Aminotransferase variant and uses thereof

Abstract

The present disclosure relates to an aminotransferase variant and uses thereof, and a microorganism expressing the aminotransferase variant exhibits excellent amino acid production capability.

Description

Aminotransferase variants and uses thereof

[0001] The present disclosure relates to aminotransferase variants and uses thereof.

[0002] Cross-citation with related application(s)

[0003] This application claims the benefit of priority based on Korean Patent Application No. 10-2024-0172347 filed November 27, 2024 and Korean Patent Application No. 10-2025-0123898 filed September 2, 2025, and all contents disclosed in the documents of said Korean patent applications are incorporated herein as part of this specification.

[0004]

[0005] L-Alanine is a colorless or white crystalline amino acid with no odor but a distinctive sweet taste, and it is widely applied in fields such as chemistry, food, and medicine. In the food industry, it is particularly utilized as a flavor enhancer or nutritional fortifier to improve umami, and it possesses various physiological effects such as promoting alcohol metabolism, protecting liver function, and stimulating insulin secretion. Compared to other amino acids, it inhibits browning reactions, so it is also used as an acidity corrector. Furthermore, it is utilized in a wide range of applications, including pharmaceuticals for benign prostatic hyperplasia and sports products. As of 2020, the annual production of alanine is approximately 500 tons, with a market size estimated at over USD 250 million. While most food additives or coloring agents are obtained through the extraction of natural resources, chemical synthesis, or biological production, L-Alanine is primarily produced through chemical synthesis or enzymatic conversion. Recently, driven by a shift in consumer awareness prioritizing a healthy lifestyle, natural substances have become more preferred over artificial products, despite their higher prices resulting from low production yields. Accordingly, various research efforts are being made to move beyond petroleum-derived L-alanine production methods, such as developing microorganisms or fermentation process technologies that produce high concentrations of amino acids.

[0006]

[0007] [Prior Art Literature]

[0008] (Patent Document 1) U.S. Published Patent Application (US 5559016 A)

[0009]

[0010] The present disclosure provides a variant polypeptide in which the amino acid corresponding to the 308th residue of the amino acid sequence of SEQ ID NO. 1 is substituted with another amino acid.

[0011] The present disclosure provides a polynucleotide encoding the variant polypeptide.

[0012] The present disclosure provides a vector comprising the polynucleotide.

[0013] The present disclosure provides a microorganism comprising one or more selected from the group consisting of the variant polypeptide, a polynucleotide encoding the variant polypeptide, and a vector comprising the polynucleotide.

[0014] The present disclosure provides a composition for producing amino acids comprising one or more selected from the group consisting of the microorganism and a culture medium in which the microorganism is cultured.

[0015] The present disclosure provides a method for producing amino acids comprising the step of culturing the microorganism in a culture medium.

[0016]

[0017] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in this disclosure may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this disclosure fall within the scope of this disclosure. Furthermore, the scope of this disclosure is not to be limited by the specific descriptions provided below. Additionally, a person skilled in the art can recognize or identify numerous equivalents to the specific aspects of this disclosure described herein using only ordinary experiments. Moreover, such equivalents are intended to be included in this disclosure.

[0018]

[0019] polypeptide

[0020] One aspect provides a variant polypeptide in which the amino acid corresponding to the 308th residue in the amino acid sequence of SEQ ID NO. 1 is substituted with another amino acid.

[0021] In this disclosure, the term “variant polypeptide” refers to a polypeptide in which one or more amino acids are conservatively substituted and / or modified, resulting in a sequence different from the amino acid sequence of the variant polypeptide before modification, while retaining functions or properties. Such variant polypeptides can generally be identified by modifying one or more amino acids in the amino acid sequence of the polypeptide and evaluating the properties of the modified polypeptide. That is, the capabilities of the variant polypeptide may be increased, unchanged, or decreased compared to the polypeptide before modification. Additionally, some variant polypeptides may include a variant polypeptide in which one or more parts, such as an N-terminal leader sequence or a transmembrane domain, have been removed. Other variant polypeptides may include a variant polypeptide in which a portion of the N- and / or C-terminus of a mature protein has been removed. The above term "variant polypeptide" may be used interchangeably with terms such as variant, modification, variant polypeptide, mutated protein, mutation, and variant (in English expressions, modification, modified polypeptide, modified protein, mutant, mutein, divergent, variant, etc.), and is not limited to any term used in the sense of being mutated. Additionally, the variant polypeptide may include deletions or additions of amino acids that have minimal effect on the properties and secondary structure of the polypeptide. For example, a signal (or leader) sequence involved in co-translational or post-translational protein translocation may be conjugated to the N-terminus of the variant polypeptide.In addition, the above variant polypeptide can be conjugated with other sequences or linkers so that it can be identified, purified, or synthesized.

[0022] In the present disclosure, 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 may involve determining a specific amino acid of a sequence that references a specific sequence.

[0023] For example, any amino acid sequence can be aligned with sequence number 1, and based on this, each amino acid residue of the said amino acid sequence can be numbered by referring to the numerical position of the amino acid residue corresponding to the amino acid residue of sequence number 1. For example, a sequence alignment algorithm such as that described in the present application can identify the position of an amino acid, or the position where modifications such as substitution, insertion, or deletion occur, by comparing with a query sequence (also referred to as a "reference sequence").

[0024] For such alignment, examples such as the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000), Trends Genet. 16: 276-277) may be used, but are not limited thereto, and sequence alignment programs and pairwise sequence comparison algorithms known in the art may be appropriately used.

[0025] The amino acid sequence of SEQ ID NO. 1 above may be a protein having aminotransferase activity.

[0026] In one example, the amino acid sequence of SEQ ID NO. 1 may be a protein having aminotransferase activity derived from a microorganism of the genus Corynebacterium. The amino acid sequence of the protein may be obtained from a known database (e.g., NCBI Reference Sequence: WP_003862691.1).

[0027] The above-mentioned microorganisms of the genus Corynebacterium are Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium pollutisoli, and Corynebacterium imitans It may be imitans), Corynebacterium testudinoris, or Corynebacterium flavescens, but is not limited thereto.

[0028] In one example, the amino acid sequence of SEQ ID NO. 1 may be a protein having aminotransferase activity derived from Corynebacterium glutamicum ATCC13869 (GenBank: ANU34753.1).

[0029] The above aminotransferase may refer to an enzyme having the function of reversibly producing L-alanine from pyruvate. L-glutamic acid may be used as a cofactor in the above reaction, and the above aminotransferase may be an enzyme having the function of catalyzing the conversion reaction between L-alanine and 2-oxoglutarate.

[0030] The above variant polypeptide may be a variant polypeptide in which the amino acid corresponding to the 308th residue of the amino acid sequence of SEQ ID NO. 1 (arginine, hereinafter the same) is substituted with another amino acid, namely glutamine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, cysteine, glycine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan.

[0031] In one example, the variant polypeptide may be a variant polypeptide in which the amino acid corresponding to the 308th residue of the amino acid sequence of SEQ ID NO. 1 is substituted with glutamine, asparagine, aspartic acid, glutamic acid, lysine, serine, cysteine, or threonine.

[0032] In one example, the variant polypeptide comprises the amino acid sequence of SEQ ID NO. 1 and 60% or more, 65% or more, 70% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 90.5% or more, 91% or more, 91.5% or more, 92% or more, 92.5% or more, 93% or more, 93.5% or more, 94% or more, 94.5% or more, 95% or more, 95.5% or more, 96% or more, 96.5% or more, 97% or more, 97.5% or more, 97.6% It includes an amino acid sequence having sequence homology of 97.7% or more, 97.8% or more, 97.9% or more, 98% or more, 98.1% or more, 98.2% or more, 98.3% or more, 98.4% or more, 98.5% or more, 98.6% or more, 98.7% or more, 98.8% or more, 98.9% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more, and

[0033] The amino acid corresponding to the 308th residue of the amino acid sequence of SEQ ID NO. 1 may be a variant polypeptide in which the amino acid is substituted with another amino acid, namely glutamine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, cysteine, glycine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan.

[0034] In this disclosure, 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 may often be used interchangeably.

[0035] Sequence homology or identity of conserved polynucleotides or polypeptides is determined by standard arrangement algorithms, and a default gap penalty established by the program used may be utilized. Practically, homologous or identical sequences can generally be hybridized with the entire sequence or a part thereof under moderate or high stringent conditions. It is evident that hybridization also includes hybridization with polynucleotides containing common codons or codons that account for codon degeneracy.

[0036] Whether any two polynucleotide or polypeptide sequences have homology or identity can be determined using a known computer algorithm, such as the “FASTA” program, using default parameters as in, for example, 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.,] (Including Academic Press, San Diego, 1994, and [CARILLO ETA / .](1988) SIAM J Applied Math 48: 1073). For example, homology or identity can be determined using BLAST or ClustalW from the National Biotechnology Information Database Center.

[0037] The homology or identity of polynucleotides or polypeptides can be determined by comparing sequence information using a GAP computer program, such as that described in, for example, Smith and Waterman, Adv. Appl. Math (1981) 2:482, or Needleman et al. (1970), J Mol Biol. 48:443. In summary, a GAP program can be defined as the total number of symbols in the shorter of the two sequences divided by the number of similarly arranged symbols (i.e., nucleotides or amino acids). The default parameters for a GAP program are (1) a binary comparison matrix (containing values ​​of 1 for identity and 0 for non-identity) and, as disclosed by Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979), or Gribskov et al. (1986) Nucl. Acids Res. 14: A weighted comparison matrix of 6745 (or an EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (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.

[0038] In the present disclosure, the phrase “a polypeptide (which may be interchangeably used with “protein”) or a polynucleotide (which may be interchangeably used with “gene”) comprises a specific nucleic acid sequence or amino acid sequence or is composed of or expressed by a specific nucleic acid sequence or amino acid sequence” may mean that the polynucleotide or polypeptide essentially comprises the specific nucleic acid sequence or amino acid sequence, and may be interpreted as comprising a “substantially equivalent sequence” in which a meaningless variation (deletion, substitution, modification, and / or addition) is applied to the specific nucleic acid sequence or amino acid sequence to the extent that the original function and / or intended function of the polynucleotide or polypeptide is maintained (or not excluding the meaningless variation).

[0039] In one example, the above variant polypeptide may be a variant polypeptide comprising the amino acid sequence of SEQ ID NO. 1, wherein the amino acid of the 308th residue in the amino acid sequence of SEQ ID NO. 1 is substituted with another amino acid, namely glutamine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, cysteine, glycine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan.

[0040] In one example, the variant polypeptide may be a variant polypeptide comprising the amino acid sequence of SEQ ID NO. 1, wherein the amino acid of the 308th residue in the amino acid sequence of SEQ ID NO. 1 is substituted with glutamine, asparagine, aspartic acid, glutamic acid, lysine, serine, cysteine, or threonine.

[0041] In one example, the variant polypeptide may include the amino acid sequence of SEQ ID NO. 3, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, or SEQ ID NO. 28, or may be composed of the said amino acid sequence, but is not limited thereto.

[0042] In one example, the variant polypeptide may be a protein having aminotransferase activity. In one example, the variant polypeptide may be a protein having pyridoxal phosphate-dependent aminotransferase activity.

[0043] The above variant polypeptide may have increased activity, and / or may have activity that increases the amino acid production capacity of a microorganism expressing the above variant polypeptide. The above amino acid may be one or more selected from the group consisting of alanine, arginine, glutamine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, cysteine, glycine, proline, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. The above amino acid may be an L-amino acid.

[0044] In one example, the variant polypeptide may have increased activity, and / or may have an activity that increases the alanine production capacity of a microorganism expressing the variant polypeptide.

[0045] It is obvious that if the above variant polypeptide contains a variant in which the amino acid corresponding to the 308th residue of the amino acid sequence of SEQ ID NO. 1 is substituted with another amino acid, then other amino acid residues other than the one corresponding to said residue may be deleted, modified, substituted, or added, provided that they exhibit aminotransferase activity. For example, this may be a case where the amino acid sequence of the above variant polypeptide has sequence additions or deletions that do not alter the activity of the polypeptide, naturally occurring mutations, silent mutations, or conservative substitutions at the N-terminus, C-terminus, and / or inside.

[0046] The aforementioned “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.

[0047]

[0048] polynucleotide

[0049] Another aspect provides a polynucleotide encoding the above variant polypeptide.

[0050] In the present disclosure, the term “polynucleotide” refers to a polymer of nucleotides in which nucleotide monomers are linked together in a long chain by covalent bonds, and may mean a DNA or RNA strand of a certain length or longer.

[0051] The above polynucleotide may refer to a gene encoding the above variant polypeptide. The above polynucleotide may be of a microorganism of the genus Corynebacterium, but is not limited thereto, and may be prepared by referring to a known codon table based on the amino acid sequence of the above variant polypeptide.

[0052] In one example, the polynucleotide codes for a variant polypeptide in which the amino acid (arginine, hereinafter the same) corresponding to the 308th residue of the amino acid sequence of SEQ ID NO. 1 is substituted with another amino acid, and

[0053] The codon coding for the amino acid corresponding to the 308th residue of the amino acid sequence of SEQ ID NO. 1 may be substituted with a codon coding for another amino acid.

[0054] In one example, the polynucleotide may be one in which the codon encoding the amino acid corresponding to the 308th residue of the amino acid sequence of SEQ ID NO. 1 is substituted with a codon encoding glutamine, asparagine, aspartic acid, glutamic acid, lysine, serine, cysteine, or threonine.

[0055] In one example, the above polynucleotide codes for a variant polypeptide in which the amino acid at the 308th residue of the amino acid sequence of SEQ ID NO. 1 is substituted with another amino acid, and

[0056] The codon coding for the amino acid of the 308th residue of the amino acid sequence of SEQ ID NO. 1 may be substituted with a codon coding for another amino acid.

[0057] In one example, the polynucleotide may be one in which the codon encoding the amino acid of the 308th residue of the amino acid sequence of SEQ ID NO. 1 is substituted with a codon encoding glutamine, asparagine, aspartic acid, glutamic acid, lysine, serine, cysteine, or threonine.

[0058] In one example, the polynucleotide comprises the nucleic acid sequence of SEQ ID NO. 2, and the 308th codon (922 to 924 nucleotides) encoding arginine in the nucleic acid sequence of SEQ ID NO. 2 may be substituted with a codon encoding another amino acid.

[0059] In one example, the polynucleotide comprises the nucleic acid sequence of SEQ ID NO. 2, and the 308th codon (922 to 924 nucleotides) encoding arginine in the nucleic acid sequence of SEQ ID NO. 2 may be substituted with a codon encoding glutamine, asparagine, aspartic acid, glutamic acid, lysine, serine, cysteine, or threonine.

[0060] It is clearly known in the art what polynucleotide sequence the codon encoding each amino acid contains. The said codon may be modified in various ways without altering the amino acid sequence of the polypeptide, taking into account the degeneracy of the codon or the codons preferred by the organism intended to express the polypeptide.

[0061] In one example, the polynucleotide may include the nucleic acid sequence of SEQ ID NO. 4, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, or SEQ ID NO. 29, or may be composed of the nucleic acid sequence, but is not limited thereto.

[0062] The polynucleotides of the present disclosure may, without limitation, include probes that can be prepared from known gene sequences, for example, sequences that can be hybridized under stringent conditions with a sequence complementary to all or part of the polynucleotide sequence of the present disclosure. The “stringent condition” 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, polynucleotides with high homology or identity, at least 60%, 65%, 70%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or more, 99.5% or more, or 99.Conditions may be listed in which polynucleotides having 9% or more homology or identity are hybridized with each other, and polynucleotides having lower homology or identity are not hybridized with each other, or conditions in which washing is performed once, specifically two to three times, at a salt concentration and temperature equivalent to the washing conditions of conventional southern hybridization, such as 60°C, 1×SSC, 0.1% SDS, specifically 60°C, 0.1×SSC, 0.1% SDS, more specifically 68°C, 0.1×SSC, 0.1% SDS.

[0063] Hybridization requires that two nucleotides have complementary sequences, but hybridized polynucleotides may contain some mismatch between bases depending on the degree of hybridization. The term “complementary” is used to describe the relationship between nucleotide bases that can hybridize with each other. For example, with respect to DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Accordingly, the polynucleotides of the present disclosure may also include substantially similar nucleic acid sequences as well as isolated nucleic acid fragments that are complementary to the entire sequence.

[0064] Specifically, a polynucleotide having homology or identity with the polynucleotide of the present disclosure can be detected using hybridization conditions including a hybridization step at a Tm value of 55 °C and using the conditions described above. Additionally, the Tm value may be 60 °C, 63 °C, or 65 °C, but is not limited thereto and can be appropriately adjusted by a person skilled in the art according to the purpose.

[0065] The appropriate strictness for hybridizing the above polynucleotides depends on the length and degree of complementarity of the polynucleotides, and the variables are well known in the art (e.g., J. Sambrook et al., i.e.).

[0066]

[0067]

[0068] vector

[0069] Another aspect provides a vector containing the above polynucleotide.

[0070] The above vector may be an insertion vector, an expression vector, or a recombination vector.

[0071] In the present disclosure, the term “vector” refers to a DNA product for delivering a target polynucleotide into a suitable host or host cell. For example, it may comprise, but is not limited to, a nucleic acid sequence of a polynucleotide encoding a target polypeptide operably linked to a suitable expression control region (or expression control sequence) so as to enable the expression of the target polypeptide within a suitable host cell. The control sequence may comprise a promoter capable of initiating transcription, any operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and / or a sequence regulating the termination of transcription and / or translation. After being transformed into a suitable host cell, the vector may be maintained independently of the host cell’s genome or inserted into the host cell’s genome. In one example, the target polynucleotide may be inserted into a chromosome via an insertion vector. The insertion of the above polynucleotide into a chromosome may be achieved by any method known in the art, for example, homologous recombination, but is not limited thereto.

[0072] The vectors available in this disclosure are not particularly limited as long as they are capable of replicating within a host cell and can be selected from all commonly used vectors. Examples of commonly used vectors include plasmids, cosmids, viruses, bacteriophages, etc., in their natural or recombinant state. For example, as the vectors, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A, etc., can be used as phage vectors or cosmid vectors, and pBR-based, pUC-based, pBluescriptII-based, pGEM-based, pTZ-based, pCL-based, and pET-based vectors, etc., can be used as plasmid vectors. Specifically, examples include pDC24 (sequence number 7), pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors, but are not limited thereto.

[0073] The above vector may additionally include a selection marker to determine whether it is introduced into transformed cells or inserted into the genome of transformed cells. The selection marker is intended to confirm whether the cells transformed by the vector or the polynucleotide has been inserted, and may be selected from genes that confer selectable phenotypes, such as drug resistance, nutritional requirements, resistance to cytotoxic agents, or the expression of surface proteins. Since only cells expressing the selection marker survive or exhibit other phenotypes in an environment treated with a selective agent, the transformed cells can be selected.

[0074] Expressing the above polypeptide in a microorganism can be performed by introducing a polynucleotide encoding the above polypeptide, or a vector containing the above polynucleotide, into a host cell and culturing a recombinant cell (e.g., a microorganism) containing the same.

[0075] The introduction of a polynucleotide encoding the above polypeptide or a vector containing said polynucleotide into a microorganism may be performed by a person skilled in the art by appropriately selecting a known transformation method. In this specification, the term "transformation" means introducing a target polynucleotide or a vector containing said polynucleotide into a host cell (microorganism) to change the genetic traits of the host cell (microorganism). The transformed polynucleotide may be inserted into the chromosomes of the host cell or located outside the chromosomes. The said polynucleotide may be introduced in an appropriate form depending on the purpose of introduction. For example, said polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a genetic structure containing all the elements necessary for self-expression. The expression cassette may include expression regulatory elements such as a promoter, a transcription termination signal, a ribosome binding site, and / or a translation termination signal, which are typically operably linked to said polynucleotide. The expression cassette may be in the form of a self-replicating expression vector. In addition, the polynucleotide may be introduced into a host cell in its own form and operably linked to a sequence required for expression in the host cell. The term "operably linked" above may mean that the polynucleotide is functionally linked to an expression regulatory element (e.g., a promoter) so as to enable transcriptional regulation (e.g., transcription initiation) of the polynucleotide. Operable linkage can be performed using gene recombination techniques known in the art.

[0076] The method of transforming the above-mentioned polynucleotide into a host cell can be carried out by any method of introducing nucleic acid into a cell (microorganism), and depending on the host cell, transformation techniques known in the art can be appropriately selected. Examples of the above-mentioned known transformation methods include, but are not limited to, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG)-mediated uptake, DEAE-dextran method, cationic liposome method, lipofection, and lithium acetate-DMSO method.

[0077]

[0078] The above polypeptide may have enhanced aminotransferase activity, and / or may have activity that enhances the amino acid production ability of a microorganism expressing the above polypeptide.

[0079] In the present disclosure, the term “enhancement” of polypeptide (protein) activity means that the activity of the polypeptide is increased compared to its intrinsic activity. Such enhancement may be used interchangeably with terms such as activation, up-regulation, overexpression, and increase. Here, activation, enhancement, up-regulation, overexpression, and increase may include exhibiting activity that was not originally possessed, or exhibiting improved activity compared to the intrinsic activity or activity prior to modification. The “intrinsic activity” refers to the activity of a specific polypeptide originally possessed by the parent strain or the non-modified microorganism prior to the change in traits caused by genetic mutations due to natural or artificial factors. This may be used interchangeably with “activity prior to modification.” The statement that the activity of a polypeptide is “enhanced,” “upregulated,” “overexpressed,” or “increased” relative to its intrinsic activity means that it has been enhanced compared to the activity and / or concentration (expression amount) of a specific polypeptide originally possessed by the parent strain or non-modified microorganism prior to transformation.

[0080] The above enhancement can be achieved by introducing an exogenous polypeptide or by enhancing the activity and / or increasing the concentration (expression amount) of the intrinsic polypeptide. Whether the activity of the polypeptide is enhanced can be confirmed by an increase in the degree of activity, expression amount, or amount of product resulting from the polypeptide activity of said polypeptide.

[0081] The enhancement of the activity of the above polypeptide may be achieved by applying various methods well known in the art, and is not limited to, as long as the activity of the target polypeptide can be enhanced compared to the microorganism before modification. Specifically, it may utilize, but is not limited to, gene engineering and / or protein engineering known to a person skilled in the art, which are routine methods of molecular biology (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.).

[0082] Specifically, the activity enhancement of the polypeptide (protein) of the present disclosure is

[0083] 1) Increase in the intracellular copy number of polynucleotides encoding polypeptides;

[0084] 2) Replace the chromosomal gene expression regulatory region encoding a polypeptide with a potent sequence;

[0085] 3) A modification of the nucleotide sequence encoding the start codon or the 5'-UTR region of the gene transcript encoding the polypeptide;

[0086] 4) Modification of the amino acid sequence of the polypeptide to enhance polypeptide activity;

[0087] 5) Modification of the nucleic acid sequence encoding the polypeptide to enhance polypeptide activity (e.g., modification of the nucleic acid sequence of the polypeptide gene to code for a polypeptide modified to enhance polypeptide activity);

[0088] 6) Introduction of a foreign polypeptide exhibiting polypeptide activity or a foreign polynucleotide encoding the same;

[0089] 7) Codon optimization of polynucleotides encoding polypeptides;

[0090] 8) Analyze the tertiary structure of the polypeptide to select and modify or chemically modify the exposed sites;

[0091] 9) Regulation of the cellular localization of polypeptides; or

[0092] 10) It may be based on two or more combinations selected from 1) to 9) above, but is not specifically limited thereto.

[0093] More specifically,

[0094] The increase in the intracellular copy number of the polynucleotide encoding the above 1) polypeptide may be achieved by introducing into a host cell a vector to which the polynucleotide encoding the said polypeptide is operably linked, which can replicate and function independently of the host. Alternatively, it may be achieved by introducing one or more copies of the polynucleotide encoding the said polypeptide into the chromosomes within the host cell. The introduction into the chromosomes may be performed by introducing into the host cell a vector capable of inserting said polynucleotide into the chromosomes within the host cell, but is not limited thereto. The said vector is as described above.

[0095] Replacing the gene expression regulatory region (or expression regulatory sequence) on the chromosome encoding the polypeptide 2) above with a sequence having potent activity may, for example, involve deletion, insertion, non-conservative or conservative substitution, or a combination thereof, to further enhance the activity of the expression regulatory region, or may involve a sequence mutation, or replacement with a sequence having stronger activity. 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 regulating the termination of transcription and translation. As an example, the original promoter may be replaced with a potent promoter, but is not limited thereto.

[0096] Examples of known strong promoters include, but are not limited to, CJ1 to CJ7 promoters (US Patent No. 7662943 B2), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13(sm3) promoter (US Patent No. 10584338 B2), O2 promoter (US Patent No. 10273491 B2), tkt promoter, yccA promoter, etc.

[0097] The above 3) modification of the nucleotide sequence encoding the start codon or 5'-UTR region of the gene transcript encoding the polypeptide may, for example, be a substitution with a nucleotide sequence encoding another start codon that has a higher polypeptide expression rate compared to the intrinsic start codon, but is not limited thereto.

[0098] The modification of the amino acid sequence or nucleic acid sequence of 4) and 5) above may be, but is not limited to, the occurrence of sequence variations in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, to enhance the activity of the polypeptide, or the replacement with an amino acid sequence or nucleic acid sequence modified to have stronger activity or an amino acid sequence or nucleic acid sequence modified to increase activity. Specifically, the replacement may be performed by inserting the polynucleotide into the chromosome by homologous recombination, but is not limited thereto. The vector used in this case may additionally include a selection marker to confirm whether the chromosome is inserted.

[0099] The introduction of an exogenous polynucleotide exhibiting the activity of the polypeptide described in 6) above may be the introduction into a host cell of an exogenous polynucleotide encoding a polypeptide that exhibits the same or similar activity as the polypeptide. As long as the exogenous polynucleotide exhibits the same or similar activity as the polypeptide, there are no restrictions on its origin or sequence. The method used for the introduction may be performed by a person skilled in the art by appropriately selecting a known transformation method, and the polypeptide may be generated and its activity increased by the expression of the introduced polynucleotide within the host cell.

[0100] The above 7) codon optimization of the polynucleotide encoding the polypeptide may be a codon optimization of the intrinsic polynucleotide such that transcription or translation increases within the host cell, or a codon optimization of the extrinsic polynucleotide such that optimized transcription and translation occur within the host cell.

[0101] 8) The above method of analyzing the tertiary structure of the polypeptide to select and modify or chemically modify an exposed site may involve, for example, determining a template protein candidate based on the degree of sequence similarity by comparing the sequence information of the polypeptide to be analyzed with a database in which sequence information of known proteins is stored, confirming the structure based on this, and selecting and modifying or modifying an exposed site to be modified or chemically modified.

[0102] The above 9) regulation of the intracellular localization of the polypeptide may involve targeting the polypeptide to a specific intracellular organelle or a specific intracellular space. For example, it may involve targeting to the periplasm or cytoplasm through the addition or removal of a leader sequence that functions for the targeting of the polypeptide, but is not limited thereto.

[0103] Such enhancement of polypeptide activity may be an increase in the activity or concentration expression of the corresponding polypeptide based on the activity or concentration of the polypeptide expressed in the wild type or the microbial strain before modification, or an increase in the amount of the product (alanine) produced from the polypeptide, but is not limited thereto.

[0104]

[0105] microorganism

[0106] Another aspect provides a microorganism comprising one or more selected from the group consisting of the polypeptide, a polynucleotide encoding the polypeptide, and a vector containing the polynucleotide.

[0107] In the present disclosure, the term “microorganism (or strain)” may include both wild-type microorganisms and microorganisms that have undergone natural or artificial genetic modification. The microorganism may be a microorganism in which a specific mechanism is enhanced or weakened due to causes such as the insertion of an external gene or the enhancement or weakening of the activity of an endogenous gene, and may be a microorganism that includes genetic modification for the production of a desired polypeptide, protein, or product (amino acid).

[0108] The microorganism (or strain, recombinant cell) of the present disclosure may be a microorganism in which the enzymatic activity of an aminotransferase is enhanced, or which has the ability to produce amino acids, or in which the ability to produce amino acids is enhanced (or increased). The amino acid may be one or more selected from the group consisting of alanine, arginine, glutamine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, cysteine, glycine, proline, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. The amino acid may be an L-amino acid. In one example, the amino acid may be alanine.

[0109] For example, the microorganism of the present disclosure may be a microorganism that is naturally non-existent in amino acid production ability or has amino acid production ability, into which one or more selected from the group consisting of the polypeptide, a polynucleotide encoding the polypeptide, and a vector containing the polynucleotide are introduced to confer or enhance amino acid production ability, but is not limited thereto.

[0110] The statement that the above microorganism has enhanced amino acid production capacity or possesses amino acid production capacity may mean that the above microorganism has improved amino acid production capacity compared to the non-modified microorganism, the cell prior to recombination, the parent strain, or the wild-type strain, or that it is endowed with amino acid production capacity unlike the non-modified microorganism, the cell prior to recombination, the parent strain, or the wild-type strain that lacks amino acid production capacity.

[0111] In the present disclosure, "non-mutated microorganism" does not exclude strains containing mutations that may naturally occur in microorganisms, and may refer to wild-type strains or natural-type strains themselves, or strains prior to genetic mutations caused by natural or artificial factors. For example, according to one example, the non-mutated microorganism may refer to strains prior to the introduction of the variant polypeptide of the present disclosure or the polynucleotide encoding said variant polypeptide. The term "non-mutated microorganism" may be used interchangeably with "pre-mutation strain," "pre-mutation microorganism," "non-mutated strain," "non-mutated microorganism," or "reference microorganism."

[0112] The microorganism may additionally include mutations that increase amino acid production, and the location of the mutation and / or the type of gene and / or protein subject to the mutation may be included without limitation as long as they increase amino acid production. The recombinant cell may be used without limitation as long as it is a cell capable of transformation.

[0113] In one example, the target strain for comparing whether the amino acid production capacity increases may be a microorganism of the genus Corynebacterium, Corynebacterium glutamicum, for example, the wild-type Corynebacterium glutamicum ATCC13869 strain or the Corynebacterium glutamicum ATCC13032 strain, but is not limited thereto.

[0114] The above microorganism may be a microorganism of the genus Corynebacterium sp. The above-mentioned microorganisms of the genus Corynebacterium are Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium pollutisoli, and Corynebacterium imitans It may be one or more microorganisms selected from the group consisting of imitans), Corynebacterium testudinoris, and Corynebacterium flavescens, but is not limited thereto. In one example, the Corynebacterium genus microorganism may be Corynebacterium glutamicum.

[0115] For example, compared to the parent strain prior to mutation or the non-mutated microorganism, the above microorganism is newly endowed with amino acid production capacity of about 3% or more, about 4% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 11% or more, about 12% or more, about 13% or more, about 14% or more, about 15% or more, about 16% or more, about 17% or more, about 18% or more, about 19% or more, about 20% or more, about 21% or more, about 22% or more, about 23% or more, about 24% or more, about 25% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 100% or more, about 150% or more, It may be increased by approximately 200% or more, approximately 250% or more, approximately 300% or more, approximately 400% or more, approximately 500% or more, approximately 600% or more, approximately 700% or more, approximately 800% or more, approximately 900% or more, approximately 1,000% or more, approximately 1,500% or more, approximately 2,000% or more, approximately 2,500% or more, or approximately 3,000% or more, but is not limited thereto.

[0116] As another example, compared to the parent strain prior to mutation or the non-mutated microorganism, the above-mentioned microorganism exhibits an amino acid production capacity of approximately 1.03 times or more, approximately 1.04 times or more, approximately 1.05 times or more, approximately 1.06 times or more, approximately 1.07 times or more, approximately 1.08 times or more, approximately 1.9 times or more, approximately 1.1 times or more, approximately 1.2 times or more, approximately 1.3 times or more, approximately 1.4 times or more, approximately 1.5 times or more, approximately 1.6 times or more, approximately 1.7 times or more, approximately 1.8 times or more, approximately 1.9 times or more, approximately 2 times or more, approximately 2.5 times or more, approximately 3 times or more, approximately 4 times or more, approximately 5 times or more, approximately 6 times or more, approximately 7 times or more, approximately 8 times or more, approximately 9 times or more, approximately 10 times or more, approximately 15 times or more, approximately 20 times or more, approximately 25 times or more, or approximately It may be 30 times or more (there is no special limit on the upper limit, for example, it may be about 1,000 times or less), but is not limited to this.

[0117] As another example, compared to the parent strain prior to mutation or the non-mutated microorganism, the above-mentioned microorganism has an amino acid production capacity of about 0.1 g / L or more, about 0.2 g / L or more, about 0.3 g / L or more, about 0.4 g / L or more, about 0.5 g / L or more, about 0.6 g / L or more, about 0.7 g / L or more, about 0.8 g / L or more, about 0.9 g / L or more, about 1 g / L or more, about 1.1 g / L or more, about 1.2 g / L or more, about 1.3 g / L or more, about 1.4 g / L or more, about 1.5 g / L or more, about 1.6 g / L or more, about 1.7 g / L or more, about 1.8 g / L or more, about 1.9 g / L or more, about 2.0 g / L or more, about 2.5 g / L or more, about 3 g / L or more, about It may be 3.5 g / L or more, about 4 g / L or more, about 4.1 g / L or more, about 4.2 g / L or more, about 4.3 g / L or more, about 4.4 g / L or more, about 4.5 g / L or more, about 4.6 g / L or more, about 4.7 g / L or more, about 4.8 g / L or more, about 4.9 g / L or more, about 5 g / L or more, about 5.5 g / L or more, about 6 g / L or more, about 7 g / L or more, about 8 g / L or more, about 9 g / L or more, about 10 g / L or more, about 15 g / L or more, about 20 g / L or more, about 25 g / L or more, about 30 g / L or more (there is no special restriction on the upper limit value, for example, it may be about 100 g / L or less), but is not limited thereto.

[0118] The above term “about” refers to a range that includes ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes, but is not limited to, all numerical values ​​within a range equivalent to or similar to the numerical value following the term “about.”

[0119]

[0120] Composition, Use, and Method

[0121] Another aspect is to provide a composition for producing amino acids comprising one or more selected from the group consisting of the microorganism and the culture medium in which the microorganism is cultured.

[0122] Another aspect provides a use for producing amino acids using one or more selected from the group consisting of the microorganism and the culture medium in which the microorganism is cultured.

[0123] Another aspect provides a use for using one or more selected from the group consisting of the microorganism and the culture medium in which the microorganism is cultured in order to produce a composition for amino acid production.

[0124] As described above, the above amino acids may be one or more selected from the group consisting of alanine, arginine, glutamine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, cysteine, glycine, proline, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan.

[0125] The composition of the present disclosure may further include any suitable excipients commonly used in compositions for producing amino acids, and such excipients may be, for example, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers or isotonic agents, but are not limited thereto.

[0126] Another aspect provides a method for producing (or manufacturing) amino acids, comprising the step of culturing the microorganism in a culture medium.

[0127] As described above, the above amino acids may be one or more selected from the group consisting of alanine, arginine, glutamine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, cysteine, glycine, proline, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan.

[0128] The method for producing amino acids of the present disclosure may include the step of culturing the microorganism in a culture medium.

[0129] In the present disclosure, "culture" means growing the microorganism, such as a strain of Corynebacterium glutamicum, under appropriately controlled environmental conditions. The culture process may be carried out according to suitable media and culture conditions known in the art. Such a culture process can be easily adjusted and used by those skilled in the art depending on the strain selected. Specifically, the culture may be batch, continuous, and / or fed-batch, but is not limited thereto.

[0130] In the present disclosure, "medium" refers to a substance mixed with nutrients as the main component required to culture the microorganism, such as a strain of Corynebacterium glutamicum, and supplies nutrients and growth factors, including water, which is indispensable for survival and growth. Specifically, any medium and other culture conditions used for culturing the microorganism of the present disclosure may be used without special limitations as long as they are media used for culturing ordinary microorganisms; however, the microorganism of the present disclosure may be cultured under aerobic conditions while controlling the temperature, pH, etc., in a conventional medium containing a suitable carbon source, nitrogen source, phosphorus, inorganic compounds, amino acids, and / or vitamins.

[0131] Specifically, culture media for the above-mentioned microorganisms, such as strains of the genus Corynebacterium, can be found in the literature ["Manual of Methods for General Bacteriology" by the American Society for Bacteriology (Washington D. Corynebacterium, USA, 1981)].

[0132] In the present disclosure, the carbon source may include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvate, lactic acid, citric acid, etc.; amino acids such as glutamic acid, methionine, lysine, etc. Additionally, natural organic nutrient sources such as starch hydrolysate, molasses (e.g., blackstrap molasses), rice bran, cassava, sugarcane residue, and corn steeping liquid may be used. Specifically, carbohydrates such as glucose and sterilized pre-treated molasses (i.e., molasses converted into reducing sugars) may be used, and other carbon sources in appropriate amounts may be used without limitation. These carbon sources may be used individually or in combination of two or more types, but are not limited thereto.

[0133] The above nitrogen sources may include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc., amino acids such as glutamic acid, methionine, glutamine, etc., peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquid, casein hydrolysate, fish or its decomposition products, defatted soybean cake or its decomposition products, etc. These nitrogen sources may be used alone or in combination of two or more, but are not limited thereto.

[0134] The above ingredients may include monopotassium phosphate, dipotassium phosphate, or corresponding sodium-containing salts. Inorganic compounds may include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc., and may also include amino acids, vitamins, and / or suitable precursors. These components or precursors may be added to the culture medium in a batch or continuous manner. However, they are not limited thereto.

[0135] In addition, during the cultivation of the microorganisms, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc., may be added to the medium in an appropriate manner to adjust the pH of the medium. In addition, during cultivation, foam generation may be suppressed by using an antifoaming agent such as fatty acid polyglycol ester. Furthermore, to maintain an aerobic state of the medium, oxygen or an oxygen-containing gas may be injected into the medium, or nitrogen, hydrogen, or carbon dioxide gas may be injected without gas injection to maintain an anaerobic and microaerobic state, but is not limited thereto.

[0136] In the culture of the present disclosure, the culture temperature may be maintained at 20 to 45°C, specifically 25 to 40°C, and culture may be carried out for about 10 to 160 hours, but is not limited thereto.

[0137] The amino acids produced by the culture of the present disclosure may be secreted into the culture medium or remain within the cell.

[0138] The method for producing amino acids of the present disclosure may additionally include, for example, the step of preparing the microorganism, the step of preparing a medium for culturing the microorganism, or a combination thereof (in any order), prior to the culturing step.

[0139] The method for producing amino acids according to the present disclosure may further include a step of recovering amino acids from a culture medium (a culture medium in which the culture is performed) or a microorganism (e.g., a strain of the genus Corynebacterium) according to the culture. The recovery step may be additionally included after the culture step.

[0140] The above recovery may involve collecting the desired amino acid using a suitable method known in the art according to the culture method of the microorganism disclosed in this disclosure, for example, batch, continuous, or fed-batch culture methods. For example, various chromatographic methods such as centrifugation, filtration, treatment with a crystallizing protein precipitating agent (salting out), extraction, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC, or a combination thereof may be used, and the desired amino acid may be recovered from the culture medium or microorganism using a suitable method known in the art.

[0141] Additionally, the amino acid production method of the present disclosure may further include a purification step. The purification may be performed using a suitable method known in the art. In one example, where the amino acid production method of the present disclosure includes both a recovery step and a purification step, the recovery step and the purification step may be performed continuously or discontinuously regardless of the order, or simultaneously or integrated into a single step, but are not limited thereto.

[0142]

[0143] The present disclosure relates to an aminotransferase variant and its use, wherein a microorganism expressing the aminotransferase variant has excellent amino acid production ability.

[0144]

[0145] The present invention will be explained in more detail below through the following examples. However, these are merely illustrative of the invention, and the scope of the invention is not limited by these examples.

[0146]

[0147] Example 1. Preparation of a strain with the alaT R308Q mutation

[0148] The wild-type strain of Corynebacterium glutamicum ATCC13869 (whole genome: CP016335.1) possesses the alaT gene (Sequence No. 2), which codes for the L-alanine biosynthetic enzyme aminotransferase (GenBank: ANU34753.1, NCBI Reference Sequence: WP_003862691.1, Sequence No. 1). The alaT gene is commonly found in microorganisms of the genus Corynebacterium, such as Corynebacterium glutamicum ATCC13869 and Corynebacterium ATCC13032.

[0149] A variant strain was constructed having a gene (Sequence No. 4) mutated to code for a variant (alaT R308Q variant, Sequence No. 3) in which arginine, the 308th amino acid residue from the N-terminus in the amino acid sequence of the above enzyme, is substituted with glutamine.

[0150] Specifically, to introduce the above alaT R308Q mutation into wild-type Corynebacterium glutamicum ATCC13869, a gene fragment containing the mutation was obtained through PCR using the primer pair of SEQ ID NO. 5 and SEQ ID NO. 6. The PCR conditions were as follows: denaturation at 95°C for 5 minutes, followed by 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 30 seconds, followed by polymerization at 72°C for 5 minutes. More specifically, an 800 bp polynucleotide was obtained by amplifying using the primer pair of SEQ ID NO. 5 and SEQ ID NO. 6. A single amino acid substitution vector containing the alaT R308Q mutation was constructed by connecting the two gene fragments obtained above to pDC24 (SEQ No. 7), which had been cleaved with restriction enzymes BamHI and SalI, using an infusion enzyme, and this was named pDC24-alaT(R308Q). The pDC24-alaT(R308Q) vector was transformed into Corynebacterium glutamicum ATCC13869 by the electro-pulse method (Appl. Microbiol. Biotechnol., 1999), and colonies were obtained by plating on compound agar plates containing 25 mg / L kanamycin. Subsequently, a final strain containing the mutation and with the introduction vector removed was obtained through the conventional secondary crossover process. The strain with the introduced mutation was finally confirmed by gene sequencing analysis using the primer pair of SEQ No. 5 and SEQ No. 6. The above strain into which the target mutation was introduced was named strain CJ0038.

[0151]

[0152] Example 2. Confirmation of L-alanine production of strain with introduced alaT R308Q mutation

[0153] To confirm the L-alanine productivity of the CJ0038 strain produced through Example 1 above and the wild-type Corynebacterium glutamicum ATCC13869, they were cultured in the following manner.

[0154] Corynebacterium glutamicum ATCC13869 (control) and the CJ0038 strain were each inoculated into a 250 ml Corner-Baffle flask containing 25 ml of production medium, and then cultured at 200 rpm for 46 hours at 30 ℃.

[0155] Production Medium

[0156] CaCO330 g / L, Sucrose 57 g / L, BM 6 g / L, MgSO40.5 g / L, (NH4)2SO450 g / L, KH2PO41 g / L, Yeast extract 2 g / L, Ammonium acetate 6.28 g / L, d-Biotin 0.05 mg / L, Thiamine-HCl 0.1 mg / L, MnSO46.7 mg / L, FeSO410 mg / L

[0157] After the culture was finished, the production of L-alanine was measured using liquid high-speed chromatography, and the concentration of L-alanine in the culture medium for each strain tested is shown in Table 1 below.

[0158] - L-Alanine Concentration (g / l) Increase in L-Alanine Concentration (%) compared to control group Corynebacterium glutamicum ATCC13869 (Control group) 6.1-CJ00388.437.70%

[0159] As can be seen in Table 1 above, the alanine production capacity of the microorganisms into which the alaT R308Q variant was introduced increased significantly compared to the control group.

[0160]

[0161] Example 3. Preparation of a strain with the alaT R308X mutation

[0162] Based on the results of Example 2 above, the following experiment was performed to determine the effect of a mutation at the 308th position of an aminotransferase derived from a microorganism of the genus Corynebacterium on the alanine production capacity of the microorganism.

[0163] A variant strain was constructed having a gene mutated to code for a variant (alaT R308X variant) in which arginine, the 308th amino acid residue in the amino acid sequence of the above aminotransferase, is substituted with another amino acid.

[0164] Specifically, to introduce the above alaT R308X mutation into wild-type Corynebacterium glutamicum ATCC13869, a gene fragment containing the corresponding mutation was obtained through PCR using (i) the primer of SEQ ID NO. 5 and the primer of SEQ ID NO. 8, and (ii) the primer of SEQ ID NO. 6 and the primer pair of SEQ ID NO. 9, 10, 11, 12, 13, 14, or 15. The PCR conditions were as follows: denaturation at 95°C for 5 minutes, followed by 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 30 seconds, followed by polymerization at 72°C for 5 minutes. More specifically, a 400 bp polynucleotide amplified using the above primer pairs was obtained. A single amino acid substitution vector containing the alaT R308X mutation was constructed by connecting each of the two gene fragments obtained above to pDC24 (Sequence No. 7), which had been cleaved with restriction enzymes BamHI and SalI, using an infusion enzyme, and was named pDC24-alaT(R308N), pDC24-alaT(R308D), pDC24-alaT(R308E), pDC24-alaT(R308K), pDC24-alaT(R308S), pDC24-alaT(R308C), and pDC24-alaT(R308T), respectively. Each of the above vectors was transformed into Corynebacterium glutamicum ATCC13869 by the electro-pulse method (Appl. Microbiol. Biotechnol., 1999), and colonies were obtained by plating on compound agar plates containing 25 mg / L of kanamycin. Subsequently, a final strain containing the mutation and with the introduction vector removed was obtained through the existing secondary crossover process. The strain with the mutation introduced was finally confirmed through gene sequence analysis using the above-mentioned primer pair.The above strains into which the target mutation was introduced were named CJ0038-N (R308N), CJ0038-D (R308D), CJ0038-E (R308E), CJ0038-K (R308K), CJ0038-S (R308S), CJ0038-C (R308C), and CJ0038-T (R308T), respectively.

[0165]

[0166] Example 4. Confirmation of L-alanine production of strains into which the alaT R308X mutation was introduced

[0167] To confirm the L-alanine productivity of each strain produced through Example 3 above and wild-type Corynebacterium glutamicum ATCC13869, they were cultured in the following manner.

[0168] Corynebacterium glutamicum ATCC13869 (control) and each of the above strains were inoculated into a 250 ml Corner-Baffle flask containing 25 ml of production medium, and then cultured at 200 rpm for 46 hours at 30 ℃.

[0169] Production Medium

[0170] CaCO3 30 g / L, Sucrose 57 g / L, BM 6 g / L, MgSO4 0.5 g / L, (NH4)2SO4 50 g / L, KH2PO4 1 g / L, Yeast extract 2 g / L, Ammonium acetate 6.28 g / L, d-Biotin 0.05 mg / L, Thiamine-HCl 0.1 mg / L, MnSO4 6.7 mg / L, FeSO4 10 mg / L

[0171] After the culture was finished, the production of L-alanine was measured using liquid high-speed chromatography, and the concentration of L-alanine in the culture medium for each strain tested is shown in Table 2 below.

[0172] L-Alanine Concentration (g / l) Increase in L-Alanine Concentration (%) compared to control group Corynebacterium glutamicum ATCC13869 (Control) 6.10 -CJ0038-N (R308N) 7.37 20.78% CJ0038-D (R308D) 7.70 26.20% CJ0038-E (R308E) 7.41 21.41% CJ0038-K (R308K) 7.00 14.70% CJ0038-S (R308S) 8.29 35.97% CJ0038-C (R308C) 7.38 20.94% CJ0038-T (R308T) 7.98 30.81%

[0173] As can be seen in Table 2 above, microorganisms to which the alaT R308X mutation was introduced showed a significant increase in alanine production capacity compared to the control group. These results indicate that an amino acid substitution mutation at the 308th position of an aminotransferase derived from a Corynebacterium genus microorganism further increases the amino acid productivity of the microorganism.

[0174]

[0175] From the foregoing description, those skilled in the art to which the present invention pertains will understand that the present invention may be implemented in other specific forms without altering its technical concept or essential features. In this regard, the embodiments described above should be understood as illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as including all modifications or variations derived from the meaning and scope of the claims set forth below and their equivalents, rather than from the detailed description above.

Claims

1. A variant polypeptide in which the amino acid corresponding to the 308th residue of the amino acid sequence of sequence no. 1 is substituted with another amino acid.

2. A variant polypeptide according to claim 1, wherein the amino acid corresponding to the 308th residue of the amino acid sequence of SEQ ID NO. 1 is substituted with glutamine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, cysteine, glycine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan.

3. In claim 1, the variant polypeptide comprises an amino acid sequence having 80% or more homology with the amino acid sequence of SEQ ID NO. 1, and is a variant polypeptide in which the amino acid corresponding to the 308th residue of the amino acid sequence of SEQ ID NO. 1 is substituted with another amino acid.

4. In claim 1, the variant polypeptide comprises the amino acid sequence of SEQ ID NO. 1, wherein the amino acid of the 308th residue in the amino acid sequence of SEQ ID NO. 1 is substituted with another amino acid.

5. In claim 1, the variant polypeptide is a variant polypeptide having aminotransferase activity.

6. The variant polypeptide of claim 1, wherein the variant polypeptide comprises the amino acid sequence of SEQ ID NO. 3, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, or SEQ ID NO.

28.

7. A polynucleotide encoding a variant polypeptide of any one of paragraphs 1 to 6.

8. A microorganism comprising one or more selected from the group consisting of a variant polypeptide in which the amino acid corresponding to the 308th residue of the amino acid sequence of SEQ ID NO. 1 is substituted with another amino acid, a polynucleotide encoding said variant polypeptide, and a recombinant vector comprising said polynucleotide.

9. In paragraph 8, the microorganism is a microorganism of the genus Corynebacterium.

10. In paragraph 9, the microorganism of the genus Corynebacterium is a microorganism that is Corynebacterium glutamicum.

11. In paragraph 8, the microorganism is a microorganism with increased amino acid production capacity.

12. In paragraph 11, the above amino acid is alanine, a microorganism.

13. A composition for producing amino acids comprising one or more selected from the group consisting of a microorganism according to any one of claims 8 to 12 and a culture medium in which said microorganism is cultured.

14. A composition for producing amino acids, wherein the amino acid in paragraph 13 is alanine.

15. A method for producing amino acids comprising the step of culturing a microorganism in a medium, the microorganism comprising one or more selected from the group consisting of a variant polypeptide in which the amino acid corresponding to the 308th residue of the amino acid sequence of SEQ ID NO. 1 is substituted with another amino acid, a polynucleotide encoding said variant polypeptide, and a recombinant vector comprising said polynucleotide.

16. A method for producing amino acids according to claim 15, further comprising, after the culturing step, a step of recovering amino acids from the cultured microorganisms, the culture medium, or both.

17. A method for producing an amino acid, wherein the amino acid in paragraph 15 is alanine.