阅读说明：本技术 新型多肽及使用其产生imp的方法 (Novel polypeptide and method for producing IMP using the same ) 是由 鲁镇雅 尹炳勋 朴素静 白珉知 李智惠 于 2019-01-04 设计创作，主要内容包括：本公开涉及具有输出5’-肌苷一磷酸的活性的新型蛋白变体,包含所述蛋白变体的微生物,和使用所述微生物制备5’-肌苷一磷酸的方法。(The present disclosure relates to novel protein variants having an activity of exporting 5 '-inosine monophosphate, a microorganism comprising the protein variants, and a method of preparing 5' -inosine monophosphate using the microorganism.)

1. A protein variant having an activity of exporting 5' -inosine monophosphate, wherein the protein variant consists of an amino acid sequence comprising, from the N-terminus of the amino acid sequence of SEQ ID No. 2, substitution of the 123 th amino acid with cysteine, substitution of the 243 th amino acid with valine, substitution of the 387 th amino acid with threonine, and substitution of the 405 th amino acid with tyrosine; substitution of threonine for amino acid 413, lysine for amino acid 458, or a combination thereof.

2. The protein variant according to claim 1, wherein the protein variant consists of an amino acid sequence further comprising, from the N-terminus of the amino acid sequence of SEQ ID NO. 2, the substitution of the 2 nd amino acid with another amino acid, the substitution of the 64 th amino acid with another amino acid, or a combination thereof.

3. A polynucleotide encoding the protein variant according to claim 1.

4. A vector comprising the polynucleotide of claim 3.

5. A microorganism of the genus Corynebacterium which produces 5' -inosine monophosphate comprising the protein variant of claim 1, the polynucleotide of claim 3 or the vector of claim 4.

6. The microorganism of the genus Corynebacterium according to claim 5, wherein the microorganism of the genus Corynebacterium is Corynebacterium parvum (Corynebacterium parvum: (Corynebacterium parvum)) (Corynebacterium parvum)Corynebacterium stationis)。

7. The microorganism of the genus Corynebacterium according to claim 5, wherein an activity of adenylosuccinate synthetase or 5' -Inosine Monophosphate (IMP) dehydrogenase is further attenuated.

8. A method for producing 5' -inosine monophosphate, which comprises culturing the microorganism belonging to the genus Corynebacterium of claim 5 in a culture medium.

9. The method of claim 8, wherein the method further comprises recovering 5' -inosine monophosphate from the microorganism or culture medium.

10. The method of claim 8, wherein the microorganism of the genus Corynebacterium is Corynebacterium parvum.

11. Use of a protein variant according to claim 1 for increasing the production of 5' -inosine monophosphate in a microorganism of the genus corynebacterium.

12. A method for increasing the export of 5' -inosine monophosphate comprising enhancing the activity of the protein consisting of SEQ ID NO. 2 in a microorganism of the genus Corynebacterium.

13. Use of a protein variant according to claim 1 for increasing the export of 5' -inosine monophosphate in a microorganism of the genus corynebacterium.

[ technical field ]

The present disclosure relates to novel protein variants having an activity of exporting 5' -inosine monophosphate, a microorganism containing the protein variants, a method for preparing 5' -inosine monophosphate using the microorganism, and a method for increasing the export of 5' -inosine monophosphate using the microorganism.

[ background art ]

5' -inosine monophosphate (hereinafter, IMP) is a nucleic acid substance, is an intermediate of a nucleic acid metabolic pathway, and is used in many fields such as foods, medicines, various medical applications, and the like. Specifically, IMP is widely used as a food seasoning or an additive for food together with 5' -guanine monophosphate (hereinafter, GMP). Although IMP itself is known to provide beef flavour, it is known to enhance the flavour of sodium glutamate (MSG) and is therefore of interest as a flavour enhancing nucleic acid based flavouring.

Examples of the method for producing IMP include a method for enzymatically degrading ribonucleic acid extracted from yeast cells (japanese patent laid-open No. 1614/1957), a method for chemically phosphorylating inosine produced by fermentation: (a method for producing IMP)Agri. Biol. Chem.36, 1511(1972), etc.), a method for culturing a microorganism which can directly produce IMP and recovering IMP in the culture solution, etc. Among these, the most frequently used method at present is a method using a microorganism capable of directly producing IMP.

Meanwhile, since enzymes do not always exhibit the optimum characteristics in nature with respect to activity, stability, substrate specificity to optical isomers, and the like required in industrial applications, various attempts have been made to improve enzymes to be suitable for the intended use by mutating their amino acid sequences, and the like. Among these, although rational design of enzymes and site-directed mutagenesis have been applied to improve enzyme functions, in many cases, these attempts have been shown to be disadvantageous because information about the structure of target enzymes is insufficient or the structure-function correlation is unclear, thus preventing their effective use. Furthermore, a method of improving enzyme activity by attempting to enhance enzymes through directed evolution for screening enzymes of desired traits from a library of modified enzymes constructed by random mutagenesis of enzyme genes has been previously reported.

[ disclosure ]

[ problem ] to

In order to produce IMP at a high yield by direct IMP production via microbial fermentation, it is necessary to smoothly perform IMP output. In order to achieve the object of the present disclosure, the inventors of the present disclosure have conducted extensive studies, and as a result, have identified proteins involved in the activity of exporting IMP, and have also found protein variants having higher activities of exporting IMP, thereby completing the present disclosure.

[ solution ]

It is an object of the present disclosure to provide protein variants having activity to export IMP.

It is another object of the present disclosure to provide polynucleotides encoding the protein variants of the present disclosure.

It is yet another object of the present disclosure to provide a vector comprising a polynucleotide of the present disclosure.

It is yet another object of the present disclosure to provide an IMP producing microorganism, wherein said microorganism comprises a protein variant of the present disclosure and a vector of the present disclosure.

It is still another object of the present disclosure to provide a method for producing IMP, which comprises culturing a microorganism of the genus corynebacterium of the present disclosure in a culture medium, and recovering IMP from the microorganism or the culture medium.

It is still another object of the present disclosure to provide a method for increasing IMP export, which comprises the step of enhancing the activity of IMP-exporting proteins in a microorganism of the genus corynebacterium.

To achieve the above object, one aspect of the present disclosure provides a protein variant having an activity of exporting IMP.

As used herein, the term "protein that exports 5' -Inosine Monophosphate (IMP)" refers to a protein that participates in the extracellular export of IMP. For the purposes of this disclosure, the term may be used interchangeably with a protein having activity to export IMP, an IMP export protein, a protein capable of exporting IMP, an IMP-export protein, and the like; in particular, the protein may be represented as ImpE, more specifically, ImpE1 or ImpE2, and even more specifically, the output protein of the present disclosure may be represented as ImpE2, but the representation of the protein is not limited thereto. Furthermore, the protein may be derived from a microorganism of the genus Corynebacterium, and in particular from Corynebacterium parvum (C.stagnanum) ((C.stagnanum))Corynebacterium stationis) However, the microorganism is not limited thereto.

The protein may be a protein comprising the amino acid sequence represented by SEQ ID NO. 2 or a protein consisting of the amino acid sequence represented by SEQ ID NO. 2, but may include, but is not limited to, any sequence having the same activity as the protein, and sequence information can be obtained from GenBank (well known database) of NCBI by one of ordinary skill in the art. Furthermore, a protein of the present disclosure that exports IMP may be a protein comprising the amino acid sequence of SEQ ID No. 2 or an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology or identity to the sequence of SEQ ID No. 2. Further, it is apparent that any protein having an amino acid sequence having a deletion, mutation, substitution or addition in a part of the sequence can also be included in the scope of the present disclosure as long as the amino acid sequence has the above-mentioned homology or identity and has an effect corresponding to the protein.

That is, although described in the present disclosure as "a protein having an amino acid sequence represented by a specific SEQ ID NO" or "a protein consisting of a specific SEQ ID NO", it is apparent that a protein having an amino acid sequence with deletion, modification, substitution, conservative substitution, or addition of some amino acids falls within the scope of the present invention as long as the protein has the same or equivalent activity as that of a protein consisting of the amino acid sequence of the corresponding SEQ ID NO. For example, the above expression does not exclude sequence additions upstream or downstream of the amino acid sequence that do not alter the function of the protein, naturally occurring mutations therein, silent mutations therein, or conservative substitutions, and even in the case of such sequence additions or mutations, as long as the protein has the same or equivalent activity as the protein variants of the present disclosure, it being apparent that the protein also falls within the scope of the present disclosure.

In the present disclosure, "homology" and "identity" refer to the degree of correlation between two given amino acid sequences or nucleotide sequences and can be expressed as a percentage.

The terms "homology" and "identity" are often used interchangeably with each other.

Sequence homology or identity of conserved polynucleotide or polypeptide sequences can be determined by standard alignment algorithms and can be used with default gap penalties established by the program used. Substantially homologous or identical polynucleotides or polypeptides can typically hybridize along the entire length or at least about 50%, about 60%, about 70%, about 80%, or about 90% or more of the entire length at moderate or high stringency. In hybridizations, polynucleotides that include degenerate codons, rather than codons, are also contemplated.

Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined, for example, using known computer algorithms, such as the "FASTA" program using default parameters (e.g., Pearson et al, (1988)Proc. Natl. Acad. Sci. USA85: 2444). Alternatively, it may be determined using: the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970,J. Mol. Biol.48:443-,Trends Genet.16:276-,Nucleic Acids Research12:387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [ S ].][F.]And the like,J Molec Bio215]403 (1990), Guide toHuge Computers, Martin J. Bishop, [ eds ],]academic Press, San Diego, 1994, and [ CARILLO ETA/.](1988)SIAM J Applied Math48: 1073). For example, the homology, similarity or identity can be determined using BLAST or ClustalW from National Center for Biotechnology Information (NCBI).

The homology, similarity or identity of polynucleotide or polypeptide sequences can be determined by using, for example, the GAP computer programs as disclosed (e.g., Smith and Waterman,Adv. Appl. Math(1981) 2:482) comparison of sequence informationAnd then determined. In short, the GAP program defines homology or identity as the value obtained by dividing the number of symbols (i.e., nucleotides or amino acids) that are similarly aligned by the total number of symbols for the shorter of the two sequences. Default parameters for the GAP program may include: (1) unary comparison matrices (containing a value of 1 for identity and 0 for non-identity) and Gribskov et al (1986)Nucl.Acids Res.14:6745, such as Schwartz and Dayhoff, eds,Atlas of Protein Sequence and Structurenational Biomedical Research Foundation, pp.353-358, 1979; (2) a penalty of 3.0 per gap and an additional penalty of 0.10 per symbol in each gap (or a gap opening penalty of 10 and a gap extension penalty of 0.5); and (3) no penalty for end gaps. Thus, as used herein, the term "homology" or "identity" refers to the correlation between sequences.

As used herein, the term "variant" refers to a polypeptide that differs from the sequence in conservative substitutions and/or changes in one or more amino acids, but that retains the function or properties of the protein. Variants differ from the identified sequence by being distinguished by several amino acid substitutions, deletions or additions. Such variants can generally be identified by altering one amino acid of the polypeptide sequence and evaluating the properties of the variant. That is, the capacity of a variant protein may be increased, unchanged, or decreased as compared to the capacity of the native protein. In addition, some altered polypeptides may include altered polypeptides in which one or more portions (e.g., N-terminal leader sequence, transmembrane domain, etc.) are removed. Other variants may include variants in which a small portion has been removed from the N-and/or C-terminus of the mature protein. As used herein, the term "conservative substitution" refers to the replacement of one amino acid with another amino acid having similar structural and/or chemical properties. The variants may have, for example, one or more conservative substitutions, while still retaining one or more biological activities. Such amino acid substitutions may generally be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature. For example, positively charged (basic) amino acids include arginine, lysine and histidine; and the negatively charged (acidic) amino acids include glutamic acid and aspartic acid; aromatic amino acids include phenylalanine, tryptophan, and tyrosine; and hydrophobic amino acids include alanine, valine, isoleucine, leucine, methionine, phenylalanine, proline, glycine, and tryptophan.

In addition, the variants may also include deletions or additions of amino acids that have minimal effect on the properties and secondary structure of the polypeptide. For example, the polypeptide may be conjugated at the N-terminus of the protein to a signal (or leader) sequence that is involved in co-translation or post-translational transfer of the protein. The polypeptide may also be conjugated to another sequence or linker to allow identification, purification, or synthesis of the polypeptide.

Specifically, the protein variant having an activity of exporting IMP of the present disclosure may be a protein variant having an amino acid sequence in which, from the N-terminus of the amino acid sequence of SEQ ID No. 2, at least one is selected from the group consisting of 123 th amino acid, 243 rd amino acid, 387 th amino acid, 405 th amino acid; the amino acids of the 413 th amino acid and the 458 th amino acid are substituted with another amino acid, but the amino acid substitution is not limited thereto.

For example, the protein variant having IMP export activity of the present disclosure may be a protein variant having IMP-export activity, which may be a protein variant having substitution of the 123 th amino acid with cysteine (i.e., F123C) from the N-terminus of the amino acid sequence of SEQ ID NO: 2; substitution of amino acid 243 with valine (i.e., I243V); substitution of the 387 amino acids with threonine (i.e., S387T); substitution of the 405 th amino acid with tyrosine (i.e., F405Y); substitution of amino acid 413 with threonine (i.e., M413T); substitution of amino acid 458 with lysine (i.e., N458K); or a combination thereof, but the amino acid substitution is not limited thereto. More specifically, the protein variant having an activity of exporting IMP may be a protein having an amino acid sequence selected from the group consisting of SEQ ID NOs 73, 74, 75, 76, 77, 78, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153 and 155, or an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more homology to these amino acid sequences. Further, it is apparent that any protein having an amino acid sequence in which a part of the amino acid sequence is deleted, modified, substituted or added may also be used as the protein of the present disclosure as long as the amino acid sequence has the above-mentioned sequence homology and shows the same action as that of the above-mentioned polypeptide.

In addition, the protein variant having an activity of exporting IMP of the present disclosure may be a protein variant consisting of an amino acid sequence further comprising, from the N-terminus of the amino acid sequence of SEQ ID NO:2, substitution of the 2 nd amino acid with another amino acid, substitution of the 64 th amino acid with another amino acid, or a combination thereof. Specifically, the protein variant having an activity of exporting IMP of the present disclosure may be a protein variant further comprising, from the N-terminus in the amino acid sequence of SEQ ID NO:2, substitution of the 2 nd amino acid with isoleucine, substitution of the 64 th amino acid with glutamic acid or aspartic acid, or a combination thereof.

The "substitution with another amino acid" is not limited as long as the other amino acid is an amino acid different from the amino acid before the substitution. For example, when the 2 nd amino acid from the N-terminus in the amino acid sequence of SEQ ID NO. 2 is substituted with another amino acid, the other amino acid is not limited as long as the other amino acid is an amino acid other than valine, and when the 64 th amino acid from the N-terminus in the amino acid sequence of SEQ ID NO. 2 is substituted with another amino acid, the other amino acid is not limited as long as the other amino acid is an amino acid other than glycine.

Another aspect of the disclosure provides a polynucleotide encoding a protein variant of the disclosure, or a vector comprising a polynucleotide of the disclosure.

As used herein, the term "polynucleotide" refers to a polymer of nucleotides in which the nucleotide monomers extend in a long chain by covalent bonds and which have a DNA or RNA strand longer than a certain length.

With respect to the polynucleotides of the present disclosure, it is apparent that, based on codon degeneracy, a protein consisting of the amino acid sequence of SEQ ID NOs 73, 74, 75, 76, 77, 78, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, or 155, or a polynucleotide that can be translated into a protein having homology to the above-mentioned protein may also be included in the scope of the present disclosure. For example, a polynucleotide of the present disclosure may be a polynucleotide sequence having a base sequence of SEQ ID NO 79, 80, 81, 82, 83, 84, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, or 156, and more specifically, a polynucleotide consisting of a base sequence of SEQ ID NO 79, 80, 81, 82, 83, 84, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, or 156. Furthermore, any sequence encoding a protein having the activity of a protein having the amino acid sequence of SEQ ID NO: 73, 74, 75, 76, 77, 78, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, or 155, as determined by hybridization under stringent conditions with a probe that can be prepared from a known gene sequence (e.g., a sequence complementary to all or part of the above-described nucleotide sequence), can be included and is not limited.

The term "stringent conditions" refers to conditions that enable specific hybridization between polynucleotides. Such conditions are specifically described in the literature references (e.g., j. Sambrook et al, supra). For example, the conditions may include hybridization between genes having high homology, 40% or more, specifically 90% or more, more specifically 95% or more, even more specifically 97% or more, and most specifically 99% or more, without hybridization between genes having homology lower than the above; or in the use of southern hybridization at 60 ℃, 1 x SSC and 0.1% SDS conventional washing conditions, specifically in corresponding to 60 ℃, 0.1 x SSC and 0.1% SDS, and more specifically 68 ℃, 0.1 x SSC and 0.1% SDS salt concentration and temperature for one, specifically two or three times hybridization.

Hybridization requires that the two nucleic acids have complementary sequences, although depending on the stringency of the hybridization, mismatches between bases may be possible. The term "complementary" is used to describe the relationship between nucleotide bases that can hybridize to each other. For example, in the case of DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. Thus, the disclosure may also include isolated nucleic acid fragments that are complementary to the entire sequence as well as substantially similar nucleic acid sequences.

Specifically, hybridization conditions including a hybridization step and T at 55 ℃ using the above conditions can be used_mPolynucleotides having homology are detected. Furthermore, T_mThe value may be 60 ℃, 63 ℃ or 65 ℃, but is not limited thereto, and may be appropriately adjusted by one of ordinary skill in the art according to the intended purpose.

The stringency suitable for hybridization of polynucleotides depends on the length and complementarity of the polynucleotides, and the relevant variables are well known in the art (see Sambrook et al, supra, 9.50 to 9.51 and 11.7 to 11.8).

In the present disclosure, the polynucleotide encoding the amino acid sequence of the protein having IMP export activity may beimpE2The genes, and the polynucleotide interpretation is as described above.

In the present disclosure, the explanation of the polynucleotide encoding the protein variant having IMP-export activity is also as described above.

As used herein, the term "vector" refers to a DNA construct comprising a nucleotide sequence of a polynucleotide encoding a target protein, wherein the target protein is operably linked to suitable control sequences such that the target protein can be expressed in a suitable host. The control sequences may include a promoter capable of initiating transcription, any operator sequence used to control transcription, sequences encoding the appropriate mRNA ribosome binding domain, and sequences which control termination of transcription and translation. Upon transformation into a suitable host cell, the vector may replicate or function independently of the host genome, or may integrate into the host genome itself.

The vector used in the present disclosure may not be particularly limited as long as the vector is replicable in host cells, and it may be constructed using any vector known in the art. Examples of vectors may include natural or recombinant plasmids, cosmids, viruses, and bacteriophages. For example, as a phage vector or cosmid vector, can be usedpWE15、M13、MBL3、MBL4、IXII、ASHII、APII、t10、t11、Charon4A、Charon21AEtc., and as the plasmid vector, those based onpBR、pUC、pBluescriptII、pGEM、pTZ、pCL、pETAnd the like. In particular, use may be made ofpDZ、pACYC177、pACYC184、pCL、pECCG117、pUC19、pBR322、pMW118、pCC1BACCarriers, and the like.

In one embodiment, a polynucleotide encoding a target protein can be replaced with an altered polynucleotide (variant) within a chromosome using a vector for insertion into the chromosome in a cell. The insertion of the polynucleotide into the chromosome can be performed using methods known in the art (e.g., by homologous recombination), but is not limited thereto. Specifically, a selection marker for confirming the insertion into the chromosome may be further included. The selectable marker is used to select for transformed cells, i.e., to confirm whether a target nucleic acid has been inserted, and markers that provide a selectable phenotype such as drug resistance, nutrient requirements, resistance to cytotoxic agents, and expression of surface proteins can be used. In the case of treatment with a selective agent, only cells capable of expressing the selectable marker can survive or express other phenotypic traits, and thus transformed cells can be easily selected.

Yet another aspect of the present disclosure provides a microorganism that can produce IMP, comprising a protein variant of the present disclosure, a polynucleotide encoding a protein variant of the present disclosure, or a vector of the present disclosure. Specifically, the microorganism of the present disclosure may be a microorganism prepared by transformation using a vector containing a polynucleotide encoding a protein variant of the present disclosure, but the microorganism is not limited thereto.

As used herein, the term "transformation" refers to the process of: introducing a vector comprising a polynucleotide encoding a target protein into a host cell, thereby enabling expression of the protein encoded by the polynucleotide in the host cell. It does not matter whether the transformed polynucleotide is inserted into and located in or extrachromosomally of the host cell, as long as the transformed polynucleotide can be expressed in the host cell. In addition, the polynucleotides include DNA and RNA encoding the target protein. The polynucleotide may be inserted in any form as long as it can be introduced into and expressed in a host cell. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a genetic construct containing all the necessary elements required for autonomous expression. The expression cassette can conventionally comprise a promoter, transcription termination signal, ribosome binding domain and translation termination signal operably linked to the polynucleotide. The expression cassette may be in the form of a self-replicating expression vector. In addition, the polynucleotide may be introduced into the host cell as it is and operably linked to a sequence necessary for its expression in the host cell, but is not limited thereto.

Furthermore, as used herein, the term "operably linked" refers to a functional linkage between a promoter sequence that initiates and mediates transcription of a polynucleotide encoding a target protein (i.e., a conjugate of the present disclosure) and the gene sequences described above.

As used herein, the term "IMP-producing microorganism" refers to a microorganism that is naturally capable of producing IMP; or a microorganism whose parent strain is not capable of naturally producing and/or exporting IMP but which is provided with the ability to produce or export IMP. In the present disclosure, the IMP producing microorganism may be used interchangeably with the IMP exporting microorganism or the microorganism having the activity of exporting IMP.

An IMP producing microorganism is a host cell or a microorganism which comprises a protein variant having an activity of exporting IMP or a polynucleotide encoding the protein variant, or which is transformed with a vector containing a polynucleotide encoding the protein variant, and thereby is capable of expressing the protein variant. Specifically, the microorganism of the present disclosure may be a microorganism of the genus Escherichia, a microorganism of the genus Serratia, a microorganism of the genus Erwinia, a microorganism of the genus Enterobacter, a microorganism of the genus Salmonella, a microorganism of the genus Streptomyces, a microorganism of the genus Pseudomonas, a microorganism of the genus Brevibacterium, a microorganism of the genus Corynebacterium, or the like, and more specifically, the microorganism of the present disclosure may be a microorganism of the genus Corynebacterium.

As used herein, the term "microorganism of the genus corynebacterium which produces IMP" refers to a microorganism of the genus corynebacterium which is naturally capable of producing IMP or capable of producing IMP by mutation. Specifically, as used herein, a microorganism of the genus corynebacterium capable of producing IMP may be a natural strain of a microorganism of the genus corynebacterium capable of producing IMP; or a microorganism of the genus Corynebacterium having an enhanced ability to produce IMP, which is prepared by inserting a gene associated with IMP production or by enhancing or attenuating an endogenous gene associated with IMP production. More specifically, in the present disclosure, the microorganism of the genus corynebacterium capable of producing IMP may be a microorganism of the genus corynebacterium having an increased ability to produce IMP by comprising a protein variant having an activity of exporting IMP or a polynucleotide encoding the protein variant or by transforming with a vector containing a polynucleotide encoding the protein variant. The "microorganism of the genus Corynebacterium having an enhanced ability to produce IMP" may be a microorganism of the genus Corynebacterium which has an enhanced ability to produce IMP as compared to its parent strain before transformation or an unmodified microorganism of the genus Corynebacterium. The "unmodified microorganism of the genus Corynebacterium" may be a natural type of microorganism of the genus Corynebacterium, or a microorganism of the genus Corynebacterium which does not contain a protein variant capable of exporting IMP, or a microorganism of the genus Corynebacterium which is not transformed with a vector containing a polynucleotide encoding a protein variant capable of exporting IMP.

In one embodiment of the present disclosure, the microorganism of the present disclosure may be a microorganism in which the activity of adenylyl succinate synthetase and/or IMP dehydrogenase is further attenuated.

Specifically, the microorganism of the present disclosure may be corynebacterium glutamicum ((c))Corynebacterium glutamicum) Corynebacterium ammoniagenes (C.ammoniagenes) (C.ammoniagenes)Corynebacterium ammoniagenes) Brevibacterium lactofermentum (A)Brevibacterium lactofermentum) Brevibacterium flavum (A)Brevibacterium flavum) Corynebacterium thermoaminogenes (C.thermophilum) ((C.thermophilum))Corynebacterium thermoaminogenes) Effective coryneform bacterium (A)Corynebacterium efficiens) Or Corynebacterium parvum (Corynebacterium stationis) However, the microorganism is not limited thereto.

Yet another aspect of the present disclosure provides a method for producing IMP, comprising culturing the IMP-producing microorganism of the present disclosure in a medium.

Specifically, the methods of the present disclosure may further comprise the step of recovering IMP from the microorganisms or the culture medium of the present disclosure.

In the above-mentioned method of the present disclosure, the culture of the microorganism may be performed in a batch method, a continuous method, a fed-batch method, etc. known in the art, but the culture method is not particularly limited thereto. Specifically, with respect to the culture conditions, the pH of the culture may be adjusted to a suitable pH (e.g., pH 5 to 9, specifically pH 6 to 8, and most specifically with an appropriate alkaline compound (e.g., sodium hydroxide, potassium hydroxide, or ammonia) or acidic compound (e.g., phosphoric acid or sulfuric acid), and aerobic conditions of the culture may be maintained by introducing oxygen or an oxygen-containing gas mixture into the culture.

In addition, examples of the carbon source to be used in the medium may include sugars and carbohydrates (e.g., glucose, sucrose, lactose, fructose, maltose, molasses, starch, and cellulose); oils and fats (e.g., soybean oil, sunflower oil, peanut oil, and coconut oil); fatty acids (e.g., palmitic acid, stearic acid, and linoleic acid); alcohols (e.g., glycerol and ethanol); and organic acids (e.g., acetic acid), but are not limited thereto. These carbon sources may be used alone or in combination, but are not limited thereto. Examples of the nitrogen source to be used in the medium may include nitrogen-containing organic compounds (e.g., peptone, yeast extract, gravy, malt extract, corn steep liquor, soybean powder, and urea) or inorganic compounds (e.g., ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate), and the like. These nitrogen sources may be used alone or in combination, but are not limited thereto. Examples of the phosphorus source to be used in the medium may include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, corresponding sodium-containing salts, and the like, but are not limited thereto. In addition, the medium may contain metal salts (e.g., magnesium sulfate or iron sulfate), amino acids, vitamins, and the like, which are essential growth promoting materials.

In the present disclosure, the method for recovering IMP produced in the step of culturing may be performed by collecting IMP from the culture broth using an appropriate method known in the art. For example, methods such as centrifugation, filtration, anion exchange chromatography, crystallization, HPLC, and the like may be used, and the desired IMP may be recovered from the culture or cultured microorganism using appropriate methods known in the art.

Further, the recovery step may include a purification process, and may be performed using an appropriate method known in the art. Thus, the IMP to be recovered may be in purified form or a microbial fermentation broth containing IMP.

Yet another aspect of the present disclosure provides a composition for producing IMP, comprising a protein variant or a polynucleotide encoding the protein variant of the present disclosure capable of exporting IMP.

The compositions of the present disclosure may further comprise, without limitation, any composition capable of manipulating the polynucleotide. In the compositions of the present disclosure, the polynucleotide may be in a form wherein the polynucleotide is contained in a vector such that an operably linked gene can be expressed in a host cell into which the polynucleotide is introduced.

In addition, the composition may further contain any suitable excipient (e.g., preservative, humectant, dispersing agent, suspending agent, buffer, stabilizer, isotonic agent, etc.) conventionally used in the composition for IMP production, but the suitable excipient is not limited thereto.

Yet another aspect of the present disclosure provides the use of a protein variant of the present disclosure for increasing IMP production in a microorganism of the genus corynebacterium.

Yet another aspect of the present disclosure provides a method for increasing the export of IMP, comprising enhancing the activity of a protein consisting of SEQ ID No. 2 in a microorganism of the genus corynebacterium. Specifically, enhancement of the activity of the protein consisting of SEQ ID NO. 2 can be performed by introducing, applying or containing a protein variant capable of exporting IMP, wherein the protein variant consists of an amino acid sequence having, from the N-terminus in the amino acid sequence of SEQ ID NO. 2, a substitution of 123 th amino acid with another amino acid, a substitution of 243 th amino acid with another amino acid, a substitution of 387 th amino acid with another amino acid, and a substitution of 405 th amino acid with another amino acid; replacing the 413 th amino acid with another amino acid, replacing the 458 th amino acid with another amino acid, or a combination thereof. The terms "protein capable of exporting IMP", "protein variant capable of exporting IMP" and "microorganism of the genus Corynebacterium" are as described above.

Yet another aspect of the present disclosure provides the use of a protein variant of the present disclosure for increasing the export of IMP in a microorganism of the genus corynebacterium.

[ advantageous effects of the invention ]

IMP can be produced in high yield by culturing a microorganism of the genus corynebacterium which produces IMP and is capable of exporting a protein variant of IMP.

[ detailed description of the invention ]

The present disclosure will be described in detail as follows. Meanwhile, each of the explanations and exemplary embodiments disclosed herein may be applied to other respective explanations and exemplary embodiments. That is, all combinations of the various factors disclosed herein are within the scope of the present disclosure. Furthermore, the scope of the present disclosure should not be limited by the specific disclosure provided below.

Example 1: discovery of IMP export proteins

Preparation of stagnant CorynebacteriumATCC6872For identifying membrane proteins of coryneform bacteria involved in export of IMP. Then, since the wild-type strain of Corynebacterium does not produce IMP or, even if it does, it produces only a small amount of IMP, it is prepared to be derived fromATCC6872Strains capable of producing IMP are referred toCJI0323For identifying the ability to produce IMP. Use ofATCC6872Genomic DNA library of the strain, preparedCJI0323The strain is used for screening membrane protein participating in IMP output. The details of the experiment are as follows.