphate is mainly taken up by cells in the form of inorganic orthophosphate (hereinafter referred to as Pi). The entry of Pi into microorganisms depends on the function of importers present in the cell membrane. Previously known Pi importers include the Pst system, which is a high-affinity Pi importer system whose expression is regulated by sensing the extracellular Pi concentration; the Pit system, which is expressed independently of the Pi concentration and injects Pi in a mixed form with metal ions; and the antiporter transport system, which injects glycerol-3-phosphate and glucose-6-phosphate in the form of organophosphorus compound, etc.

Several types of methods used in the production of amino acids by regulating the Pit system among the Pi importer systems have been reported, but the effect of the Pit system on the production of nucleic acids has not been reported. In addition, regarding the increase in the production of amino acids, it has been confirmed that the weakening or strengthening of the expression of the Pit system leads to different results for each microorganism and amino acid. In the case of amino acid production, it can be confirmed that the regulation of the Pi supply system in a certain direction does not have the same effect on increasing or decreasing the amount of production (US 9506094 B2, US 9873898 B2).

Description of the invention

Technical task

The problem solved by the present invention consists in proposing a microorganism Corynebacterium stationis, which has the ability to produce purine nucleotides, in which the activity of the phosphate importer system (Pit system) is enhanced, and a method for producing purine nucleotides using this microorganism.

Technical solution

The aim of the present invention is to propose a microorganism Corynebacterium stationis, which has the ability to produce nucleotides, and in which the activity of the phosphate importer system (Pit system) is enhanced.

Another objective of the present invention is to provide a method for producing purine nucleotides, comprising: culturing the microorganism Corynebacterium stationis, which has the ability to produce purine nucleotides, in a medium.

Another object of the present invention is to provide a composition for producing purine nucleotides, comprising a microorganism Corynebacterium stationis having the ability to produce nucleotides; a medium on which the microorganism is cultured; or a combination thereof.

Another object of the present invention is to provide the use of the microorganism Corynebacterium stationis, which has the ability to produce nucleotides, for the production of purine nucleotides.

Beneficial effects

The supply of phosphate, a key component in the production of purine nucleotides, to microorganisms can be facilitated by enhancing the activity of the phosphate importer according to the present invention. As a result, a microorganism having the ability to produce purine nucleotides can efficiently produce purine nucleotides and can significantly contribute to reducing the cost of producing purine nucleotides on an industrial scale.

Detailed Description of Preferred Embodiments

Below, the present invention will be described in detail. In this regard, each description and embodiment disclosed herein can be applied to other descriptions and embodiments, respectively. That is, all combinations of various elements disclosed herein fall within the scope of the present invention. In addition, the scope of the present invention is not limited to the specific description given below. In addition, a number of articles and patent documents are indicated in the present description. The contents of these articles and patent documents are incorporated into this invention by reference in their entirety, and the level of the technical field to which the present invention pertains and the contents of the present invention will be described more clearly.

In one aspect of the present invention, there is provided a Corynebacterium stationis microorganism having the ability to produce purine nucleotides, in which the activity of the phosphate importer system (Pit system) encoded by the pit gene is enhanced.

As used herein, the term "phosphate importer system" refers to a polypeptide having the function of introducing phosphate into a cell or to a system containing it. The phosphate importer system may include a phosphate importer. For the purposes of the present invention, the phosphate importer system may be a Pit system.

As used herein, the term “phosphate importer” refers to a polypeptide that plays a role in the intracellular uptake of phosphate. Phosphate is mainly taken up by cells in the form of inorganic orthophosphate (hereinafter referred to as Pi) and is known to be dependent on the function of importers present in the cell membrane. Specifically, previously known Pi importers include the Pst system, the Pit system, and the antiporter transport system that brings in glycerol-3-phosphate and glucose-6-phosphate in the form of organophosphorus, etc., but are not limited to them.

When used herein, the term “Pit system” refers to a phosphate transporter importer (low-affinity phosphate transporter; tellurite importer; hereafter referred to as Pit) that exhibits low affinity for Pi. Expression of the Pit system is independent of extracellular Pi concentration, and Pi is known to be introduced into cells as a metal cation complex {JOURNAL OF BACTERIOLOGY, Sept. 2001, p.5008-5014 Vol. 183, No. 17).

The Pit system may comprise at least one polypeptide encoded by at least one gene selected from pit, pit1, pit2, pit3, pitA, pitB and pitC, or a Pit, Pit1, Pit2, Pit3, PitA, PitB and PitC polypeptide, and for the purposes of the present invention, the Pit system may be a polypeptide encoded by the pitA (inorganic phosphate transporter) gene or a PitA polypeptide, but is not limited to them.

The Pit system may include a polypeptide represented by the amino acid sequence SEQ ID NO: 1, but this system or polypeptide is not limited to this, since the amino acid sequence of the enzyme exhibiting the activity may differ depending on the species or strain of microorganisms.

The PitA polypeptide or the polypeptide encoded by the pitA gene may consist of, but is not limited to, the polypeptide represented by the amino acid sequence SEQ ID NO: 1.

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

In one embodiment, the polypeptide encoded by the pitA gene may include 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, but is not limited thereto.

The amino acid sequence of SEQ ID NO: 1 can be obtained from various databases, such as NCBI GenBank, a known database, etc., but not limited thereto. In one example, the amino acid sequence can be obtained from Escherichia coli, but not limited thereto, and can include any sequence having the same activity as the above amino acid sequence, without limitation. In addition, although the polypeptide having the activity of a phosphate importer, the Pit system or PitA of the present invention is defined as a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, it is not excluded that a nonsense sequence is added thereto before or after the amino acid sequence of SEQ ID NO: 1, a natural mutation or a silent mutation, and it is obvious to those skilled in the art that as long as the polypeptide has an activity identical to or corresponding to the activity of the polypeptide comprising the amino acid sequence of SEQ ID NO: 1, it may refer to the polypeptide having the activity of a phosphate importer, the Pit system or PitA of the present invention.

In a specific embodiment, a polypeptide having the phosphate importer, Pit system or PitA activity 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 a homology or identity of 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more.

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

Furthermore, it is obvious that any polypeptide having an amino acid sequence with a deletion, modification, substitution or addition in part of the sequence may also be included within the scope of the polypeptide of the present invention, provided that the amino acid sequence has such homology or identity and exhibits an efficacy corresponding to that of the polypeptide.

Although the present invention describes it as a "polypeptide comprising an amino acid sequence of a certain SEQ ID NO," a "polypeptide consisting of an amino acid sequence of a certain SEQ ID NO," or a "polypeptide having an amino acid sequence of a certain SEQ ID NO," it is obvious that any polypeptide having an amino acid sequence with a deletion, modification, substitution, conservative substitution or addition in a part of the sequence can also be used in the present invention, as long as this polypeptide can have an activity identical to or corresponding to the activity of a polypeptide consisting of an amino acid sequence with the corresponding SEQ ID NO. For example, it can include sequence additions that do not change the function of the protein of the present invention, at the N-terminus and/or C-terminus of the amino acid sequence, naturally occurring mutations, silent mutations therein or conservative substitutions.

As used herein, the term "conservative substitution" refers to the replacement of one amino acid with another amino acid having similar structural and/or chemical properties. Such amino acid substitution may typically be based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residue. Typically, conservative substitutions may have little or no effect on the activity of the polypeptide.

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

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

In the present invention, the amino acid sequence of SEQ ID NO: 1 can be encoded by a polynucleotide comprising, for example, the nucleic acid sequence of SEQ ID NO: 2.

As used herein, a "polynucleotide" which is a polymer of nucleotides consisting of nucleotide monomers linked into a long chain by a covalent bond is a strand of DNA or RNA having at least a specific length. In the present invention, the polynucleotide may encode a polypeptide having phosphate importer activity or a Pit system having the amino acid sequence of SEQ ID NO: 1, but is not limited thereto.

The polynucleotide may include any polynucleotide without limitation, provided that it is a polynucleotide encoding a polypeptide having the activity of a phosphate importer, a Pit system or PitA according to the present invention. In the present invention, the gene encoding the amino acid sequence of a phosphate importer or a Pit system may be any 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.

Specifically, a polynucleotide encoding a polypeptide having phosphate importer, Pit system, or PitA activity may include a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1.

The polynucleotide may be subject to various modifications in the coding region without changing the amino acid sequence of the polypeptide due to codon degeneracy or taking into account the codons preferred in the organism in which the polypeptide is to be expressed. The polynucleotide may include, for example, the nucleotide sequence of SEQ ID NO: 2 and may consist of a nucleotide sequence having a homology or identity of 80%, in particular 90% or more, more in particular 95% or more, 96% or more, 97% or more, or 98% or more, or even more in particular 99% or more of this, but not limited thereto.

In addition, a probe may be included that may be derived from a known gene sequence, such as any nucleotide sequence that hybridizes to a sequence complementary to all or part of a nucleotide sequence under stringent conditions to encode the amino acid sequence of SEQ ID NO: 1, without limitation. "Stringent conditions" refer to conditions that allow specific hybridization between polynucleotides. Such conditions are described in detail in the literature (e.g., in J. Sambrook et al., supra). For example, stringent conditions may include conditions in which polynucleotides having high homology or identity, polynucleotides having a homology or identity of 40% or more, in particular 90% or more, more in particular 95% or more, 96% or more, 97% or more, 98% or more, and especially in particular 99% or more, hybridize with each other, while polynucleotides having a homology or identity lower than the above homology or identity do not hybridize with each other; or may include conventional washing conditions for Southern hybridization, i.e., washing once, specifically two or three times, with a salt concentration and temperature corresponding to 60°C, 1×SSC (citrate-saline solution), 0.1% SDS (sodium dodecyl sulfate), specifically 60°C, 0.1×SSC, 0.1% SDS, and more specifically 68°C, 0.1×SSC, 0.1% SSD.

Hybridization requires that two nucleic acids have complementary sequences, although, depending on the stringency of hybridization, mismatches between bases are possible. The term "complementary" is used to describe the relationship between nucleotide bases that can hybridize to each other. With respect to, for example, DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Thus, the present invention may also include an isolated fragment of nucleic acid complementary to the entire sequence, as well as a nucleic acid sequence substantially similar thereto.

Specifically, polynucleotides having homology or identity can be detected using hybridization conditions including a hybridization step at a _Tm value of 55°C under the conditions described above. In addition, the _Tm value may be 60°C, 63°C, or 65°C, but is not limited thereto, and can be appropriately adjusted by a person skilled in the art depending on the purpose thereof.

The appropriate stringency for polynucleotide hybridization depends on the length and degree of complementarity of the polynucleotides, and these variables are well known in the art (see Sambrook et al., supra, 9.50-9.51, 11.7-11.8).

When used here, the term "homology" or "identity" refers to the degree of relatedness between two given amino acid sequences or nucleotide sequences and may be expressed as a percentage. The terms homology and identity can often be used interchangeably with each other.

Sequence homology or identity of conserved polynucleotides or polypeptides can be determined by standard alignment algorithms and can be used in conjunction with the default gap penalty set by the program used. As such, homologous or identical sequences are typically expected to hybridize over the entire length or at least to about 50%, 50%, 60%, 70%, 80%, or 90% of the entire length of the sequences under moderate or very stringent conditions. Polynucleotides that contain degenerate codons in place of codons in the hybridizing polynucleotides can also be considered.

Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can, for example, be determined by a known computer algorithm such as the “FASTA” program (Pearson et al, (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444) using default parameters. Alternatively, it can be determined by the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), which is performed using the Needleman program from 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 software package (Devereux, J., et al., Nucleic Acids Research 12: 387 (1984)), B LAS TP, BLASTN, FASTA (Atschul, S. F., et al., J. MOLECBIOL 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 from the National Center for Biotechnology Information.

Homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using, for example, the GAP computer program such as Needleman et al. (1970) J Mol Biol. 48:443, which is described in Smith and Waterman, Adv. Appl. Math (1981) 2:482. In general, the GAP program defines homology, similarity, or identity as a value obtained by dividing the number of similarly aligned characters (i.e., nucleotides or amino acids) by the total number of characters in the shorter of the two sequences. Default parameters for the GAP program may include (1) a binary comparison matrix (containing the value 1 for identity and 0 for non-identity) and a weight comparison matrix (or EDNAFULL substitution matrix (EMBOSS version of NCBI NUC4.4)) in Gribskov et al. (1986) Nucl Acids Res. 14:6745, which is described in 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 penalty of 0.10 for each character in each gap (or a gap-opening penalty of 10 and a gap-extending penalty of 0.5); and (3) no penalty for trailing spaces.

Furthermore, whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined by comparing the sequences using Southern hybridization experiments under certain stringent conditions, and the appropriate hybridization conditions to be determined can be established by a method within the scope of the present invention that is known to one skilled in the art (e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2 ^nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, NY, 1989; F. M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York).

In one embodiment, enhancing the activity of the phosphate importer system may be, but is not limited to, enhancing the activity of a polypeptide encoded by the pitA gene.

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 a microorganism into which a foreign pitA gene or a foreign pit polypeptide has been introduced, but not limited thereto. Furthermore, it includes those microorganisms in which the introduced foreign genes or polypeptides have been enhanced. Specifically, the foreign pitA gene may be derived from Escherichia sp. or Escherichia coli, but not limited thereto.

As used herein, the term “enhancement of polypeptide activity” means that the activity of the polypeptide is increased relative to its endogenous activity. Enhancement may be used interchangeably with terms such as activation, up-regulation, overexpression, increase, etc. In particular, the terms activation, enhancement, up-regulation, overexpression, and increase may include both cases in which an activity that was not originally present is exhibited or the activity is enhanced relative to the endogenous or pre-modification activity. “Endogenous activity” refers to the activity of a particular polypeptide that was originally possessed by the parent strain prior to transformation, or by the unmodified microorganism when the trait is altered by genetic variation due to a natural or artificial factor. Endogenous activity may also be used interchangeably with “pre-modification activity.” “Enhancement,” “upregulation,” “overexpression,” or “increase” of the activity of a polypeptide relative to the endogenous activity means that the activity of the polypeptide is enhanced relative to the activity of the particular polypeptide originally possessed by the parent strain prior to transformation, or by the unmodified microorganism.

Enhancement may be achieved by introducing a foreign polypeptide or enhancing the activity and/or concentration (expression level) of an endogenous polypeptide. Whether or not the activity of a polypeptide is enhanced may be confirmed by the activity or expression level of the corresponding polypeptide or by an increase in the amount of product obtained from the corresponding polypeptide.

The enhancement of the activity of the polypeptide can be caused by various methods well known in the art, and is not limited as long as it can enhance the activity of the target polypeptide compared to the activity of the microorganism before the modification. Specifically, genetic engineering and/or protein engineering well known to those skilled in the art, which is a common method of molecular biology, can be used, but the method 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.).

Specifically, enhancing the polypeptide activity of the present invention can be achieved by:

1) an increase in the number of intracellular copies of the polynucleotide encoding the polypeptide;

2) replacement of the region that regulates the expression of a gene encoding a polypeptide on a chromosome with a sequence that has strong activity;

3) modifications of the nucleotide sequence encoding the initiation codon or 5'-UTR of the transcript of the gene encoding the polypeptide;

4) modifications of the amino acid sequence of the polypeptide in such a way that the activity of the polypeptide is enhanced;

5) modifications of a polynucleotide sequence encoding a polypeptide such that the activity of the polypeptide is enhanced (e.g. modifications of the polynucleotide sequence of a polypeptide gene encoding a polypeptide that has been modified to enhance the activity of that polypeptide);

6) introduction of a foreign polypeptide exhibiting polypeptide activity, or a foreign polynucleotide encoding such a polypeptide;

7) codon optimization of the polynucleotide encoding the polypeptide;

8) analysis of the tertiary structure of the polypeptide and, thereby, selection and modification of the exposed site, or chemical modification of the same; or

9) combinations of two or more selected from items 1-8 above, but not specifically limited to these.

More specifically:

1) A method for increasing the number of intracellular copies of a polynucleotide encoding a polypeptide can be carried out by introducing into a host cell a vector that is operably linked to a polynucleotide encoding a polypeptide and is capable of replicating and functioning independently of the host cell. Alternatively, the method can be carried out by introducing one copy or two copies of polynucleotides encoding a polypeptide into a chromosome of a host cell. Introduction into a chromosome can be carried out by introducing a vector that is capable of integrating a polynucleotide into a chromosome of a host cell into a host cell, but is not limited thereto. The vector is as described above.

2) The method of replacing an expression regulatory region (or an expression regulatory sequence) of a gene encoding a polypeptide on a chromosome with a sequence having a strong activity can be carried out, for example, by inducing a modification 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 by replacing the sequence with a sequence having a stronger activity. The expression regulatory region may include a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating the termination of transcription and translation, etc., but is not particularly limited thereto. In one example, the method may include replacing an original promoter with a strong promoter, but is not limited thereto.

Examples of known strong promoters may include CJ1-CJ7 promoters (US 7662943 B2), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13 (sm3) promoter (US 10584338 B2), 02 promoter (US 10273491 B2), tkt promoter, yccA promoter, etc., but the strong promoter is not limited to these.

3) A method for modifying a nucleotide sequence encoding an initiation codon or the 5'-UTR of a transcript of a gene encoding a polypeptide can be carried out, for example, by replacing the nucleotide sequence with a nucleotide sequence encoding another initiation codon that has a higher degree of expression of the polypeptide compared to the endogenous initiation codon, but is not limited to this.

Methods 4) and 5) of modifying an amino acid sequence or a polynucleotide sequence can be carried out by inducing a modification of the sequence by deletion, insertion, non-conservative or conservative substitution of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide, or a combination thereof to enhance the activity of the polypeptide, or by replacing the sequence with an amino acid sequence or a polynucleotide sequence modified to impart a stronger activity, or an amino acid sequence or a polynucleotide sequence modified to enhance the activity, but are not limited thereto. The substitution can be carried out specifically by inserting the polynucleotide into the chromosome by homologous recombination, but is not limited thereto. The vector used in the present invention may further comprise a selectable marker for confirming the insertion into the chromosome. The selectable marker is as described above.

6) The method of introducing a foreign polynucleotide exhibiting the activity of a polypeptide can be carried out by introducing into a host cell a foreign polynucleotide encoding a polypeptide that exhibits the same/similar activity as the polypeptide. The foreign polynucleotide can be used without limitation, regardless of its origin or sequence, provided that it exhibits the same/similar activity as the polypeptide. The introduction can be carried out by ordinary persons skilled in the art by appropriately selecting a transformation method known in the art, and the expression of the introduced polynucleotide in the host cell allows the production of the polypeptide, thereby increasing its activity.

7) A method for codon-optimizing a polynucleotide encoding a polypeptide can be carried out by codon-optimizing an endogenous polynucleotide to increase transcription or translation in a host cell or by optimizing its codons such that optimized transcription and translation of a foreign polynucleotide in a host cell can be achieved.

In addition, 8) the method of analyzing the tertiary structure of a polypeptide and thereby selecting and modifying an exposed region or chemically modifying it can be carried out, for example, by comparing the sequence information of the polypeptide to be analyzed with a database in which the sequence information of known proteins is stored to determine candidate template proteins according to the degree of sequence similarity and thus confirming the structure based on this information, thereby selecting and transforming or modifying the exposed site to be modified or chemically modified.

Such an increase in the activity of a polypeptide may mean that the activity or concentration (expression level) of the corresponding polypeptide is increased relative to the activity or concentration of the polypeptide expressed in the wild-type strain or in the microorganism before modification, or that the amount of product obtained from the polypeptide is increased, but is not limited to this.

The modification of a part or the whole polynucleotide in the microorganism of the present invention can be carried out by (a) homologous recombination using a vector for chromosomal insertion into the microorganism or genome editing using an engineered nuclease (e.g., CRISPR-Cas9), and/or (b) can be induced by light such as ultraviolet rays and radiation, etc., and/or chemical treatment, but not limited thereto. The method for modifying a part or the whole gene can include a method using recombinant DNA technology. For example, a part or the whole gene can be deleted by injecting a nucleotide sequence or a vector containing a nucleotide sequence homologous to a target gene into a microorganism to induce homologous recombination. The introduced nucleotide sequence or vector can include a dominant selectable marker, but not limited thereto.

In the present invention, the polypeptide intended to enhance the activity may be a phosphate importer, and specifically, it may be a Pit system or a polypeptide encoded by the pitA gene, or a PitA polypeptide. For the purposes of the present invention, the enhancement of the activity may be achieved by introducing a foreign Pit system, a foreign polypeptide encoded by the pitA gene, or a foreign PitA polypeptide, but is not limited to this.

For the purposes of the present invention, enhancing the activity of the phosphate importer system may include, but is not limited to, introducing a foreign pitA gene or a polypeptide encoded by a foreign pitA.

As used herein, the term "introducing a polypeptide" may mean exhibiting an activity of a specific polypeptide that the microorganism did not originally possess, or exhibiting an improved activity compared to the endogenous activity or the activity prior to modification of the polypeptide. For example, a specific polypeptide may be introduced, a polynucleotide encoding a specific polypeptide may be introduced into a chromosome within a microorganism, or a vector containing a polynucleotide encoding a specific polypeptide may be introduced into a microorganism to exhibit its activity.

As used herein, the term “foreign gene” refers to a non-native (unnatural) gene included in a microorganism. For example, it may be (a) a gene that cannot be found naturally in a microorganism (exogenous), (b) a gene that can be found naturally in a microorganism (endogenous) but transcription or translation of that gene in the microorganism results in an unnatural amount (greater or lesser amount compared to the amount present in nature), (c) a polypeptide sequence that is endogenously present in the microorganism but the sequence of the gene encoding it is different, and/or (d) a combination of two or more of the above in a microorganism, but not limited to these.

For the purposes of the present invention, since the pit system does not exist in the microorganism Corynebacterium stationis, the foreign gene may be a foreign pitA gene, may be a pitA gene from Escherichia sp., or may be a pitA gene derived from Escherichia coli, but is not limited thereto.

The vector of the present invention may comprise a DNA construct comprising a nucleotide sequence of a polynucleotide encoding a target polypeptide operably linked to a suitable region regulating expression (expression regulating sequence) so as to be able to express the target polypeptide in a suitable host cell. The expression regulating region may comprise a promoter capable of initiating transcription, any operator sequence for regulating transcription, a sequence encoding a suitable mRNA binding site for the ribosome, and a sequence for regulating the termination of transcription and translation. After transformation into a suitable host cell, the vector may replicate or function independently of the host genome or may integrate into its genome.

The vector used in the present specification is not particularly limited, and any vector known in the art can be used. Examples of the vector commonly used may include natural or recombinant plasmids, cosmids, viruses, and bacteriophages. For example, as the phage vector or the cosmid vector, pWE15, M13, MBL3, MBL4, IXII, ASHII, ARP, t10, t11, Charon4A, and Charon21A, etc. can be used; and as the plasmid vector, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, and pET-based vectors, etc. can be used. Specifically, pDZ, pDC, pdc2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC, etc. vector can be used.

In one example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome using a vector for intracellular chromosomal insertion. The insertion of the polynucleotide into the chromosome can be accomplished by any method known in the art, such as, but not limited to, homologous recombination. The vector can further comprise a selectable marker to confirm the insertion into the chromosome. The selectable marker is intended to select cells transformed by the vector, i.e., to confirm whether the target nucleic acid molecule has been introduced, and markers can be used that provide selectable phenotypes, such as drug resistance, auxotrophy, resistance to cellular toxic agents, or expression of surface polypeptides. Only cells expressing the selectable marker are able to survive or exhibit different phenotypes in a medium treated with a selective agent, and thus transformed cells can be selected.

As used herein, the term "transformation" refers to the introduction of a vector containing a polynucleotide encoding a target polypeptide into a host cell or microorganism such that the polypeptide encoded by the polynucleotide can be expressed in the host cell. As long as the transformed polynucleotide can be expressed in the host cell, it does not matter whether the transformed polynucleotide is integrated into and localized within the chromosome of the host cell or is localized extrachromosomally, and both cases can be included. In addition, the polynucleotide can include DNA and/or RNA encoding the target polypeptide. The polynucleotide can be introduced in any form, so long as it can be introduced into the host cell and expressed therein. For example, the polynucleotide can be introduced into the host cell in the form of an expression cassette, which is a gene construct that includes all the elements necessary for its autonomous expression. An expression cassette may typically include a promoter operably linked to a polynucleotide, a transcriptional terminator, a ribosome binding site, or a translation terminator. The expression cassette may be in the form of a self-replicating expression vector. Additionally, the polynucleotide may be introduced into the host cell as is and operably linked to sequences necessary for expression in the host cell, but is not limited to this.

Furthermore, when used herein, the term "operably linked" means that the polynucleotide sequence is operably linked to a promoter sequence that initiates and mediates transcription of the polynucleotide encoding the target variant of the present invention.

As used herein, the term “strain (or microorganism)” includes all wild-type microorganisms or naturally or artificially genetically modified microorganisms, and may be a microorganism in which a particular mechanism is weakened or strengthened due to the insertion of a foreign gene, or the enhancement or inactivation of the activity of an endogenous gene, etc., and may be a microorganism comprising genetic modification to produce a desired polypeptide, protein or product.

As used herein, the term "a Corynebacterium stationis microorganism having the ability to produce purine nucleotides" refers to a microorganism that has the ability to produce and accumulate purine nucleotides within the body as a target product, or to secrete or accumulate purine nucleotides in a medium when the microorganism is cultured in the medium. In one embodiment, the term "having the ability to produce purine nucleotides" can be used interchangeably with "producing purine nucleotides". The ability to produce purine nucleotides can be a property of a wild-type strain of the Corynebacterium stationis microorganism, and can be imparted or enhanced by genetic modification.

The microorganism having the ability to produce purine nucleotides according to the present invention may be a recombinant strain having an increased ability to produce purine nucleotides by enhancing the activity of the phosphate importer system (Pit system) encoded by the pit gene. The recombinant strain having an increased ability to produce purine nucleotides may have an increased productivity of purine nucleotides compared with a natural wild-type microorganism or a microorganism with an unmodified phosphate importer system (i.e., a microorganism expressing a wild-type phosphate importer system, a microorganism without an enhanced phosphate importer system, or a microorganism that does not express a phosphate importer system), but is not limited thereto. In one example, a microorganism with an unmodified phosphate importer system, a target strain to be compared with respect to whether the ability to produce purine nucleotides is increased, may be CJX1664 (KCCM12285P, KR 10-1950141 B1, US 2020-0392478 A1), CJX1665 (KCCM12286P, KR 10-1950141 B1, US 2020-0392478 A1), CJI2332 (KCCM12277P, KR 10-1950141 B1, US 2020-0392478 A1) or CJI2335 (KCCM12278P, KR 10-1956510 B1, EP 3705572 A1), but not limited to them.

In one example, a recombinant strain having an increased producing ability may have an ability to produce purine nucleotides increased by 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 11.5% or more, about 12% or more, or about 13% or more (the upper limit is not particularly limited, such as 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 ability of the parent strain before modification or the unmodified microorganism, but not limited thereto, as long as it has an increased + value compared to the ability production of the parent strain before modification or the unmodified microorganism. In another example, a recombinant strain having an increased production ability may have an ability to produce purine nucleotides increased by about 1.01 times or more, about 1.02 times or more, about 1.03 times or more, about 1.05 times or more, about 1.06 times or more, about 1.07 times or more, about 1.08 times or more, about 1.09 times or more, about 1.10 times or more, about 1.11 times or more, about 1.12 times or more, or about 1.13 times or more (the upper limit is not particularly limited, such as about 10 times or less, about 5 times or less, about 3 times or less, or about 2 times or less) compared to the original strain before modification or the unmodified microorganism, but not limited thereto. When used herein, the term “about” refers to a range that includes all of ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all values that are equivalent or similar to the values specified below, but the range is not limited thereto.

A microorganism having the ability to produce the purine nucleotides of the present invention may be a microorganism having the ability to produce a target polypeptide or a target product, involved in the production of the target polypeptide by including one or more of the pit system, the PitA polypeptide; a polynucleotide encoding them; and a vector containing said polynucleotide, but not limited to this. The microorganism may be 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, but not limited to this.

As used herein, the term "unmodified microorganism" does not exclude a strain containing a mutation that may occur naturally in the microorganism, and may refer to a wild-type strain or the wild-type strain itself, or to a strain before a property has changed due to genetic modification caused by natural or artificial factors. For example, an unmodified microorganism may refer to a strain in which the phosphate importer system described herein has not been introduced or enhanced, or to a strain before it has been enhanced or introduced. "Unmodified microorganism" may be used interchangeably with "pre-modification strain," "pre-modification microorganism," "non-mutant strain," "non-modified strain," "non-mutant microorganism," or "reference microorganism."

As used herein, a “purine nucleotide” may be any one or more nucleotides selected from the group consisting of 5'-inosine monophosphate (IMP), 5'-xanthosine monophosphate (XMP), and 5'-guanosine monophosphate (GMP). IMP is a deaminated adenine compound and refers to a nucleotide consisting of one molecule of hypoxanthine, ribose, and phosphate. IMP can be biosynthesized from 5'-phosphoribosyl-1-pyrophosphate (PRPP), and specifically, it can be formed by substituting a pyrophosphate group bonded to the first carbon of PRPP with a nitrogen atom and by forming an imidazole ring and a pyrimidine ring in nine steps, but is not limited thereto. XMP refers to a nucleotide produced by the dehydrogenation of IMP and can be synthesized from IMP by, but is not limited to, inosine 5'-monophosphate dehydrogenase. GMP refers to a nucleotide having a structure in which the phosphate group forms an ester bond in the ribose of a guanosine molecule. GMP can be synthesized by adding ammonia molecules to XMP by, but is not limited to, 5'-guanylic acid biosynthase (GMP synthase).

In another aspect of the present invention, a method for producing purine nucleotides is provided, comprising culturing the microorganism Corynebacterium stationis, which has the ability to produce purine nucleotides, in which the activity of the phosphate importer system (Pit system) is enhanced, in a medium.

The phosphate importer system, the Pit system, the enhancement, the purine nucleotide and the microorganism Corynebacterium stationis, etc. are the same as described in the other aspects above.

As used herein, the term "culturing" means that the microorganism is grown under appropriately controlled environmental conditions. The culturing process of the present invention can be carried out in a suitable culture medium and culturing conditions known in the art. Such a culturing process can be easily adapted for use by those skilled in the art according to the selected strain. Specifically, the culturing can be a batch culturing, a continuous culturing and/or a fed-batch culturing, but is not limited thereto.

As used herein, the term "medium" refers to a mixture of substances that contains nutrients necessary for culturing a microorganism as a main ingredient, and it supplies nutrients and growth factors, together with water, which are necessary for survival and growth. Specifically, the medium and other culture conditions used for culturing the microorganism of the present invention may be any medium used for conventional microorganism culturing, without particular limitation. However, the microorganism of the present invention can be cultured under aerobic conditions in a conventional medium containing a suitable carbon source, a nitrogen source, a phosphorus source, an inorganic compound, an amino acid and/or a vitamin, while adjusting the temperature, pH, etc.

In the present invention, the carbon source may include carbohydrates such as glucose, sucrose, lactose, fructose, sucrose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvic acid, lactic acid, citric acid, etc.; amino acids such as glutamic acid, methionine, lysine, etc. Further, the carbon source may include natural organic nutrients such as starch hydrolysate, molasses, black molasses, rice bran, cassava, sugarcane molasses, corn steep liquor, etc. Specifically, carbohydrates such as glucose and sterilized pre-treated molasses (i.e., molasses converted into reducing sugar) can be used, and further, various other carbon sources can be used in an appropriate amount without limitation. These carbon sources may be used individually or in combination of two or more types, but are not limited thereto.

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

The phosphorus source may include monopotassium phosphate, dipotassium phosphate or corresponding sodium-containing salts, etc. Examples of inorganic compounds may include sodium chloride, calcium chloride, ferric chloride, magnesium sulfate, ferrous sulfate, manganese sulfate, calcium carbonate, etc. In addition, amino acids, vitamins and/or corresponding precursors, etc. may be included. These constituent ingredients or precursors may be added to the medium in a batch or continuous manner, but the said phosphorus sources are not limited thereto.

In addition, the pH of the medium can be adjusted appropriately by adding a compound such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. during the cultivation of the microorganism. In addition, the formation of bubbles can be prevented during the cultivation by using an antifoaming agent such as fatty acid polyglycol ester. In addition, oxygen gas or a gas containing oxygen can be introduced into the medium to maintain an aerobic condition of the medium; or nitrogen gas, hydrogen gas, or carbon dioxide can be introduced to maintain anaerobic or microaerobic conditions without introducing gas, but the gas is not limited to this.

The temperature of the environment may be in the range of 27°C to 37°C, specifically 30°C to 33°C, but not limited to this. Cultivation may be continued until the production of the useful product reaches the desired level. Specifically, cultivation may be continued for 20 to 120 hours, but not limited to this.

Specifically, the nutrient medium for the microorganism of the genus Corynebacterium can be found in the literature (“Manual of Methods for General Bacteriology” by the American Society for Bacteriology (Washington D.C., USA, 1981)).

The purine nucleotides obtained by culturing according to the present invention may be released into the medium or remain in the cells.

The method for producing purine nucleotides according to the present invention may further include a step of obtaining the microorganism Corynebacterium stationis according to the present invention, a step of preparing a medium for culturing the microorganism, or a combination thereof (irrespective of the order, in any order), for example, before the culturing step.

The method for producing purine nucleotides according to the present invention may further comprise a step of isolating purine nucleotides from a nutrient medium (a medium in which the culture was grown) or a microorganism (a microorganism from which the culture was grown). The extraction step may further be included after the culturing step.

In the isolation step, the desired purine nucleotides can be collected by using the microorganism culturing method of the present invention, for example, by using a suitable method known in the art according to a batch culturing method, a continuous culturing method, or a fed-batch culturing method. For example, methods such as centrifugation, filtration, treatment with a precipitant for crystallizing protein (salting out method), extraction, ultrasonic disruption, ultrafiltration, dialysis, various types of chromatography such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, etc., HPLC or a combination thereof can be used, and the desired purine nucleotides can be isolated from the medium or the microorganisms using suitable methods known in the art.

In addition, the method for producing purine nucleotides of the present invention may further include a purification step, which can be performed using a suitable method known in the art. In one example, when the method for producing purine nucleotides of the present invention includes both an isolation step and a purification step, the isolation step and the purification step can be performed continuously or intermittently regardless of the order or simultaneously, or can be combined into one step, but the method is not limited thereto.

Specifically, the method for producing 5'-guanosine monophosphate (GMP) may further include a step of converting 5'-xanthosine monophosphate (XMP) into GMP in the culturing step. In the method for producing GMP of the present invention, the conversion step may be further included after the culturing step or the extraction step. The conversion step can be performed using a suitable method known in the art. For example, the conversion can be performed using coryneform microorganisms, Escherichia coli, or XMP aminase (KR 10-0655902 B1), but is not limited thereto.

In another aspect of the present invention, there is provided a composition for producing purine nucleotides comprising the microorganism Corynebacterium stationis of the present invention; a medium in which the microorganism is cultured; or a combination thereof.

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

In the composition of the present invention, the microorganism, the medium and the purine nucleotide, etc. are the same as described in other aspects above.

In another aspect of the present invention, there is provided the use of the microorganism Corynebacterium stationis according to the present invention for the production of purine nucleotides.

When used to produce purine nucleotides, the microorganism and the purine nucleotide, etc. are as described in other aspects above.

Embodiment of the invention

The present invention will now be described in detail by means of Examples. However, these Examples are only preferred Examples provided for illustrative purposes, and it is obvious to those skilled in the art to which the present invention pertains that the scope of the present invention is not intended to be limited by these Examples.

Example 1: Production of a strain with introduced foreign pitA and evaluation of IMP productivity

1-1: Preparation of a recombinant vector for introducing a foreign pitA gene and obtaining a strain producing IMP

The wild-type Corynebacterium stationis strains lack the pit system. Accordingly, a vector for introducing the foreign Pit system into Corynebacterium stationis was obtained. In addition, the CJ1 promoter (Korean Patent No. 10-0620092, SEQ ID No.: 3), a known strong promoter among the promoters of the genus Corynebacterium, was conjugated with the foreign pitA gene. A vector for replacing the fepB gene, one of the periplasm binding proteins of the genus Corynebacterium, was obtained.

Specifically, to obtain an insertion vector capable of deleting the fepB gene of Corynebacterium stationis, the chromosomal gene of strain ATCC6872, which is the wild-type Corynebacterium stationis, was isolated using a G-spin total DNA extraction mini kit (Cat. No. 17045, Intron) according to the protocol provided in the kit, and PCR was performed using it as a template. High-fidelity DNA polymerase (Intron) from Maxime PCR PreMix (i-pfu) was used as the polymerase. PCR was performed as follows: 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 different PCR products were obtained. (fepBdel-A, fepBdel-B). fepB-A was 1038 bp in size and was amplified using SEQ ID NO: 4 and SEQ ID NO: 5 as primers. fepB-B was 1111 bp in size and was amplified using SEQ ID NO: 6 and SEQ ID NO: 7 as primers. Secondary PCR was performed using these two kinds of amplified products as templates by the cross-linking PCR method. PCR was performed under the same conditions as described above, and a 2131 bp PCR product was obtained in which the fepB gene was deleted and which included the SpeI and NotI restriction enzyme sites in the middle. Then, the amplified products were digested using the restriction sites (fepBdel-A: HindIII, fepBdel-B: HindIII), located at both ends, and then cloned into the pDZ vector by T4 ligase activity, resulting in the pDZ-fepBdel vector for deletion of the fepB gene. The sequences of the primers used above are as follows.

The vector in which the nucleic acid region of the fepB gene deleted from the pDZ-fepBdel vector was replaced with foreign pitA was prepared as follows. Specifically, chromosomes of the wild-type strain Corynebacterium stationis ATCC6782 and E coli MG1655 were isolated, and PCR was performed using the chromosomes as a template and high-fidelity DNA polymerase (Intron) from Maxime PCR premix (i-pfu) as a polymerase. PCR was performed as follows: 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 2 minutes and 30 seconds. As a result of PCR, two PCR products (pCJ1-pitA-A, pCJ1-pitA-B) of the pitA gene conjugated with the CJ1 promoter were obtained. pCJ1-pitA-A, which is the PCR product of the promoter region, had a size of 341 bp and was amplified using SEQ ID NO: 8 and SEQ ID NO: 9 as primers. pCJ1-pitA-B, which is the PCR product of the pitA gene, had a size of 1537 bp and was amplified using SEQ ID NO: 10 and SEQ ID NO: 11 as primers. Secondary PCR was performed using two kinds of amplification products as templates by the cross-linking PCR method. PCR was performed under the same conditions as described above, resulting in the pitA gene conjugated to the pCJ1 promoter containing nucleotide sequences homologous to the pDZ-fepBdel vector at both ends. This gene was cloned into the pDZ-fepBdel vector digested with SpeI and NotI restriction enzymes using homologous recombinase, yielding the pDZ-ΔfepB::pCJ1/pitA(Eco) vector. The sequences of the primers used above are as follows.

Then, the above-obtained pDZ-ΔfepB::pCJ1/pitA(Eco) vector was transformed into each strain CJI2332, which is an IMP-producing strain (IMP-producing strain KCCM12277P derived from ATCC6872 Corynebacterium stationis, Korean Patent No. 10-1950141, US 2020-0392478 Al) and CL2335 (IMP-producing strain KCCM12278P derived from Corynebacterium stationis ATCC6872, Korean Patent No. 10-1956510, EP 3705572 A1) by electroporation to obtain strains containing the CJ1 promoter-pitA gene in the middle of the endogenous fepB gene on the chromosome, through the second crossing over process. The newly inserted CJ1 promoter-pitA gene was confirmed using SEQ ID NO: 12 and SEQ ID NO: 13 as primers that can amplify the region where both ends of the inserted gene and the fepB gene meet. In the same manner as above, two strains, CJI2332_pCJ1/pitA and CJI2335_pCJ1/pitA, were obtained, which are strains that constitutively overexpress pitA introduced exogenously via the CJ1 promoter.

1-2. Evaluation of IMP productivity of strain with included foreign pitA

In order to study the improvement in the productivity of IMP strain CJI2332_pCJ1/pitA and strain CJI2335_pCJ1/pitA obtained in Example 1-1, the following experiment was performed.

Each of CJI2332_pCJ1/pitA, CJI2332, CJI2335_pCJ1/pitA, and CJI2335 was seeded in 5 ml of seed medium in an autoclavable test tube having a diameter of 18 mm and cultured with shaking at 30°C for 24 hours and used as the seed culture broth. 29 ml of the fermentation medium was poured into a 250 ml shaking Erlenmeyer flask and then autoclaved at 121°C for 15 minutes. Then, 2 ml of the seed culture broth was seeded into it and cultured for 3 days. The culture conditions were controlled at 170 rpm, 30°C, and pH 7.5. The compositions of the seed medium and the fermentation medium were as follows.

IMP seeding medium

1% glucose, 1% peptone, 1% meat extract, 1% yeast extract, 0.25% sodium chloride, 100 mg/l adenine, 100 mg/l guanine, pH 7.2 (based on 1 l of medium)

Fermentation medium in IMP flask

0.1% sodium glutamate, 1% ammonium chloride, 1.2% magnesium sulfate, 0.01% calcium chloride, 20 mg/l ferrous sulfate, 20 mg/l manganese sulfate, 20 mg/l zinc sulfate, 5 mg/l copper sulfate, 23 mg/l L-cysteine, 24 mg/l alanine, 8 mg/l nicotinic acid, 45 μg/l biotin, 5 mg/l thiamine-HCl, 30 mg/l adenine, 1.9% phosphoric acid (85%), 4.2% glucose, 2.4% raw sugar (based on 1 l of medium).

The results of IMP production measurement by the HPLC method after completion of the culture are shown in Table 4 below.

Table 4

As shown in Table 4, it was confirmed that under the same conditions, the accumulation yield of IMP produced by the CJI2332_pCJ1/pitA strain and the CJI2335_pCJ1/pitA strain increased by about 13% and about 12.5%, respectively, compared with the parental strains CJI2332 and CJI2335. CJI2332_pCJ1/pitA was named CJI2630 and deposited at the Korea Culture Center for Microorganisms (KCCM), the depository authority under the Budapest Treaty, on April 13, 2021, with the accession number KCCM12974P.

CJI2335_pCJ1/pitA was named CJI2631 and deposited at the Korea Culture Center for Microorganisms (KCCM), the depository authority under the Budapest Treaty, on April 13, 2021, with the accession number KCCM12975P.

Example 2: Production of a strain with introduced foreign pitA and evaluation of HMP productivity

2-1: Obtaining a strain with introduced foreign pitA

pDZ-ΔfepB::pCJ1/pitAA(Eco) was transformed into each of CJX1664, which is a XMP-producing strain (XMP-producing strain KCCM12285P derived from Corynebacterium stationis, Korean Patent No. 10-1950141, US 2020-0392478 Al), and CJX1665, which is a purA (G85S) variant strain of CJX1664 (KCCM12286P, Korean Patent No. 10-1950141, US 2020-0392478 A1), by electroporation to obtain the CJX1664_pCJ1/pitA strain and the CJX1665_pCJ1/pitA strain containing the pCJ1-pitA gene in the middle endogenous fepB gene on the chromosome through a second crossing over process. The newly inserted pCJ1-pitA gene was confirmed using SEQ ID NO: 12 and SEQ ID NO: 13 as primers that can amplify the region where both ends of the inserted gene and the fepB gene meet.

Evaluation of the productivity of XMP of a foreign gene with introduced pitA

To measure the productivity of XMP in the strain CJX1664_pCJ1/pitA and the strain CJX1665_pCJ1/pitA obtained in Example 2-1, a flask test was performed. Each of CJX1664, CJX1664_pCJ1/pitA, CJX1665, and CJX1665_pCJ1/pitA was seeded in a 14 mL tube containing 2.5 mL of the following seeding medium, and cultured with shaking at 30°C and 170 rpm for 24 hours. 0.7 ml of seed culture broth was inoculated into a 250 ml angle-baffled flask containing 32 ml (24 ml of main medium +8 ml of separate sterile medium) of the following production medium and cultured with shaking at 30°C and 170 rpm for 75 hours. The compositions of the seed medium, main medium and separate sterile medium were as follows.

Medium for sowing in a flask XMR

30 g/l glucose, 15 g/l peptone, 15 g/l yeast extract, 2.5 g/l sodium chloride, 3 g/l urea, 150 mg/l adenine, 150 mg/l guanine, pH 7.0 (based on 1 l of medium)

Medium for the production of HMR in a flask (basic medium)

50 g/l glucose, 10 g/l magnesium sulfate, 100 mg/l calcium chloride, 20 mg/l iron sulfate, 10 mg/l manganese sulfate, 10 mg/l zinc sulfate, 0.8 mg/l copper sulfate, 20 mg/l histidine, 15 mg/l cysteine, 15 mg/l beta-alanine, 100 μg/l biotin, 5 mg/l thiamine, 50 mg/l adenine, 25 mg/l guanine, 15 mg/l niacin, pH 7.0 (based on 1 l of medium)

Medium for the production of HMR in a flask (separate sterile medium)

18 g/l of monobasic potassium phosphate, 42 g/l of dibasic potassium phosphate, 7 g/l of urea, 5 g/l of ammonium sulfate (based on 1 l of medium)

The results of measuring the HMP production by the HPLC method after completion of the culture are shown in Table 5 below.

As shown in Table 5 above, the concentration of XMP produced by CJX1664_pCJ1/pitA and CJX1665_pCJ1/pitA, which are strains that constitutively express pita E coli, increased by 6.7% and 6.4%, respectively, compared with the parental strains CJX1664 and CJX1665.

Based on the above, a person skilled in the art to which the present invention pertains can understand that the present invention can be embodied in other specific forms without changing the technical concepts or essential characteristics of the present invention. In this regard, the exemplary embodiments disclosed herein are intended for illustrative purposes only and should not be construed as limiting the scope of the present invention. On the contrary, it is intended that the present invention cover not only the exemplary embodiments but also various alternatives, modifications, equivalents and other embodiments that may be included within the spirit and scope of the present invention as defined by the appended claims.

SEQUENCE LIST

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<110>CJ CheilJedangCorporation

<120> MICROORGANISM PRODUCING PURINE NUCLEOTIDES AND METHOD

OBTAINING PURINE NUCLEOTIDES WITH ITS USE

<130>OPA22030

<150> KR 10-2021-0065740

<151> 2021-05-21

<160> 13

<170>KoPatentIn 3.0

<210> 1

<211> 499

<212> PRT

<213> Artificial Sequence

<220>

<223>PitA

<400> 1

Met Leu His LeuPhe Ala GlyLeu Asp Leu His ThrGlyLeuLeuLeu

1 5 10 15

LeuLeu Ala Leu Ala Phe Val LeuPhe Tyr Glu Ala Ile AsnGlyPhe

20 25 30

His Asp Thr Ala Asn Ala Val Ala Thr Val Ile Tyr ThrArg Ala Met

35 40 45

ArgSerGlnLeu Ala Val Val Met Ala Ala Val PheAsnPheLeuGly

50 55 60

Val LeuLeuGlyGlyLeuSer Val Ala Tyr Ala Ile Val His Met Leu

65 70 75 80

Pro Thr Asp LeuLeuLeuAsn Met GlySerSer His GlyLeu Ala Met

85 90 95

Val PheSer Met LeuLeu Ala Ala Ile IleTrpAsnLeuGlyThrTrp

100 105 110

Tyr PheGlyLeu Pro Ala SerSerSer His ThrLeu Ile Gly Ala Ile

115 120 125

Ile Gly Ile GlyLeuThrAsn Ala Leu Met ThrGlyThrSer Val Val

130 135 140

Asp Ala LeuAsn Ile Pro Lys Val LeuSer Ile PheGlySerLeu Ile

145 150 155 160

Val Ser Pro Ile Val GlyLeu Val Phe Ala GlyGlyLeu Ile PheLeu

165 170 175

LeuArgArg Tyr TrpSerGlyThr Lys LysArg Ala Arg Ile His Leu

180 185 190

Thr Pro Ala GluArgGlu Lys Lys Asp Gly Lys LysLys Pro ProPhe

195 200 205

TrpThrArg Ile Ala Leu Ile LeuSer Ala Ile Gly Val Ala PheSer

210 215 220

His Gly Ala Asn Asp GlyGln Lys Gly Ile GlyLeu Val Met Leu Val

225 230 235 240

Leu Ile Gly Val Ala Pro Ala GlyPhe Val ValAsn Met Asn Ala Thr

245 250 255

Gly Tyr Glu Ile ThrArgThrArg Asp Ala Ile AsnAsn Val Glu Ala

260 265 270

Tyr PheGluGln His Pro Ala LeuLeu Lys Gln Ala ThrGly Ala Asp

275 280 285

GlnLeu Val Pro Ala Pro Glu Ala Gly Ala ThrGln Pro Ala GluPhe

290 295 300

His Cys His Pro SerAsnThr Ile Asn Ala LeuAsnArgLeu Lys Gly

305 310 315 320

Met LeuThrThr Asp Val GluSer Tyr Asp Lys LeuSerLeu Asp Gln

325 330 335

ArgSerGln Met ArgArg Ile Met LeuCys Val Ser Asp Thr Ile Asp

340 345 350

Lys Val Val Lys Met Pro Gly Val Ser Ala Asp AspGlnArgLeuLeu

355 360 365

Lys LysLeu Lys Ser Asp Met LeuSerThr Ile Glu Tyr Ala Pro Val

370 375 380

Trp Ile Ile Met Ala Val Ala Leu Ala LeuGly Ile GlyThr Met Ile

385 390 395 400

GlyTrpArgArg Val Ala ThrThr Ile GlyGlu Lys Ile Gly Lys Lys

405 410 415

Gly Met Thr Tyr Ala GlnGly Met Ser Ala Gln Met Thr Ala Ala Val

420 425 430

Ser Ile GlyLeu Ala Ser Tyr ThrGly Met Pro Val SerThrThr His

435 440 445

Val LeuSerSerSer Val Ala GlyThr Met Val Val Asp GlyGlyGly

450 455 460

LeuGlnArg Lys Thr Val ThrSer Ile Leu Met Ala Trp Val PheThr

465 470 475 480

Leu Pro Ala Ala Val LeuLeuSerGlyGlyLeu Tyr TrpLeuSerLeu

485 490 495

GlnPheLeu

<210> 2

<211> 1500

<212> DNA

<213> Artificial Sequence

<220>

<223>pitA

<400> 2

atgctacatttgtttgctggcctggatttgcataccgggctgttattattgcttgcactg 60

gcttttgtgctgttctacgaagccatcaatggtttccatgacacagccaacgccgtggca 120

accgttatctatacccgcgcgatgcgttctcagctcgccgtggttatggcggcggtattc 180

aactttttgggtgttttgctgggtggtctgagtgttgcctatgccattgtgcatatgctg 240

ccgacggatctgctgcttaatatgggatcgtctcatggccttgccatggtgttctctatg 300

ttgctggcggcgattatctggaacctgggtacctggtactttggtttacctgcatccagc 360

tctcatacgctgattggcgcgatcatcgggattggtttaaccaatgcgttgatgaccggg 420

acgtcagtggtggatgcactcaatatcccgaaagtattaagtattttcggttctctgatc 480

gtttcccctattgtcggcctggtgtttgctggcggtctgattttcttgctgcgtcgctac 540

tggagcggcaccaagaaacgcgcccgtatccacctgaccccagcggagcgtgaaaagaaa 600

gacggcaagaaaaagccgccgttctggacgcgtattgcgctgatcctttccgctatcggc 660

gtggcgttttcgcacggcgcgaacgatggtcagaaaggcattggtctggttatgttggta 720

ttgattggcgtcgcgccagcaggcttcgtggtgaacatgaatgccactggctacgaaatc 780

acccgtacccgtgatgccatcaacaacgtcgaagcttactttgagcagcatcctgcgctg 840

ctcaaacaggctaccggtgctgatcagttagtaccggctccggaagctggcgcaacgcaa 900

cctgcggagttccactgccatccgtcgaataccattaacgcgctcaaccgcctgaaaggt 960

atgttgaccaccgatgtggaaagctacgacaagctgtcgcttgatcaacgtagccagatg 1020

cgccgcattatgctgtgcgtttctgacactatcgacaaagtggtgaagatgcctggcgtg 1080

agtgctgacgatcagcgcctgttgaagaaactgaagtccgacatgcttagcaccatcgag 1140

tatgcaccggtgtggatcatcatggcggtcgcgctggcgttaggtatcggtacgatgatt 1200

ggctggcgccgtgtggcaacgactatcggtgagaaaatcggtaagaaaggcatgacctac 1260

gctcaggggatgtctgcccagatgacggcggcagtgtctatcggcctggcgagttatacc 1320

gggatgccggtttccactactcacgtactctcctcttctgtcgcggggacgatggtggta 1380

gatggtggcggcttacagcgtaaaaccgtgaccagcattctgatggcctgggtgtttacc 1440

cttccggctgcggtactgctttccggcgggctgtactggctctccttgcagttcctgtaa 1500

1500

<210> 3

<211> 301

<212> DNA

<213> Artificial Sequence

<220>

<223>CJ1 promoter

<400> 3

caccgcgggcttattccattacatggaatgaccaggaatggcagggaatgcgacgaaatt 60

gactgttgtcgggagcttctgatccgatgctgccaaccaggagagaaaataatgacatgt 120

gcaggcacgctggtgagctggagattatgatctcaagtaccttttttcttgcactcgag 180

ggggctgagtgccagaatggttgctgacaccaggttgaggttggtacacactcaccaatc 240

ctgccgtcgcgggcgcctgcgtggaacataaaccttgagtgaaacccaatctaggagatt 300

a 301

<210> 4

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223>fepBdel-A-F

<400> 4

cccaagcttccggtgttcagaatcgctccg

30

<210> 5

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223>fepBdel-A-R

<400> 5

gcggccgcaaaggactagtcctccggcattcagtcaggtc 40

<210> 6

<211> 56

<212> DNA

<213> Artificial Sequence

<220>

<223>fepBdel-B-F

<400> 6

ctagtcctttgcggccgcccattccgcccttcaaccttccgcctagattacttctc 56

<210> 7

<211> 32

<212> DNA

<213> Artificial Sequence

<220>

<223>fepBdel-B-R

<400> 7

cccaagcttgtgcaagctgtggatcgtctt cc

32

<210> 8

<211> 49

<212> DNA

<213> Artificial Sequence

<220>

<223> pCJ1-pitA-A-F

<400> 8

ttcgacctgactgaatgccggaggaaccgcgggcttattccattacatg 49

<210> 9

<211> 46

<212> DNA

<213> Artificial Sequence

<220>

<223> pCJ1-pitA-A-R

<400> 9

caaacaaatgtagcatttaatctcctagattgggtttcactcaagg 46

<210> 10

<211> 43

<212> DNA

<213> Artificial Sequence

<220>

<223> pCJ1-pitA-B-F

<400> 10

acccaatctaggagattaaatgctacatttgtttgctggcctg 43

<210> 11

<211> 43

<212> DNA

<213> Artificial Sequence

<220>

<223> pCJ1-pitA-B-R

<400> 11

cggaaggttgaaggcggattacaggaactgcaagggagagc cag 43

<210> 12

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223>fepBdel-check-F

<400> 12

gacctgactgaatgccggag g

21

<210> 13

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223>fepBdel-check-R

<400> 13

ggaaggttgaaggcggagtg g

21

<---

Claims (10)

1. The microorganism Corynebacterium stationis , which has the ability to produce purine nucleotides, in which the activity of the phosphate importer system encoded by the pit gene is enhanced, and where the enhancement of the activity of the phosphate importer system is an enhancement of the activity of the polypeptide encoded by the pitA gene. 2. The microorganism according to claim 1, wherein the pitA gene is a foreign pitA gene. 3. The microorganism according to item 2, wherein the foreign gene pitA is obtained from E. coli . 4. The microorganism of claim 1, wherein the polypeptide encoded by the pitA gene comprises a polypeptide having 99% or more identity with the amino acid sequence of SEQ ID NO: 1. 5. The microorganism of claim 1, wherein the purine nucleotide is any one or more selected from the group consisting of 5'-inosine monophosphate, 5'-xanthosine monophosphate, and 5'-guanosine monophosphate. 6. A method for obtaining a purine nucleotide, comprising culturing a microorganism according to any of paragraphs 1-5 in a medium. 7. The method according to claim 6, which further comprises isolating purine nucleotides from the cultured microorganism or medium. 8. The method of claim 6, wherein the purine nucleotide is any one or more selected from the group consisting of 5'-inosine monophosphate, 5'-xanthosine monophosphate, and 5'-guanosine monophosphate. 9. A composition for producing a purine nucleotide, comprising at least one material selected from a microorganism according to any one of claims 1-5 and a medium on which the microorganism according to any one of claims 1-5 is cultivated, and excipients. 10. Use of a microorganism according to any one of paragraphs 1-5 for producing a purine nucleotide.