Mutant prolyl isomerase and method for producing glutamate series amino acids using the same

Abstract

This application relates to a mutant prolyl isomerase and a method for producing glutamate series amino acids using the same.

Description

Technical Field

[0001] This application relates to a mutant prolyl isomerase and a method for producing glutamate series amino acids using the same.

Background Art

[0002] Glutamate is one of the proteinaceous amino acids widely found in plants, animals, and microorganisms, and is converted and metabolized into ornithine (L-ornithine), citrulline (L-citrulline), arginine (L-arginine), glutamine (L-glutamine), etc. in internal organs.

[0003] Glutamate has a unique and distinctive taste and is widely used not only in the food field but also in the pharmaceutical field and other animal feed fields. Ornithine is used as a nutritional supplement because it is effective in muscle formation and reduction of body fat, and is also used as a pharmaceutical for improving cirrhosis and liver dysfunction. Citrulline is known to have physiological activities such as promoting ammonia metabolism, improving blood flow by vasodilation, lowering blood pressure, neurotransmission, enhancing immunity, and scavenging reactive oxygen species. Arginine is used as a pharmaceutical such as a liver function promoter, a brain function promoter, and a comprehensive amino acid preparation, and is also used as a food additive such as kamaboko additive, health drink additive, and salt substitute for hypertensive patients.

[0004] Enhancing the glutamic acid biosynthesis pathway can increase not only glutamic acid but also glutamic acid-series amino acids such as ornithine, citrulline, arginine, and glutamine. Methods for producing such glutamic acid mainly involve fermentation using Corynebacterium and Corynebacterium species and their mutants (Amino Acid Fermentation, Gakkai Shuppan Center: 195-215, 1986). Other known methods include using microorganisms such as Escherichia coli, Bacillus subtilis, Streptomyces, Penicillium, Klebsiella, Erwinia, and Pantoea (US Patent Publication No. 6682912). [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] U.S. Patent Publication No. 6682912 [Patent Document 2] U.S. Registered Patent US 7662943 B2 [Patent Document 3] U.S. Registered Patent US 10584338 B2 [Patent Document 4] U.S. Registered Patent US 10273491 B2 [Patent Document 5] US 2023-0134555 A1 [Patent Document 6] Registered Patent No. 10-0048440 of the Republic of Korea [Non-patent literature]

[0006] [Non-Patent Document 1] Amino Acid Fermentation, Gakkai Shuppan Center: 195-215, 1986 [Non-Patent Document 2] Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]:2444 [Non-licensed document 3] Rice et al., 2000, Trends Genet. 16:276-277

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Non-licensed literature 9

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[0007] This application relates to a mutant prolyl isomerase and a method for producing glutamate series amino acids using the same. [[Means for Solving the Problems]]

[0008] This application provides a mutant prolyl isomerase in which the amino acid corresponding to the 95th position in the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid and has at least 90% sequence identity with SEQ ID NO: 1.

[0009] This application provides a polynucleotide encoding the mutant prolyl isomerase of this application.

[0010] This application provides a microorganism containing the mutant prolyl isomerase of this application; or a polynucleotide encoding the same.

[0011] This application provides a method for producing glutamate series amino acids, which includes culturing a microorganism containing the mutant prolyl isomerase of this application; or a polynucleotide encoding the same; in a medium. [[Effects of the Invention]]

[0012] When culturing a microorganism that produces glutamate series amino acids using the mutant prolyl isomerase of this application, the production of glutamate series amino acids containing ornithine, citrulline, arginine, and glutamine increases compared to a microorganism having an existing wild-type prolyl isomerase. [Modes for carrying out the invention]

[0013] This can be explained in more detail as follows: On the other hand, each description and embodiment disclosed in this application also applies 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. Furthermore, the categories of this application are not limited by the specific descriptions described below. In addition, numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated as a whole into this specification by reference to more clearly explain the level of the technical field to which this application belongs and the content of this application.

[0014] One aspect of this application provides a mutant prolyl isomerase in which the amino acid corresponding to the 95th position in the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid, and which has at least 90% sequence identity with SEQ ID NO: 1.

[0015] In this application, the term "mutant prolyl isomerase" means any polypeptide or prolyl isomerase having the function of regulating prolyl isomerase activity, and includes a variant in which the amino acid corresponding to the 95th position from the N-terminus of SEQ ID NO: 1 is replaced with another amino acid. That is, in a mutant prolyl isomerase in which the amino acid corresponding to the 95th position in the amino acid sequence of SEQ ID NO: 1 of this application is replaced with another amino acid, SEQ ID NO: 1 is a reference sequence, which is a sequence used to determine the position of an amino acid within the amino acid sequence of any polypeptide or prolyl isomerase having prolyl isomerase activity. The aforementioned "mutant prolyl isomerase" may also be referred to as "prolyl isomerase variant," "fkpA variant," "mutant fkpA," "FkpA variant," "mutant FkpA," "PPIase variant," "mutant PPIase," etc.

[0016] The proteins to which mutations are introduced in this application may be proteins having prolyl isomerase activity. Specifically, the protein may, but is not limited to, a protein containing the amino acid sequence of SEQ ID NO: 1 and having prolyl isomerase activity. Any protein that has the same or corresponding activity as a protein containing the amino acid sequence of SEQ ID NO: 1 is eligible for mutation introduction in this application, excluding the addition of meaningless sequences before or after the amino acid sequence of SEQ ID NO: 1, naturally occurring mutations, or silent mutations thereof. For example, the proteins to which mutations are introduced in this application may be proteins containing the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity thereto, or proteins consisting of or substantially composed of such homologous or identical amino acid sequences. Furthermore, it is obvious that proteins having amino acid sequences in which some sequences are deleted, modified, substituted, or added, as long as they have such homology or identity and exhibit the efficacy corresponding to the aforementioned protein, are also included within the range of proteins targeted by mutation in this application.

[0017] In this application, the term "prolyl isomerase (prolyl cis / trans isomerase)" refers to an enzyme expressed in clusters with citrate synthase (CS) within microorganisms, which delays the aggregation of citrate synthase. In addition, it is known that the N-terminal domain of prolyl isomerase, which possesses chaperone function, exhibits effective chaperone function within microorganisms under high-temperature conditions. The aforementioned prolyl isomerase may be used interchangeably with "peptidyl-prolyl cis-trans isomerase," "FKBP-type peptidyl-prolyl cis-trans isomerase," "FkpA," or "PPIase." The amino acid sequence of FkpA can be obtained from known databases such as NCBI's Genebank.

[0018] For example, the FkpA protein of this application may be of microorganism origin, specifically prokaryotic or eukaryotic, and more specifically, may be derived from microorganisms of the genus Corynebacterium, but is not limited thereto.

[0019] As another example, the FkpA protein may be NCgl0796 (Cg0950) derived from a microorganism of the genus Corynebacterium, but it is obvious that it includes proteins with prolyl isomerase activity from a variety of origins.

[0020] In this application, the amino acid in the original amino acid sequence of the FkpA protein to be mutated, corresponding to position 95 of SEQ ID NO: 1, may be tyrosine (Y).

[0021] The mutant prolyl isomerase of this application may be one in which the amino acid at the position corresponding to position 95 in the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid.

[0022] As an example, the mutant prolyl isomerase may be such that the amino acid corresponding to the 95th position in the amino acid sequence of SEQ ID NO: 1 is substituted with one or more amino acids selected from the group consisting of methionine, serine, threonine, asparagine, cysteine, histidine, lysine, aspartic acid, alanine, valine, leucine, glutamine, glycine, proline, glutamic acid, arginine, isoleucine, phenylalanine, and tryptophan. As another example, the mutant prolyl isomerase may be such that the amino acid corresponding to the 95th position in the amino acid sequence of SEQ ID NO: 1 is substituted with one or more amino acids selected from the group consisting of methionine, serine, threonine, asparagine, cysteine, histidine, lysine, aspartic acid, alanine, valine, leucine, glutamine, glycine, proline, and glutamic acid. As yet another example, the mutant prolyl isomerase may be such that the amino acid corresponding to the 95th position in the amino acid sequence of SEQ ID NO: 1 is substituted with an amino acid other than an aromatic amino acid. As another example, the mutant prolyl isomerase may be one in which the amino acid corresponding to the 95th position in the amino acid sequence of SEQ ID NO: 1 is replaced with an amino acid having a nonpolar, polar, or charged side chain.

[0023] As another example, the mutant prolyl isomerase of this application may have, include, or consist of the amino acid sequence described in SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, or SEQ ID NO: 31, or may substantially consist of the said amino acid sequence.

[0024] As an example, the mutant prolyl isomerase of this application may be one in which the amino acid at position 95 in the amino acid sequence of SEQ ID NO: 1 is replaced with an amino acid other than tyrosine.

[0025] The mutant prolyl isomerase of this application is an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.1% homology or identity with the amino acid sequence described in Sequence ID No. 1, or consisting of such homologous or identical amino acid sequences, or substantially consisting of such amino acid sequences. Furthermore, it is obvious that mutant prolyl isomerases having amino acid sequences in which some sequences are deleted, modified, substituted, conservatively substituted, or added, as long as they have such homology or identity and exhibit efficacy corresponding to the mutant prolyl isomerase of this application, are also included within the scope of this application.

[0026] For example, the amino acid sequence may have additions or deletions of sequences that do not alter the function of the mutant prolyl isomerase of this application, spontaneous mutations, silent mutations, or conservative substitutions at its N-terminus, C-terminus, and / or within it.

[0027] The term "conservative substitution" refers to the substitution of one amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions can generally occur based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. Typically, conservative substitutions have little to no effect on the activity of a protein or polypeptide.

[0028] For example, among amino acids having electrically charged side chains, positively charged (basic) amino acids include arginine, lysine, and histidine; negatively charged (acidic) amino acids include glutamic acid and aspartic acid; amino acids having nonpolar side chains (nonpolar amino acids) include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline; amino acids having polar or hydrophilic side chains (polar amino acids) include serine, threonine, cysteine, tyrosine, asparagine, and glutamine; and amino acids having uncharged side chains include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Furthermore, aromatic amino acids can be classified into those containing phenylalanine, tryptophan, and tyrosine.

[0029] In this application, the terms "mutant protein" or "variant" refer to a polypeptide in which one or more amino acids are conservatively substituted and / or modified, resulting in a sequence that differs from the amino acid sequence of the variant before the mutation, but in which the functions or properties are maintained. Such variants may 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 capabilities of the variant may be increased, unchanged, or decreased compared to the original polypeptide. Some variants may also include those in which one or more parts, such as the N-terminal leader sequence or the transmembrane domain, are removed. Other variants may include those in which parts of the N and / or C-terminus of a mature protein are removed. The term "mutant protein" may be used interchangeably with other terms such as mutant, modified, mutant polypeptide, mutated protein, mutation, and divergent (in English, these may be expressed as modification, modified polypeptide, modified protein, mutant, mutein, divergent, etc.), and is not limited to any other term used to mean mutated. For example, the divergent may be a polypeptide in which the amino acid corresponding to the 95th position of the amino acid sequence of Sequence ID No. 1 is substituted with methionine, serine, threonine, asparagine, cysteine, histidine, lysine, aspartic acid, alanine, valine, leucine, glutamine, glycine, proline, glutamic acid, arginine, isoleucine, phenylalanine, or tryptophan. As another example, the variant may be a polypeptide in which the amino acid corresponding to the 95th position of the amino acid sequence of SEQ ID NO: 1 is substituted with methionine, serine, threonine, asparagine, cysteine, histidine, lysine, aspartic acid, alanine, valine, leucine, glutamine, glycine, proline, or glutamic acid.As another example, the variant may be a polypeptide comprising the amino acid sequence described in SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, or SEQ ID NO: 31, but is not limited thereto.

[0030] Furthermore, the mutants may include the deletion or addition of amino acids that have minimal impact on the polypeptide's properties and secondary structure. For example, the N-terminus of the mutant can be conjugated with a signal (or leader) sequence that is involved in protein translocation co-translationally or post-translationally. The mutants can also be conjugated with other sequences or linkers for confirmation, purification, or synthesis.

[0031] In this application, the terms "homology" or "identity" refer to the degree of similarity between two given amino acid sequences or base sequences, and may be expressed as a percentage. The terms homology and identity are often used interchangeably.

[0032] The homology or identity of sequences of conserved polynucleotides or polypeptides is determined by standard sequencing algorithms, which may also be used in conjunction with a default gap penalty established by the program used. Substantially homologous or identical sequences are generally hybridizable in whole or in part with other sequences under moderate to high stringent conditions. It is obvious that hybridization also includes hybridization with polynucleotides containing codons in general or codon degeneracy in polynucleotides.

[0033] Whether any two polynucleotide or polypeptide sequences are homologous, similar, or identical can be determined using known computer algorithms such as the “FASTA” program with default parameters, for example, as in Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]:2444. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), as 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) (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 (Including Diego, 1994, and [CARILLO et al] (1988) SIAM J Applied Math 48:1073). For example, homology, similarity, or identity can be determined using BLAST or ClustalW from the National Center for Biotechnology Information Databases.

[0034] The homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using a GAP computer program, such as Needleman et al. (1970), J Mol Biol. 48:443, as is publicly known, for example, in Smith and Waterman, Adv. Appl. Math (1981) 2:482. In summary, the GAP program can be defined as the total number of symbols in the shorter of two sequences divided by the number of similarly sequenced symbols (i.e., nucleotides or amino acids). Default parameters for the GAP program may include (1) a unitary matrix (containing values ​​of 1 for identity and 0 for non-identity) and a weighted comparison matrix of Gribskov et al (1986) Nucl. Acids Res. 14:6745 (or EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix) as disclosed by Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or a gap opening penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for terminal gaps.

[0035] As an example of this application, the mutant prolyl isomerase of this application has the activity to increase the glutamate series amino acid production capacity compared to wild-type polypeptides that have prolyl isomerase activity.

[0036] In this application, the term "corresponding to" refers to an amino acid residue at a position listed in the polypeptide, or an amino acid residue that is similar, identical, or homologous to a residue listed in the polypeptide. Identifying the amino acid at the corresponding position can also determine the specific amino acid in the sequence referencing a particular sequence. As used in this application, "corresponding region" generally refers to a similar or corresponding position in the related protein or reference protein.

[0037] For example, any amino acid sequence can be aligned with sequence number 1, and based on this, each amino acid residue in the amino acid sequence can be numbered by referring to the numerical position of the amino acid residue corresponding to the amino acid residue in sequence number 1. For example, a sequence alignment algorithm such as the one described in this application can be used to verify the position of amino acids, or the position where deformations such as substitution, insertion, or deletion occur, by comparing them with a query sequence (also called a "reference sequence").

[0038] For such sorting, 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) can be used, but are not limited to these. Any sequence sorting program or pairwise sequence comparison algorithm known in this field can be appropriately used.

[0039] Another aspect of this application is to provide a polynucleotide encoding the mutant prolyl isomerase of this application.

[0040] The FkpA protein of this application may be encoded by the fkpA gene.

[0041] For example, the fkpA gene may, but is not limited to, a polynucleotide encoding NCgl0796 (Cg0950) derived from a microorganism of the genus Corynebacterium. For another example, the fkpA gene may, but is not limited to, a fkpA gene derived from a microorganism of the genus Corynebacterium, and it is obvious that it may include fkpA genes of diverse origins encoding proteins with FkpA protein activity.

[0042] In this application, the term "polynucleotide" means a polymer of nucleotides in which nucleotide units (monomers) are covalently linked together in a long chain, and is a DNA or RNA chain of a certain length or longer. More specifically, it means a polynucleotide fragment encoding the mutant prolyl isomerase.

[0043] The polynucleotides encoding mutant prolyl isomerase in this application may include a base sequence encoding mutant prolyl isomerase in which the amino acid corresponding to position 95 in the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid, or a base sequence in which the codons corresponding to positions 283-285 in the nucleotide sequence of SEQ ID NO: 2 are substituted with codons encoding other amino acids. For example, the polynucleotides in this application may include a base sequence encoding the amino acid sequence described in SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, or SEQ ID NO: 31. As a more specific example of this application, the polynucleotides in this application may have or include the sequence of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, or SEQ ID NO: 32. Furthermore, the polynucleotide of this application may consist of, or substantially constitute, the sequence of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, or SEQ ID NO: 32.

[0044] The polynucleotides of this application may have various modifications to their coding regions, taking into consideration the degeneracy of the codons or the preferred codons in organisms that intend to express the mutant prolyl isomerase of this application, as long as these modifications do not alter the amino acid sequence of the mutant prolyl isomerase of this application. Specifically, the polynucleotides of this application may have, or include, a base sequence that is homologous or identical to the sequence of SEQ ID NO: 2 by 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%, or consist of, or substantially constitute, a base sequence that is homologous or identical to the sequence of SEQ ID NO: 2 by 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%. In this case, in the homologous or identical sequence, the codon encoding the amino acid corresponding to the 95th position of Sequence ID No. 1 may be one of the codons encoding an amino acid other than tyrosine, for example, methionine, serine, threonine, asparagine, cysteine, histidine, lysine, aspartic acid, alanine, valine, leucine, glutamine, glycine, proline, or glutamic acid.

[0045] Furthermore, the polynucleotides of this application may include, without limitation, any probes produced from known gene sequences, such as sequences that can hybridize under stringent conditions with complementary sequences to all or part of the polynucleotide sequences of this application. “Stringent conditions” means conditions that enable specific hybridization between polynucleotides. Such conditions are specifically described in the literature (see J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989; FM Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50-9.51, 11.7-11.8). For example, we can list conditions in which polynucleotides with high homology or identity hybridize with each other, with homology or identity levels of 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 99% or more, but do not hybridize with polynucleotides with lower homology or identity levels. Alternatively, we can list conditions in which the polynucleotides are washed once, specifically two to three times, at a salt concentration and temperature equivalent to the washing conditions of normal Southern hybridization: 60°C, 1×SSC, 0.1% SDS, more specifically 60°C, 0.1×SSC, 0.1% SDS, or more specifically 68°C, 0.1×SSC, 0.1% SDS.

[0046] Hybridization requires that two nucleic acids have complementary sequences, even if mismatches between bases are possible depending on the stringency of the hybridization. The term "complementary" is used to describe the relationships between nucleotide bases that can hybridize with each other. For example, in DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Thus, the polynucleotides of this application may also include not only substantially similar nucleic acid sequences, but also isolated nucleic acid fragments that are complementary to the overall sequence.

[0047] Specifically, polynucleotides homologous or identical to the polynucleotides of this application can be detected using hybridization conditions that include a hybridization step at a Tm value of 55°C, and under the conditions described above. The Tm value may also be 60°C, 63°C, or 65°C, but is not limited thereto and may be appropriately adjusted by those skilled in the art depending on the purpose.

[0048] The appropriate stringency for hybridizing the aforementioned polynucleotides depends on the length and degree of complementarity of the polynucleotides, and these variables are well known in the art (e.g., J. Sambrook et al., ibid.).

[0049] Another aspect of this application is to provide a vector comprising the polynucleotide of this application.

[0050] The vector may, but is not limited to, be an expression vector for expressing the polynucleotide in a host cell.

[0051] The vector of this application may include a DNA product comprising a polynucleotide sequence encoding the target polypeptide, operably linked to a suitable regulatory region (or regulatory sequence) so as to enable the expression of the target polypeptide in a suitable host. The regulatory 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 sequences regulating the termination of transcription and decoding. After being transformed into a suitable host cell, the vector can replicate or function independently of the host genome and integrate into the genome itself.

[0052] The vectors used in this application are 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 their natural or recombinant state. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage vectors or cosmid vectors, and pDZ, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, and pET can be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC vectors can be used.

[0053] As an example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome via a chromosome insertion vector within a cell. The insertion of the polynucleotide into the chromosome may be carried out by any method known in the art, such as homologous recombination, but is not limited thereto. A selection marker may further be included to confirm the presence or absence of the chromosome insertion. The selection marker is for selecting cells transformed with the vector, i.e., confirming the presence or absence of the target nucleic acid molecule insertion, and may be a marker that confers a selectable phenotype, such as drug resistance, nutritional requirements, resistance to cytotoxic agents, or expression of a surface polypeptide. Transformed cells can be selected because, in an environment treated with a selective agent, only cells expressing the selection marker survive or exhibit other phenotypes.

[0054] In this application, the term "transformation" means introducing a vector containing 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 or outside the chromosomes of the host cell, as long as they can be expressed in the host cell. The polynucleotide also includes DNA and / or RNA encoding the target polypeptide. The polynucleotide may be introduced into the host cell in any form that can be introduced into the host cell and expressed. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a gene structure containing all the elements necessary for its expression. The expression cassette may typically include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the polynucleotide. The expression cassette may also be in the form of a self-replicating expression vector. The polynucleotide may also be introduced into the host cell in its own form and operably linked to the sequences necessary for expression in the host cell, but is not limited thereto.

[0055] Furthermore, the term "operably linked" in the foregoing means that the polynucleotide sequence is functionally linked to a promoter sequence that initiates and mediates the transcription of the polynucleotide encoding the target mutant prolyl isomerase of this application.

[0056] Another aspect of this application is to provide a microorganism containing the mutant prolyl isomerase or the polynucleotide of this application.

[0057] The bacterial strain of this application may include the mutant prolyl isomerase of this application, the polynucleotide encoding the polypeptide, or a vector containing the polynucleotide of this application.

[0058] In this application, the term "microorganism (or strain)" includes all wild-type microorganisms and microorganisms that have undergone natural or artificial genetic modification, and is a microorganism in which a specific mechanism is weakened or strengthened due to causes such as the insertion of external genes or the enhancement or inactivation of the activity of endogenous genes, and may be a microorganism that includes genetic modification for the production of a target polypeptide, protein, or product.

[0059] The strains of this application may be microorganisms that naturally possess the ability to produce glutamate-series amino acids or microorganisms that have been conferred the ability to produce glutamate-series amino acids to strains that do not possess this ability. For example, the microorganisms may be microorganisms in which the mutant prolyl isomerase of this application or the polynucleotide encoding it has been introduced, thereby improving the ability to produce glutamate-series amino acids, but are not limited thereto.

[0060] In this application, the term "glutamate series amino acids" can include glutamic acid and all L-amino acids that can be biosynthesized using glutamic acid as a precursor. Examples of L-amino acids that can be produced through the glutamate biosynthesis pathway using glutamic acid as a precursor include glutamine, ornithine, citrulline, and arginine. In addition, any other L-amino acid that can be biosynthesized using glutamic acid as a precursor may be included in the scope of this application. Furthermore, this application can include glutamic acid that can be produced with the involvement of the prolyl isomerase of this application and substances synthesized using it as a precursor without limitation.

[0061] On the other hand, in this application, the term "L-amino acid" includes all proteinaceous and non-proteinaceous amino acids.

[0062] The strain of this application may be a microorganism in which glutamate series amino acid production ability is improved compared to a parent strain or wild-type Corynebacterium strain that does not contain the mutant prolyl isomerase of this application. The said microorganism may have improved glutamate series amino acid production ability due to the introduction of the mutant prolyl isomerase of this application.

[0063] As an example, the prolyl isomerase-non-mutated microorganism used as a control strain to compare the presence or absence of increased glutamate series amino acid production capacity may be, but is not limited to, C. glutamicum ATCC 13869 strain, C. gl::argR*_argG* strain producing citrulline, KFCC10680 strain producing glutamine, C. gl::argR*_argF* strain producing ornithine, or C. gl::argR* strain producing arginine.

[0064] As an example, the recombinant strain with improved production capacity has approximately 1% or more of the glutamate series amino acid production capacity compared to the parent strain or non-mutated microorganism before mutation. Specifically, this includes approximately 2% or more, approximately 5% or more, approximately 10% or more, approximately 15% or more, approximately 20% or more, approximately 25% or more, approximately 30% or more, approximately 31% or more, approximately 31.9% or more, approximately 32% or more, approximately 32.9% or more, approximately 33% or more, approximately 33.8% or more, approximately 34% or more, approximately 35% or more, approximately 36% or more, and approximately 36% or more. The increase may be 0.4% or more, approximately 37% or more, approximately 37.2% or more, approximately 38% or more, approximately 38.8% or more, approximately 39% or more, or approximately 39.5% or more (there are no special restrictions on the upper limit; for example, it may be approximately 200% or less, approximately 150% or less, approximately 100% or less, or approximately 50% or less), but it is not limited to this as long as it has a positive increase compared to the production capacity of the parent strain before mutation, the non-myxoid microorganism, or the prolyl isomerase non-myxoid microorganism. In other examples, the recombinant strains with improved glutamate series amino acid production capacity showed glutamate series amino acid production capacity of approximately 1.01 times or more, approximately 1.02 times or more, approximately 1.05 times or more, approximately 1.1 times or more, approximately 1.15 times or more, approximately 1.2 times or more, approximately 1.25 times or more, approximately 1.3 times or more, approximately 1.31 times or more, approximately 1.319 times or more, and approximately 1.32 times or more compared to the original parent strain, non-myxoid microorganisms, or prolyl isomerase non-myxoid microorganisms. The improvement may be approximately 1.329 times or more, approximately 1.33 times or more, approximately 1.338 times or more, approximately 1.34 times or more, approximately 1.35 times or more, approximately 1.36 times or more, approximately 1.364 times or more, approximately 1.37 times or more, approximately 1.372 times or more, approximately 1.38 times or more, approximately 1.388 times or more, approximately 1.39 times or more, or approximately 1.395 times or more (there is no special limit on the upper limit; for example, it may be approximately 10 times or less, or approximately 5 times or less), but it is not limited to these.

[0065] In this application, the term "non-metamorphic microorganism" means a wild-type or naturally occurring strain itself, or a strain before its characteristics are altered by genetic mutations due to natural or artificial factors, and does not exclude strains containing naturally occurring mutations in microorganisms. Furthermore, the term "prolyl isomerase non-metamorphic microorganism" in this application means a strain in which the prolyl isomerase variants described herein have not been introduced, or before they have been introduced. The prolyl isomerase non-metamorphic microorganisms in this application do not exclude strains containing variants of other proteins or other genes other than the prolyl isomerase or the polynucleotide encoding it.

[0066] In this application, the term "non-mutant microorganism" may be used interchangeably with "pre-deformation strain," "pre-deformation microorganism," "non-mutant strain," "non-mutant myxomycete strain," "non-mutant microorganism," or "reference microorganism."

[0067] The microorganisms of this application may, but are not limited to, microorganisms containing mutant prolyl isomerase or the polynucleotide encoding it; or microorganisms genetically modified to contain mutant prolyl isomerase or the polynucleotide encoding it (e.g., recombinant microorganisms). “Intrinsic activity” means the activity of a particular polypeptide that was originally present in the parental strain, wild-type, or non-mutant microorganism before the trait change due to a genetic mutation caused by natural or anthropogenic factors. This may be used interchangeably with “pre-modification activity.”

[0068] As another example of this application, the microorganisms of this application are Corynebacterium glutamicum, Corynebacterium stationis, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium singulare, Corynebacterium halotolerans, and Corynebacterium striatum. It may also be Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris, or Corynebacterium flavescens, and more specifically, it may be Corynebacterium glutamicum, but is not limited thereto.

[0069] As yet another example of this application, the recombinant microorganisms of this application may further be microorganisms in which the activity of a portion of a protein in the glutamate series amino acid biosynthesis pathway is enhanced, or the activity of a portion of a protein in the glutamate series amino acid degradation pathway is weakened, thereby enhancing the glutamate series amino acid production capacity.

[0070] As yet another example of this application, the recombinant microorganism of this application may be a strain in which the ability to produce ornithine, one of the glutamate amino acids, is enhanced.

[0071] In any one of the aforementioned embodiment examples, the recombinant microorganism of this application may be modified to enhance the biosynthetic pathway from glutamate to ornithine by enhancing the activity of one or more enzymes selected from the group consisting of acetylglutamate synthase (ArgJ), which converts glutamate to acetylglutamate (N-acetylglutamate); ornithine acetyltransferase (ArgJ), which converts acetylornithine to ornithine; acetylglutamate kinase (ArgB), which converts acetylglutamyl phosphate (N-acetylglutamyl phosphate); acetylgamma-glutamyl phosphate reductase (ArgC), which converts acetylglutamyl phosphate to acetylglutamate semialdehyde; and acetylornithine aminotransferase (ArgD), which converts acetylglutamate semialdehyde to acetylornithine, compared to its intrinsic activity, thereby improving its ornithine production capacity.

[0072] As yet another example of this application, the recombinant microorganism of this application may be a strain in which the ability to produce citrulline, one of the glutamate series amino acids, is enhanced.

[0073] In any one of the aforementioned embodiment examples, the recombinant microorganism of this application may be modified to enhance the biosynthetic pathway from ornithine to citrulline by increasing the activity of ornithine carbamoyltransferase (ArgF), which converts ornithine to citrulline, compared to its endogenous activity, thereby improving its citrulline production capacity. Alternatively, the recombinant microorganism of this application may be further modified to enhance the biosynthetic pathway from glutamic acid to ornithine, as described above, thereby improving its ability to produce ornithine, a precursor of citrulline, and thus improving its citrulline production capacity.

[0074] As yet another example of this application, the recombinant microorganism of this application may be a strain in which the production capacity of arginine, one of the glutamate amino acids, has been improved.

[0075] In any one of the embodiments described above, the recombinant microorganism of this application may be modified to enhance the biosynthetic pathway from ornithine to arginine by enhancing the activity of one or more enzymes selected from the group consisting of argininosuccinate synthase (argG), argininosuccinate degrading enzyme (argH), aspartate ammonia lyase (aspA), and aspartate aminotransferase (aspB) that are involved in arginine synthesis, thereby improving its arginine production capacity. Alternatively, the recombinant microorganism of this application may be further modified, as described above, to enhance the biosynthetic pathway from glutamate to ornithine, thereby improving its arginine production capacity by improving its ability to produce ornithine, a precursor of citrulline.

[0076] As yet another example of this application, the recombinant microorganism of this application may be a strain in which the ability to produce glutamine, one of the glutamate amino acids, is improved.

[0077] In any one of the aforementioned embodiments, the recombinant microorganism of this application may be modified to enhance the biosynthetic pathway from glutamic acid to glutamine by increasing the activity of glutamine synthetase, which is involved in glutamine synthesis, compared to its endogenous activity, thereby improving its glutamine production capacity. Alternatively, the recombinant microorganism of this application may be further modified, as described above, to enhance the biosynthetic pathway from glutamic acid to glutamine, thereby improving its ability to produce glutamic acid, a precursor of glutamine, and thus improving its glutamine production capacity.

[0078] In this application, the term “weakening” of the activity of a polypeptide (including, for example, the protein specified in the name of each enzyme) encompasses all concepts of reduced or absent activity compared to its endogenous activity. Such weakening may be used interchangeably with terms such as inactivation, deficiency, down-regulation, decline, reduce, and attenuation.

[0079] The aforementioned weakening may include cases where the activity of the polypeptide itself is reduced or eliminated compared to the polypeptide activity originally possessed by the microorganism due to mutations in the polynucleotide encoding the polypeptide, cases where the overall polypeptide activity and / or concentration (expression level) in the cell is lower than that of the natural strain due to inhibition of the expression of the gene encoding the polynucleotide or inhibition of translation into the polypeptide, cases where the polynucleotide is not expressed at all, and / or cases where there is no polypeptide activity despite the expression of the polynucleotide. "Inactivation, deficiency, reduction, downregulation, decrease, and attenuation" of polypeptide activity compared to its endogenous activity means that it has decreased compared to the activity of the specific polypeptide originally possessed by the parent strain or non-mutant microorganism before the trait change.

[0080] The weakening of the activity of such polypeptides can be achieved by any method known in the art, but is not limited to these methods, and can be achieved by applying a variety of well-known methods in the field (e.g., 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.).

[0081] Specifically, the weakening of the activity of the polypeptide in this application is, 1) Deletion of all or part of the gene encoding the polypeptide; 2) Modification of the gene expression regulatory region (or gene expression regulatory sequence) so that the expression of the polypeptide-coding gene is reduced; 3) Modification of the amino acid sequence constituting the polypeptide (e.g., deletion / substitution / addition of one or more amino acids in the amino acid sequence) so as to remove or weaken the activity of the polypeptide; 4) Modification of the gene sequence encoding the polypeptide so that the polypeptide activity is removed or weakened (for example, deletion / substitution / addition of one or more nucleic acid bases in the nucleic acid sequence of the polypeptide gene so that it encodes a polypeptide that has been modified so that the polypeptide activity is removed or weakened); 5) Modifications of the nucleotide sequence encoding the start codon or 5'-UTR region of a polypeptide-encoding gene transcript; 6) Introduction of an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the transcript of the gene encoding the polypeptide; 7) Addition of a sequence complementary to the Shine-Dalgarno sequence to the leading end of the Shine-Dalgarno sequence in a polypeptide-encoding gene to form a secondary structure that cannot be attached to ribosomes; 8) Addition of a promoter that is transcribed in the opposite direction to the 3' end of the ORF (open reading frame) of the polypeptide-coding gene sequence (Reverse transcription engineering, RTE); 9) Regulation of the intracellular localization of proteins (polypeptides); or 10) A combination of two or more selected from items 1) to 9) above is also acceptable, but is not particularly limited thereto.

[0082] for example, The deletion of part or all of the gene encoding the polypeptide described in 1) above may be the removal of the entire polynucleotide encoding the endogenous target polypeptide within the chromosome, replacement with a polynucleotide in which some nucleotides are deleted, or replacement with a marker gene.

[0083] Furthermore, the modification of the expression regulatory region (or expression regulatory sequence) described in 2) above may involve deletion, insertion, non-conservative or conservative substitution, or a combination thereof, resulting in a mutation on the expression regulatory region (or expression regulatory sequence), or replacement with a sequence having weaker activity. The expression regulatory region includes, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence that regulates the termination of transcription and decoding.

[0084] The modifications of the amino acid sequence or polynucleotide sequence described in 3) and 4) above may be, but are not limited to, the deletion, insertion, non-conservative or conservative substitution, or combination thereof of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide, thereby causing a sequence mutation, or replacement with an amino acid sequence or polynucleotide sequence modified to have weaker activity or an amino acid sequence or polynucleotide sequence modified to have no activity, in order to weaken the activity of the polypeptide. For example, gene expression can be inhibited or weakened by introducing a mutation in the polynucleotide sequence to form a termination codon, but is not limited to this.

[0085] The sequence modification encoding the start codon or 5'-UTR region of the polypeptide-encoding gene transcript described in 5) above may, but is not limited to, substitution with a sequence encoding another start codon that has a lower polypeptide expression rate compared to the endogenous start codon.

[0086] The introduction of an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the transcript of the gene encoding the polypeptide (6) can be done by referring to, for example, the literature [Weintraub, H. et al., Antisense-RNA as a molecular tool for genetic analysis, Reviews - Trends in Genetics, Vol. 1(1) 1986].

[0087] 7) In order to form a secondary structure that ribosomes cannot attach to, the addition of a sequence complementary to the Shine-Dalgarno sequence to the leading end of the Shine-Dalgarno sequence of the polypeptide-encoding gene may make mRNA translation impossible or reduce its rate.

[0088] Furthermore, the addition of a promoter that is transcribed in the opposite direction to the 3' end of the ORF (open reading frame) of the gene sequence encoding the polypeptide (Reverse transcription engineering, RTE) may weaken the activity by creating complementary antisense nucleotides in the transcript of the gene encoding the polypeptide.

[0089] The 9) intracellular positional regulation of proteins (polypeptides) described above may involve targeting the proteins (polypeptides) to specific organelles or specific intracellular spaces within the cell. For example, this may involve targeting the periplasm or cytoplasm through the addition or removal of a leader sequence that functions in targeting the proteins (polypeptides), but is not limited thereto.

[0090] Such weakening of polypeptide activity may be, but is not limited to, a reduction in the activity or concentration expression level of the corresponding polypeptide relative to the activity or concentration of the polypeptide expressed in the wild-type or pre-deformation microbial strain, or a reduction in the amount of product produced from the polypeptide.

[0091] In this application, the term “enhancement” of polypeptide activity means that the polypeptide activity increases compared to its endogenous activity. This enhancement may be used interchangeably with terms such as activation, upregulation, overexpression, and increase. Here, activation, enhancement, upregulation, overexpression, and increase can all include exhibiting activity that was not originally present, or exhibiting improved activity compared to the endogenous activity or the activity before the mutation. “Enhancement,” “upregulation,” “overexpression,” or “increase” of polypeptide activity compared to its endogenous activity means that the activity and / or concentration (expression level) of a particular polypeptide that was originally present in the parental strain or non-mutant microorganism before the mutation is improved.

[0092] The aforementioned enhancement can be achieved by introducing an exogenous polypeptide or by enhancing the activity and / or concentration (expression level) of an endogenous polypeptide. Whether or not the polypeptide's activity has been enhanced can be confirmed by an increase in the polypeptide's activity level, expression level, or the amount of product excreted from the polypeptide.

[0093] The enhancement of the polypeptide activity can be achieved by applying a variety of methods well known in the field, and is not limited as long as it can enhance the activity of the target polypeptide compared to the microorganism before deformation. Specifically, this may involve, but is not limited to, the use of genetic engineering and / or protein engineering, which are routine methods in molecular biology and are well known to ordinary technicians in this field (e.g., Sitnicka et al. Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol. 2. 1-16, Sambrook et al. Molecular Cloning 2012, etc.).

[0094] Specifically, the enhancement of the activity of the polypeptide in this application is 1) Increase in the intracellular copy number of polynucleotides encoding polypeptides; 2) Replacement of gene expression regulatory regions on chromosomes encoding polypeptides with highly active sequences; 3) Modifications of the nucleotide sequence encoding the start codon or 5'-UTR region of a polypeptide-encoding gene transcript; 4) Modification of the amino acid sequence of the polypeptide so as to enhance polypeptide activity; 5) Modification of the polynucleotide sequence encoding the polypeptide so as to enhance polypeptide activity (for example, modification of the polynucleotide sequence of the polypeptide gene so as to encode a polypeptide modified to enhance polypeptide activity); 6) Introduction of a foreign polypeptide exhibiting polypeptide activity or a foreign polynucleotide encoding it; 7) Codon optimization of polynucleotides encoding polypeptides; 8) Analyze the tertiary structure of the polypeptide, select exposed areas, and deform or chemically modify them; 9) Regulation of the cellular localization of proteins (polypeptides); or 10) A combination of two or more selected from items 1) to 9) above is also acceptable, but is not particularly limited thereto.

[0095] More specifically, The increase in the intracellular copy number of the polynucleotide encoding the polypeptide described in 1) above may be achieved by introducing into the host cell a vector that can replicate and function independently of the host, on which the polynucleotide encoding the polypeptide is operably linked. Alternatively, it may be achieved by introducing one or more copies of the polynucleotide encoding the polypeptide into the chromosomes within the host cell. The introduction into the chromosomes can be, but is not limited to, by introducing into the host cell a vector that can insert the polynucleotide into the chromosomes within the host cell. The vector is as described above.

[0096] The replacement of the gene expression regulatory region (or expression regulatory sequence) on the chromosome encoding the polypeptide with a more potent sequence may, for example, involve the generation of a sequence mutation by deletion, insertion, non-conservative or conservative substitution or a combination thereof, or replacement with a sequence having stronger activity, in order to further enhance the activity of the expression regulatory region. The expression regulatory region may include, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence that regulates the termination of transcription and decoding. For example, the original promoter may be replaced with a potent promoter, but is not limited to this.

[0097] Examples of well-known strong promoters include, but are not limited to, the CJ1-CJ7 promoters (US Registered Patent US 7662943 B2), the lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13 (sm3) promoter (US Registered Patent US 10584338 B2), O2 promoter (US Registered Patent US 10273491 B2), tkt promoter, and yccA promoter.

[0098] The sequence modification encoding the start codon or 5'-UTR region of the polypeptide-encoding gene transcript described in 3) above may, but is not limited to, substitution with a sequence encoding another start codon that has a higher polypeptide expression rate compared to the endogenous start codon.

[0099] The modifications of the amino acid sequence or polynucleotide sequence described in 4) and 5) above may be, but are not limited to, deletion, insertion, non-conservative or conservative substitution or combination thereof of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide, thereby causing a sequence mutation, or replacement with an improved amino acid sequence or polynucleotide sequence that has stronger activity or an improved amino acid sequence or polynucleotide sequence that has increased activity, in order to enhance the activity of the polypeptide. Specifically, such replacement can be carried out by inserting a polynucleotide into the chromosome by homologous recombination, but is not limited to this. The vector used in this case may further include a selection marker for confirming the presence or absence of chromosomal insertion.

[0100] The introduction of a foreign polynucleotide exhibiting polypeptide activity (6) above may be the introduction of a foreign polynucleotide encoding a polypeptide exhibiting the same or similar activity as the polypeptide into the host cell. The foreign polynucleotide is not restricted in its origin or sequence, as long as it exhibits the same or similar activity as the polypeptide. The method used for the introduction can be appropriately selected by those skilled in the art from known transformation methods, and the introduction of the polynucleotide in the host cell may generate a polypeptide and increase its activity.

[0101] The codon optimization of the polynucleotide encoding the polypeptide described in 7) above may be codon optimization of the endogenous polynucleotide so that transcription or translation increases in the host cell, or the codon of the exogenous polynucleotide so that optimized transcription or translation occurs in the host cell.

[0102] 8) Analyzing the tertiary structure of a polypeptide and selecting exposed sites to deform or chemically modify may, for example, involve comparing the sequence information of the polypeptide to be analyzed with a database containing sequence information of known proteins to determine candidate template proteins according to the degree of sequence similarity, confirming the structure based on that, and selecting exposed sites to deform or chemically modify.

[0103] The 9) intracellular positional regulation of proteins (polypeptides) described above may involve targeting the proteins (polypeptides) to specific organelles or specific intracellular spaces within the cell. For example, this may involve targeting the periplasm or cytoplasm through the addition or removal of a leader sequence that functions in targeting the proteins (polypeptides), but is not limited thereto.

[0104] Such enhancement of polypeptide activity may be achieved by increasing the activity or concentration of the corresponding polypeptide based on the activity or concentration of the polypeptide expressed in the wild-type or pre-deformation microbial strain, or by increasing the amount of product produced from the polypeptide, but is not limited to these methods.

[0105] Modification of some or all of the polynucleotides in the microorganism of this application may be induced by (a) homologous recombination using a chromosome insertion vector within the microorganism or genome editing using an engineered nuclease (e.g., CRISPR-Cas9) and / or (b) treatment with light such as ultraviolet light and radiation and / or chemical substances, but are not limited to these. The method for modifying some or all of the genes may include methods using DNA recombination technology. For example, deletion of some or all of the genes may be achieved by injecting a nucleotide sequence or vector containing a nucleotide sequence homologous to the target gene into the microorganism to induce homologous recombination. The injected nucleotide sequence or vector may include, but are not limited to, a dominant selection marker.

[0106] In the microorganism of this application, the mutant prolyl isomerase, polynucleotides, and glutamic acid series amino acids are as described in the other aspects mentioned above.

[0107] Another aspect of this application provides a method for producing glutamate-series amino acids, comprising the step of culturing a microorganism containing the mutant prolyl isomerase or polynucleotide of this application in a culture medium.

[0108] The glutamate series amino acid production method of this application may include the step of culturing a microorganism containing the mutant prolyl isomerase of this application, or the polynucleotide of this application, or the vector of this application, in a culture medium.

[0109] In this application, the term "culture" means growing the microorganisms of this application under appropriately controlled environmental conditions. The culture process of this application can be carried out according to suitable culture media and culture conditions known in the art. Such a culture process can be easily adapted and used by those skilled in the art depending on the selected microorganisms. Specifically, the culture may be batch, continuous, and / or fed-batch.

[0110] In this application, the term "culture medium" means a substance mixed primarily with nutrients necessary for culturing the microorganisms of this application, supplying nutrients and growth factors, including water, which are essential for survival and growth. Specifically, the culture medium and other culture conditions used for culturing the microorganisms of this application can be any culture medium used for culturing ordinary microorganisms without any special restrictions, but the microorganisms of this application can be cultured under aerobic conditions in an ordinary culture medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids and / or vitamins, while adjusting the temperature, pH, etc.

[0111] Specifically, culture media for microorganisms of the genus Corynebacterium can be found in the literature ["Manual of Methods for General Bacteriology" by the American Society for Bacteriology (Washington DC, USA, 1981)].

[0112] In this application, the carbon source may include carbohydrates such as glucose, sucrose, lactose, fructose, sucrose, and maltose; sugar alcohols such as mannitol and sorbitol; organic acids such as pyruvic acid, lactic acid, and citric acid; and amino acids such as glutamic acid, methionine, and lysine. In addition, natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn maceration can be used. Specifically, carbohydrates such as glucose and sterilized pre-treated molasses (i.e., molasses converted to reducing sugars) can be used, and other appropriate amounts of carbon sources can be used in a variety of ways without limitation. These carbon sources may be used alone or in combination of two or more, and are not limited to these uses.

[0113] The nitrogen sources used include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; and organic nitrogen sources such as amino acids like glutamic acid, methionine, and glutamine, peptone, NZ-amine, meat extracts, yeast extracts, malt extracts, corn maceration, casein hydrolysates, fish or their decomposition products, and defatted soybean cake or its decomposition products. These nitrogen sources may be used individually or in combination of two or more, and are not limited to these uses.

[0114] The phosphorus source may include monopotassium phosphate, dipotassium phosphate, or their corresponding sodium-containing salts. Inorganic compounds such as sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, and calcium carbonate may be used, and other components such as amino acids, vitamins, and / or appropriate precursors may also be included. These components or precursors can be added to the culture medium in batches or continuously, but are not limited to these methods.

[0115] Furthermore, during the cultivation of the microorganisms of this application, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid can be added to the culture medium in an appropriate manner to adjust the pH of the culture medium. In addition, during cultivation, antifoaming agents such as fatty acid polyglycol esters can be used to suppress the formation of bubbles. Furthermore, in order to maintain an aerobic state in the culture medium, oxygen or oxygen-containing gas can be injected into the culture medium, or in order to maintain an anaerobic and microaerobic state, nitrogen, hydrogen, or carbon dioxide gas can be injected without gas injection, or by injection.

[0116] In the culture described in this application, the culture temperature can be maintained at 20-45°C, specifically 25-40°C, and the culture can be performed for approximately 10-160 hours, but is not limited to this.

[0117] The glutamate series amino acids produced by the culture described in this application are secreted into the culture medium or remain within the cells. At that time, the glutamate series amino acids are as described above.

[0118] The method for producing glutamic acid series amino acids of this application may further include, for example, the step of preparing the microorganism of this application, the step of preparing a culture medium for culturing the microorganism, or a combination thereof (in any order), for example, before the culturing step.

[0119] The glutamate series amino acid production method of this application may further include a step of recovering glutamate series amino acids from the culture medium (the culture medium in which the culture was performed) or from the Corynebacterium glutamicum strain. The recovery step may further include a step after the culture step.

[0120] The aforementioned recovery may involve collecting the target glutamate series amino acids using appropriate methods known in the art, such as the microorganism culture methods of this application, for example, batch, continuous, or fed-batch culture methods. For example, various chromatography methods such as centrifugation, filtration, treatment with a crystallizing protein precipitant (salting-out method), extraction, sonication, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC, or a combination thereof can be used to recover the target glutamate series amino acids from the culture medium or microorganisms using appropriate methods known in the art.

[0121] Furthermore, the glutamate series amino acid production method of this application may further include a purification step. The purification can be carried out using a suitable method known in the art. For example, if the glutamate series amino acid production method of this application includes both a recovery step and a purification step, the recovery step and the purification step can be carried out sequentially or discontinuously, regardless of the procedure, or simultaneously or integrated into a single step, but are not limited thereto.

[0122] In the method of this application, the mutant prolyl isomerase, polynucleotide, vector, bacterial strain, and glutamate series amino acids are as described in the other aspects described above.

[0123] Another aspect of this application is to provide a composition for the production of glutamate series amino acids comprising the mutant prolyl isomerase of this application, a polynucleotide encoding it, a vector containing the polynucleotide or a microorganism containing the polynucleotide of this application; a culture medium in which the microorganism is cultured; or a combination of two or more of these.

[0124] The composition of this application may further contain any suitable excipients commonly used in compositions for L-amino acid production, such excipients may include, but are not limited to, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, or isotonic agents.

[0125] Another aspect of this application provides the use of the mutant prolyl isomerase of this application for the production of glutamate series amino acids.

[0126] Another aspect of this application provides the use of the mutant prolyl isomerase of this application, the polynucleotide encoding it, a vector containing the polynucleotide, or a microorganism containing the polynucleotide of this application for the production of glutamate series amino acids.

[0127] The aforementioned prolyl isomerase, mutant prolyl isomerase, polynucleotide, vector, bacterial strain, culture medium, and glutamic acid series amino acids are as described in the other sections above.

[0128] The present application will be described in more detail below with reference to experimental examples. However, the following embodiments are merely preferred embodiments for illustrative purposes of the present application and are not intended to limit the scope of the rights of this application. On the other hand, technical matters not described herein can be easily understood and performed by a person of ordinary skill who is skilled in the art of this application or a similar art. [Examples]

[0129] Example 1: Production of a mutant prolyl isomerase expression vector Vectors were constructed to introduce point mutations Y95M, Y95S, Y95T, Y95N, Y95C, Y95H, Y95K, Y95D, Y95A, Y95V, Y95L, Y95Q, Y95G, Y95P, and Y95E into the fkpA gene (SEQ ID NO: 2), which encodes prolyl isomerase (FkpA), in wild-type Corynebacterium glutamicum.

[0130] To construct vectors for introducing each mutation, the genome of wild-type Corynebacterium glutamicum ATCC 13869 was used as a template to amplify the homologous recombinant A arm and homologous recombinant B arm. The primer pairs used for each substituted residue are shown in Table 1 below.

[0131] [Table 1]

[0132] The PCR conditions involved denaturation at 95°C for 5 minutes, followed by denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, polymerization at 72°C for 1 minute, repeated 30 times, and then polymerization at 72°C for 5 minutes. Subsequently, PCR fragments were extracted using a gel purification kit (QIAGEN). The homologous recombinant A arm and B arm gene fragments obtained above were ligated with the vector pDCM2 (US 2023-0134555 A1), which had been cleaved with BamHI and XbaI restriction enzymes, using the Gibson assembly method (DG Gibson et al., NATURE METHODS, VOL.6 NO.5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix). These were then used to transform E. coli DH5α, and the resulting mixtures were streaked onto LB solid medium containing 25 mg / L kanamycin. To select transformed colonies, PCR was performed using primer pairs of SEQ ID NOs. 51 and 52. Plasmids were obtained from the selected colonies using commonly known plasmid extraction methods, and the obtained plasmids were designated as pDCM2-fkpA(Y95M), pDCM2-fkpA(Y95S), pDCM2-fkpA(Y95T), pDCM2-fkpA(Y95N), pDCM2-fkpA(Y95C), pDCM2-fkpA(Y95H), and pD These were named CM2-fkpA(Y95K), pDCM2-fkpA(Y95D), pDCM2-fkpA(Y95A), pDCM2-fkpA(Y95V), pDCM2-fkpA(Y95L), pDCM2-fkpA(Y95Q), pDCM2-fkpA(Y95G), pDCM2-fkpA(Y95P), and pDCM2-fkpA(Y95E).

[0133] The primer sequences used here are shown in Table 2 below.

[0134] [Table 2]

[0135] Example 2: Evaluation of L-citrulline production capacity Example 2-1. Preparation of the control group To prepare a control strain for evaluating L-citrulline production capacity, a vector was constructed in which the glutamic acid at position 47 of the argR (ANU33619.1) protein sequence was replaced with a termination codon. Using the genome of wild-type C. glutamicum ATCC 13869 as a template, the homologous recombinant A arm was amplified using primer pairs of SEQ ID NOs. 53 and 54, and the homologous recombinant B arm was amplified using primer pairs of SEQ ID NOs. 55 and 56. Subsequently, a plasmid was obtained using the same method as in Example 1, and this plasmid was named pDCM2-argR(E47*).

[0136] Furthermore, in order to produce microorganisms with improved L-citrulline production capacity, a vector was created in which the phenylalanine at position 68 of the argG (ANU33620.1) protein sequence was replaced with a termination codon. Using the genome of C. glutamicum ATCC13869 as a template, the homologous recombinant A arm was amplified using primers SEQ ID NOs. 57 and 58, and the homologous recombinant B arm was amplified using primers SEQ ID NOs. 59 and 60. Subsequently, a plasmid was obtained using the method described in Example 1, and this plasmid was named pDCM2-argG(F68*).

[0137] The primer sequences used here are shown in Table 3 below.

[0138] [Table 3]

[0139] Wild-type C. glutamicum ATCC 13869 was transformed using the fabricated pDCM2-argR(E47*) vector via electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541-545). Following a secondary cross-reaction, a strain was obtained in which the 139th base sequence of argR was replaced from guanine (g) to thymine (t), and the 47th protein sequence was replaced with a termination codon. The genetic manipulation was confirmed by PCR and sequence analysis using primer pairs of sequence numbers 53 and 56, which can amplify adjacent regions including the insertion site of the gene. The resulting microorganism was named C. gl::argR*.

[0140] To produce a microorganism with improved citrulline production in the C.gl::argR* obtained above, a microorganism was obtained using the pDCM2-argG(F68*) vector in a similar manner. PCR and nucleotide sequence analysis were performed using primer pairs of sequence numbers 57 and 60, which can amplify the adjacent region including the insertion site of the gene, to confirm the genetic manipulation. The microorganism thus obtained was named C.gl::argR*_argG*.

[0141] Example 2-2. Production of mutant prolyl isomerase-expressing bacterial strains To produce mutant strains with point mutations introduced into prolyl isomerase based on the C.gl::argR*_argG* strain obtained in Example 2-1, the following were used: pDCM2-fkpA(Y95M), pDCM2-fkpA(Y95S), pDCM2-fkpA(Y95T), pDCM2-fkpA(Y95N), pDCM2-fkpA(Y95C), pDCM2-fkpA(Y95H), pDCM2-fkpA(Y95K), pDCM2-fkpA(Y95D), pDCM2-fkpA(Y95A), pDCM2-fkpA(Y95V), pDCM2-fkpA(Y95L) After transforming the C.gl::argR*_argG* strain by electroporation using pDCM2-fkpA(Y95Q), pDCM2-fkpA(Y95G), pDCM2-fkpA(Y95P), and pDCM2-fkpA(Y95E), strains were obtained in which the 95th amino acid from the N-terminus of prolyl isomerase, tyrosine, was replaced with methionine, serine, threonine, asparagine, cysteine, histidine, lysine, aspartic acid, alanine, valine, leucine, glutamine, glycine, proline, or glutamic acid, respectively, via a secondary crossover process. DNA fragments containing the fkpA gene within the chromosome were PCR-amplified using primer pairs of SEQ ID NOs: 33 and 50 from the genome of the strains obtained above. The PCR conditions involved denaturation at 95°C for 10 minutes, followed by denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, polymerization at 72°C for 2 minutes, repeated 30 times, and then polymerization at 72°C for 10 minutes. Analysis of the nucleotide sequences of the amplified genes confirmed that mutations were introduced in the nucleotide sequence from the ORF start codon downwards to positions 283-285 of the fkpA gene in each strain (Table 4).

[0142] [Table 4]

[0143] This yields mutant strains based on the C.gl::argR*_argG* strain in which the 95th amino acid from the N-terminus of prolyl isomerase, tyrosine, is replaced with methionine, serine, threonine, asparagine, cysteine, histidine, lysine, aspartic acid, alanine, valine, leucine, glutamine, glycine, proline, or glutamic acid. These mutant strains are then named C.gl::argR*_argG*_fkpA(Y95M), C.gl::argR*_argG*_fkpA(Y95S), C.gl::argR*_argG*_fkpA(Y95T), C.gl::argR*_argG*_fkpA(Y95N), and C.gl::argR*_arg G*_fkpA(Y95C), C.gl::argR*_argG*_fkpA(Y95H), C.gl::argR*_argG*_fkpA(Y95K), C.gl: :argR*_argG*_fkpA(Y95D), C.gl::argR*_argG*_fkpA(Y95A), C.gl::argR*_argG*_fkpA(Y 95V), C.gl::argR*_argG*_fkpA(Y95L), C.gl::argR*_argG*_fkpA(Y95Q), C.gl::argR*_ar They were named gG*_fkpA(Y95G), C.gl::argR*_argG*_fkpA(Y95P), and C.gl::argR*_argG*_fkpA(Y95E).

[0144] Examples 2-3. Evaluation of citrulline production capacity C.gl::argR*_argG*_fkpA(Y95M), C.gl::argR*_argG*_fkpA(Y95S), C.gl::argR*_argG*_fkpA(Y95T), C.gl::argR*_argG*_fkpA(Y95N), C.gl::argR*_argG*_fkpA(Y95C), C.gl::argR*_argG*_fkpA(Y95H), C.gl::argR*_argG*_fkpA(Y95K), C.gl::argR*_argG*_fkpA(Y95D), C.gl::argR The L-citrulline production capacity of the strains *_argG*_fkpA(Y95A), C.gl::argR*_argG*_fkpA(Y95V), C.gl::argR*_argG*_fkpA(Y95L), C.gl::argR*_argG*_fkpA(Y95Q), C.gl::argR*_argG*_fkpA(Y95G), C.gl::argR*_argG*_fkpA(Y95P), and C.gl::argR*_argG*_fkpA(Y95E) was compared with the control strain C.gl::argR*_argG* by culturing them in the following production medium. Each strain was inoculated into a 250 mL corner baffle flask containing 25 mL of production medium and cultured at 33 °C for 48 hours with shaking at 200 rpm. After the culture was completed, the citrulline concentration was measured using HPLC, and the results are shown in Table 5 below.

[0145] <Production culture medium> Raw sugar 50g, (NH4)2SO4 30g, yeast extract 1g, KH2PO4 1.1g, MgSO4·7H2O 1.2g, L-arginine 0.2g, biotin 1mg, thiamine hydrochloride 5mg, calcium pantothenate 5mg, nicotinamide 15mg, MnSO4 10mg, FeSO4 10mg, ZnSO4 0.5mg, CuSO4 0.5mg, CaCO3 30g, pH 7.2 (based on 1 liter of distilled water)

[0146] [Table 5]

[0147] As a result, as shown in Table 5 above, strains in which the 95th amino acid from the N-terminus of prolyl isomerase, tyrosine, was replaced with methionine, serine, threonine, asparagine, cysteine, histidine, lysine, aspartic acid, alanine, valine, leucine, glutamine, glycine, proline, or glutamic acid all showed increased citrulline production and yield compared to the control strain C.gl::argR*_argG*.

[0148] Example 3: Evaluation of L-glutamine production capacity Example 3-1. Establishment of a control group and production of a mutant prolyl isomerase-expressing bacterial strain. KFCC10680 (Registered Patent No. 10-0048440 in the Republic of Korea) was used as a control strain for evaluating L-glutamine production capacity.

[0149] To produce mutant strains in which a point mutation has been introduced into prolyl isomerase based on the aforementioned KFCC10680 strain, the following strains were used: pDCM2-fkpA(Y95M), pDCM2-fkpA(Y95S), pDCM2-fkpA(Y95T), pDCM2-fkpA(Y95N), pDCM2-fkpA(Y95C), pDCM2-fkpA(Y95H), pDCM2-fkpA(Y95K), pDCM2-fkpA(Y95D), pDCM2-fkpA(Y95A), pDCM2-fkpA(Y95V), pDCM2-fkpA(Y95L), pDCM2-fkpA(Y95Q), pD KFCC10680 strains were transformed by electroporation using pDCM2-fkpA(Y95G), pDCM2-fkpA(Y95P), and pDCM2-fkpA(Y95E), specifically pDCM2-fkpA(Y95V) with nonpolar amino acid substitution, pDCM2-fkpA(Y95T) with polar amino acid substitution, and pDCM2-fkpA(Y95D) with charged amino acid substitution. After secondary cross-reactivity, strains were obtained in which the tyrosine at the 95th amino acid from the N-terminus of prolyl isomerase was substituted with valine, threonine, and aspartic acid, respectively. DNA fragments containing the fkpA gene within the chromosome were PCR-amplified using primer pairs of SEQ ID NOs: 33 and 50 from the genome of the strains obtained above. The PCR conditions involved denaturation at 95°C for 10 minutes, followed by denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, polymerization at 72°C for 2 minutes, repeated 30 times, and then polymerization at 72°C for 10 minutes. Analysis of the nucleotide sequences of the amplified genes confirmed that mutations were introduced in the nucleotide sequence from the ORF start codon downwards to positions 283-285 of the fkpA gene in each strain (Table 6).

[0150] [Table 6]

[0151] As a result, mutant strains were obtained in which the 95th amino acid from the N-terminus of prolyl isomerase, tyrosine, was replaced with valine, threonine, and aspartic acid, respectively, based on the KFCC10680 strain. These mutant strains were named KFCC10680::fkpA(Y95V), KFCC10680::fkpA(Y95T), and KFCC10680::fkpA(Y95D), respectively.

[0152] Example 3-2. Evaluation of glutamine production capacity The L-glutamine production capacity of the KFCC10680::fkpA(Y95V), KFCC10680::fkpA(Y95T), and KFCC10680::fkpA(Y95D) strains from Example 3-1 and the control strain KFCC10680 was compared by culturing them using the following production medium. Each strain was inoculated into a 250 mL corner baffle flask containing 25 mL of production medium and cultured at 32 °C for 48 hours with shaking at 200 rpm. After the culture was completed, the concentration of glutamine was measured using HPLC, and the results are shown in Table 7 below.

[0153] <Production culture medium> Raw sugar 60g, (NH4)2SO4 30g, yeast extract 1g, KH2PO4 0.6g, MgSO4·7H2O 0.6g, biotin 0.8mg, thiamine hydrochloride 4mg, calcium pantothenate 4mg, nicotinamide 20mg, MnSO4 8mg, FeSO4 8mg, ZnSO4 0.4mg, CuSO4 0.4mg, CaCO3 30g, pH 6.8 (based on 1 liter of distilled water)

[0154] [Table 7]

[0155] As a result, as shown in Table 7 above, all strains in which the 95th amino acid from the N-terminus of prolyl isomerase, tyrosine, was substituted with valine, threonine, and aspartic acid, respectively, showed increased glutamine production and yield compared to the control strain KFCC10680.

[0156] Example 4: Evaluation of L-ornithine production capacity Example 4-1. Preparation of the control group To prepare a control strain for evaluating L-ornithine production capacity, a vector was constructed in which the serine at position 55 of the ArgF (ANU33618.1) protein sequence was replaced with a termination codon. Using the genome of wild-type C. glutamicum ATCC13869 as a template, the homologous recombinant A arm was amplified using the primer pair of SEQ ID NOs. 61 and 62, and the homologous recombinant B arm was amplified using the primer pair of SEQ ID NOs. 63 and 64. Subsequently, a plasmid was obtained using the same method as in Example 1 and named pDCM2-argF(S55*).

[0157] Furthermore, in order to produce microorganisms with improved L-ornithine production capacity, a vector was created in which the glutamic acid at position 47 of the ArgR (ANU33619.1) protein sequence was replaced with a termination codon. Using the genome of wild-type C. glutamicum ATCC 13869 as a template, the homologous recombinant A arm was amplified using the primer pair of SEQ ID NOs. 53 and 54, and the homologous recombinant B arm was amplified using the primer pair of SEQ ID NOs. 55 and 56. Subsequently, a plasmid was obtained using the same method as in Example 1, and this plasmid was named pDCM2-argR(E47*).

[0158] The primer sequences used here are shown in Table 8 below.

[0159] [Table 8]

[0160] Wild-type C. glutamicum ATCC 13869 was transformed using the fabricated pDCM2-argF(S55*) vector via electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541-545). Following a secondary cross-reaction, a microorganism was obtained in which the 164th base sequence of argF was replaced from cytosine (c) to adenine (a), and the 55th protein sequence was replaced with a termination codon. The genetic manipulation was confirmed by PCR and sequence analysis using primer pairs of sequence numbers 61 and 64, which can amplify the adjacent region including the insertion site of the gene. The microorganism thus obtained was named C. gl::argF*.

[0161] To produce a microorganism with improved ornithine production from the C.gl::argF* obtained above, the microorganism was obtained using the pDCM2-argR(E47*) vector by the method described above. PCR and nucleotide sequence analysis were performed using primer pairs of sequence numbers 53 and 56, which can amplify the adjacent region including the insertion site of the gene, to confirm the genetic manipulation. The microorganism thus obtained was named C.gl::argR*_argF*.

[0162] Example 4-2. Production of mutant prolyl isomerase-expressing bacterial strains To produce mutant strains with point mutations introduced into prolyl isomerase based on the C.gl::argR*_argF* strain obtained in Example 4-1, the C.gl::argR*_argF* strain was transformed by electroporation using pDCM2-fkpA(Y95V), pDCM2-fkpA(Y95T), and pDCM2-fkpA(Y95D) from Example 1. After a secondary crossover, strains were obtained in which the tyrosine at the 95th amino acid from the N-terminus of prolyl isomerase was replaced with valine, threonine, and aspartic acid, respectively. DNA fragments containing the fkpA gene within the chromosome were PCR-amplified using primer pairs of SEQ ID NOs. 33 and 50 from the genome of the obtained strains. The PCR conditions involved denaturation at 95°C for 10 minutes, followed by denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, polymerization at 72°C for 2 minutes, repeated 30 times, and then polymerization at 72°C for 10 minutes. Analysis of the amplified gene sequences confirmed that mutations were introduced in the fkpA gene from the ORF start codon to the lower 283-285 nucleotide sequences in each strain (Table 9).

[0163] [Table 9]

[0164] This resulted in the acquisition of mutant strains based on the C.gl::argR*_argF* strain in which the 95th amino acid from the N-terminus of prolyl isomerase, tyrosine, was substituted with valine, threonine, and aspartic acid, respectively. These mutant strains were named C.gl::argR*_argF*_fkpA(Y95V), C.gl::argR*_argF*_fkpA(Y95T), and C.gl::argR*_argF*_fkpA(Y95D), respectively.

[0165] Example 4-3. Evaluation of ornithine production capacity The L-ornithine production capacity of the C.gl::argR*_argF*_fkpA(Y95V), C.gl::argR*_argF*_fkpA(Y95T), and C.gl::argR*_argF*_fkpA(Y95D) strains from Example 4-2 was compared with the control strain C.gl::argR*_argF* by culturing them using the following production medium. Each strain was inoculated into a 250 mL corner baffle flask containing 25 mL of production medium and cultured at 32 °C for 48 hours with shaking at 200 rpm. After the culture was completed, the ornithine concentration was measured using HPLC, and the results are shown in Table 10 below.

[0166] <Production culture medium> Raw sugar 50g, (NH4)2SO4 30g, yeast extract 1g, KH2PO4 1.1g, MgSO4·7H2O 1.2g, L-arginine 0.2g, biotin 1mg, thiamine hydrochloride 5mg, calcium pantothenate 5mg, nicotinamide 15mg, MnSO4 10mg, FeSO4 10mg, ZnSO4 0.5mg, CuSO4 0.5mg, CaCO3 30g, pH 7.2 (based on 1 liter of distilled water)

[0167] [Table 10]

[0168] As a result, as shown in Table 10 above, all strains in which the 95th amino acid from the N-terminus of prolyl isomerase, tyrosine, was substituted with valine, threonine, and aspartic acid, respectively, showed increased ornithine production and yield compared to the control strain C.gl::argR*_argF*.

[0169] Example 5: Evaluation of L-arginine production capacity Example 5-1. Preparation of the control group To prepare a control strain for evaluating L-arginine production capacity, a vector was constructed in which the glutamic acid at position 47 of the ArgR (ANU33619.1) protein sequence was replaced with a termination codon. Using the genome of wild-type C. glutamicum ATCC 13869 as a template, the homologous recombinant A arm was amplified using primer pairs of SEQ ID NOs. 53 and 54, and the homologous recombinant B arm was amplified using primer pairs of SEQ ID NOs. 55 and 56. Subsequently, a plasmid was obtained using the same method as described above, and this plasmid was named pDCM2-argR(E47*).

[0170] The primer sequences used here are shown in Table 11 below.

[0171] [Table 11]

[0172] Wild-type C. glutamicum ATCC 13869 was transformed using the fabricated pDCM2-argR(E47*) vector via electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541-545). Following a secondary cross-reaction, a strain was obtained in which the 139th base sequence of argR was replaced from guanine (g) to thymine (t), and the 47th protein sequence was replaced with a termination codon. The genetic manipulation was confirmed by PCR and sequence analysis using primer pairs of sequence numbers 53 and 56, which can amplify adjacent regions including the insertion site of the gene. The resulting microorganism was named C. gl::argR*.

[0173] Example 5-2. Production of mutant prolyl isomerase-expressing bacterial strains To produce mutant strains with point mutations introduced into prolyl isomerase based on the C.gl::argR* strain obtained in Example 5-1, the C.gl::argR* strain was transformed by electroporation using pDCM2-fkpA(Y95V), pDCM2-fkpA(Y95T), and pDCM2-fkpA(Y95D) from Example 1. After a secondary crossover, strains were obtained in which the tyrosine at the 95th amino acid from the N-terminus of prolyl isomerase was replaced with valine, threonine, and aspartic acid, respectively. DNA fragments containing the fkpA gene within the chromosome were PCR-amplified using primer pairs of SEQ ID NOs. 33 and 50 in the genome of the obtained strains. The PCR conditions were: denaturation at 95°C for 10 minutes, denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, polymerization at 72°C for 2 minutes, repeated 30 times, followed by polymerization at 72°C for 10 minutes. Analysis of the amplified gene sequences revealed that mutations were introduced in the fkpA gene at positions 283-285 below the ORF start codon in each strain (Table 12).

[0174] [Table 12]

[0175] This resulted in the acquisition of mutant strains based on the C.gl::argR* strain in which the 95th amino acid from the N-terminus of prolyl isomerase, tyrosine, was substituted with valine, threonine, and aspartic acid, respectively. These mutant strains were named C.gl::argR*_fkpA(Y95V), C.gl::argR*_fkpA(Y95T), and C.gl::argR*_fkpA(Y95D), respectively.

[0176] Example 5-3. Evaluation of Arginine Production Capacity The L-arginine production capacity of the C.gl::argR*_fkpA(Y95V), C.gl::argR*_fkpA(Y95T), and C.gl::argR*_(Y95D) strains from Example 5-2 and the control strain C.gl::argR* was compared by culturing them using the following production medium. Each strain was inoculated into a 250 mL corner baffle flask containing 25 mL of production medium and cultured at 32 °C for 48 hours with shaking at 200 rpm. After the culture was completed, the arginine concentration was measured using HPLC, and the results are shown in Table 13 below.

[0177] <Production culture medium> Raw sugar 50g, (NH4)2SO4 30g, yeast extract 1g, KH2PO4 1.1g, MgSO4·7H2O 1.2g, L-arginine 0.2g, biotin 1mg, thiamine hydrochloride 5mg, calcium pantothenate 5mg, nicotinamide 15mg, MnSO4 10mg, FeSO4 10mg, ZnSO4 0.5mg, CuSO4 0.5mg, CaCO3 30g, pH 7.2 (based on 1 liter of distilled water)

[0178] [Table 13]

[0179] As shown in Table 13 above, strains in which the 95th amino acid from the N-terminus of prolyl isomerase, tyrosine, was substituted with valine, threonine, and aspartic acid, respectively, all showed increased arginine production and yield compared to the control strain C.gl::argR*.

[0180] From the above description, a person skilled in the art to which this application pertains will understand that this application can be implemented in other specific forms without altering its technical idea or essential features. In this regard, it should be understood that the embodiments described above are merely illustrative and not limiting. The scope of this application should be interpreted as encompassing all modified or altered forms derived from the meaning and scope of the claims, as described below, and their equivalent concepts, rather than from the above detailed description.

Claims

1. A mutant prolyl isomerase in which the amino acid corresponding to position 95 in the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid, and which has at least 90% sequence identity with SEQ ID NO:

1.

2. The mutant prolyl isomerase according to claim 1, wherein the other amino acid is one or more selected from the group consisting of methionine, serine, threonine, asparagine, cysteine, histidine, lysine, aspartic acid, alanine, valine, leucine, glutamine, glycine, proline, and glutamic acid.

3. The mutant prolyl isomerase according to claim 1, wherein the mutant prolyl isomerase has an amino acid sequence consisting of SEQ ID NO: 1 in which amino acid position 95 is replaced with another amino acid.

4. A polynucleotide encoding a mutant prolyl isomerase according to any one of claims 1 to 3.

5. A microorganism comprising one or more selected from the following: a mutant prolyl isomerase having at least 90% sequence identity with SEQ ID NO: 1, in which the amino acid corresponding to the 95th position in the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid; a polynucleotide encoding the same; and a vector containing the polynucleotide.

6. The microorganism according to claim 5, wherein the microorganism has improved glutamate series amino acid production capacity compared to a Corynebacterium microorganism containing wild-type prolyl isomerase having the amino acid sequence of Sequence ID No. 1 or a polynucleotide encoding it.

7. The microorganism according to claim 6, wherein the glutamate series amino acid is one or more selected from the group consisting of glutamic acid, citrulline, glutamine, ornithine, and arginine.

8. The microorganism according to claim 5, wherein the microorganism is a microorganism of the genus Corynebacterium.

9. The microorganism according to claim 8, wherein the Corynebacterium genus microorganism is Corynebacterium glutamicum.

10. A method for producing glutamic acid series amino acids, comprising the step of culturing a microorganism containing a mutant prolyl isomerase having at least 90% sequence identity with SEQ ID NO: 1, in which the amino acid corresponding to the 95th position in the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid, or a polynucleotide encoding the same, in a culture medium.

11. The method according to claim 9, wherein the glutamate series amino acid is one or more selected from the group consisting of glutamic acid, citrulline, glutamine, ornithine, and arginine.

12. A composition for producing glutamate series amino acids, comprising: a mutant prolyl isomerase having at least 90% sequence identity with SEQ ID NO: 1, in which the amino acid corresponding to the 95th position in the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid; a polynucleotide encoding the same; a vector containing the polynucleotide or a microorganism containing the polynucleotide; a culture medium for the same; or a combination of two or more of these.

13. Use of a mutant prolyl isomerase having at least 90% sequence identity with SEQ ID NO: 1, a polynucleotide encoding the same, a vector containing the polynucleotide, or a microorganism containing the polynucleotide for the production of glutamate series amino acids.

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