WO2022231370A1 - Novel bifunctional phosphoribosylaminoimidazolecarboxamide formyltransferase/imp cyclohydrolase variant and method for producing imp using same - Google Patents

Abstract

The present application relates to a novel bifunctional phosphoribosylaminoimidazolecarboxamide formyltransferase/IMP cyclohydrolase variant, a Corynebacterium stationis strain comprising the variant, and a method for producing IMP using the strain.

Description

Novel bifunctional phosphoribosylaminoimidazolecarboxamide formyltransferase/IMP cyclohydrolase variant and IMP production method using the same

The present application is a novel bifunctional phosphoribosylaminoimidazolecarboxamide formyltransferase / IMP cyclohydrolase (bifunctional phosphoribosylaminoimidazolecarboxamide formyltransferase / IMP cyclohydrolase) variant, Corynebacterium stasis strain comprising the variant And it relates to a method for producing IMP using the strain.

In order to produce IMP and other useful substances, various studies are being conducted for the development of high-efficiency production microorganisms and fermentation process technology. For example, a target substance-specific approach such as increasing the expression of a gene encoding an enzyme involved in IMP biosynthesis or removing a gene unnecessary for biosynthesis is mainly used (EP 3722430 A1, US 2020-0347346) A1). The enzyme involved in the IMP biosynthesis, for example, bifunctional phosphoribosylaminoimidazolecarboxamide formyltransferase / IMP cyclohydrolase (bifunctional phosphoribosylaminoimidazolecarboxamide formyltransferase / IMP cyclohydrolase) and the like (https: / /www.uniprot.org/uniprot/P15639).

However, as the demand for IMP increases, there is still a need for research to increase the effective production capacity of IMP.

The present inventors provide a novel bifunctional phosphoribosylaminoimidazolecarboxamide formyltransferase/IMP cyclohydrolase variant that enhances IMP production, a Corynebacterium stasis strain comprising the variant, and the strain This application was completed by developing an IMP production method using

One object of the present application is a bifunctional phosphory, consisting of the amino acid sequence set forth in SEQ ID NO: 1, in which alanine, an amino acid corresponding to position 351 of the amino acid sequence of SEQ ID NO: 3, is substituted with valine It is to provide a mutant of bolusaminoimidazolecarboxamide formyltransferase/IMP cyclohydrolase (bifunctional phosphoribosylaminoimidazolecarboxamide formyltransferase/IMP cyclohydrolase).

Another object of the present application is to provide a polynucleotide encoding the variant of the present application.

Another object of the present application is a variant of the present application or a polynucleotide encoding the variant, and having IMP (5'-inosine monophosphate) producing ability, Corynebacterium stationis ( Corynebacterium stationis ) strain is to provide

Another object of the present application is to include the variant of the present application or a polynucleotide encoding the variant, and having an IMP-producing ability, including the step of culturing a Corynebacterium stasis strain in a medium, IMP to provide a production method.

Another object of the present application is a microorganism comprising a variant of the present application, a polynucleotide encoding the variant, a vector including the polynucleotide or the polynucleotide of the present application; the culture medium; Or to provide a composition for producing IMP comprising a combination of two or more of them.

Another object of the present application is that alanine, which is an amino acid corresponding to position 351 of the amino acid sequence of SEQ ID NO: 3, is substituted with valine, consisting of the amino acid sequence set forth in SEQ ID NO: 1, bifunctional To provide an IMP production use of phosphoribosylaminoimidazolecarboxamide formyltransferase/IMP cyclohydrolase variants.

When culturing a Corynebacterium stationionis strain, including the bifunctional phosphoribosylaminoimidazolecarboxamide formyltransferase/IMP cyclohydrolase variant of the present application, microorganisms having an existing unmodified polypeptide It is possible to produce high-yield IMP.

This will be described in detail as follows. Meanwhile, each description and embodiment disclosed in the present application may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. In addition, it cannot be seen that the scope of the present application is limited by the detailed description described below. In addition, a number of papers and patent documents are referenced throughout this specification and their citations are indicated. The disclosure contents of the cited papers and patent documents are incorporated herein by reference in their entirety to more clearly describe the level of the technical field to which the present invention pertains and the content of the present invention.

One aspect of the present application is bifunctional phosphoribosylaminoimidazolecarboxa, consisting of the amino acid sequence set forth in SEQ ID NO: 1, in which alanine, an amino acid corresponding to position 351 of the amino acid sequence of SEQ ID NO: 3, is substituted with valine A mutant of the mid formyltransferase/IMP cyclohydrolase (bifunctional phosphoribosylaminoimidazolecarboxamide formyltransferase/IMP cyclohydrolase) is provided.

The variant of the present application may have, include, or consist of the amino acid sequence set forth in SEQ ID NO: 1, or may consist essentially of the amino acid sequence.

In addition, in the variant of the present application, in the amino acid sequence set forth in SEQ ID NO: 1, the amino acid corresponding to position 351 based on the amino acid sequence of SEQ ID NO: 3 is valine, and at least 70%, 75 with the amino acid sequence set forth in SEQ ID NO: 1 %, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or an amino acid sequence having at least 99.9% homology or identity. In addition, as long as it is an amino acid sequence having such homology or identity and exhibiting efficacy corresponding to the variant of the present application, variants having an amino acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted or added are also included within the scope of the present application. is self-evident

For example, sequence additions or deletions, naturally occurring mutations, silent mutations or conservation within the N-terminus, C-terminal and/or within the amino acid sequence that do not alter the function of the variants of the present application It is a case of having an enemy substitution.

The term “conservative substitution” means substituting an amino acid for another amino acid having similar structural and/or chemical properties. Such amino acid substitutions may generally occur based on similarity in the polarity, charge, solubility, hydrophobicity, hydrophilicity and/or amphipathic nature of the residues. Typically, conservative substitutions may have little or no effect on the activity of the protein or polypeptide.

As used herein, the term "variant" means that one or more amino acids are conservatively substituted and/or modified so that they differ from the amino acid sequence before the mutation of the variant, but have functions or properties. refers to a polypeptide that is maintained. Such variants can generally be identified by modifying one or more amino acids in the amino acid sequence of the polypeptide and evaluating the properties of the modified polypeptide. That is, the ability of the variant may be increased, unchanged, or decreased compared to the polypeptide before the mutation. In addition, some variants may include variants in which one or more portions, such as an N-terminal leader sequence or a transmembrane domain, have been removed. Other variants may include variants in which a portion is removed from the N- and/or C-terminus of the mature protein. The term "variant" may be used interchangeably with terms such as mutant, modified, mutant polypeptide, mutated protein, mutant and mutant (in English, modified, modified polypeptide, modified protein, mutant, mutein, divergent, etc.) and, as long as it is a term used in a mutated sense, it is not limited thereto. For the purposes of the present application, the variant may be a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 in which alanine, which is an amino acid corresponding to position 351 of the amino acid sequence of SEQ ID NO: 3, is substituted with valine.

In addition, variants may include deletions or additions of amino acids that have minimal effect on the properties and secondary structure of the polypeptide. For example, a signal (or leader) sequence involved in protein translocation may be conjugated to the N-terminus of the mutant, either co-translationally or post-translationally. The variants may also be conjugated with other sequences or linkers for identification, purification, or synthesis.

As used herein, the term 'homology' or 'identity' 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.

Sequence homology or identity of a conserved polynucleotide or polypeptide is determined by standard alignment algorithms, with default gap penalties established by the program used may be used. Substantially homologous or identical sequences are generally capable of hybridizing with all or part of a sequence under moderate or high stringent conditions. It is apparent that hybridization also includes hybridization with polynucleotides containing common codons or codons taking codon degeneracy into account in the polynucleotide.

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

Homology, similarity or identity of polynucleotides or polypeptides is described, for example, in Smith and Waterman, Adv. Appl. Math (1981) 2:482, eg, Needleman et al. (1970), J Mol Biol. can be determined by comparing sequence information using a GAP computer program such as 48:443. In summary, a GAP program can be defined as the total number of symbols in the shorter of the two sequences divided by the number of similarly aligned symbols (ie, nucleotides or amino acids). Default parameters for the GAP program are: (1) a binary comparison matrix (containing values of 1 for identity and 0 for non-identity) and Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation , pp. 353-358 (1979), Gribskov et al (1986) Nucl. Acids Res. 14: weighted comparison matrix of 6745 (or EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap (or a gap open penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for end gaps.

As an example of the present application, the variant of the present application may have bifunctional phosphoribosylaminoimidazolecarboxamide formyltransferase/IMP cyclohydrolase activity. In addition, the variant of the present application may have an activity to increase IMP production capacity compared to a wild-type polypeptide having a bifunctional phosphoribosylaminoimidazolecarboxamide formyltransferase/IMP cyclohydrolase activity.

As used herein, the term "bifunctional phosphoribosylaminoimidazolecarboxamide formyltransferase/IMP cyclohydrolase" refers to 10-formyltetrahydrofolate + 5-amino-1-(5 Phosphoribosylaminoimidazolecarboxamide formyltransferase, which catalyzes the chemical reaction of -phospho-D-ribosyl)imidazole-4-carboxamide ⇔ tetrahydrofolate + 5-formamido-1-(5-phospho-D-ribosyl)imidazole-4-carboxamide (EC:2.1.2.3) and IMP + H ₂ O ⇔ 5-formamido-1-(5-phospho-D-ribosyl)imidazole-4-carboxamide catalyzing the chemical reaction of IMP cyclohydrolase (EC:3.5.4.10) containing two domains of activity. have Specifically, the bifunctional phosphoribosylaminoimidazolecarboxamide formyltransferase/IMP cyclohydrolase of the present application may be used in combination with PurH. In the present application, the sequence of the bifunctional phosphoribosylaminoimidazolecarboxamide formyltransferase/IMP cyclohydrolase can be obtained from GenBank of NCBI, a known database. Specifically, it may be a polypeptide having bifunctional phosphoribosylaminoimidazolecarboxamide formyltransferase/IMP cyclohydrolase activity encoded by purH , but is not limited thereto.

As used herein, the term "corresponding to" refers to an amino acid residue at a listed position in a polypeptide, or to an amino acid residue that is similar, identical or homologous to a listed residue in a polypeptide. Identifying an amino acid at a corresponding position may be determining a specific amino acid in a sequence that refers to a specific sequence. As used herein, "corresponding region" generally refers to a similar or corresponding position in a related protein or reference protein.

For example, any amino acid sequence is aligned with SEQ ID NO: 3, and based on this, each amino acid residue of the amino acid sequence can be numbered with reference 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 such as that described in this application can identify the position of an amino acid, or a position at which modifications, such as substitutions, insertions, or deletions, occur compared to a query sequence (also referred to as a "reference sequence").

Such alignments include, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), the Needleman program in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al. , 2000), Trends Genet. 16: 276-277), etc., but is not limited thereto, and a sequence alignment program known in the art, a pairwise sequence comparison algorithm, etc. may be appropriately used.

Another aspect of the present application is to provide a polynucleotide encoding the variant of the present application.

As used herein, the term "polynucleotide" refers to a DNA or RNA strand of a certain length or longer as a polymer of nucleotides in which nucleotide monomers are linked in a long chain by covalent bonds, and more specifically, encoding the variant. polynucleotide fragments.

The polynucleotide encoding the variant of the present application may include a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 1. As an example of the present application, the polynucleotide of the present application may have or include the sequence of SEQ ID NO: 2. In addition, the polynucleotide of the present application may consist of, or consist essentially of, the sequence of SEQ ID NO: 2.

In consideration of codon degeneracy or preferred codons in organisms that want to express the variants of the present application, the polynucleotides of the present application are various in the coding region within the range that does not change the amino acid sequence of the variants of the present application. Deformation can be made. Specifically, the polynucleotide of the present application has 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% homology or identity to the sequence of SEQ ID NO: 2 Having or including a nucleotide sequence that is more than, 98% or more, and less than 100%, or homology or identity with the sequence of SEQ ID NO: 2 is 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% of the nucleotide sequence may consist of or consist essentially of, but is not limited thereto. In this case, in the sequence having the homology or identity, the codon encoding the amino acid corresponding to position 351 of SEQ ID NO: 1 may be one of the codons encoding valine.

In addition, the polynucleotide of the present application may be included without limitation as long as it can hybridize under stringent conditions with a probe that can be prepared from a known gene sequence, for example, a sequence complementary to all or part of the polynucleotide sequence of the present application. can The "stringent condition" means a condition that enables specific hybridization between polynucleotides. These conditions are described in J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; F.M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50-9.51, 11.7-11.8). For example, polynucleotides with high homology or identity, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or a condition in which polynucleotides having 99% or more homology or identity hybridize with each other and polynucleotides with lower homology or identity do not hybridize, or a washing condition of conventional Southern hybridization at 60°C, 1 ХSSC, 0.1% SDS, specifically 60° C., 0.1ХSSC, 0.1% SDS, more specifically 68° C., 0.1ХSSC, 0.1% SDS at a salt concentration and temperature equivalent to one wash, specifically two to three times conditions can be enumerated.

Hybridization requires that two nucleic acids have complementary sequences, although mismatch between bases is possible depending on the stringency of hybridization. The term "complementary" is used to describe the relationship between nucleotide bases capable of hybridizing to each other. For example, with respect to DNA, adenine is complementary to thymine and cytosine is complementary to guanine. Accordingly, the polynucleotides of the present application may also include substantially similar nucleic acid sequences as well as isolated nucleic acid fragments complementary to the overall sequence.

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

The appropriate stringency for hybridizing the polynucleotides depends on the length of the polynucleotides and the degree of complementarity, and the parameters are well known in the art (eg, J. Sambrook et al., supra).

Another aspect of the present application is to provide a vector comprising the polynucleotide of the present application. The vector may be an expression vector for expressing the polynucleotide in a host cell, but is not limited thereto.

The vector of the present application may include a DNA preparation comprising the nucleotide sequence of a polynucleotide encoding the target polypeptide operably linked to a suitable expression control region (or expression control sequence) so that the target polypeptide can be expressed in a suitable host. can The expression control region may include a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence regulating the termination of transcription and translation. After transformation into an appropriate host cell, the vector can replicate or function independently of the host genome, and can be integrated into the genome itself.

The vector used in the present application is not particularly limited, and any vector known in the art may be used. Examples of commonly used vectors include plasmids, cosmids, viruses and bacteriophages in a natural or recombinant state. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A may be used as phage vectors or cosmid vectors, and pDZ-based, pBR-based, and pUC-based plasmid vectors may be used. , pBluescript II-based, pGEM-based, pTZ-based, pCL-based, pET-based and the like can be used. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors and the like can be used.

For example, a polynucleotide encoding a target polypeptide may be inserted into a chromosome through a vector for intracellular chromosome insertion. The insertion of the polynucleotide into the chromosome may be performed by any method known in the art, for example, homologous recombination, but is not limited thereto. It may further include a selection marker (selection marker) for confirming whether the chromosome is inserted. The selection marker is used to select cells transformed with the vector, that is, to determine whether a target nucleic acid molecule is inserted, and selectable phenotypes such as drug resistance, auxotrophy, resistance to cytotoxic agents, or surface polypeptide expression. Markers to be given can be used. In an environment treated with a selective agent, only the cells expressing the selectable marker survive or exhibit other expression traits, so that the transformed cells can be selected.

As used herein, the term "transformation" refers to introducing a vector including a polynucleotide encoding a target polypeptide into a host cell or microorganism so that the polypeptide encoded by the polynucleotide can be expressed in the host cell. The transformed polynucleotide may include all of them regardless of whether they are inserted into the chromosome of the host cell or located outside the chromosome, as long as they can be expressed in the host cell. In addition, the polynucleotide includes DNA and/or RNA encoding a target polypeptide. The polynucleotide may be introduced in any form as long as it can be introduced and expressed into a host cell. For example, the polynucleotide may be introduced into a host cell in the form of an expression cassette, which is a gene construct including all elements necessary for self-expression. The expression cassette 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 in the form of an expression vector capable of self-replication. In addition, the polynucleotide may be introduced into a host cell in its own form and operably linked to a sequence required for expression in the host cell, but is not limited thereto.

In addition, the term “operably linked” as used herein means that a promoter sequence that initiates and mediates transcription of a polynucleotide encoding the target variant of the present application and the polynucleotide sequence are functionally linked.

Another aspect of the present application is to provide a strain of Corynebacterium stationis ( Corynebacterium stationis ) comprising a variant of the present application or a polynucleotide of the present application.

The strain of the present application may include a vector comprising the mutant polypeptide of the present application, a polynucleotide encoding the polypeptide, or the polynucleotide of the present application.

As used herein, the term "strain (or microorganism)" includes both wild-type microorganisms and microorganisms in which genetic modification has occurred naturally or artificially. As a result, a specific mechanism is weakened or enhanced as a microorganism, and may be a microorganism including genetic modification for the production of a desired polypeptide, protein or product.

The strain of the present application includes a strain comprising any one or more of a mutant of the present application, a polynucleotide of the present application, and a vector including the polynucleotide of the present application; a strain modified to express a variant of the present application or a polynucleotide of the present application; a variant of the present application, or a strain expressing the polynucleotide of the present application (eg, a recombinant strain); Or it may be a strain having the mutant activity of the present application (eg, a recombinant strain), but is not limited thereto.

The strain of the present application may be a strain having IMP-producing ability.

The strain of the present application is a microorganism having a naturally bifunctional phosphoribosylaminoimidazolecarboxamide formyltransferase/IMP cyclohydrolase or IMP-producing ability, or a bifunctional phosphoribosylaminoimidazolecarboxa Mid formyltransferase / IMP cyclohydrolase or IMP-producing ability is introduced into the parent strain without the mutant of the present application or a polynucleotide encoding it (or a vector including the polynucleotide) and / or IMP-producing ability is endowed It may be a microorganism, but is not limited thereto.

As an example, the strain of the present application is transformed with a vector containing the polynucleotide of the present application or a polynucleotide encoding the variant of the present application, and expresses the variant of the present application as a cell or microorganism, The strain of the application may include all microorganisms capable of producing IMP, including the mutant of the present application. For example, the strain of the present application is a bifunctional phosphoribosylaminoimidazolecarboxamide formyltransferase/IMP by introducing a polynucleotide encoding a variant of the present application into a natural wild-type microorganism or a microorganism producing IMP. The cyclohydrolase mutant is expressed, and it may be a recombinant strain having an increased IMP-producing ability. The recombinant strain with increased IMP-producing ability is a natural wild-type microorganism or bifunctional phosphoribosylaminoimidazolecarboxamide formyltransferase/IMP cyclohydrolase unmodified microorganism (ie, wild-type bifunctional phosphoribosyl) A microorganism having an increased IMP production ability compared to a microorganism expressing aminoimidazolecarboxamide formyltransferase/IMP cyclohydrolase (SEQ ID NO: 3) or a mutant (SEQ ID NO: 1) protein that does not express it. It may be a microorganism However, the present invention is not limited thereto. As an example, the strain with increased IMP-producing ability of the present application may be a microorganism having increased IMP-producing ability compared to Corynebacterium stasis comprising the polypeptide of SEQ ID NO: 3 or a polynucleotide encoding the same, but is not limited thereto does not For example, the target strain, bifunctional phosphoribosylaminoimidazolecarboxamide formyltransferase/IMP cyclohydrolase unmodified microorganism, is the CJI2332 strain (KCCM12277P, KR 10 -1950141 B1), but is not limited thereto.

For example, the recombinant strain with increased production capacity is about 1% or more, specifically about 1% or more, about 2.5% or more, about 5% or more, about 6% compared to the IMP production capacity of the parent strain or unmodified microorganism before mutation. or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 11% or more, about 11.5% or more, about 12% or more, about 12.5% or more, about 13% or more, about 13.5% or more, about 14% or more, about 14.5% or more, about 15% or more, about 15.5% or more, about 16% or more, about 16.5% or more, about 17% or more, about 17.5% or more, about 18% or more, about 18.5% or more, about 19% or more, about 19.5% or more, about 20% or more, about 20.1% or more, or about 20.2% or more (the upper limit is not particularly limited, for example, about 200% or less, about 150% or less, about 100% or less, about 50% or less, about 40% or less, about 30% or less, or about 25% or less) may be increased, but as long as it has an increase in + value compared to the production capacity of the parent strain or unmodified microorganism before mutation , but not limited thereto. In another example, the recombinant strain with increased production capacity has an IMP production capacity of about 1.01 times or more, about 1.02 times or more, about 1.03 times or more, about 1.05 times or more, about 1.06 times or more, compared to the parent strain or unmodified microorganism before mutation. , about 1.07 times or more, about 1.08 times or more, about 1.09 times or more, about 1.10 times or more, about 1.11 times or more, about 1.12 times or more, about 1.13 times or more, about 1.14 times or more, about 1.15 times or more, about 1.16 times or more , about 1.17 times or more, about 1.18 times or more, about 1.19 times or more, or about 1.2 times or more (the upper limit is not particularly limited, for example, about 10 times or less, about 5 times or less, about 3 times or less, or about 2 times or more. may be below) may be increased, but is not limited thereto. The term “about” is a range including all of ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc. not limited

As used herein, the term "unmodified microorganism" does not exclude a strain containing a mutation that can occur naturally in a microorganism, it is a wild-type strain or a natural-type strain itself, or a genetic variation caused by natural or artificial factors. It may mean the strain before being changed. For example, the unmodified microorganism may refer to a strain in which the bifunctional phosphoribosylaminoimidazolecarboxamide formyltransferase/IMP cyclohydrolase variant described herein is not introduced or before it is introduced. The "unmodified microorganism" may be used interchangeably with "strain before modification", "microbe before modification", "unmodified strain", "unmodified strain", "unmodified microorganism" or "reference microorganism".

In another example of the present application, the microorganism of the present application is Corynebacterium glutamicum ( Corynebacterium glutamicum ), Corynebacterium crudilactis ), Corynebacterium deserti ( Corynebacterium deserti ), Cory Nebacterium efficiens ( Corynebacterium efficiens ), Corynebacterium callunae ), Corynebacterium stationis , Corynebacterium stationis ), Corynebacterium singulare ( Corynebacterium singulare ), Corynebacterium halo Tolerans ( Corynebacterium halotolerans ), Corynebacterium striatum ( Corynebacterium striatum ), Corynebacterium ammoniagenes ( Corynebacterium ammoniagenes ), Corynebacterium pollutisoli ( Corynebacterium pollutisoli ), Corynebacterium imitans imitans ), Corynebacterium testudinoris ) or Corynebacterium flavescens ).

As used herein, the term “attenuation” of polypeptide activity is a concept that includes both reduced or no activity compared to intrinsic activity. The attenuation may be used interchangeably with terms such as inactivation, deficiency, down-regulation, decrease, reduce, attenuation, and the like.

The attenuation is when 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, etc. When the overall polypeptide activity level and/or concentration (expression amount) in the cell is lower than that of the native strain due to (translation) inhibition, etc., when the expression of the polynucleotide is not made at all, and/or when the expression of the polynucleotide is Even if there is no activity of the polypeptide, it may also be included. The "intrinsic activity" refers to the activity of a specific polypeptide originally possessed by the parent strain, wild-type or unmodified microorganism before transformation when the trait is changed due to genetic mutation caused by natural or artificial factors. This may be used interchangeably with "activity before modification". "Inactivation, deficiency, reduction, downregulation, reduction, attenuation" of the activity of the polypeptide compared to the intrinsic activity means that the activity of the specific polypeptide originally possessed by the parent strain or unmodified microorganism before transformation is lowered.

Attenuation of the activity of such a polypeptide may be performed by any method known in the art, but is not limited thereto, and may be achieved by application of various methods well known in the art (eg, Nakashima N et al., Bacterial cellular engineering by genome editing and gene silencing. Int J Mol Sci. 2014;15(2):2773-2793, Sambrook et al. Molecular Cloning 2012, etc.).

Specifically, the attenuation of the polypeptide activity of the present application is

1) deletion of all or part of a gene encoding a polypeptide;

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

3) modification of the amino acid sequence constituting the polypeptide such that the activity of the polypeptide is eliminated or attenuated (eg, deletion/substitution/addition of one or more amino acids on the amino acid sequence);

4) modification of the gene sequence encoding the polypeptide such that the activity of the polypeptide is eliminated or attenuated (eg, one or more nucleobases on the nucleotide sequence of the polypeptide gene to encode a polypeptide modified such that the activity of the polypeptide is eliminated or attenuated) deletion/replacement/addition of);

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

6) introduction of an antisense oligonucleotide (eg, antisense RNA) that complementarily binds to the transcript of said gene encoding the polypeptide;

7) adding a sequence complementary to the Shine-Dalgarno sequence in front of the Shine-Dalgarno sequence of the gene encoding the polypeptide to form a secondary structure that cannot be attached to the ribosome;

8) addition of a promoter transcribed in the opposite direction to the 3' end of the open reading frame (ORF) of the gene sequence encoding the polypeptide (Reverse transcription engineering, RTE); or

9) It may be a combination of two or more selected from 1) to 8) above, but is not particularly limited thereto.

for example,

1) The deletion of a part or all of the gene encoding the polypeptide may be the removal of the entire polynucleotide encoding the endogenous target polypeptide in the chromosome, replacement with a polynucleotide in which some nucleotides are deleted, or replacement with a marker gene.

In addition, the above 2) modification of the expression control region (or expression control sequence), deletion, insertion, non-conservative or conservative substitution, or a combination thereof, mutation in the expression control region (or expression control sequence) occurs, or weaker replacement with an active sequence. The expression control region includes, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating the termination of transcription and translation.

In addition, 5) the base sequence modification encoding the start codon or 5'-UTR region of the gene transcript encoding the polypeptide is, for example, a base encoding another start codon having a lower expression rate of the polypeptide compared to the intrinsic start codon It may be substituted with a sequence, but is not limited thereto.

In addition, the modification of the amino acid sequence or polynucleotide sequence of 3) and 4) above deletes, inserts, non-conservative or conservative substitution of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide so as to weaken the activity of the polypeptide. Or a combination thereof may result in a mutation in sequence, or replacement with an amino acid sequence or polynucleotide sequence improved to have weaker activity or an amino acid sequence or polynucleotide sequence improved to have no activity, but is not limited thereto. For example, by introducing a mutation in the polynucleotide sequence to form a stop codon, the expression of a gene may be inhibited or attenuated, but is not limited thereto.

6) The introduction of an antisense oligonucleotide (eg, antisense RNA) that complementarily binds to the transcript of the gene encoding the polypeptide is described, for example, in Weintraub, H. et al., Antisense-RNA as a molecular tool. for genetic analysis, Reviews - Trends in Genetics, Vol. 1(1) 1986].

7) Addition of a sequence complementary to the Shine-Dalgarno sequence in front of the Shine-Dalgarno sequence of the gene encoding the polypeptide to form a secondary structure that cannot be attached to the ribosome is mRNA translation It may make it impossible or slow it down.

8) the addition of a promoter transcribed in the opposite direction to the 3' end of the open reading frame (ORF) of the gene sequence encoding the polypeptide (Reverse transcription engineering, RTE) is an antisense complementary to the transcript of the gene encoding the polypeptide It may be to attenuate activity by making nucleotides.

As used herein, the term "enhancement" of a polypeptide activity means that the activity of the polypeptide is increased compared to the intrinsic activity. The reinforcement may be used interchangeably with terms such as activation, up-regulation, overexpression, and increase. Herein, activation, enhancement, up-regulation, overexpression, and increase may include all of those exhibiting an activity that was not originally possessed, or exhibiting an improved activity compared to an intrinsic activity or an activity prior to modification. The “intrinsic activity” refers to the activity of a specific polypeptide originally possessed by the parent strain or unmodified microorganism before transformation when the trait is changed due to genetic mutation caused by natural or artificial factors. "Enhance", "up-regulation", "overexpression" or "increase" in the activity of a polypeptide compared to its intrinsic activity means the activity of a specific polypeptide originally possessed by the parent strain or unmodified microorganism before transformation. And / or concentration (expression amount) means improved compared to.

The enrichment can be achieved by introducing an exogenous polypeptide, or by enhancing the activity and/or concentration (expression amount) of the endogenous polypeptide. Whether or not the activity of the polypeptide is enhanced can be confirmed from the increase in the level of activity, expression level, or the amount of product excreted from the polypeptide.

The enhancement of the activity of the polypeptide can be applied by various methods well known in the art, and is not limited as long as it can enhance the activity of the target polypeptide compared to the microorganism before modification. Specifically, it may be one using genetic engineering and/or protein engineering well known to those skilled in the art, which is a routine method of molecular biology, but is not limited thereto (eg, Sitnicka et al. Functional Analysis of Genes. Advances in Cell). Biology 2010, Vol. 2. 1-16, Sambrook et al. Molecular Cloning 2012, etc.).

Specifically, the enhancement of the polypeptide activity of the present application is

1) increasing the intracellular copy number of a polynucleotide encoding the polypeptide;

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

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

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

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

6) introduction of a foreign polypeptide exhibiting the activity of the polypeptide or a foreign polynucleotide encoding the same;

7) codon optimization of the polynucleotide encoding the polypeptide;

8) by analyzing the tertiary structure of the polypeptide to select an exposed site for modification or chemical modification; or

9) It may be a combination of two or more selected from 1) to 8) above, but is not particularly limited thereto.

More specifically,

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

2) Replacing the gene expression control region (or expression control sequence) on the chromosome encoding the polypeptide with a sequence with strong activity is, for example, deletion, insertion, non-conservative or Conservative substitution or a combination thereof may result in a mutation in the sequence, or replacement with a sequence having a stronger activity. The expression control region is not particularly limited thereto, but may include a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence controlling the termination of transcription and translation. As an example, the original promoter may be replaced with a strong promoter, but is not limited thereto.

Examples of known strong promoters include CJ1 to CJ7 promoters (US 7662943 B2), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13 (sm3) promoter (US Patent US 10584338 B2), O2 promoter (US Patent US 10273491 B2), tkt promoter, yccA promoter, etc., but is not limited thereto.

3) Modification of the nucleotide sequence encoding the start codon or 5'-UTR region of the gene transcript encoding the polypeptide is, for example, a nucleotide sequence encoding another start codon having a higher expression rate of the polypeptide compared to the intrinsic start codon. It may be a substitution, but is not limited thereto.

The modification of the amino acid sequence or polynucleotide sequence of 4) and 5) above may include deletion, insertion, non-conservative or conservative substitution of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide to enhance the activity of the polypeptide; A combination thereof may result in sequence mutation, or replacement with an amino acid sequence or polynucleotide sequence improved to have stronger activity or an amino acid sequence or polynucleotide sequence improved to increase activity, but is not limited thereto. The replacement may be specifically performed by inserting a polynucleotide into a chromosome by homologous recombination, but is not limited thereto. In this case, the vector used may further include a selection marker for confirming whether or not the chromosome is inserted. The selection marker is the same as described above.

6) The introduction of the foreign polynucleotide exhibiting the activity of the polypeptide may be the introduction of the foreign polynucleotide encoding the polypeptide exhibiting the same/similar activity as the polypeptide into a host cell. The foreign polynucleotide is not limited in its origin or sequence as long as it exhibits the same/similar activity as the polypeptide. The method used for the introduction can be performed by appropriately selecting a known transformation method by those skilled in the art, and the introduced polynucleotide is expressed in a host cell to generate a polypeptide and increase its activity.

7) Codon optimization of the polynucleotide encoding the polypeptide is codon-optimized so that the transcription or translation of the endogenous polynucleotide is increased in the host cell, or the transcription and translation of the foreign polynucleotide is optimized in the host cell. It may be that its codons are optimized so that the

8) Selecting an exposed site by analyzing the tertiary structure of the polypeptide and modifying or chemically modifying it is, for example, by comparing the sequence information of the polypeptide to be analyzed with a database in which sequence information of known proteins is stored to determine the degree of sequence similarity. Accordingly, it may be to determine a template protein candidate, check the structure based on this, and select an exposed site to be modified or chemically modified and modified or modified.

Such enhancement of polypeptide activity is to increase the activity or concentration of the corresponding polypeptide based on the activity or concentration of the polypeptide expressed in the wild-type or pre-modified microbial strain, or increase the amount of product produced from the polypeptide. However, the present invention is not limited thereto.

Modification of part or all of the polynucleotide in the microorganism of the present application is (a) homologous recombination using a vector for chromosome insertion in the microorganism or genome correction using engineered nuclease (e.g., CRISPR-Cas9) and/or (b) It may be induced by light and/or chemical treatments such as, but not limited to, ultraviolet and radiation. The method for modifying part or all of the gene may include a method by DNA recombination technology. For example, by injecting a nucleotide sequence or a vector containing a nucleotide sequence homologous to a target gene into the microorganism to cause homologous recombination, a part or all of the gene may be deleted. The injected nucleotide sequence or vector may include a dominant selection marker, but is not limited thereto.

In the microorganism of the present application, variants, polynucleotides, IMPs, and the like are as described in the other aspects above.

Another aspect of the present application provides a method for producing IMP, comprising the step of culturing a Corynebacterium stationionis strain comprising a mutant of the present application or a polynucleotide of the present application in a medium.

The method for producing IMP of the present application may include culturing a Corynebacterium stationionis strain comprising the mutant of the present application or the polynucleotide of the present application or the vector of the present application in a medium.

In the present application, the term "cultivation" means growing the Corynebacterium stasis strain of the present application under moderately controlled environmental conditions. The culture process of the present application may be performed according to a suitable medium and culture conditions known in the art. Such a culture process can be easily adjusted and used by those skilled in the art according to the selected strain. Specifically, the culture may be a batch, continuous and/or fed-batch, but is not limited thereto.

As used herein, the term "medium" refers to a material in which nutrients required for culturing the Corynebacterium stasis strain of the present application are mixed as a main component, and nutrients including water essential for survival and development and growth factors. Specifically, any medium and other culture conditions used for culturing the Corynebacterium stasis strain of the present application may be used without particular limitation as long as it is a medium used for culturing conventional microorganisms, but the Corynebacterium of the present application Rium stasis strains can be cultured while controlling temperature, pH, etc. under aerobic conditions in a conventional medium containing an appropriate carbon source, nitrogen source, phosphorus, inorganic compound, amino acid and/or vitamin.

Specifically, the culture medium for the Corynebacterium sp. strain 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 application, the carbon source includes carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, and the like; sugar alcohols such as mannitol and sorbitol; organic acids such as pyruvic acid, lactic acid, citric acid and the like; amino acids such as glutamic acid, methionine, lysine, and the like may be included. In addition, natural organic nutrient sources such as starch hydrolyzate, molasses, blackstrap molasses, rice winter, cassava, sugar cane offal and corn steep liquor can be used, specifically glucose and sterilized pre-treated molasses (i.e., converted to reducing sugar). molasses) may be used, and other appropriate amounts of carbon sources may be variously used without limitation. These carbon sources may be used alone or in combination of two or more, but is not limited thereto.

Examples of the nitrogen source include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, anmonium carbonate, and ammonium nitrate; Amino acids such as glutamic acid, methionine, glutamine, and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or degradation products thereof, defatted soybean cake or degradation products thereof, etc. can be used These nitrogen sources may be used alone or in combination of two or more, but is not limited thereto.

The phosphorus may include potassium first potassium phosphate, second potassium phosphate, or a sodium-containing salt corresponding thereto. As the inorganic compound, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc. may be used, and in addition, amino acids, vitamins and/or suitable precursors may be included. These components or precursors may be added to the medium either batchwise or continuously. However, the present invention is not limited thereto.

In addition, by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. to the medium in an appropriate manner during the culture of the Corynebacterium stasis strain of the present application, the pH of the medium may be adjusted. In addition, during culturing, an antifoaming agent such as fatty acid polyglycol ester may be used to suppress bubble formation. In addition, in order to maintain the aerobic state of the medium, oxygen or oxygen-containing gas may be injected into the medium, or nitrogen, hydrogen or carbon dioxide gas may be injected without or without gas to maintain anaerobic and microaerobic conditions, it is not

In the culture of the present application, the culture temperature may be maintained at 20 to 45° C., specifically, 25 to 40° C., and may be cultured for about 10 to 160 hours, but is not limited thereto.

The IMP produced by the culture of the present application may be secreted into the medium or may remain in the cell.

The IMP production method of the present application includes the steps of preparing the Corynebacterium stasis strain of the present application, preparing a medium for culturing the strain, or a combination thereof (regardless of the order, in any order) , for example, prior to the culturing step, may further include.

The IMP production method of the present application may further include recovering the IMP from the culture medium (cultured medium) or the Corynebacterium stationionis strain of the present application. The recovering step may be further included after the culturing step.

The recovery may be to collect the desired IMP using a suitable method known in the art according to the culture method of the microorganism of the present application, for example, a batch, continuous or fed-batch culture method. For example, centrifugation, filtration, treatment with a crystallized protein precipitating agent (salting out method), extraction, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity Various chromatography such as island chromatography, HPLC, or a combination thereof may be used, and a desired IMP may be recovered from a medium or a microorganism using a suitable method known in the art.

In addition, the IMP production method of the present application may additionally include a purification step. The purification may be performed using a suitable method known in the art. In one example, when the IMP production method of the present application includes both the recovery step and the purification step, the recovery step and the purification step are performed continuously or discontinuously, regardless of the order, or performed simultaneously or integrated into one step may be, but is not limited thereto.

In the method of the present application, variants, polynucleotides, vectors, strains, and the like are as described in the other aspects above.

Another aspect of the present application provides a variant of the present application, a polynucleotide encoding the variant, a vector including the polynucleotide or a Corynebacterium stasis strain comprising the polynucleotide of the present application; the culture medium; Or to provide a composition for producing IMP comprising a combination of two or more of them.

The composition of the present application may further include any suitable excipients commonly used in compositions for IMP production, and these excipients may be, for example, preservatives, wetting agents, dispersing agents, suspending agents, buffering agents, stabilizing agents or isotonic agents, etc. However, the present invention is not limited thereto.

In the composition of the present application, variants, polynucleotides, vectors, strains, media and IMPs are the same as those described in the other aspects above.

Another object of the present application is that alanine, which is an amino acid corresponding to position 351 of the amino acid sequence of SEQ ID NO: 3, is substituted with valine, consisting of the amino acid sequence set forth in SEQ ID NO: 1, bifunctional To provide an IMP production use of phosphoribosylaminoimidazolecarboxamide formyltransferase/IMP cyclohydrolase variants.

For use in the present application, variants and IMPs and the like are as described in the other aspects above.

Hereinafter, the present application will be described in more detail by way of Examples. However, the following examples are merely preferred embodiments for illustrating the present application, and therefore, are not intended to limit the scope of the present application thereto. On the other hand, technical matters not described in this specification can be sufficiently understood and easily implemented by those skilled in the art or similar technical fields of the present application.

Example 1: Construction of a vector for the expression of bifunctional phosphoribosylaminoimidazolecarboxamide formyltransferase/IMP cyclohydrolase variants in microorganisms

A mutant (A351V; SEQ ID NO: 1) in which alanine at position 351 of the bifunctional phosphoribosylaminoimidazolecarboxamide formyltransferase/IMP cyclohydrolase amino acid sequence (SEQ ID NO: 3) is substituted with valine (A351V; SEQ ID NO: 1) produces IMP In order to confirm the effect on the expression strain, the vector for the production of the expression strain was prepared as follows using the plasmid pDCM2 (Korea Publication No. 10-2020-0136813) for the insertion and replacement of genes in the Corynebacterium chromosome.

PCR was performed using the primer pair of the sequences of SEQ ID NOs: 5 and 6 and the primer pair of the sequences of SEQ ID NOs: 7 and 8 using gDNA (genomic DNA) of wild-type Corynebacterium stasis ATCC6872 as a template, respectively. Using the mixture of the two fragments obtained above as a template, overlapping PCR was performed again using a pair of primers of SEQ ID NO: 5 and SEQ ID NO: 8 to obtain a fragment. After denaturation at 94°C for 5 minutes, PCR was repeated 30 times at 94°C for 30 seconds, at 55°C for 30 seconds, and at 72°C for 1 minute and 30 seconds, and then at 72°C for 5 minutes. The pDCM2 vector was treated with SmaI and the PCR product obtained above was fusion cloned. Fusion cloning was performed using the In-Fusion® HD cloning kit (Clontech). The resulting plasmid was named pDCM2-purH (A351V).

Example 2: Evaluation of IMP production ability of microorganisms expressing bifunctional phosphoribosylaminoimidazole carboxamide formyltransferase/IMP cyclohydrolase mutant

2-1. Bifunctional phosphoribosylaminoimidazole carboxamide formyltransferase/IMP cyclohydrolase Production of mutant expression strains

The vector prepared in Example 1 was transformed into Corynebacterium stasis CJI2332 strain (KCCM12277P, KR 10-1950141 B1).

In the strain in which the homologous recombination occurred, the strain into which the mutant was introduced was selected using SEQ ID NOs: 9 and 10. The selected strain was named CJI2332_purH_A351V.

2-2. Comparison of IMP production capacity of bifunctional phosphoribosylaminoimidazole carboxamide formyltransferase/IMP cyclohydrolase mutant expression strains

IMP production ability was analyzed by evaluating the flask fermentation titer of the strain prepared in Example 2-1 and the control parent strain.

First, each strain was inoculated into a test tube with a diameter of 18 mm containing 2 ml of the seed medium, and cultured with shaking at 30 ° C. for 24 hours was used as a seed culture medium. 2 ml of the seed culture solution was inoculated into a 250ml corner-baffle flask containing 29ml of production medium (24ml of main medium + 5ml of separate sterile medium) and cultured at 30°C for 72 hours at 170rpm. After completion of the culture, IMP production capacity was measured by HPLC. IMP concentration and concentration increase rate in the culture medium for each strain tested are shown in Table 1 below.

<Seed medium (pH 7.5)>

Glucose 1%, peptone 1%, broth 1%, yeast extract 1%, sodium chloride 0.25%, adenine 100mg/l, guanine 100mg/l (based on 1 liter of distilled water)

<Production medium - main medium (pH 7.5)>

Yeast extract 0.2%, sodium glutamate 0.1%, magnesium sulfate 1.2%, calcium chloride 0.01%, iron sulfate 20mg/l, manganese sulfate 20mg/l, zinc sulfate 20mg/l, copper sulfate 5mg/l, L-cysteine 23mg/l, beta Alanine 24mg/l, nicotinic acid 8mg/l, biotin 45㎍/l, thiamine hydrochloride 5mg/l, adenine 30mg/l, glucose 2.55%, fructose 1.45% (based on 1 liter of distilled water)

<Production medium - separate sterilization medium (pH 7.5)>

Phosphoric acid (85%) 2.3%, potassium hydroxide (45%) 2.55% (based on 1 liter of distilled water)

The experiment was repeated 3 times, and the average value of the analysis results is shown in Table 1 below.

Comparison of IMP production capacity

strain	IMP concentration (g/L)	IMP concentration increase rate (%)
CJ2332	1.83	-
CJI2332_purH_A351V	2.20	20.2

As shown in Table 1, the CJI2332_purH_A351V strain exhibited a 20.2% increased IMP-producing ability compared to the control.

From the above description, those skilled in the art to which the present application pertains will be able to understand that the present application may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present application should be construed as including all changes or modifications derived from the meaning and scope of the claims described below, rather than the above detailed description, and equivalent concepts thereof, to be included in the scope of the present application.

Claims (5)

1. Bifunctional phosphoribosylaminoimidazole carboxamide formyltransferase/IMP cyclohydrolase consisting of the amino acid sequence shown in SEQ ID NO: 1, in which alanine, an amino acid corresponding to position 351 of SEQ ID NO: 3, is substituted with valine variant.
2. A polynucleotide encoding the variant of claim 1 .
3. A Corynebacterium stasis strain comprising the variant of claim 1 or a polynucleotide encoding the variant.
4. According to claim 3, wherein the strain has increased IMP production capacity compared to Corynebacterium stasis comprising the polypeptide of SEQ ID NO: 3 or a polynucleotide encoding it, the strain.
5. A method for producing IMP, comprising the step of culturing a Corynebacterium stasis strain comprising the mutant of claim 1 or a polynucleotide encoding the mutant in a medium.