RU2804010C1 - New variant of glutamine hydrolyzing gmp synthase and a method of producing purine nucleotide using it - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: following is proposed: a polypeptide involved in the production of a purine nucleotide selected from the group XMP (xanthosine monophosphate) or GMP (guanosine monophosphate), in which the amino acid corresponding to position 29 in the following SEQ ID NO: 3 is replaced by another amino acid selected from glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine and histidine. Also the following is proposed: a polynucleotide encoding the specified polypeptide, a microorganism for producing a purine nucleotide selected from the XMP or GMP group, containing the specified polypeptide or a polynucleotide encoding the specified polypeptide, a method for producing a purine nucleotide selected from the XMP or GMP group. Also the following is proposed: a composition for producing a purine nucleotide selected from the XMP or GMP group, and a method of increasing the ability of a microorganism to produce XMP or GMP.

EFFECT: invention makes it possible to obtain purine nucleotides with high efficiency.

8 cl, 9 tbl, 4 ex

Description

FIELD OF THE INVENTION

The present disclosure relates to a variant of glutamine hydrolyzing GMP synthase (guanosine monophosphate synthase) and a method for producing a purine nucleotide using it.

BACKGROUND ART

Purine nucleotides, which are a type of nucleic acid-based substances, are widely used for seasoning or food and have attracted attention as flavor-enhancing nucleic acid-based seasonings.

To obtain purine nucleotides, various studies have been carried out to develop microorganisms with highly efficient products and fermentation process technology. For example, target-specific approaches are mainly used, such as increasing the expression of genes encoding enzymes involved in the biosynthesis of purine nucleotides, or removing genes that are not necessary for biosynthesis (EP 3722430 A1 and US 2020-0347346 A1).

As the demand for purine nucleotides increases, research into the efficient production of purine nucleotides is still needed.

DESCRIPTION OF THE INVENTION

TECHNICAL PROBLEM

According to the present invention, a variant of glutamine hydrolyzing GMP synthase is provided.

TECHNICAL SOLUTION

One aspect of the present invention is to provide a variant of glutamine hydrolyzing GMP synthase.

Another aspect of the present invention is the provision of a polynucleotide encoding this variant.

Yet another aspect of the present invention is to provide a microorganism containing a variant of a polynucleotide encoding the variant.

Yet another aspect of the present invention is to provide a method for producing a purine nucleotide, comprising culturing the microorganism.

Beneficial effects

Variants of the present invention can be used to produce purine nucleotides with high efficiency.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will be specifically described as follows. Each description and embodiment disclosed in this invention may also apply to other descriptions and embodiments. That is, all combinations of different elements disclosed in this invention fall within the scope of the present invention. Moreover, the scope of the present invention is not limited to the specific description below. In addition, reference and citations are made to numerous articles and patent documents throughout this specification. The inventions of the cited articles and patent documents are incorporated herein in their entirety by reference, and the level of technical field within which the present disclosure falls and the details of the present invention are more clearly explained.

According to one aspect of the present invention, a variant of glutamine hydrolyzing GMP synthase is provided in which the amino acid corresponding to position 29 in SEQ ID NO: 3 is replaced by another amino acid.

The term “glutamine hydrolyzing GMP synthase variant” refers to a variant comprising the replacement of the amino acid corresponding to position 29 from the N terminus of SEQ ID NO: 3 with another amino acid in a polypeptide having glutamine hydrolyzing GMP synthase activity or in a glutamine hydrolyzing GMP synthase .

In the present invention, “glutamine hydrolyzing GMP synthase” is an enzyme involved in the conversion of 5'-xanthosine monophosphate (XMP) to 5'-guanosine monophosphate (hereinafter GMP).

The glutamine hydrolyzing GMP synthase of the present invention may be a glutamine hydrolyzing GMP synthase or a polypeptide having glutamine hydrolyzing GMP synthase activity, which are subject to modification to obtain the glutamine hydrolyzing GMP synthase variant of the present invention.

In particular, the polypeptide may be a naturally occurring polypeptide or a wild-type polypeptide, or a mature polypeptide thereof, and may include a variant or functional fragment thereof, but any polypeptide is included without limitation, provided that it may be the parent of a glutamine hydrolyzing variant GMP synthases according to the present invention.

In one embodiment, the glutamine hydrolyzing GMP synthase of the present invention may be derived from, but is not limited to, the genus Corynebacterium and, more specifically, Corynebacterium stationis.

In one embodiment, the glutamine hydrolyzing GMP synthase of the present invention may be a polypeptide of SEQ ID NO: 3. In one embodiment, the glutamine hydrolyzing GMP synthase of the present invention may be a polypeptide having at least about 60%, 70% new, 75%, 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98% or 99% similar sequence identity to the polypeptide of SEQ ID NO: 3, and includes any polypeptide that has activity identical to or corresponding to that of a polypeptide consisting of the amino acid sequence of SEQ ID NO: 3, without limitation, within the range of glutamine hydrolyzing GMP synthases. In particular, the glutamine hydrolyzing GMP synthase may have, contain, or essentially consist of the amino acid sequence set forth in SEQ ID NO: 3, but is not limited to it.

The glutamine hydrolyzing GMP synthase to be mutated in the present invention may be a glutamine hydrolyzing GMP synthase derived from Corynebacterium species, and in particular, a polypeptide/protein containing the amino acid sequence set forth in SEQ ID NO: 3, or any polypeptide /protein having the same activity as this synthesis may be included without limitation. For example, a given polypeptide/protein may comprise, but is not limited to, the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence having at least 80% homology or identity thereto. In particular, the polypeptide/protein may comprise an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology or identity with SEQ ID NO: 3. A polypeptide/protein having a deletion, modification, substitution or insertion in part of its sequence is obviously included in the present invention within the range of polypeptides/proteins subject to mutation, provided that it is a polypeptide/protein having such homology or identity and exhibiting glutamine hydrolyzing GMP synthase activity.

The sequence of the glutamine hydrolyzing GMP synthase of the present invention may be available in NCBI (National Center for Biotechnology Information) GenBank, a well-known database. For example, the glutamine hydrolyzing GMP synthase of the present invention may be a polypeptide encoded by the guaA gene. For example, the glutamine hydrolyzing GMP synthase of the present invention may be derived from Corynebacterium stationis, Corynebacterium casei, or Corynebacterium ammoniagenes, and examples thereof may be, but are not limited to, the enzyme expressed by the NCBI reference sequence WP_194285183.1, WP_006823296.1, or WP_040355890.1 .

The term "homology" or "identity" as used herein refers to the degree of similarity 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.

Homology or sequence identity of conserved polynucleotides or polypeptides can be determined by a standard alignment algorithm, and default gap penalties set by the program can be shared. Substantially homologous or identical sequences can generally hybridize to each other in whole or in part under moderate to highly stringent conditions. It will be appreciated that hybridization also includes hybridization with a polynucleotide containing regular codons or codons that take into account the degeneracy of codons in the polynucleotide.

Homology, similarity or identity between any two polynucleotide or polypeptide sequences can be determined using known computer algorithms, such as the FASTA program, using default parameters as in Pearson et al., (1988) Proc. Natl. Acad. Sci. USA 85: 2444. Alternatively, homology, similarity or identity can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-4153), which is implemented in the Needleman program of the European package Molecular Biology Open Software Suite (EMBOSS) (including Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later) (GCG software package (Devereux, J., et at, Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, S.F., et at, JMOLEC BIOL 215:403 (1990); Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994; and CARILLO et at (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 database.

Homology, similarity or identity between polynucleotides or polypeptides can be determined by comparing sequence information using the GAP computer program, such as those introduced by Needleman et at (1970), J Mol Biol. 48: 443, as disclosed by Smith and Waterman, Adv. Appl. Math (1981) 2: 482. Briefly, the GAP program defines homology, similarity, or identity as the value obtained by dividing the number of similar aligned characters (i.e., nucleotides or amino acids) by the total number of characters in the shorter of two sequences. Default parameters for the GAP program may include: (1) a binary comparison matrix (containing a value of 1 for identity and 0 for non-identity) and a weighted comparison matrix Gribskov et al., (1986) Nucl. Acids Res. 14: 6745, as disclosed in Schwartz and Dayhofr", eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979) (or EDNAFULL substitution matrix (EMBOSS NCBI version NUC4.4)); (2) a 3.0 penalty for each gap and an additional 0.10 penalty for each character in each gap (or a 10 penalty for opening a gap and a 0.5 penalty for extending a gap); and (3) no penalty for ending gaps.

The term “variant” as used herein refers to a polypeptide that has a different sequence but retains functions or properties comparable to the amino acid sequence before the mutation through conservative substitution and/or modification of at least one amino acid. Such a variant can typically be identified by modifying one or more amino acids in the amino acid sequence of a given polypeptide and evaluating the properties of the modified polypeptide. That is, the ability of a given variant may be increased, remain unchanged, or decreased compared to the ability of the polypeptide before the mutation. Some variants may include a variant in which at least one portion, such as an N-terminal leader sequence or transmembrane domain, is deleted. Other options may include one in which part of the N-terminus and/or C-terminus of the mature protein is removed. The term "variant" may also be used interchangeably with "modification", "modified protein", "modified polypeptide", "mutant", "mutein", "divergent" or the like, and any term that is used in the sense of mutation may be used without its limitation.

In addition, this variant may include deletions or insertions of amino acids that have minimal impact on the properties and secondary structures of the polypeptide. For example, a signal (or leader) sequence of the N-terminus of a protein may be conjugated to the N-terminus of a given variant, which co-translationally or post-translationally directs the transfer of the protein. In addition, a given variant may also be conjugated to another sequence or linker such that the variant is identified, purified or synthesized.

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

This “other amino acid” is not limited, provided that the amino acid is different from the amino acid before the substitution. The expression “replaced by a particular amino acid” in the present invention is understood to mean a replacement with a different amino acid from the amino acid before the replacement, even if “replacement with a different amino acid” is not specifically stated.

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

In one embodiment, the glutamine hydrolyzing GMP synthase variant of the present invention may comprise an amino acid sequence in which the amino acid corresponding to position 29 in the amino acid sequence set forth in SEQ ID NO: 3 is an amino acid other than arginine, and which has at least 70 %, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99 .5%, 99.7% or 99.8% homology or identity with the amino acid sequence set forth in SEQ ID NO: 3.

For example, a variant glutamine hydrolyzing GMP synthase of the present invention may consist of the amino acid sequence of SEQ ID NO: 1 or may contain an amino acid sequence having at least 70%, 75%, 80%, 85% , 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.8% a new identity with it, but not limited to it.

It is clear that even a variant having an amino acid sequence with a deletion, modification, substitution, conservative substitution or insertion in part thereof may also be included within the scope of the present invention, provided that the amino acid sequence has such homology or identity and exhibits activity , corresponding to an embodiment of the present invention.

For example, there may be an insertion or deletion of a sequence, a naturally occurring mutation, a silent mutation, or a conservative substitution that does not alter the function of a variant of the present invention, at the N-terminus, C-terminus, and/or within a given amino acid sequence.

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 can generally occur based on similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or amphipathic nature of the residues. Typically, a conservative substitution may have little or no effect on the activity of the protein or polypeptide.

The term “corresponding” as used herein refers to an amino acid residue at a position listed in a polypeptide, or an amino acid residue similar, identical or homologous to a residue listed in a polypeptide. Identification of an amino acid at a corresponding position may constitute identification of a particular amino acid of a sequence related to a particular sequence. The term "corresponding region" as used herein generally refers to a similar or corresponding site in a related protein or reference protein.

For example, any amino acid sequence is aligned to SEQ ID NO: 3, and based on this, each amino acid residue of that amino acid sequence can be numbered relative to the numerical position of the amino acid residue corresponding to the amino acid residue of SEQ ID NO: 3. For example, a sequence alignment algorithm as described in of the present invention, can identify the position of an amino acid or the position of occurrence of modifications, such as substitution, insertion or deletion, compared to a query sequence (also referred to as a “reference sequence”).

For such alignment, for example, one can use the Needleman Wunsch algorithm (Needleman and Wunsch, 1970, J. Mot Biol. 48: 443-453) or the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), but not limited to, a sequence alignment program, a pairwise sequence comparison algorithm, or the like, which are known in the art, can be used, as appropriate.

According to another aspect of the present invention, a polynucleotide encoding a variant of the present invention is provided.

The term "polynucleotide" as used herein refers to a strand of DNA or RNA having more than a certain length, in the form of a nucleotide polymer, which is a long chain of nucleotide monomers linked by covalent bonds.

For example, a polynucleotide encoding a glutamine hydrolyzing GMP synthase of the present invention may have or contain a sequence encoding the nucleotide sequence of SEQ ID NO: 4. For example, a polynucleotide encoding a glutamine hydrolyzing GMP synthase may consist of or substantially consist of the sequence SEQ ID NO: 4. NO: 4.

The polynucleotide of the present invention may be variously modified in its coding region within the range in which the amino acid sequence of the variant of the present invention is not changed, taking into account codon degeneracy or codon preferences of the organism in which the variant of the present invention is to be expressed.

In particular, a polynucleotide encoding a glutamine hydrolyzing GMP synthase variant of the present invention may have or contain a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99 % and less than 100% homology or identity to the sequence of SEQ ID NO: 4, or may consist of or substantially consists of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least at least 98% and less than 100% homology or identity with, but not limited to, the sequence of SEQ ID NO: 4. In sequences having such homology or identity, the codon encoding the amino acid corresponding to position 29 in SEQ ID NO: 3 may be one of the codons encoding any amino acids other than arginine.

In addition, the polynucleotide of the present invention may include, without limitation, a sequence that can hybridize under stringent conditions with a probe that can be derived from a known gene sequence, for example, a sequence complementary to or the entire sequence of the polynucleotide of the present invention. The term "stringent conditions" refers to conditions that ensure specific hybridization between polynucleotides. These conditions are described in detail in the literature (see J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989 and F. M. Ausubel et at, Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50-9.51, 11.7-11.8). For example, such conditions may include conditions in which polynucleotides having high homology or identity, such as polynucleotides having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% - high homology or identity between them hybridize with each other, but polynucleotides having less homology or identity do not hybridize with each other; or wash conditions for a typical Southern hybridization, in which the wash is carried out at a salt concentration and temperature corresponding to 60°C, 1xSSC (salt citrate), 0.1% SDS (sodium dodecyl sulfate); specifically 60×C, 0.1×SSC, 0.1% SDS; and more particularly 68°C, 0.1×SSC, 0.1% SDS, once, specifically twice or three times.

Hybridization requires that the two nucleic acids have complementary sequences, although mismatches between bases may be possible, depending on the stringency of hybridization. The term "complementarity" is used to describe the relationship between nucleotide bases that can hybridize with each other. For example, for DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Therefore, the polynucleotide of the present invention may also include isolated nucleic acid fragments complementary to the entire sequence, as well as substantially similar nucleic acid sequences.

In particular, a polynucleotide having homology or identical to the polynucleotide of the present invention can be detected using hybridization conditions including a hybridization step at a T _m (melting temperature) of 55° C. and using the conditions described above. In addition, the T _m value may be 60°C, 63°C or 65°C, but not limited to, and can be suitably controlled by one skilled in the art according to the purpose.

The appropriate stringency for polynucleotide hybridization depends on the length of the polynucleotides and the degree of complementarity, and its variables are well known in the art (eg, Sambrook et al., supra).

According to yet another aspect of the present invention, there is provided a vector comprising a polynucleotide encoding a variant of the present invention. The vector may be, but is not limited to, an expression vector for expressing a polynucleotide in a host cell.

A “vector” of the present invention may include a DNA construct containing the nucleotide sequence of a polynucleotide encoding a target polypeptide operably linked to a suitable expression control region (or expression control sequence) so as to express the target polypeptide in a suitable host. This expression control region may include a promoter capable of initiating transcription, any operator sequence for controlling such transcription, a sequence for encoding a suitable ribosome binding site for the mRNA, and a sequence for controlling termination of transcription and translation. This vector, after transformation into a suitable host cell, can replicate or function independently of the host genome or can itself integrate into the host genome.

The vector used in the present invention is not particularly limited, and any vector known in the art can be used. Examples of a vector that is commonly used may 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 vectors or cosmid vectors, and pBR-based, pUC-based, pBluescript II based, pGEM based, pTZ based, pCL based and pET based. In particular, the vectors pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118 and pCC1BAC can be used.

For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome via a chromosomal insertion vector in a cell. Insertion of a given polynucleotide into a chromosome can be accomplished using any method known in the art, such as, but not limited to, homologous recombination. The vector may further include a selectable marker to test for chromosomal integration or non-integration. This selectable marker serves to select cells transformed with the vector, that is, to identify the insertion of a target nucleic acid molecule, and markers that confer a selectable phenotype, such as drug resistance, auxotrophy, resistance to cytotoxic agents, or surface protein expression, can be used. Under the circumstances of treatment with a selective agent, only cells expressing the selective markers may survive or exhibit other phenotypic characteristics, so that transformed cells can be selected.

The term "transformation" as used herein means that a vector containing a polynucleotide encoding a target polypeptide is introduced into a host cell or microorganism to cause the host cell to express the polypeptide encoded by the polynucleotide. Examples of the transformed polynucleotide may include any polypeptide as long as it can be expressed in a host cell, whether it is inserted and located on the chromosome of the host cell or located extrachromosomally. Further, examples of the polynucleotide include DNA and/or RNA encoding a target protein. The polynucleotide can be introduced in any form, provided that the polynucleotide can be introduced into a host cell and can be expressed. For example, a given polynucleotide can be introduced as an expression cassette, which is a genetic construct containing all the factors required for autonomous expression. The expression cassette typically may include a promoter operably linked to the polynucleotide, a transcription termination signal, a ribosome binding site, and a translation termination signal. The expression cassette may be an expression vector capable of autonomous replication. In addition, the polynucleotide may be introduced into a host cell as is to bind in a functional manner to, but not limited to, a sequence required for expression in the host cell.

As used above, the term “functionally linked” refers to a functional relationship between a promoter sequence for initiating and mediating transcription of a polynucleotide encoding a variant of the present invention and the sequence of that polynucleotide.

According to yet another aspect of the present invention, a microorganism containing a variant of the present invention or a polynucleotide encoding the specified variant is provided.

The microorganism of the present invention may contain a variant of the present invention, a polynucleotide encoding the specified variant, or a vector including the specified polynucleotide.

The term "microorganism (or strain)" as used herein, covers all wild type microorganisms or naturally or artificially genetically modified microorganisms, refers to a microorganism in which a specific mechanism is weakened or enhanced due to the insertion of an exogenous gene or enhancement of activity , or inactivation of an endogenous gene, and may be a microorganism that includes genetic modification to produce a target polypeptide, protein or product.

The microorganism of the present invention may be a microorganism comprising at least one variant of the present invention, a polynucleotide encoding the variant of the present invention, and a vector including a polynucleotide encoding the variant of the present invention; a microorganism modified to express a variant of the present invention or a polynucleotide encoding a variant of the present invention; a microorganism (eg, a recombinant strain) expressing a variant of the present invention or a polynucleotide encoding a variant of the present invention; or a microorganism (eg, a recombinant strain) having the activity of a variant of the present invention, but is not limited to them.

The microorganisms of the present invention may have the ability to produce purine nucleotides.

The microorganism of the present invention may be a microorganism having the natural ability to produce glutamine hydrolyzing GMP synthase or purine nucleotides, or a microorganism obtained by introducing a variant of the present invention or a polynucleotide encoding it (or a vector comprising the polynucleotide) into a parent strain without the ability to producing glutamine hydrolyzing GMP synthase or purine nucleotide, and/or conferring, but not limited to, the ability of the parent strain to produce purine nucleotide. For example, the microorganism of the present invention may be a microorganism having the ability to produce a purine nucleotide conferred by the introduction of a variant of the glutamine hydrolyzing GMP synthase of the present invention. For example, the microorganism of the present invention may be a microorganism having a purine nucleotide producing ability enhanced by the introduction of a glutamine hydrolyzing GMP synthase variant of the present invention. However, the microorganism of the present invention is not limited to this.

For example, a strain of the present invention is a cell or microorganism transformed with a vector comprising a polynucleotide of the present invention or a polynucleotide encoding a variant of the present invention, and thus expresses a variant of the present invention, and, for purposes of the present invention, a strain of the present invention The invention covers all microorganisms that can produce purine nucleotide by including the variant of the present invention. For example, the strain of the present invention may be a recombinant strain having an enhanced ability to produce purine nucleotide by expressing a variant of glutamine hydrolyzing GMP synthase by introducing a polynucleotide encoding the variant of the present invention into a naturally occurring wild-type microorganism or a purine nucleotide-producing microorganism. This recombinant strain has an increased ability to produce purine nucleotide compared to a natural wild-type microorganism or a microorganism not modified with glutamine hydrolyzing GMP synthase (i.e., a microorganism expressing wild-type glutamine hydrolyzing GMP synthase (SEQ ID NO: 3), or a microorganism that does not express the modified protein), but is not limited to this.

The term "unmodified microorganism" as used herein may refer to a wild-type or natural strain as it occurs in nature, or to a strain before transformation due to genetic mutation caused by natural or man-made factors , not excluding strains that include mutations that can occur in microorganisms in nature. For example, the unmodified microorganism may be a strain into which the glutamine hydrolyzing GMP synthase variant described herein has not been introduced, or before its introduction. The term "unmodified microorganism" may be used interchangeably with the terms "strain before modification", "microorganism before modification", "unmodified strain", "unmodified strain", "unmodified microorganism" or "reference microorganism".

In one embodiment, the microorganism of the present invention may be a microorganism of the genus Corynebacterium. For example, the microorganism of the present invention may be Corynebacterium stationis, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium glutamicum, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans , Corynebacterium testudinoris or Corynebacterium flavescens . In particular, the microorganism of the present invention may be, but is not limited to, Corynebacterium stationis.

The term "purine nucleotide" as used herein refers to a nucleotide containing a purine-based structure. The purine nucleotide may be XMP (xanthosine monophosphate), IMP (inosine monophosphate), GMP (guanosine monophosphate) or AMP (adenosine monophosphate) and in particular IMP, XMP or GMP. More specifically, the purine nucleotide may be, but is not limited to, XMP or GMP.

"Inosine 5'-monophosphate or 5'-inosinic acid (IMP)" and "guanosine 5'-monophosphate or 5'-guanylic acid (GMP)" of the present invention, which are intermediates in metabolic systems of nucleic acid biosynthesis, have a physiologically important role in the body and are widely used in foods, pharmaceuticals and the like. In particular, IMP itself is known to impart a beef flavor, and GMP derived from CMP is known to impart a mushroom flavor. Both substances are known to enhance the flavor of monosodium glutamate (MSG) and have thus received considerable attention as nucleic acid flavor-enhancing seasonings.

In one embodiment, GMP can be obtained by conversion from, but not limited to, CMP. For example, the level of GMP production can be increased by converting overproduced XMP to GMP, and thus GMP is also included within the scope of the present invention.

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

The term "purine nucleotide-producing microorganism" as used herein includes all wild-type microorganisms or naturally or artificially genetically modified microorganisms and refers to a microorganism that has a specific mechanism weakened or enhanced due to the insertion of an exogenous gene or enhancement or inactivation of the activity of an endogenous gene, where the microorganism may have a genetic mutation or increased activity in relation to the production of the target purine nucleotide.

The purine nucleotide producing microorganism of the present invention may retain an increased ability to produce the target purine nucleotide by containing a variant of the glutamine hydrolyzing GMP synthase of the present invention.

In one embodiment, the purine nucleotide-producing microorganism, or the microorganism having the ability to produce purine nucleotide, in the present invention may be a microorganism in which some of the genes involved in the biosynthesis of purine nucleotides are enhanced or weakened, or some of the genes involved in Purine nucleotide degradation pathways are enhanced or weakened.

The term "reducing" the activity of a polypeptide, as used herein, has the idea of a reduction in activity or lack of activity compared to endogenous activity. The term "decrease" may be used interchangeably with the terms "inactivation", "deficiency", "down-regulation", "reduction", "fall", "attenuation" or the like.

Reduction may also include: a case where the activity of the polypeptide itself is reduced or eliminated compared to the activity of the polypeptide possessed by the original microorganism due to mutation of the polynucleotide encoding the polypeptide or the like; a case where the activity and/or concentration (expression level) of all polypeptides in the cell is lower compared to the natural strain due to inhibition of expression of the gene of the polynucleotide encoding the polypeptide, or inhibition of translation into the polypeptide encoding the polynucleotide, or inhibition of translation into this polypeptide; a case where the expression of a given polynucleotide is not carried out; and/or a case where the polypeptide has no activity despite expression of the polynucleotide. The term "intrinsic activity" refers to the activity of a particular polypeptide originally possessed by the parent strain before modification or by a wild-type microorganism or an unmodified microorganism when the strain or microorganism is transformed by a genetic mutation caused by natural or artificial factors. This term can be used interchangeably with the phrase "activity before modification". The terms "inactivation, deficiency, reduction, down-regulation, reduction or attenuation" of the activity of a polypeptide relative to its own activity means that the activity of the polypeptide is reduced compared to the activity of the particular polypeptide originally possessed by the parent strain before transformation or by the unmodified microorganism.

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

In particular, reducing the activity of the polypeptide of the present invention can be achieved by:

1) deletion of part or all of the gene encoding this polypeptide;

2) modification of the expression control region (or expression control sequence) to reduce expression of the gene encoding the polypeptide;

3) modification of the amino acid sequence constituting the polypeptide to eliminate or reduce the activity of the polypeptide (eg, deletion/replacement/insertion of at least one amino acid in the amino acid sequence);

4) modification of the sequence of the gene encoding the given polynucleotide to eliminate or reduce the activity of the given polypeptide (for example, deletion/replacement/insertion of at least one nucleotide in the nucleotide sequence of the gene of the given polypeptide to encode a polypeptide modified to eliminate or reduce the activity of the given polypeptide);

5) modification of the nucleotide sequence encoding the start codon of the 5'-UTR (untranslated) region of the transcript of the gene encoding this polypeptide;

6) introducing an antisense oligonucleotide (for example, antisense RNA) that complementarily binds to the transcript of the gene encoding the given polypeptide;

7) adding a sequence complementary to the Shine Dalgarno sequence of the gene encoding the polypeptide above the Shine Dalgarno sequence with the formation of a secondary structure that makes it impossible for ribosomes to attach;

8) adding a promoter that is subject to reverse transcription to the 3' end of the open reading frame (ORF) sequence of the gene encoding the polypeptide (reverse transcription engineering (RTE); or

9) combinations of two or more than two selected from, but not specifically limited to, paragraphs (1)-(8).

For example, they are described as follows.

Deletion of part or all of the gene encoding the polypeptide in paragraph (1) may be the elimination of the entire polynucleotide encoding the endogenous target protein in the chromosome, replacement with a polynucleotide with deletion of some nucleotides, or replacement with a marker gene.

The modification of the expression control region (or expression control sequence) in (2) may be a mutation of the expression control region (or expression control sequence) by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, or substitution with a sequence having lower activity. The expression control region includes, but is not limited to, a promoter, an operator sequence, a sequence for encoding a ribosome binding site, and a sequence for controlling transcription and translation termination.

Modification of the amino acid sequence or polynucleotide sequence according to claims. (3) and (4) there may be a sequence modification by deletion, insertion, non-conservative or conservative substitution, or combination thereof in the amino acid sequence of the polypeptide or polynucleotide sequence encoding the polypeptide, or substitution with an amino acid sequence or polynucleotide sequence modified so as to have a more reduced activity, or an amino acid sequence, or a polynucleotide sequence improved to have no activity, so as to reduce the activity of a given polypeptide. For example, the expression of a given gene can be inhibited or attenuated by introducing a mutation into a given polynucleotide sequence to form a termination codon.

Modification of the nucleotide sequence encoding the start codon or 5'-UTR region of the transcript of the gene encoding the polypeptide of claim (5) may be, for example, the replacement of the encoding nucleotide sequence, rather than the endogenous start codon, with another start codon having a lower rate of expression polypeptide, but not limited to them.

The introduction of an antisense oligonucleotide (for example, antisense RNA) complementarily binding to the gene transcript encoding the polypeptide according to claim (6), for example, can be cited in the literature (Weintraub, H. et al., Antisense-RNA as a molecular tool for genetic analysis, Reviews - Trends in Genetics, Vol. 1(1) 1986].

The addition of a sequence complementary to the Shine-Dalgarno sequence of the gene encoding a given polypeptide to the upstream Shine-Dalgarno sequence so as to form a secondary structure that prevents the attachment of ribosomes at (7) may prevent translation of the mRNA or reduce its rate .

The addition of a promoter that is subject to reverse transcription to the 3' end of the open reading frame (ORF) of the gene encoding the polypeptide (reverse transcription engineering (RTE)) in paragraph (8) can make the antisense nucleotide complementary to the transcript of the gene encoding the polypeptide. polypeptide, thereby reducing the activity of the polypeptide.

The term "enhancing" the activity of a polypeptide, as used herein, refers to increasing the activity of a given polypeptide compared to its own activity. The term "enhancement" may be used interchangeably with "activation", "upregulation", "overexpression", "increase" or the like. Here, “activation,” “enhancement,” “upregulation,” “overexpression,” and “increase” may include all of exhibiting activity not originally possessed or exhibiting increased activity relative to intrinsic activity or activity before modification. The term "intrinsic activity" refers to the activity of a particular polypeptide originally possessed by the parent strain before its transformation, or by an unmodified microorganism when that strain or microorganism is transformed by a genetic mutation caused by natural or artificial factors. This term can be used interchangeably with "activity before modification". “Amplification,” “upregulation,” “overexpression,” or “increase” in the activity of a polypeptide relative to its intrinsic activity means an improvement in the activity and/or concentration (level of expression) of a particular polypeptide originally possessed by the pre-transformation parent strain or unmodified microorganism.

This enhancement can be achieved by introducing an exogenous polypeptide or increasing the activity and/or concentration (expression level) of an endogenous polypeptide. Increased activity of a given polypeptide can be identified by an increase in the activity level or expression level of the corresponding polypeptide or the amount of product produced from the corresponding polypeptide.

Enhancement of the activity of a given polypeptide can be achieved using various methods well known in the art, and is not limited to, provided that the method can increase the activity of the target polypeptide compared to the microorganism before transformation. In particular, genetic engineering and/or protein engineering well known to one skilled in the art, which is a traditional method of molecular biology, can be used (for example, Sitnicka et al. Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol. 2. 1-16, Sambrook et al. Molecular Cloning 2012 or the like).

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

1) increasing the number of intracellular copies of the polynucleotide encoding this polypeptide;

2) replacing the gene expression control region on the chromosome encoding the given polypeptide with a sequence having higher activity;

3) modification of the nucleotide sequence encoding the start codon or 5'-UTR region of the transcript of the gene encoding this polypeptide;

4) modification of the amino acid sequence of the polypeptide to enhance the activity of this polypeptide;

5) modification of a polynucleotide sequence encoding a polypeptide to enhance the activity of a given polypeptide (for example, modification of a polynucleotide sequence of a polypeptide gene to encode a modified polypeptide to enhance the activity of a given polypeptide);

6) introduction of an exogenous polypeptide demonstrating the activity of this polypeptide, or an exogenous polynucleotide encoding it;

7) optimization of codons of the polynucleotide encoding this polypeptide;

8) modification or chemical modification of the exposed site selected by analysis of the tertiary structure of the polypeptide; or

9) combinations of two or more than two selected from paragraphs. (1) - (8), but not specifically limited to them.

More specifically, polypeptide enhancement is described as follows.

Increasing the number of intracellular copies of a polynucleotide encoding a polypeptide according to claim (1) can be achieved by introducing into a host cell a vector in which the polynucleotide encoding the corresponding polypeptide is functionally linked and which can replicate and function independently of the host. Alternatively, the enhancement of claim (1) may be achieved by introducing one or more copies of the polynucleotide encoding the corresponding polypeptide into a chromosome in the host cell. Introduction into a chromosome can be accomplished by introducing into a host cell a vector capable of inserting a given polynucleotide into a chromosome in the host cell. This polynucleotide is as described above.

Replacement of a gene expression control region on a chromosome encoding a polypeptide with a sequence having the potent activity of claim (2), for example, may be a modification in the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, or substitution with a sequence having a stronger activity, to further enhance the activity of the expression control region. This expression control region may include, but is not particularly limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, a transcription and translation termination control sequence, and the like.

For example, it may involve, but is not limited to, replacing the original promoter with a stronger promoter.

Examples of known stronger promoter may be 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 promoter ( sm3) (US 10584338 B2), O2 promoter (US 10273491 B2), tkt promoter, USE A promoter and the like, but not limited to.

Modification of the nucleotide sequence encoding the start codon or the 5'-UTR region of the transcript of the gene encoding the polypeptide of claim 3) may be, for example, the replacement of the encoding nucleotide sequence, rather than the endogenous start codon, with another start codon having a higher rate of expression polypeptide, but is not limited to them.

Modification of the amino acid sequence or polynucleotide sequence in claims. (4) and (5) may be a sequence modification by deletion, insertion, non-conservative or conservative substitution, or combination thereof, in the amino acid sequence of a polypeptide or polynucleotide sequence encoding the polypeptide, or substitution with an amino acid sequence or polynucleotide sequence modified to have stronger activity, or an amino acid sequence or polynucleotide sequence modified to have increased activity to enhance the activity of the polypeptide. In particular, this replacement can be carried out by the insertion of a polynucleotide into the chromosome through homologous recombination, but is not limited to it. The vector used here may further include a selectable marker to verify insertion into the chromosome. This selectable marker is as described above.

The introduction of an exogenous polypeptide that exhibits the activity of the polypeptide in claim (6) may be the introduction into a host cell of an exogenous polynucleotide encoding a polypeptide that exhibits the same/similar activity to the polypeptide. The exogenous polynucleotide is not limited in its origin or sequence, as long as the exogenous polynucleotide exhibits the same/similar activity to the polynucleotide. Administration can be carried out by any known transformation method suitably selected by one skilled in the art, and by expressing the introduced polynucleotide in the host cell, the polypeptide is produced and its activity can be increased.

The codon optimization of the polynucleotide encoding the polypeptide in (7) may be codon optimization of an endogenous polynucleotide to enhance its transcription or translation in the host cell, or codon optimization of an exogenous polynucleotide to ensure its optimized transcription or translation in the host cell.

Modification or chemical modification of the exposed site selected by analysis of the tertiary structure of the polypeptide in paragraph (8) may be, for example, modification or chemical modification of the exposed site to be modified or chemically modified by comparing the sequence information of the polynucleotide to be analyzed with the base data, which stores sequence information of existing proteins, identifying a template candidate protein according to sequence similarity and identifying a structure based on it.

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

Modification of part or all of a polynucleotide in a microorganism of the present invention can be induced by the following: (a) genome editing using homologous recombination or a genetically modified nuclease (eg, CRISPR-Cas9) using a vector for chromosomal insertion into the microorganism and/or (b) treatment with light such as, but not limited to, ultraviolet light and radiation, and/or chemicals. The method of modifying part or all of a gene may include a genetic engineering method. For example, a nucleotide sequence or vector containing a nucleotide sequence homologous to a target gene can be introduced into a microorganism to effect homologous recombination, resulting in the deletion of part or all of the gene. The nucleotide sequence or vector to be introduced may include, but is not limited to, a dominant selection marker.

According to yet another aspect of the present invention, there is provided a method for producing a purine nucleotide, comprising cultivating a microorganism comprising a variant of the present invention or a polynucleotide encoding the variant.

The method for producing a purine nucleotide of the present invention may include culturing in a medium a microorganism containing a variant of the present invention, a polynucleotide encoding it, or a vector comprising said polynucleotide.

The term "cultivation" as used herein refers to growing the microorganism of the present invention under suitably adjusted environmental conditions. The culture method of the present invention can be carried out according to suitable media or culture conditions known in the art. Such cultivation can easily be adjusted according to the selected strain by one skilled in the art. In particular, this cultivation may be, but is not limited to, batch type, continuous type and/or fed-batch type.

The term "medium" as used herein refers to a mixture containing, as main ingredients, nutrients required for the cultivation of a microorganism, wherein the medium supplies nutrients, growth factors and the like, including water, which are important for survival and growth. In particular, with regard to the medium and other culture conditions used for cultivating the microorganism of the present invention, any medium that is used for conventional cultivation of microorganisms can be used without particular limitation. However, the microorganisms of the present invention can be cultivated under aerobic conditions in a conventional medium containing suitable carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids and/or vitamins, while temperature, pH and the like are adjusted.

In particular, the culture medium for a strain 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)).

In the present invention, the carbon source may include carbohydrates such as glucose, sucrose, lactose, fructose, sugar 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. In addition, natural sources of organic nutrients such as starch hydrolysates, molasses, raw molasses, rice bran, cassava, sugar cane bagasse and liquid corn extract can be used, and in particular carbohydrates such as glucose and sterile pre-processed molasses can be used (ie molasses converted to reducing sugars), and suitable amounts of other carbon sources can be used without limitation. These carbon sources may be used alone or in combination of two or more, but are not limited to.

In terms of nitrogen sources, inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate and ammonium nitrate can be used; amino acids such as glutamic acid, methionine and glutamine; and organic nitrogen sources such as peptone, NZ-amine, meat extracts, yeast extracts, malt extracts, liquid corn extract, casein hydrolysates, fish or its degradation products, defatted soybean meal or its degradation products, and the like. These nitrogen sources may be used alone or in combination of two or more of them, but are not limited to them.

Examples of phosphate sources may include monobasic potassium phosphate, dibasic potassium phosphate, and their corresponding sodium salts. With regard to inorganic compounds, sodium chloride, calcium chloride, ferric chloride, magnesium sulfate, ferrous sulfate, manganese sulfate, calcium carbonate and the like can be used, and in addition amino acids, vitamins and/or suitable precursors can be included. These constituent ingredients or precursors can be added to the medium in batches or in a continuous manner. However, the present disclosure is not limited to them.

The pH of the medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid to the medium in a suitable manner during the cultivation of the microorganism of the present invention. In addition, an antifoam agent such as a polyglycol fatty acid ester may be added to suppress foam formation during culture. In addition, oxygen or an oxygen-containing gas may be injected into the medium to maintain an aerobic state of the medium, or nitrogen, hydrogen or carbon dioxide gas may be injected, or no gas may be injected to maintain an anaerobic or non-aerobic state of the medium, but is not limited to.

In the cultivation of the present invention, the culture temperature can be maintained at 20° C. to 45° C., in particular 25° C. to 40° C., and the culture can be carried out for about, but not limited to, 10 hours to 160 hours.

The purine nucleotide produced by the cultivation of the present invention can be released into the medium or can be stored in cells without being released.

The method for producing a purine nucleotide of the present invention may further include obtaining a microorganism of the present invention, preparing a medium for culturing the microorganism, or a combination of these steps (regardless of the order, in any order), for example, before or after the culture step.

The method for producing a purine nucleotide of the present invention may further include isolating the purine nucleotide from a culture medium (culture medium) or from a microorganism. An isolation step may further be included after the culture step.

The isolation may be the selection of the desired purine nucleotide using a suitable method known in the art, according to the microorganism culture method of the present invention, for example, batch, continuous or fed-batch culture. For example, centrifugation, filtration, treatment with a crystallized protein precipitating agent (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 can be used. chromatography, HPLC (high performance liquid chromatography) and a combination of these methods, and the desired purine nucleotide can be isolated from the environment or microorganism using a suitable method known in the art.

The method for producing a purine nucleotide of the present invention may further include a purification step. Purification can be carried out using a suitable method known in the art. In a typical embodiment, when the purine nucleotide production method of the present invention includes both an isolation step and a purification step, the isolation step and the purification step can be carried out continuously or intermittently, regardless of the order, or can be carried out simultaneously or integrated into one step, but is not limited to .

In a typical embodiment, the method for producing GMP of the present invention may further comprise converting XMP to GMP. In the GMP production method of the present invention, a conversion step may be further included after the cultivation step or the conversion step. This conversion step can be carried out using a suitable method known in the art. For example, the conversion may be carried out using, but is not limited to, corynebacterium, E. coli, or 5'-xanthyl acid aminase (KR 10-0655902 B1, US 2013-0095529 A1 or the like).

In the method of the present invention, the variant, polynucleotide, vector, strain, purine nucleotide and the like are as described in other aspects above.

Yet another aspect of the present invention is to provide a composition for producing a purine nucleotide comprising a variant of the present invention, a polynucleotide encoding the variant, a microorganism including a vector comprising the polynucleotide, or a polynucleotide of the present invention; medium obtained by cultivating this microorganism; or its compositions.

The composition of the present invention may further contain any suitable excipient that is typically used in the composition to produce a purine nucleotide, and examples of this excipient may include, but are not limited to, a preservative, humectant, dispersant, suspending agent, buffer, stabilizer or isotonic agent.

In the composition of the present invention, the variant, polynucleotide, vector, microorganism, medium, purine nucleotide and the like are as described in other aspects above.

Yet another aspect of the present invention is to provide a method of increasing the ability of a microorganism to produce a purine nucleotide, comprising modifying said microorganism to express a variant of the present invention.

In the method of the present invention, the variant, polynucleotide, vector, microorganism, medium, purine nucleotide and the like are as described in other aspects above.

According to yet another aspect of the present invention, the use of a variant of the present invention for the production of a purine nucleotide is provided.

According to yet another aspect of the present invention, the use of a variant of the present invention, a polynucleotide encoding said variant, or a microorganism comprising a vector containing said polynucleotide is provided.

The variant, polynucleotide, vector, microorganism, and the like are as described in other aspects above.

Method for carrying out the invention

Below, the present disclosure will be described in detail with reference to examples and experimental examples. However, these examples and test examples are provided to specifically illustrate the present invention, and the scope of the present invention is not limited to them.

Example 1: generation of a variant with a weakened guaA synthase

To detect the guaA variant for the production of XMP, a library of mutant guaA, the gene encoding this variant, was constructed.

Example 1-1: Preparation of a vector containing guaA

To generate the guaA mutant library, a recombinant vector containing guaA was first constructed. PCR (polymerase chain reaction) was performed using the chromosome of Corynebacterium stationis ATCC6872 as a template, along with primers SEQ ID NO: 5 and SEQ ID NO: 6, and the amplification product was cloned into the pCR2.1 vector using the TOPO cloning kit (Invitrogen) , and the resulting vector was named pCR-guaA.

Example 1-2: Construction of a guaA mutant library

A guaA mutant library was constructed using the pCR-guaA vector constructed in Example 1-1 as a template. This library was prepared using a reduced fidelity error-tolerant PCR kit (Clontech Diversify® Random PCR Mutagenesis Kit) and generated guaA mutant PCR products with different sequences by random mutagenesis using primers SEQ ID NO: 7 and 8 according to the manufacturer's manual. Amplification products were cloned using the TOPO cloning kit (Invitrogen), then transformed into E. coli DH5α and spread on solid LB medium (lysogeny medium) containing kanamycin (25 mg/L). Transformed E. coli colonies were collected to isolate plasmids, which were called pCR-guaA library.

Example 1-3: Constructed Library Evaluation and Strain Selection

The pCR-guaA library obtained in Example 1-2 was transformed into the wild-type Corynebacterium stationis strain ATCC6872 by electroporation, and then this strain was distributed on a nutrient medium containing 25 mg/l kanamycin, obtaining 5000 colonies of the strain into which the mutant was inserted gene. The corresponding colonies were named C.st ATCC6872_pCR_guaA(mt)1 to C.st ATCC6872_pCR_guaA(mt)5000.

- Nutrient medium: 1% peptone, 1% beef extract, 0.25% sodium chloride, 1% yeast extract, 150 mg/l adenine, 150 mg/l guanine, 2% agar, pH 7.2 (based on 1 l distilled water).

Each of the 5000 colonies obtained was autoclaved under pressure, inoculated in 100 μl of seed medium containing kanamycin (25 mg/l), cultured in a 96-deep well plate with shaking at 30°C, 1200 rpm for 24 hours using a shaker. for microplates (TAITAC) and then used as seed culture. 320 µl of autoclaved fermentation medium (240 µl main medium plus 80 µl separate sterilized medium) was dispensed into a 96-deep well plate and then inoculated with every 15 µl of seed culture, followed by shaking culture under the same conditions as above, in within 72 hours.

To analyze the amount of CMP produced in the culture, 640 μl of sterile water was dispensed into the culture after completion of culture, followed by centrifugation at 4000 rpm for 15 minutes, and then 3 μl of the supernatant was transferred to a 96-well UV (ultraviolet) plate, in which was dosed with 297 μl of distilled water. A microplate reader was then used to shake for 30 seconds, and a spectrophotometer was used to measure absorbance at 25°C and a wavelength of 260 nm. Ten colonies of mutant strains showing increased uptake compared to the wild-type strain were then selected. Other colonies showed similar or reduced uptake levels compared to controls.

Ten selected strains were repeatedly measured for uptake by the above method and examined for the level of CMP production, and one strain (C.st ATCC6872_pCR_guaA(mt)726) having a significantly increased ability to produce CMP was selected as compared to the wild type strain.

To confirm the correctness of the ultimately selected strain, fermentation titer analysis was performed.

The wild type Corynebacterium stationis strain ATCC6872 and the selected strain C.st ATCC6872_pCR_guaA(mt)726 were inoculated into 14 ml tubes containing 2.5 ml of the seed medium below and cultured with shaking at 170 rpm for 24 hours at 30° WITH. A 250 ml corner baffled flask containing 32 ml of the following production medium (24 ml basal medium plus 8 ml separate sterile medium) was inoculated with 0.7 ml of seed culture and cultured with shaking at 170 rpm for 75 hours at 30°C.

The compositions of inoculum medium, basal medium and separate sterile medium are as follows.

CMR seed medium for flask

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 liter of distilled water).

CMR production medium for flask (basic medium)

50 g/l glucose, 10 g/l magnesium sulfate, 3 g/l yeast extract, 100 mg/l calcium chloride, 20 mg/l ferrous sulfate, 10 mg/l magranz sulfate, 10 mg/l zinc sulfate, 0, 8 mg/l copper sulfate, 20 mg/l histidine, 15 mg/l cystine, 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 distilled water).

CMP production medium for flask (separate sterile medium)

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

The results of measuring the level of CMP production by the HPLC method after completion of cultivation are shown in Table 3.

As shown in Table 3, the concentration of CMP produced was 4.83 g/L in C.st ATCC6872_pCR guaA(mt)726 compared with the wild-type strain.

Example 1-4: Mutation Identification by Gene Sequencing

To identify the sequence of the guaA variant of the C.st strain ATCC6872_pCR_guaA(mt)726, this C.st strain ATCC6872_pCR_guaA(mt)726 was subjected to PCR using primers SEQ ID NO: 8 and 9, and sequencing. As a result of comparison with the guaA gene sequence of the wild type strain, it was identified that the C.st strain ATCC6872_pCR_guaA(mt)726 includes a mutation in which the amino acid at position 29 in the guaA gene is replaced from arginine to cysteine (the nucleotide at position 85 is replaced from c to t: SEQ ID NO: 2).

This identified sequence variant is shown in Table 4.

The following examples examined whether the guaA mutation had an effect on CMP production.

Example 2: Identifying the impact of the guaA variant

Example 2-1: Construction of a guaA Variant Expression Vector

To study the effect on the production of CMF variant (R29C; SEQ ID NO: 1), in which arginine at position 29 of the amino acid sequence of the guaA enzyme was replaced by cysteine, a vector was constructed to obtain its expression strain as follows using the plasmid pDCM2 (Korean publication No. 10- 2020-0136813) for gene insertion and replacement in the Corynebacterium chromosome.

PCR was performed using the Corynebacterium stationis ATCC6872 chromosome as a template, along with primer pairs SEQ ID NOs: 10 and 11, and SEQ ID NOs: 12 and 13. Solg™ Pfu-X DNA polymerase was used as the polymerase, and PCR was performed at following PCR amplification conditions: denaturation at 95°C for 5 minutes, 30 cycles of denaturation at 95°C for 30 seconds, annealing at 58°C for 30 seconds, polymerization at 72°C for 60 seconds and then reaction polymerization at 72°C for 5 minutes to thereby obtain each PCR product. The amplification product was mixed with the pDCM2 vector, obtained previously by digestion with the restriction enzyme SmaI, and cloned using the Gibson assembly method (DG Gibson et al., NATURE METHODS, Vol. 6 No. 5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix) to obtain a recombinant plasmid, which was named pDCM2-guaA(R29C). Cloning was carried out by mixing the Gibson assembly reagent and each of the gene fragments at the calculated number of moles, followed by preservation at 50°C for 1 hour.

Example 2-2: Preparation of a strain expressing the guaA variant

The vector pDCM2-guaA(R29C) constructed in the above example was transformed into Corynebacterium stationis strain ATCC6872 by electroporation, and strains having this vector inserted into the chromosome by recombination of homologous sequences were selected on medium containing 25 mg/L kanamycin. Selected primary strains were subjected to secondary crossing over to select a strain having a mutation introduced into the target gene. The introduction of the gene mutation into the final transformed strain was examined by PCR using the primer pair SEQ ID NO: 14 and SEQ ID NO: 15 and sequencing, and the confirmed strain was named C.st ATCC6872::guaA(R29C).

Example 2-3: Comparison of the CMP production ability of a strain expressing the guaA variant

The CMP production abilities of the C.st strain ATCC6872::guaA(R29C) obtained in Example 2-2 and the wild-type strain were examined by the fermentation titer evaluation method in Example 1-3. After completion of cultivation, the CMP production level was measured by HPLC, and the cultivation results are shown in Table 5.

This analysis was repeated three times, and the means of the assay results are shown in Table 5.

As shown in Table 5, the concentration of CMP produced in C.st strain ATCC6872::guaA(R29C) was 5.21 g/L.

This strain of C.st ATCC6872::guaA(R29C) was designated CN02-2299 and was deposited at the Korea Microbial Conservation Center, a depositary institution under the Budapest Agreement, on August 9, 2021, and assigned accession number KCCM13029P.

Example 3: Replacement of an amino acid with other amino acids in the guaA mutation

Example 3-1: Construction of an amino acid replacement and insertion vector for the guaA mutation

Through the above example, it was identified that XMP could be obtained by introducing a mutation in guaA(R29C). To investigate the positional importance of the guaA mutation, vectors were constructed to replace the amino acid at position 29 with other amino acids and the effect on the ability to produce CMP was examined. Site-directed mutagenesis was carried out using the vector pDCM2-guaA(R29C) constructed in Example 2-1 as a template.

Specifically, PCR was performed using primer pairs SEQ ID NOs: 10 and 16, and SEQ ID NOs: 17 and 13, primer pairs SEQ ID NOs: 10 and 18, and SEQ ID NOs: 19 and 13, primer pairs SEQ ID NO : 10 and 20, and SEQ ID NO: 21 and 13, primer pairs SEQ ID NO: 10 and 22, and SEQ ID NO: 23 and 13, primer pairs SEQ ID NO: 10 and 24, and SEQ ID NO: 25 and 13, primer pairs SEQ ID NOs: 10 and 26, and SEQ ID NOs: 27 and 13, primer pairs SEQ ID NOs: 10 and 28, and SEQ ID NOs: 29 and 13, primer pairs SEQ ID NOs: 10 and 30, and SEQ ID NOs: 31 and 13, primer pairs SEQ ID NOs: 10 and 32, and SEQ ID NOs: 33 and 13, primer pairs SEQ ID NOs: 10 and 34, and SEQ ID NOs: 35 and 13, primer pairs SEQ ID NOs: 10 and 36, and SEQ ID NOs: 37 and 13, primer pairs SEQ ID NOs: 10 and 38, and SEQ ID NOs: 39 and 13, primer pairs SEQ ID NOs: 10 and 40, and SEQ ID NO: 41 and 13, primer pairs SEQ ID NOs: 10 and 42, and SEQ ID NOs: 43 and 13, primer pairs SEQ ID NOs: 10 and 44, and SEQ ID NOs: 45 and 13, primer pairs SEQ ID NOs: 10 and 46, and SEQ ID NOs: 47 and 13, primer pairs SEQ ID NOs: 10 and 48, and SEQ ID NOs: 49 and 13, and primer pairs SEQ ID NOs: 10 and 50, and SEQ ID NOs: 51 and 13. Solg™ Pfu-X DNA polymerase was used as the polymerase, and PCR was carried out under the following PCR amplification conditions: denaturation at 95°C for 5 minutes, 30 cycles of denaturation at 95°C for 30 seconds, annealing at 58°C C for 30 seconds, polymerize at 72°C for 60 seconds and then polymerize at 72°C for 5 minutes to thereby obtain each PCR product. The corresponding amplification products were mixed with the pDCM2 vector obtained previously by digestion with the restriction enzyme SmaI and cloned by the Gibson assembly method to obtain recombinant plasmids, and the information of the plasmids thus obtained is shown in Table 6. Cloning was carried out by mixing the Gibson assembly reagent and the corresponding fragments at calculated number of moles, followed by canning at 50°C for 1 hour.

Example 3-2: Obtaining strains having mutations in the form of substitutions in the guaA variant with other amino acids and comparing the ability to produce CMP

The 18 mutation vectors obtained in Example 3-1 were transformed into wild-type Corynebacterium stationis strain ATCC6872, and strains having vectors inserted into the chromosome by recombination of homologous sequences were selected on medium containing 25 mg/L kanamycin. Selected primary strains were subjected to secondary crossing over to select strains having a mutation introduced into the target gene. The introduction of gene mutation into the finally transformed strains was examined by PCR using the primer pair SEQ ID NO: 14 and SEQ ID NO: 15 and sequencing, and the names of the strains according to the inserted mutations are shown in Table 7 below.

The resulting strains, C.st strain ATCC6872::guaA(R29C) and the wild-type strain, were cultured and analyzed for CMP concentration by the fermentation titer evaluation method in Example 1-3. This analysis was repeated three times, and the means of the assay results are shown in Table 8.

Referring to Table 8, it was identified that strains including guaA, in which the amino acid at position 29 in the amino acid sequence encoded by the guaA gene is replaced by other amino acids except arginine, produce CMP compared to the wild type strain. That is, the amino acid at position 29 in the amino acid sequence encoded by the guaA gene corresponded to the position of the major mutation in the production of XMP and, more specifically, when the amino acid at position 29 in the amino acid sequence encoded by the guaA gene was replaced by amino acids other than arginine (cysteine, glycine, phenylalanine, serine, tyrosine, tryptophan, isoleucine, valine, proline, threonine, alanine, aspartic acid, asparagine, histidine, leucine, methionine, glutamine, lysine and glutamic acid), the amounts of CMP produced by microorganisms containing such a mutation may be significantly increased.

Therefore, it can be seen that variants having substitutions of the amino acid at position 29 in guaA with other amino acids can be effectively used in the production of purine nucleotide.

Example 4: Identification of GMP Producing Capacity of a Strain Expressing the quaA Variant

GMP production was examined using the CMP culture of C.st ATCC6872::guaA(R29C) as the CMP-producing strain through the following method (see Korean Patent No. KR 10-0655902 B1 and US Patent Publication No. US 2013-0095529 A1).

C.st strain ATCC6872::guaA(R29C) was cultivated by the fermentation titer evaluation method in Example 1-3. After completion of cultivation, the level of CMP production was measured by a method using HPLC. To convert the produced CMP to GMP, the following conversion reaction additives and E. coli CMP-aminase were added to the fermentation liquid in a corner baffled flask to carry out the conversion reaction at 40° C. for 2.5 hours.

Conversion reaction additives

1.8 g/l phytic acid, 4.8 g/l magnesium sulfate, 3 ml/l nymeen, 2% xylene, 100 mg/l adenine, 7.7 g/l dibasic sodium phosphate (Na ₂ HPO ₄ ), 2 g/l glutamine, 46 g/l glucose.

As a result of this analysis, the conversion rate indicating the level of GMP production compared to the level of CMP consumption is shown in Table 9.

As shown in Table 9, 3.54 g/L GMP was produced through the conversion reaction from CMP produced by C.st strain ATCC6872::guaA(R29C).

Therefore, it was identified that a variant having a substitution of amino acid at position 29 in guaA with another amino acid increased the production of GMP, and thus it can be seen that the variant proposed in the present invention can be effectively used in the production of purine nucleotide.

As set forth above, one skilled in the art to which this disclosure relates will appreciate that the present disclosure may be embodied in other specific forms without departing from its technical spirit or essential characteristics. Accordingly, the embodiments described above should be construed as illustrative by example and not limiting of the present disclosure. It should be understood that, within the scope of the present invention, all changes or modifications derived from the definitions and scope of the claims and their equivalents will fall within the scope of the present invention.

--->

LIST OF SEQUENCES

<110> CJ CheilJedang Corporation

<120> NEW VARIANT OF HYDROLYZING GLUTAMINE GMP SYNTHASE AND METHOD

OBTAINING PURINE NUCLEOTIDE USING IT

<130> OPA21333

<150> KR 10-2021-0125841

<151> 2021-09-23

<160> 51

<170> KoPatentIn 3.0

<210> 1

<211> 524

<212>PRT

<213> Artificial Sequence

<220>

<223>guaA_R29C

<400> 1

Met Thr Gln Pro Ala Thr Thr Pro Arg Pro Val Leu Val Val Asp Phe

1 5 10 15

Gly Ala Gln Tyr Ala Gln Leu Ile Ala Arg Arg Val Cys Glu Ala Ser

20 25 30

Ile Tyr Ser Glu Val Val Pro His Ser Ala Thr Val Lys Glu Ile Lys

35 40 45

Ala Lys Asn Pro Ala Ala Leu Ile Leu Ser Gly Gly Pro Ser Ser Val

50 55 60

Tyr Ala Asp Gly Ala Pro Gln Leu Lys Pro Glu Leu Leu Glu Leu Gly

65 70 75 80

Val Pro Val Phe Gly Ile Cys Tyr Gly Phe Gln Ala Met Asn His Ala

85 90 95

Leu Gly Gly Asn Val Ala Gln Thr Gly Asp Arg Glu Tyr Gly Arg Thr

100 105 110

Glu Ile Thr His Thr Gly Gly Val Leu His Asp Gly Leu Glu Glu Asn

115 120 125

His Lys Val Trp Met Ser His Gly Asp Ala Val Asp Lys Ala Pro Glu

130 135 140

Gly Phe Thr Val Thr Ala Ser Ser Ala Gly Ala Pro Val Ala Ala Met

145 150 155 160

Glu Cys Val Ala Lys Gln Met Ala Gly Val Gln Tyr His Pro Glu Val

165 170 175

Met His Ser Pro His Gly Gln Glu Val Leu Val Arg Phe Leu Thr Glu

180 185 190

Val Ala Gly Leu Glu Gln Thr Trp Thr Ser Ala Asn Ile Ala Gln Gln

195 200 205

Leu Ile Asp Asp Val Arg Ala Gln Ile Gly Pro Glu Gly Arg Ala Ile

210 215 220

Cys Gly Leu Ser Gly Gly Val Asp Ser Ala Val Ala Ala Ala Leu Val

225 230 235 240

Gln Arg Ala Ile Gly Asp Arg Leu Thr Cys Val Phe Val Asp His Gly

245 250 255

Leu Leu Arg Ala Gly Glu Arg Glu Gln Val Glu Lys Asp Phe Val Ala

260 265 270

Ser Thr Gly Ala Lys Leu Ile Thr Ala His Glu Ala Asp Ala Phe Leu

275 280 285

Ser Lys Leu Ala Gly Val Thr Asp Pro Glu Ala Lys Arg Lys Ala Ile

290 295 300

Gly Ala Glu Phe Ile Arg Ser Phe Glu Arg Ala Val Ala Gln Ala Leu

305 310 315 320

Glu Glu Ser Pro Glu Asp Ser Thr Val Asp Phe Leu Val Gln Gly Thr

325 330 335

Leu Tyr Pro Asp Val Val Glu Ser Gly Gly Gly Asp Gly Thr Ala Asn

340 345 350

Ile Lys Ser His His Asn Val Gly Gly Leu Pro Asp Asp Val Glu Phe

355 360 365

Glu Leu Val Glu Pro Leu Arg Leu Leu Phe Lys Asp Glu Val Arg Ala

370 375 380

Val Gly Arg Glu Leu Gly Leu Pro Glu Glu Ile Val Ala Arg Gln Pro

385 390 395 400

Phe Pro Gly Pro Gly Leu Gly Ile Arg Ile Ile Gly Glu Val Thr Glu

405 410 415

Glu Arg Leu Glu Ile Leu Arg Gln Ala Asp Leu Ile Ala Arg Thr Glu

420 425 430

Leu Thr Asn Ala Gly Leu Asp Gly Asp Ile Trp Gln Cys Pro Val Val

435 440 445

Leu Leu Ala Asp Val Arg Ser Val Gly Val Gln Gly Asp Gly Arg Thr

450 455 460

Tyr Gly His Pro Ile Val Leu Arg Pro Val Ser Ser Glu Asp Ala Met

465 470 475 480

Thr Ala Asp Trp Thr Arg Val Pro Tyr Asp Val Leu Glu Lys Ile Ser

485 490 495

Thr Arg Ile Thr Asn Glu Val Asn Asp Val Asn Arg Val Val Val Asp

500 505 510

Ile Thr Ser Lys Pro Pro Gly Thr Ile Glu Trp Glu

515 520

<210> 2

<211> 1575

<212> DNA

<213> Artificial Sequence

<220>

<223> guaA_c85t

<400> 2

gtgactcaac ctgcaacaac tccgcgccca gtcctcgtgg tggatttcgg tgcccaatac 60

gcacagctga ttgctcgtcg cgtatgtgag gcatcgattt actccgaggt agtcccacat 120

tccgccaccg ttaaagagat taaagctaaa aaccctgcag ctttgatttt gtccggtggc 180

ccgtcctctg tttatgccga tggcgccccg caattaaagc ctgaactgct cgagcttggt 240

gtgccagtct ttggcatctg ctacggcttc caagccatga accatgcttt gggtggcaac 300

gttgcgcaaa ccggtgaccg tgaatacggc cgcaccgaaa tcacccatac cggtggtgtg 360

ctgcacgacg gcttagaaga aaaccacaag gtctggatgt cccacggtga tgctgtggat 420

aaggcacctg agggctttac cgtgaccgca tcgtcggctg gtgcgccggt tgcagcgatg 480

gaatgcgtgg ccaagcaaat ggctggtgtg caataccacc ccgaggttat gcattcccca 540

cacggacagg aagtactcgt tcgcttcctc accgaggtag cagggctaga gcagacctgg 600

acctcggcaa atattgcgca gcagcttatc gatgatgtcc gcgcgcaaat cggccctgaa 660

ggccgcgcta tttgtggcct gtcgggcggc gtggactccg cagtcgctgc agcgctcgtg 720

cagcgcgcca ttggcgaccg tttgacctgt gtgttcgtgg accacggtct gctgcgcgcc 780

ggtgagcgtg agcaggtaga aaaggacttc gtggcttcga ctggtgcgaa gctgattacc 840

gcgcatgaag ctgatgcttt cttgtctaag ctcgccggtg ttaccgatcc tgaggctaag 900

cgcaaggcta tcggcgcgga attcatccgt tcctttgagc gtgctgtggc acaggctttg 960

gaagaatctc ctgaagactc cacagtggac ttcctggtcc agggcacctt gtatccggat 1020

gtggttgaat ccggcggcgg tgacggcacc gcaaatatca agtcccacca caatgttggc 1080

ggcctgccag acgatgtcga attcgaactc gtcgagccac tacgcctgct gtttaaggac 1140

gaagtccgtg ccgtcggccg cgagctcggc ctgcctgagg aaatcgttgc ccgccagcca 1200

ttccctggcc ctggcctagg catccgcatc atcggtgaag tcaccgagga gcgtctagaa 1260

atcctgcgtc aagcagacct gattgcgcgt accgagctga ccaacgctgg cctcgacggt 1320

gatatctggc agtgcccagt cgtactgctt gccgatgtcc gctccgtcgg agtccaaggc 1380

gacggccgca cctacggcca cccaatcgtg ctgcgcccag tgtcatccga ggatgccatg 1440

accgccgact ggacccgcgt tccttacgac gtcctagaga aaatctccac ccgcattacc 1500

aacgaagtca acgacgtcaa ccgcgtggtc gtcgacatca cctccaagcc accgggaacc 1560

atcgagtggg agtaa 1575

<210> 3

<211> 524

<212>PRT

<213>Unknown

<220>

<223> Corynebacterium stationis

<400> 3

Met Thr Gln Pro Ala Thr Thr Pro Arg Pro Val Leu Val Val Asp Phe

1 5 10 15

Gly Ala Gln Tyr Ala Gln Leu Ile Ala Arg Arg Val Arg Glu Ala Ser

20 25 30

Ile Tyr Ser Glu Val Val Pro His Ser Ala Thr Val Lys Glu Ile Lys

35 40 45

Ala Lys Asn Pro Ala Ala Leu Ile Leu Ser Gly Gly Pro Ser Ser Val

50 55 60

Tyr Ala Asp Gly Ala Pro Gln Leu Lys Pro Glu Leu Leu Glu Leu Gly

65 70 75 80

Val Pro Val Phe Gly Ile Cys Tyr Gly Phe Gln Ala Met Asn His Ala

85 90 95

Leu Gly Gly Asn Val Ala Gln Thr Gly Asp Arg Glu Tyr Gly Arg Thr

100 105 110

Glu Ile Thr His Thr Gly Gly Val Leu His Asp Gly Leu Glu Glu Asn

115 120 125

His Lys Val Trp Met Ser His Gly Asp Ala Val Asp Lys Ala Pro Glu

130 135 140

Gly Phe Thr Val Thr Ala Ser Ser Ala Gly Ala Pro Val Ala Ala Met

145 150 155 160

Glu Cys Val Ala Lys Gln Met Ala Gly Val Gln Tyr His Pro Glu Val

165 170 175

Met His Ser Pro His Gly Gln Glu Val Leu Val Arg Phe Leu Thr Glu

180 185 190

Val Ala Gly Leu Glu Gln Thr Trp Thr Ser Ala Asn Ile Ala Gln Gln

195 200 205

Leu Ile Asp Asp Val Arg Ala Gln Ile Gly Pro Glu Gly Arg Ala Ile

210 215 220

Cys Gly Leu Ser Gly Gly Val Asp Ser Ala Val Ala Ala Ala Leu Val

225 230 235 240

Gln Arg Ala Ile Gly Asp Arg Leu Thr Cys Val Phe Val Asp His Gly

245 250 255

Leu Leu Arg Ala Gly Glu Arg Glu Gln Val Glu Lys Asp Phe Val Ala

260 265 270

Ser Thr Gly Ala Lys Leu Ile Thr Ala His Glu Ala Asp Ala Phe Leu

275 280 285

Ser Lys Leu Ala Gly Val Thr Asp Pro Glu Ala Lys Arg Lys Ala Ile

290 295 300

Gly Ala Glu Phe Ile Arg Ser Phe Glu Arg Ala Val Ala Gln Ala Leu

305 310 315 320

Glu Glu Ser Pro Glu Asp Ser Thr Val Asp Phe Leu Val Gln Gly Thr

325 330 335

Leu Tyr Pro Asp Val Val Glu Ser Gly Gly Gly Asp Gly Thr Ala Asn

340 345 350

Ile Lys Ser His His Asn Val Gly Gly Leu Pro Asp Asp Val Glu Phe

355 360 365

Glu Leu Val Glu Pro Leu Arg Leu Leu Phe Lys Asp Glu Val Arg Ala

370 375 380

Val Gly Arg Glu Leu Gly Leu Pro Glu Glu Ile Val Ala Arg Gln Pro

385 390 395 400

Phe Pro Gly Pro Gly Leu Gly Ile Arg Ile Ile Gly Glu Val Thr Glu

405 410 415

Glu Arg Leu Glu Ile Leu Arg Gln Ala Asp Leu Ile Ala Arg Thr Glu

420 425 430

Leu Thr Asn Ala Gly Leu Asp Gly Asp Ile Trp Gln Cys Pro Val Val

435 440 445

Leu Leu Ala Asp Val Arg Ser Val Gly Val Gln Gly Asp Gly Arg Thr

450 455 460

Tyr Gly His Pro Ile Val Leu Arg Pro Val Ser Ser Glu Asp Ala Met

465 470 475 480

Thr Ala Asp Trp Thr Arg Val Pro Tyr Asp Val Leu Glu Lys Ile Ser

485 490 495

Thr Arg Ile Thr Asn Glu Val Asn Asp Val Asn Arg Val Val Val Asp

500 505 510

Ile Thr Ser Lys Pro Pro Gly Thr Ile Glu Trp Glu

515 520

<210> 4

<211> 1575

<212> DNA

<213>Unknown

<220>

<223> Corynebacterium stationis

<400> 4

gtgactcaac ctgcaacaac tccgcgccca gtcctcgtgg tggatttcgg tgcccaatac 60

gcacagctga ttgctcgtcg cgtacgtgag gcatcgattt actccgaggt agtcccacat 120

tccgccaccg ttaaagagat taaagctaaa aaccctgcag ctttgatttt gtccggtggc 180

ccgtcctctg tttatgccga tggcgccccg caattaaagc ctgaactgct cgagcttggt 240

gtgccagtct ttggcatctg ctacggcttc caagccatga accatgcttt gggtggcaac 300

gttgcgcaaa ccggtgaccg tgaatacggc cgcaccgaaa tcacccatac cggtggtgtg 360

ctgcacgacg gcttagaaga aaaccacaag gtctggatgt cccacggtga tgctgtggat 420

aaggcacctg agggctttac cgtgaccgca tcgtcggctg gtgcgccggt tgcagcgatg 480

gaatgcgtgg ccaagcaaat ggctggtgtg caataccacc ccgaggttat gcattcccca 540

cacggacagg aagtactcgt tcgcttcctc accgaggtag cagggctaga gcagacctgg 600

acctcggcaa atattgcgca gcagcttatc gatgatgtcc gcgcgcaaat cggccctgaa 660

ggccgcgcta tttgtggcct gtcgggcggc gtggactccg cagtcgctgc agcgctcgtg 720

cagcgcgcca ttggcgaccg tttgacctgt gtgttcgtgg accacggtct gctgcgcgcc 780

ggtgagcgtg agcaggtaga aaaggacttc gtggcttcga ctggtgcgaa gctgattacc 840

gcgcatgaag ctgatgcttt cttgtctaag ctcgccggtg ttaccgatcc tgaggctaag 900

cgcaaggcta tcggcgcgga attcatccgt tcctttgagc gtgctgtggc acaggctttg 960

gaagaatctc ctgaagactc cacagtggac ttcctggtcc agggcacctt gtatccggat 1020

gtggttgaat ccggcggcgg tgacggcacc gcaaatatca agtcccacca caatgttggc 1080

ggcctgccag acgatgtcga attcgaactc gtcgagccac tacgcctgct gtttaaggac 1140

gaagtccgtg ccgtcggccg cgagctcggc ctgcctgagg aaatcgttgc ccgccagcca 1200

ttccctggcc ctggcctagg catccgcatc atcggtgaag tcaccgagga gcgtctagaa 1260

atcctgcgtc aagcagacct gattgcgcgt accgagctga ccaacgctgg cctcgacggt 1320

gatatctggc agtgcccagt cgtactgctt gccgatgtcc gctccgtcgg agtccaaggc 1380

gacggccgca cctacggcca cccaatcgtg ctgcgcccag tgtcatccga ggatgccatg 1440

accgccgact ggacccgcgt tccttacgac gtcctagaga aaatctccac ccgcattacc 1500

aacgaagtca acgacgtcaa ccgcgtggtc gtcgacatca cctccaagcc accgggaacc 1560

atcgagtggg agtaa 1575

<210> 5

<211> 18

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 5

gcgatgtgat tgccggtg 18

<210> 6

<211> 18

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 6

gctctaggaa gtcagcag 18

<210> 7

<211> 19

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 7

tgcccaatac gcacagctg 19

<210> 8

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 8

ggctggggct ttgtcc 16

<210> 9

<211> 18

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 9

ggcttagcaa gacctggc 18

<210> 10

<211> 37

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 10

gtgaattcga gctcggtacc cgcaccgcag ttagccc 37

<210> 11

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 11

taaatcgatg cctcacatac gcgacgagca atc 33

<210> 12

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 12

gattgctcgt cgcgtatgtg aggcatcgat tta 33

<210> 13

<211> 39

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 13

ggtcgactct agaggatccc ccgaggtcca ggtctgctc 39

<210> 14

<211> 17

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 14

ggaatccgaa gcggagc 17

<210> 15

<211> 18

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 15

caggccacaa atagcgcg 18

<210> 16

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 16

taaatcgatg cctcgcctac gcgacgagca atc 33

<210> 17

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 17

gattgctcgt cgcgtaggcg aggcatcgat tta 33

<210> 18

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 18

taaatcgatg cctcgaatac gcgacgagca atc 33

<210> 19

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 19

gattgctcgt cgcgtattcg aggcatcgat tta 33

<210> 20

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 20

taaatcgatg cctcggatac gcgacgagca atc 33

<210> 21

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 21

gattgctcgt cgcgtatccg aggcatcgat tta 33

<210> 22

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 22

taaatcgatg cctcgtatac gcgacgagca atc 33

<210> 23

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 23

gattgctcgt cgcgtatacg aggcatcgat tta 33

<210> 24

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 24

taaatcgatg cctcccatac gcgacgagca atc 33

<210> 25

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 25

gattgctcgt cgcgtatggg aggcatcgat tta 33

<210> 26

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 26

taaatcgatg cctcgattac gcgacgagca atc 33

<210> 27

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 27

gattgctcgt cgcgtaatcg aggcatcgat tta 33

<210> 28

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 28

taaatcgatg cctccactac gcgacgagca atc 33

<210> 29

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 29

gattgctcgt cgcgtagtgg aggcatcgat tta 33

<210> 30

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 30

taaatcgatg cctctggtac gcgacgagca atc 33

<210> 31

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 31

gattgctcgt cgcgtaccag aggcatcgat tta 33

<210> 32

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 32

taaatcgatg cctcggttac gcgacgagca atc 33

<210> 33

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 33

gattgctcgt cgcgtaaccg aggcatcgat tta 33

<210> 34

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 34

taaatcgatg cctctgctac gcgacgagca atc 33

<210> 35

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 35

gattgctcgt cgcgtagcag aggcatcgat tta 33

<210> 36

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 36

taaatcgatg cctcatctac gcgacgagca atc 33

<210> 37

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 37

gattgctcgt cgcgtagatg aggcatcgat tta 33

<210> 38

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 38

taaatcgatg cctcgtttac gcgacgagca atc 33

<210> 39

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 39

gattgctcgt cgcgtaaacg aggcatcgat tta 33

<210> 40

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 40

taaatcgatg cctcgtgtac gcgacgagca atc 33

<210> 41

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 41

gattgctcgt cgcgtacacg aggcatcgat tta 33

<210> 42

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 42

taaatcgatg cctccagtac gcgacgagca atc 33

<210> 43

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 43

gattgctcgt cgcgtactgg aggcatcgat tta 33

<210> 44

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 44

taaatcgatg cctccattac gcgacgagca atc 33

<210> 45

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 45

gattgctcgt cgcgtaatgg aggcatcgat tta 33

<210> 46

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 46

taaatcgatg cctcctgtac gcgacgagca atc 33

<210> 47

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 47

gattgctcgt cgcgtacagg aggcatcgat tta 33

<210> 48

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 48

taaatcgatg cctcctttac gcgacgagca atc 33

<210> 49

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 49

gattgctcgt cgcgtaaagg aggcatcgat tta 33

<210> 50

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 50

taaatcgatg cctcttctac gcgacgagca atc 33

<210> 51

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 51

gattgctcgt cgcgtagaag aggcatcgat tta 33

<---

Claims (8)

1. A polypeptide involved in the production of a purine nucleotide selected from the group XMP (xanthosine monophosphate) or GMP (guanosine monophosphate), in which the amino acid corresponding to position 29 in SEQ ID NO: 3 is replaced by another amino acid selected from glycine, alanine, valine, leucine , isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine and histidine. 2. Polynucleotide encoding the polypeptide according to claim 1. 3. A microorganism for producing a purine nucleotide selected from the group XMP or GMP, containing a polypeptide in which the amino acid corresponding to position 29 in SEQ ID NO: 3 is replaced by another amino acid selected from glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine and histidine, or a polynucleotide encoding said polypeptide. 4. The microorganism according to claim 3, wherein said microorganism is Corynebacterium stationis . 5. A method for obtaining a purine nucleotide selected from the XMP or GMP group, including cultivating a microorganism according to claim 3 or 4. 6. The method of claim 5, further comprising isolating XMP or GMP from the culture medium or said microorganism. 7. A composition for producing a purine nucleotide selected from the group XMP or GMP, containing an excipient typically used in the composition for producing a purine nucleotide, and at least one of the following: a polypeptide, in which the amino acid corresponding to position 29 in SEQ ID NO: 3 , is replaced by another amino acid selected from glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine and histidine; and a microorganism containing said polypeptide. 8. A method of increasing the ability of a microorganism to produce XMP or GMP, comprising introducing the polypeptide according to claim 1 or a polynucleotide encoding it into the specified microorganism.