Variant of low-specific threonine aldolase and use thereof

Abstract

The present disclosure provides: a variant of a low-specific threonine aldolase; a microorganism comprising the variant; a microorganism comprising the variant and exhibiting enhanced aldehyde dehydrogenase activity; a method for producing at least one amino acid selected from the group consisting of glycine, alanine, and valine, the method comprising a step of culturing the microorganism; and a composition for producing the at least one amino acid, the composition comprising the microorganism. The microorganism exhibits an excellent ability to produce at least one amino acid selected from the group consisting of glycine, alanine, and valine.

Description

Variants of low-specificity threonine aldolase and their uses

[0001] A method for producing one or more amino acids selected from the group consisting of a variant of low-specificity threonine aldolase, a microorganism comprising said variant, a microorganism comprising said variant with enhanced aldehyde dehydrogenase activity, and a step of culturing said microorganism, and a composition for producing said amino acids comprising said microorganism are provided.

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

[0003] This application claims the benefit of priority based on Korean Patent Application No. 10-2024-0166465 filed November 20, 2024 and Korean Patent Application No. 10-2025-0102642 filed July 28, 2025, and all contents disclosed in the documents of said Korean patent applications are incorporated herein as part of this specification.

[0004]

[0005] L-amino acids are the basic building blocks of proteins and are used as important materials for pharmaceutical raw materials, food additives, animal feed, nutritional supplements, insecticides, and fungicides. Among them, glycine is mainly used as a seasoning in the food industry to provide sweetness, and is used in combination with natural seasonings to enhance flavor. It is also utilized for antioxidant and buffering functions, and in the pharmaceutical field, it is used in intravenous fluids, antacids, comprehensive amino acid preparations, and nutritional supplements.

[0006] Various studies are being conducted to efficiently produce amino acids, such as efforts to develop microorganisms capable of high-efficiency amino acid production or fermentation process technologies. Specifically, target-specific approaches have been developed, such as increasing the expression of genes encoding enzymes involved in amino acid biosynthesis in Corynebacterium strains or removing genes unnecessary for amino acid synthesis. In addition to these methods, techniques to remove genes not involved in amino acid production or to eliminate genes with unknown specific functions in amino acid production are also being utilized. However, there is still a growing need for research on methods capable of producing amino acids efficiently and with high yields.

[0007]

[0008] [Prior Art Literature]

[0009] (Patent Document 1) U.S. Registered Patent Publication (US 11661616 B2)

[0010]

[0011] One example of the present disclosure provides a variant polypeptide in which an amino acid corresponding to one or more residues selected from the group consisting of the 29th, 126th, and 289th residues of SEQ ID NO. 1 is substituted with another amino acid.

[0012] Another example of the present disclosure provides a polynucleotide encoding the variant polypeptide.

[0013] Another example of the present disclosure provides a vector comprising the polynucleotide.

[0014] Another example of the present disclosure provides a microorganism comprising a variant polypeptide in which an amino acid corresponding to one or more residues selected from the group consisting of the 29th, 126th, and 289th residues of SEQ ID NO. 1 is substituted with another amino acid, or a polynucleotide encoding the same.

[0015] Another example of the present disclosure provides a method for producing one or more amino acids selected from the group consisting of glycine, alanine, and valine, comprising the step of culturing the microorganism in a culture medium.

[0016] Another example of the present disclosure provides a composition for producing one or more amino acids selected from the group consisting of glycine, alanine, and valine, comprising the microorganism.

[0017] Another example of the present disclosure provides a use for producing one or more amino acids selected from the group consisting of glycine, alanine, and valine of the variant polypeptide, the polynucleotide, the vector, and / or the microorganism.

[0018] Another example of the present disclosure provides a use for using the variant polypeptide, the polynucleotide, the vector and / or the microorganism in the preparation of a composition for producing one or more amino acids selected from the group consisting of glycine, alanine and valine.

[0019]

[0020] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in this application may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the scope of this application 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 application described in this application using only ordinary experiments. Moreover, such equivalents are intended to be included in this application.

[0021]

[0022] One aspect provides a variant polypeptide in which an amino acid corresponding to one or more residues selected from the group consisting of the 29th, 126th, and 289th residues of SEQ ID NO. 1 is substituted with another amino acid.

[0023] In this application, 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 variant polypeptides 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 variant polypeptides in which a portion has been removed from the N- and / or C-terminus of a mature protein. The term "variant polypeptide" mentioned above 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.

[0024] 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. Additionally, the variant polypeptide may be conjugated with another sequence or linker so that it can be identified, purified, or synthesized.

[0025] The polypeptide into which the mutation of the present application is introduced may be a protein having low-specificity threonine aldolase activity. Threonine aldolase activity may refer to enzymatic activity that decomposes threonine into acetaldehyde and glycine (e.g., EC 4.1.2.48). The low-specificity threonine aldolase activity means that, unlike enzymes with high substrate selectivity, it can exhibit enzymatic reactions on various similar substrates, and the protein having low-specificity threonine aldolase activity means that it can exhibit enzymatic reactions using amino acids having a structure similar to L-threonine, such as L-allo-threonine, L-threophenylserine, and / or L-erythro-phenylserine, as substrates. The low-specificity threonine aldolase activity may refer to enzymatic activity that decomposes L-threonine and / or L-allo-threonine into acetaldehyde and glycine. In addition, the low-specificity threonine aldolase activity may refer to enzymatic activity that degrades L-threophenylserine and / or L-erythrophenylserine using them as substrates.

[0026] Specifically, the polypeptide subject to the introduction of the above mutation is the amino acid sequence of SEQ ID NO. 1 or at least 60%, 65%, 70%, 75%, 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%, It may have, include, be composed of, or essentially be composed of, but is not limited to, an amino acid sequence having sequence homology or identity of 97.6% or more, 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. That is, meaningless addition of sequences before or after the amino acid sequence of SEQ ID NO. 1, naturally occurring mutations, or silent mutations thereof are not excluded, and if a protein having the same or corresponding activity as the protein containing the amino acid sequence of SEQ ID NO. 1 is present, it may be a protein subject to the introduction of mutation in this application.For example, the protein subject to the introduction of the mutation in this application has at least 60%, 65%, 70%, 75%, 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%, and 97.5% of the amino acid sequence of SEQ ID NO. 1. It may be a protein composed of an amino acid sequence having 97.6% or more, 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 homology or identity. In addition, if the amino acid sequence has such homology or identity and exhibits efficacy corresponding to the said protein, a protein having an amino acid sequence in which some sequences are deleted, modified, substituted, or added may also be included within the scope of the protein subject to modification in this application.

[0027] In this specification, 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.

[0028] 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.

[0029] 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.,] Homology or identity can be determined, for example, using BLAST from the National Biotechnology Information Database Center or ClustalW.

[0030] 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.

[0031] In this specification, 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.

[0032] 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").

[0033] 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.

[0034] In one example, the polypeptide to which the above mutation is introduced may be encoded by a polynucleotide containing the nucleic acid sequence of SEQ ID NO. 2.

[0035]

[0036] The above variant polypeptide may have low-specificity threonine aldolase activity and / or activity that increases the production capacity of one or more amino acids selected from the group consisting of glycine, alanine, and valine in microorganisms into which the above variant polypeptide or a polynucleotide encoding it has been introduced.

[0037] In one example, the above variant polypeptide may be one in which the amino acid corresponding to the 29th residue in the amino acid sequence of SEQ ID NO. 1 is substituted with another amino acid.

[0038] The amino acid corresponding to the 29th residue in the amino acid sequence of SEQ ID NO. 1 is valine, and in this case, the other substituted amino acid may be any one amino acid selected from the group consisting of glycine, arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. In one example, the amino acid corresponding to the 29th residue of SEQ ID NO. 1 may be substituted with glycine.

[0039] In one example, the above variant polypeptide may be one in which the amino acid corresponding to the 126th residue in the amino acid sequence of SEQ ID NO. 1 is substituted with another amino acid.

[0040] The amino acid corresponding to the 126th residue of the above sequence number 1 is histidine, and in this case, the other substituted amino acid may be any one amino acid selected from the group consisting of leucine, arginine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, valine, isoleucine, methionine, phenylalanine, tyrosine, and tryptophan.

[0041] In one example, the above variant polypeptide may be one in which the amino acid corresponding to the 289th residue in the amino acid sequence of SEQ ID NO. 1 is substituted with another amino acid.

[0042] The amino acid corresponding to the 289th residue of SEQ ID NO. 1 is alanine, and in this case, the other substituted amino acid may be any one amino acid selected from the group consisting of threonine, arginine, histidine, lysine, aspartic acid, glutamic acid, serine, asparagine, glutamine, cysteine, glycine, proline, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan.

[0043] In one embodiment, the amino acid corresponding to the 289th residue of SEQ ID NO. 1 may be substituted with threonine.

[0044]

[0045] In one example, the above variant polypeptide may be (i) the amino acid corresponding to the 29th residue in the amino acid sequence of SEQ ID NO. 1 is substituted with another amino acid; and (ii) the amino acid corresponding to the 126th residue in the amino acid sequence of SEQ ID NO. 1 is substituted with another amino acid.

[0046] The above 29th residue, 126th residue, and each other amino acid are as described above.

[0047] In one example, the above variant polypeptide may be (i) the amino acid corresponding to the 29th residue in the amino acid sequence of SEQ ID NO. 1 is substituted with another amino acid; and (ii) the amino acid corresponding to the 289th residue in the amino acid sequence of SEQ ID NO. 1 is substituted with another amino acid.

[0048] The 29th residue, the 289th residue, and each other amino acid are as described above.

[0049] In one example, the above variant polypeptide may be (i) the amino acid corresponding to the 126th residue in the amino acid sequence of SEQ ID NO. 1 is substituted with another amino acid; and (ii) the amino acid corresponding to the 289th residue in the amino acid sequence of SEQ ID NO. 1 is substituted with another amino acid.

[0050] The above 126th residue, 289th residue, and each other amino acid are as described above.

[0051]

[0052] In one example, the above variant polypeptide may be (i) the amino acid corresponding to the 29th residue in the amino acid sequence of SEQ ID NO. 1 is substituted with another amino acid; (ii) the amino acid corresponding to the 126th residue in the amino acid sequence of SEQ ID NO. 1 is substituted with another amino acid; and (iii) the amino acid corresponding to the 289th residue in the amino acid sequence of SEQ ID NO. 1 is substituted with another amino acid.

[0053] The 29th residue, 126th residue, 289th residue, and each other amino acid are as described above.

[0054]

[0055] The above variant polypeptide is,

[0056] Having low-specificity L-threonine aldolase activity; and / or

[0057] The amino acid sequence of SEQ ID NO. 1 and at least 60%, 65%, 70%, 75%, 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%, 97.6%, 97.7%, Having, including, being composed of, or essentially composed of, an amino acid sequence having homology or identity of 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

[0058] The amino acid corresponding to one or more residues selected from the group consisting of the 29th, 126th, and 289th residues of sequence number 1 may be substituted with another amino acid.

[0059] In addition, any polypeptide having such homology or identity and exhibiting activity corresponding to that of the variant polypeptide may be included in the variant polypeptide of the present application even if some sequences have deletions, modifications, substitutions, conservative substitutions, and / or added amino acid sequences. For example, this may be the case where the amino acid sequence of the variant polypeptide of the present application has sequence additions or deletions, naturally occurring mutations, silent mutations, or conservative substitutions at the N-terminus, C-terminus, and / or within it that do not alter the activity of the variant polypeptide.

[0060] 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.

[0061] In one example, the variant polypeptide may have, include, be composed of, or essentially be composed of an amino acid sequence selected from the group consisting of SEQ ID NOs 8 to 32.

[0062] In one example, the above variant polypeptide may be encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 33 to 57.

[0063] In one example, the variant polypeptide may have, include, be composed of, or essentially be composed of an amino acid sequence selected from the group consisting of SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 31 and SEQ ID NO. 32.

[0064] In one example, the variant polypeptide may have, include, be composed of, or essentially be composed of an amino acid sequence selected from the group consisting of SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, and SEQ ID NO. 31.

[0065] In one example, the variant polypeptide may have, include, be composed of, or essentially be composed of an amino acid sequence selected from the group consisting of SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31 and SEQ ID NO. 32.

[0066]

[0067] Another aspect provides a polynucleotide encoding (or encoding) the above variant polypeptide.

[0068] In this application, the term "polynucleotide" refers to a polymer of nucleotides in which nucleotide monomers are linked together in a long chain by covalent bonds, and means a DNA or RNA strand of a certain length or longer.

[0069] In one example, the polynucleotide of the present application may code for a variant polypeptide in which an amino acid corresponding to one or more residues selected from the group consisting of the 29th, 126th, and 289th residues of SEQ ID NO. 1 is substituted with another amino acid.

[0070] In one example, the polynucleotide of the present application may comprise a nucleic acid sequence encoding the variant polypeptide, wherein a codon corresponding to one or more codons selected from the group consisting of (i) a codon corresponding to the 85th to 87th position in the nucleic acid sequence of SEQ ID NO. 2; (ii) a codon corresponding to the 376th to 378th position; and (iii) a codon corresponding to the 865th to 867th position is substituted with a codon encoding a different amino acid.

[0071] Since the codons corresponding to positions 85 to 87 in the nucleic acid sequence of SEQ ID NO. 2 are codons coding for valine, codons coding for amino acids other than valine can be identified by referring to the microorganism intended to express the polynucleotide and a known codon table.

[0072] Since the codons corresponding to positions 376 to 378 in the nucleic acid sequence of SEQ ID NO. 2 are codons coding for histidine, codons coding for amino acids other than histidine can be identified by referring to the microorganism intended to express the polynucleotide and a known codon table.

[0073] Since the codons corresponding to positions 865 to 867 in the nucleic acid sequence of SEQ ID NO. 2 are codons coding for alanine, codons coding for amino acids other than alanine can be identified by referring to the microorganism intended to express the polynucleotide and a known codon table.

[0074] The polynucleotide of the present application may have various modifications made to its coding region within a range that does not alter the amino acid sequence of the variant polypeptide of the present application, taking into account the degeneracy of codons or the codons preferred by the organism intended to express the variant polypeptide of the present application.

[0075] In one example, the polynucleotide comprises the nucleic acid sequence of SEQ ID NO. 2 or at least 60%, 65%, 70%, 75%, 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%, 97.6%, Having, including, being composed of, or essentially composed of, a nucleic acid sequence having sequence homology or identity 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, or comprising, said nucleic acid sequence, said nucleic acid sequence, and

[0076] (i) a codon corresponding to the 85th to 87th position in the nucleic acid sequence of SEQ ID NO. 2; (ii) a codon corresponding to the 376th to 378th position; and (iii) a codon corresponding to the 865th to 867th position. A codon corresponding to one or more codons selected from the group consisting of these may be substituted with a codon coding for another amino acid.

[0077] In one example, the polynucleotide of the present application may have, include, be composed of, or essentially be composed of a nucleic acid sequence encoding an amino acid sequence described by any one of SEQ ID NOs 8 to 32.

[0078] In one example, the polynucleotide may have, include, be composed of, or essentially be composed of any one of the nucleic acid sequences selected from SEQ ID NOs 33 to 57.

[0079] Polynucleotides having, including, being composed of, or essentially composed of a nucleic acid sequence selected from any one of SEQ ID NOs 33 to 57 may each code for an amino acid sequence described by any one of SEQ ID NOs 8 to 32.

[0080] The polynucleotide of the present application 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 application. 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%, 75%, 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%, 97.6%, 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.Conditions may be listed in which polynucleotides having homology or identity of 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 are hybridized with each other, and polynucleotides having homology or identity of less than that are not hybridized with each other, or conditions in which the washing conditions of normal southern hybridization are 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, are washed once, specifically two to three times, at a salt concentration and temperature equivalent to the washing conditions of normal southern hybridization.

[0081] 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 this application may also include substantially similar nucleic acid sequences as well as isolated nucleic acid fragments that are complementary to the entire sequence.

[0082] Specifically, a polynucleotide having homology or identity with the polynucleotide of the present application can be detected using hybridization conditions including a hybridization step at a Tm value of 55 ℃ and using the conditions described above. Additionally, the Tm value may be 60 ℃, 63 ℃, or 65 ℃, but is not limited thereto and can be appropriately adjusted by a person skilled in the art according to the purpose.

[0083] 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.).

[0084]

[0085] Another aspect provides a vector comprising a polynucleotide encoding the variant polypeptide. The vector may be an insertion vector or an expression vector.

[0086] In this specification, 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 base 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 in a suitable host cell. The expression control region 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. For 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.

[0087] The vectors available in this specification are not particularly limited as long as they are replicable 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, but are not limited to, vectors such as pCES208, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC.

[0088] 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.

[0089] Expressing the above variant polypeptide in a microorganism can be performed by introducing a polynucleotide encoding the above variant polypeptide, or a vector containing the same, into a host cell and culturing a recombinant cell (e.g., a microorganism) containing the same.

[0090] The introduction of a polynucleotide encoding the above-mentioned variant polypeptide or a vector containing the same into a microorganism may be carried out 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 the same into a host cell (microorganism) to change the genetic traits of the host cell (microorganism). The transformed polynucleotide may be inserted into or located outside the chromosome of the host cell. The polynucleotide may be introduced in an appropriate form depending on the purpose of introduction. For example, the 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 typically include expression regulatory elements such as a promoter, a transcription termination signal, a ribosome binding site, and / or a translation termination signal, which are operably linked to the polynucleotide. The expression cassette may be in the form of a self-replicating expression vector. In addition, the polynucleotide may be introduced into 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.

[0091] 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.

[0092]

[0093] In this application, the term “enhancement” of polypeptide (protein) activity means that the activity of the polypeptide is increased compared to its intrinsic activity. The term “enhancement” may be used interchangeably with terms such as activation, up-regulation, overexpression, and increase. The term “enhancement” may include, but is not limited to, exhibiting activity that was not originally possessed, or exhibiting improved activity compared to the intrinsic activity or activity prior to modification. The term “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,” “activated,” “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.

[0094] Whether the activity of the above polypeptide is enhanced can be confirmed from the increase in the degree of activity, expression amount, or amount of product resulting from the activity of the polypeptide.

[0095] 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 such methods as long as the activity of the target polypeptide can be enhanced compared to that of the microorganism before modification. Such enhancement may be achieved by introducing an exogenous polypeptide or by enhancing the activity and / or increasing the concentration (expression level) of the intrinsic polypeptide. Specifically, it may be achieved by using, but is not limited to, genetic engineering and / or protein engineering, which are routine methods of molecular biology and are well known to those skilled in the art (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.).

[0096] Specifically, the activity enhancement of the polypeptide (protein) of the present application is

[0097] 1) Increase in the intracellular copy number of polynucleotides encoding polypeptides;

[0098] 2) Replace the chromosomal gene expression regulatory region encoding a polypeptide with a potent sequence;

[0099] 3) A modification of the nucleotide sequence encoding the start codon or the 5'-UTR region of the gene transcript encoding the polypeptide;

[0100] 4) Modification of the amino acid sequence of the polypeptide to enhance polypeptide activity;

[0101] 5) Modification of the polynucleotide sequence encoding the polypeptide to enhance polypeptide activity (e.g., modification of the polynucleotide sequence of the polypeptide gene to code for a polypeptide modified to enhance polypeptide activity);

[0102] 6) Introduction of a foreign polypeptide exhibiting polypeptide activity or a foreign polynucleotide encoding the same;

[0103] 7) Codon optimization of polynucleotides encoding polypeptides;

[0104] 8) Analyze the tertiary structure of the polypeptide to select and modify or chemically modify the exposed sites;

[0105] 9) Regulation of the cellular localization of polypeptides; or

[0106] 10) It may be based on two or more combinations selected from 1) to 9) above, but is not specifically limited thereto.

[0107] More specifically,

[0108] 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.

[0109] 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.

[0110] 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.

[0111] 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.

[0112] The modification of the amino acid sequence or polynucleotide 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 polynucleotide sequence modified to have stronger activity or an amino acid sequence or polynucleotide 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 has been inserted.

[0113] 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.

[0114] 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.

[0115] 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.

[0116] 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.

[0117] Such enhancement of polypeptide activity may involve 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-modification microbial strain, or increasing the amount of the product produced from said polypeptide, but is not limited thereto.

[0118]

[0119] In this application, the term “weakening” of the activity of a polypeptide is a concept that encompasses both a decrease in activity and the absence of activity relative to the intrinsic activity. The term “weakening” may be used interchangeably with terms such as inactivation, deficiency, down-regulation, decrease, reduce, and attenuation.

[0120] The above-mentioned 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, etc.; cases where the overall polypeptide activity and / or concentration (expression amount) within the cell is lower than that of the natural strain due to inhibition of gene expression of the polynucleotide encoding it or inhibition of translation into the polypeptide; cases where the expression of the polynucleotide does not occur at all; and / or cases where the polypeptide is inactive even if the polynucleotide is expressed. The above-mentioned “intrinsic activity” refers to the activity of a specific polypeptide originally possessed by the parent strain, wild-type, or non-modified microorganism prior to the change in trait due to genetic mutation caused by natural or artificial factors. This may be used interchangeably with “activity before modification.” The statement that the activity of a polypeptide is “inactivated, deficient, reduced, downregulated, lowered, or attenuated” relative to its intrinsic activity means that the activity of a specific polypeptide has decreased compared to the activity originally possessed by the parent strain or non-transformed microorganism prior to the transformation.

[0121] The attenuation of the activity of such polypeptides can be performed by any method known in the art, but is not limited thereto, and can be achieved by the application of various methods well known in the art (e.g., Nakashima N et al., Bacterial cellular engineering by genome editing and gene silencing. Int J Mol Sci. 2014;15(2):2773-2793, Sambrook et al. Molecular Cloning 2012 et al.).

[0122] Specifically, the weakening of polypeptide (protein) activity is

[0123] 1) Deletion of all or part of the gene encoding a polypeptide;

[0124] 2) Modification of the expression regulatory region (or expression regulatory sequence) to reduce the expression of the gene encoding the polypeptide;

[0125] 3) Modification of the amino acid sequence constituting the polypeptide so as to remove or weaken the activity of the polypeptide (e.g., deletion / substitution / addition of one or more amino acids in the amino acid sequence);

[0126] 4) Modification of the gene sequence encoding the polypeptide so as to remove or weaken the activity of the polypeptide (e.g., deletion / substitution / addition of one or more nucleotide bases on the nucleotide base sequence of the polypeptide gene to code for a polypeptide modified so as to remove or weaken the activity of the polypeptide);

[0127] 5) A modification of the nucleotide sequence encoding the start codon or the 5'-UTR region of the gene transcript encoding the polypeptide;

[0128] 6) Introduction of an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the transcript of the gene encoding the polypeptide;

[0129] 7) Addition of a sequence complementary to the Shine-Dalgarno sequence to the upstream end of the Shine-Dalgarno sequence of a polypeptide-coding gene to form a secondary structure incapable of ribosome attachment;

[0130] 8) Addition of a reverse-transcribed promoter to the 3' end of the ORF (open reading frame) of a gene sequence encoding a polypeptide (Reverse transcription engineering, RTE);

[0131] 9) Regulation of the cellular localization of polypeptides; or

[0132] 10) It may be based on two or more combinations selected from 1) to 9) above, but is not specifically limited thereto.

[0133] for example,

[0134] The deletion of part or all of the gene encoding the polypeptide mentioned above 1) may be the removal of the entire polynucleotide encoding the intrinsic target polypeptide within the chromosome, replacement with a polynucleotide in which some nucleotides have been deleted, or replacement with a marker gene.

[0135] Additionally, modification of the expression regulatory region (or expression regulatory sequence) described in 2) above may be a 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 regulating the termination of transcription and translation.

[0136] In addition, 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 lower polypeptide expression rate compared to the intrinsic start codon, but is not limited thereto.

[0137] In addition, modifications to the amino acid sequences or polynucleotide sequences of 4) and 5) above may involve 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 weaken the activity of the polypeptide, or may involve 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, but are not limited thereto. For example, gene expression may be inhibited or weakened by introducing a variation within the polynucleotide sequence to form a stop codon, but are not limited thereto. The "stop codon" is a codon on the mRNA that does not specify an amino acid and acts as a signal indicating that the protein synthesis process has ended; generally, three types, UAA, UAG, and UGA, may be used as stop codons.

[0138] For the introduction of an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the transcript of the gene encoding the polypeptide 6) above, refer to the literature [Weintraub, H. et al., Antisense-RNA as a molecular tool for genetic analysis, Reviews - Trends in Genetics, Vol. 1(1) 1986].

[0139] 7) In order to form a secondary structure in which ribosome attachment is impossible, the addition of a sequence complementary to the Shine-Dalgarno sequence to the front of the Shine-Dalgarno sequence of a gene encoding a polypeptide may make mRNA translation impossible or slow it down.

[0140] Reverse transcription engineering (RTE) 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 above may weaken the activity by creating an antisense nucleotide complementary to the transcript of the gene encoding the polypeptide.

[0141] 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.

[0142] Such weakening of polypeptide activity may involve a reduction in the activity or concentration expression of the corresponding polypeptide relative to the activity or concentration of the polypeptide expressed in the wild-type or pre-modification microbial strain, or an increase in the amount of product produced from said polypeptide, but is not limited thereto.

[0143]

[0144] Another aspect provides a microorganism comprising a variant polypeptide in which an amino acid corresponding to one or more residues selected from the group consisting of the 29th, 126th, and 289th residues of SEQ ID NO. 1 is substituted with another amino acid, or a polynucleotide encoding the same.

[0145] The above variant polypeptide and polynucleotide are as described above.

[0146] In this application, 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 (e.g., one or more amino acids selected from the group consisting of glycine, alanine, and valine).

[0147] The microorganism (or strain, recombinant cell) of the present application may have the ability (or production volume) to produce one or more amino acids selected from the group consisting of glycine, alanine, and valine, or may be a microorganism in which the ability to produce said amino acids is enhanced (or increased).

[0148] The statement that the above microorganism has an enhanced ability to produce one or more amino acids selected from the group consisting of glycine, alanine, and valine, or possesses the ability to produce said amino acids, may mean that the above microorganism has an enhanced ability to produce one or more amino acids selected from the group consisting of glycine, alanine, and valine compared to a non-modified microorganism, a cell prior to recombination, a parental wild-type microorganism, and / or a microorganism to which a pre-mutation polypeptide (e.g., low-specificity threonine aldolase derived from E. coli or a polypeptide containing the amino acid sequence of SEQ ID NO. 1) has been introduced, or that the above amino acid production ability is conferred unlike a non-modified microorganism, a cell prior to recombination, a parental strain, a wild-type microorganism, and / or a microorganism to which a pre-mutation polypeptide has been introduced that lacks the ability to produce said amino acids.

[0149] In this application, "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, the "non-mutated microorganism" may, in one example, refer to a strain in which the variant polypeptide of this application or a polynucleotide encoding said variant polypeptide has not been introduced, or prior to such introduction. The "non-mutated microorganism" may be used interchangeably with "pre-mutation strain," "pre-mutation microorganism," "non-mutated strain," "non-mutated strain," "non-mutated microorganism," or "reference microorganism."

[0150] The microorganism may additionally include a mutation that increases the production of one or more amino acids selected from the group consisting of glycine, alanine, and valine, 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 it increases the production of said amino acids. The recombinant cell may be used without limitation as long as it is a cell capable of transformation.

[0151] In one example, the microorganism may be a microorganism having increased glycine production capacity, comprising one or more selected from the group consisting of (i) a variant polypeptide having, containing, being composed of, or essentially composed of said amino acid sequence selected from the group consisting of SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 31, and SEQ ID NO. 32, (ii) a polynucleotide encoding (or coding) said variant polypeptide, and (iii) a vector containing said polynucleotide.

[0152] The above-mentioned microorganisms with increased glycine production capacity may mean that their glycine production capacity is enhanced compared to non-modified microorganisms, pre-recombination cells, parent strains, wild-type microorganisms, and / or microorganisms to which the pre-mutation polypeptide was introduced, or that they are endowed with glycine production capacity unlike non-modified microorganisms, pre-recombination cells, parent strains, wild-type microorganisms, and / or microorganisms to which the pre-mutation polypeptide was introduced.

[0153] In one example, the microorganism may be a microorganism with increased alanine production capacity, comprising one or more selected from the group consisting of (i) a variant polypeptide having, containing, being composed of, or essentially composed of said amino acid sequence selected from the group consisting of SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, and SEQ ID NO. 31, (ii) a polynucleotide encoding (or coding) said variant polypeptide, and (iii) a vector containing said polynucleotide.

[0154] The above-mentioned microorganisms with increased alanine production capacity may mean that their alanine production capacity is enhanced compared to non-modified microorganisms, pre-recombination cells, parent strains, wild-type microorganisms, and / or microorganisms to which the pre-mutation polypeptide was introduced, or that they are endowed with alanine production capacity unlike non-modified microorganisms, pre-recombination cells, parent strains, wild-type microorganisms, and / or microorganisms to which the pre-mutation polypeptide was introduced.

[0155] In one example, the microorganism may be a microorganism having increased valine production capacity and comprising one or more selected from the group consisting of (i) a variant polypeptide having, containing, being composed of, or essentially composed of, said amino acid sequence selected from the group consisting of SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31, and SEQ ID NO. 32, (ii) a polynucleotide encoding (or coding) said variant polypeptide, and (iii) a vector containing said polynucleotide.

[0156] The microorganism with increased valine production capacity mentioned above may mean that its valine production capacity is enhanced compared to non-modified microorganisms, pre-recombination cells, parent strains, wild-type microorganisms, and / or microorganisms to which the pre-mutation polypeptide was introduced, or that it is endowed with valine production capacity unlike non-modified microorganisms, pre-recombination cells, parent strains, wild-type microorganisms, and / or microorganisms to which the pre-mutation polypeptide was introduced.

[0157] 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.

[0158] In one example, the microorganism may be Corynebacterium glutamicum.

[0159] In one example, the microorganism may be Corynebacterium glutamicum ATCC13032.

[0160] The above microorganism may be a microorganism of the genus Bacillus. The above microorganism of the genus Bacillus may be a microorganism selected from the group consisting of Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus methylotrophicus, Bacillus licheniformis, Bacillus velezensis, Bacillus sonorensis, and Bacillus valismortis, but is not limited thereto.

[0161] The above microorganism may be a microorganism of the genus Escherichia. The above microorganism of the genus Escherichia may be Escherichia coli, but is not limited thereto. In one example, the above microorganism may be a microorganism in which the activity of aldehyde dehydrogenase is further enhanced.

[0162] The above aldehyde dehydrogenase may mean a protein having aldehyde dehydrogenase activity. The above aldehyde dehydrogenase activity may mean an enzyme activity that catalyzes a reaction converting aldehyde or acetaldehyde into a carboxylic acid (EC 1.2.1.3). In one example, the above aldehyde dehydrogenase may have acetaldehyde dehydrogenase activity.

[0163] A microorganism that produces one or more amino acids selected from the group consisting of glycine, alanine, and valine, with the activity of the above aldehyde dehydrogenase further enhanced, may be a microorganism into which the aldehyde dehydrogenase or a polynucleotide encoding it has been introduced. The above aldehyde dehydrogenase may be an endogenous protein of the microorganism to be introduced or an exogenous protein.

[0164] In one example, the aldehyde dehydrogenase is,

[0165] Sequence No. 76 or therewith, at least 60%, 65%, 70%, 75%, 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%, 97.6%, 97.7%, It may have, include, be composed of, or essentially be composed of an amino acid sequence having sequence homology or identity of 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.

[0166] In addition, if a protein possesses such homology or identity and exhibits aldehyde dehydrogenase activity, variants of the aldehyde dehydrogenase having amino acid sequences in which some sequences are deleted, modified, substituted, conservedly substituted, or added may also be included in the said aldehyde dehydrogenase. For example, this includes cases where there are sequence additions or deletions that do not alter aldehyde dehydrogenase activity at the N-terminus, C-terminus, and / or within the said amino acid sequence, naturally occurring mutations, silent mutations, or conserved substitutions.

[0167] In one example, the aldehyde dehydrogenase and / or the polynucleotide encoding it may be of origin from a microorganism selected from the group consisting of microorganisms of the genus Escherichia, microorganisms of the genus Saccharomyces, microorganisms of the genus Zymomonas and microorganisms of the genus Corynebacterium.

[0168] In one example, the aldehyde dehydrogenase derived from a microorganism of the genus Saccharomyces and / or the polynucleotide encoding it may be derived from a microorganism selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces boulardii, and Saccharomyces pastorianus, but is not limited thereto.

[0169] In one example, the aldehyde dehydrogenase derived from a microorganism of the genus Saccharomyces and / or the polynucleotide encoding it may be derived from Saccharomyces cerevisiae. In one example, the aldehyde dehydrogenase derived from Saccharomyces cerevisiae may be the ALDH1 protein. In one example, the aldehyde dehydrogenase derived from Saccharomyces cerevisiae comprises the amino acid of SEQ ID NO. 76 or at least 60%, 65%, 70%, 75%, 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%, It may have, include, be composed of, or be essentially composed of an amino acid sequence having sequence homology or identity of 97.5% or more, 97.6% or more, 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.

[0170] The polynucleotide encoding the above aldehyde dehydrogenase may be of a microorganism of the genus Saccharomyces, but is not limited thereto, and may be prepared by referring to a known codon table based on the amino acid sequence of the aldehyde dehydrogenase of the microorganism of the genus Saccharomyces.

[0171] In one example, the polynucleotide encoding an aldehyde dehydrogenase derived from Saccharomyces cerevisiae is the nucleic acid sequence of SEQ ID NO. 77 or at least 60%, 65%, 70%, 75%, 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%, It may have, include, be composed of, or be essentially composed of a nucleic acid sequence having sequence homology or identity of 97% or more, 97.5% or more, 97.6% or more, 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.

[0172] In one example, a microorganism that produces one or more amino acids selected from the group consisting of glycine, alanine, and valine, in which the activity of the aldehyde dehydrogenase is further enhanced, may be a microorganism into which an aldehyde dehydrogenase derived from a microorganism of the genus Saccharomyces or a polynucleotide encoding the same has been introduced.

[0173] In one example, the above microorganism is,

[0174] (a) one or more selected from the group consisting of (i) a variant polypeptide having, including, being composed of, or essentially composed of, said amino acid sequence selected from the group consisting of SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 31 and SEQ ID NO. 32, (ii) a polynucleotide encoding (or coding) said variant polypeptide, and (iii) a vector comprising said polynucleotide; and

[0175] (b) comprising one or more selected from the group consisting of (i) a polypeptide having, containing, being composed of, or essentially composed of the amino acid sequence of SEQ ID NO. 76, (ii) a polynucleotide encoding (or coding) the polypeptide, and (iii) a vector comprising the polynucleotide, and

[0176] It may be a microorganism with increased ability to produce one or more amino acids selected from the group consisting of glycine, alanine, and valine.

[0177] Microorganisms with increased amino acid production capacity selected from the group consisting of glycine, alanine, and valine may mean that the amino acid production capacity is enhanced compared to non-modified microorganisms, pre-recombinant cells, parent strains, wild-type microorganisms, and / or microorganisms to which pre-mutation polypeptides are introduced, or that the amino acid production capacity is conferred unlike non-modified microorganisms, pre-recombinant cells, parent strains, wild-type microorganisms, and / or microorganisms to which pre-mutation polypeptides are introduced that lack the amino acid production capacity.

[0178]

[0179] In one example, a microorganism comprising (i) one or more selected from the group consisting of the variant polypeptide, a polynucleotide encoding the variant polypeptide, and a vector containing the polynucleotide, or (ii) one or more selected from the group consisting of the variant polypeptide, a polynucleotide encoding the variant polypeptide, and a vector containing the polynucleotide, wherein the aldehyde dehydrogenase activity is further enhanced, is newly endowed with the ability to produce one or more amino acids selected from the group consisting of glycine, alanine, and valine compared to the parent strain before mutation, the non-modified microorganism, and / or the microorganism to which the pre-mutation polypeptide was introduced, or is about 0.4% or more, about 0.5% or more, about 1% or more, about 2% or more, about 3% or more, about 4% or more, about 5% or more, about 8% or more, about 9% or more, about 10% or more, about 20% or more, about It may be increased by 25% or more, approximately 30% or more, approximately 40% or more, approximately 50% or more, approximately 60% or more, approximately 70% or more, approximately 80% or more, approximately 90% or more, approximately 100% or more, approximately 150% or more, 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, approximately 3,000% or more, approximately 3,500% or more, or approximately 4,000% or more, but is not limited thereto.

[0180] As another example, (i) 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, or (ii) 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, wherein the aldehyde dehydrogenase activity is further enhanced, compared to the parent strain before mutation, the non-modified microorganism, and / or the microorganism to which the pre-mutation polypeptide was introduced, the production capacity of one or more amino acids selected from the group consisting of glycine, alanine, and valine is about 1.04 times or more, about 1.05 times or more, about 1.01 times or more, about 1.02 times or more, about 1.03 times or more, about 1.04 times or more, about 1.05 times or more, about 1.08 times or more, or about 1.09 times or more It may be about 1.1 times or more, about 1.2 times or more, about 1.3 times or more, about 1.4 times or more, about 1.5 times or more, about 1.6 times or more, about 1.7 times or more, about 1.8 times or more, about 1.9 times or more, about 2 times or more, about 2.5 times or more, about 3 times or more, about 4 times or more, about 5 times or more, about 6 times or more, about 7 times or more, about 8 times or more, about 9 times or more, about 10 times or more, about 15 times or more, about 20 times or more, about 25 times or more, or about 30 times or more (there is no special restriction on the upper limit value, for example, it may be about 1,000 times or less), but is not limited thereto.

[0181] As another example, a microorganism comprising one or more selected from the group consisting of the variant polypeptide, a polynucleotide encoding the variant polypeptide, and a vector containing the polynucleotide, or a microorganism comprising one or more selected from the group consisting of the variant polypeptide, a polynucleotide encoding the variant polypeptide, and a vector containing the polynucleotide, wherein the aldehyde dehydrogenase activity is further enhanced, compared to the parent strain before mutation, the non-mutated microorganism, and / or the microorganism to which the pre-mutation polypeptide was introduced, has a production capacity of one or more amino acids selected from the group consisting of glycine, alanine, and valine of about 0.03 g / L or more, about 0.04 g / L or more, about 0.05 g / L or more, about 0.06 g / L or more, about 0.07 g / L or more, about 0.08 g / L or more, about 0.09 g / L or more, or about 0.1 g / L. Above, approximately 0.2 g / L or more, approximately 0.3 g / L or more, approximately 0.4 g / L or more, approximately 0.5 g / L or more, approximately 0.6 g / L or more, approximately 0.7 g / L or more, approximately 0.8 g / L or more, approximately 0.9 g / L or more, approximately 1 g / L or more, approximately 1.1 g / L or more, approximately 1.2 g / L or more, approximately 1.3 g / L or more, approximately 1.4 g / L or more, approximately 1.5 g / L or more, approximately 1.6 g / L or more, approximately 1.7 g / L or more, approximately 1.8 g / L or more, approximately 1.9 g / L or more, approximately 2.0 g / L or more, approximately 2.1 g / L or more, approximately 2.2 g / L or more, approximately 2.3 g / L or more, approximately 2.4 g / L or more, approximately 2.5 g / L or more, approximately 2.6 g / L or more, approximately 2.7 g / L or more, approximately 2.8 g / L or more, approximately 2.9 g / L or more, approximately 3 g / L or more, approximately 3.1 g / L or more, approximately 3.2 g / L or more, approximately 3.3 g / L or more, approximately 3.4 g / L or more, approximately 3.5 g / L or more, approximately 3.6 g / L or more, approximately 3.7 g / L or more, approximately 3.8 g / L or more, approximately 3.It may be 9 g / L or more, about 4 g / L or more, about 4.5 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, for example, it may be about 100 g / L or less), but is not limited thereto.

[0182] 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.”

[0183]

[0184] Another aspect provides a method for producing one or more amino acids selected from the group consisting of glycine, alanine, and valine, comprising the step of culturing the microorganism in a culture medium.

[0185] In this application, "culture" means growing the microorganism under appropriately controlled environmental conditions. The culture process of this application 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 selected strain. Specifically, the culture may be batch, continuous, and / or fed-batch, but is not limited thereto.

[0186] In this application, "medium" refers to a substance mixed with nutrients as the main component required to culture the microorganism, and supplies nutrients and growth factors, including water, which is indispensable for survival and growth. Specifically, the medium and other culture conditions used for culturing the microorganism of this application may be any medium used for culturing microorganisms without special limitations, provided that the microorganism of this application is 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.

[0187] Specifically, the culture medium for the microorganism of the present application, such as a strain of the genus Corynebacterium, can be found in the literature ["Manual of Methods for General Bacteriology" by the American Society for Bacteriology (Washington D. Corynebacterium, USA, 1981)].

[0188] In the present application, 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.; and 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 steep 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.

[0189] 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.

[0190] 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.

[0191] In addition, during the cultivation of the microorganism of the present application, 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, an antifoaming agent such as fatty acid polyglycol ester may be used to suppress the formation of bubbles. 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.

[0192] In the culture of the present application, the culture temperature can be maintained at 20 to 45°C, specifically 25 to 40°C, and culture can be performed for about 10 to 160 hours, but is not limited thereto.

[0193] One or more amino acids selected from the group consisting of glycine, alanine, and valine produced by the culture of the present application may be secreted into the culture medium or remain in the cell.

[0194] The method for producing one or more amino acids selected from the group consisting of glycine, alanine, and valine of the present application may additionally include, for example, the step of preparing a microorganism of the present application, the step of preparing a medium for culturing said microorganism, or a combination thereof (in any order), prior to the culturing step.

[0195] The method for producing one or more amino acids selected from the group consisting of glycine, alanine, and valine according to the present application may further include a step of recovering one or more amino acids selected from the group consisting of glycine, alanine, and valine from a culture medium (a culture medium in which culture is performed) or microorganisms according to the culture. The recovery step may be additionally included after the culture step.

[0196] The above recovery may involve collecting one or more amino acids selected from the group consisting of glycine, alanine, and valine using a suitable method known in the art according to the culture method of the microorganism of the present application, such as a batch, continuous, or fed-batch culture method. For example, various chromatographic methods such as centrifugation, filtration, treatment with a crystallizing protein precipitating agent (salting out method), 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 one or more amino acids selected from the group consisting of glycine, alanine, and valine may be recovered from the culture medium or microorganism using a suitable method known in the art.

[0197] In addition, the method for producing one or more amino acids selected from the group consisting of glycine, alanine, and valine according to the present application may additionally include a purification step. The purification may be performed using a suitable method known in the art. In one example, if the method for producing one or more amino acids selected from the group consisting of glycine, alanine, and valine according to the present application 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.

[0198]

[0199] Another aspect provides a composition for producing one or more amino acids selected from the group consisting of glycine, alanine, and valine, containing the above-mentioned microorganism.

[0200] The above composition may further include any suitable solvent or excipient commonly used in compositions for producing one or more amino acids selected from the group consisting of glycine, alanine, and valine, and such excipients may be, for example, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, or isotonic agents, but are not limited thereto.

[0201] Another aspect provides a use for the microorganism to be used for producing one or more amino acids selected from the group consisting of glycine, alanine, and valine.

[0202] Another aspect provides a use for the microorganism to be used in the preparation of a composition for producing one or more amino acids selected from the group consisting of glycine, alanine, and valine.

[0203] The above microorganisms are as described above.

[0204]

[0205] The present application provides a variant of low-specificity threonine aldolase, a microorganism comprising said variant, a microorganism comprising said variant with enhanced aldehyde dehydrogenase activity, a method for producing one or more amino acids selected from the group consisting of glycine, alanine, and valine comprising the step of culturing said microorganism, and a composition for producing said amino acids comprising said microorganism. said microorganism exhibits excellent production ability of one or more amino acids selected from the group consisting of glycine, alanine, and valine.

[0206]

[0207] 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.

[0208]

[0209] Example 1. Low-specificity threonine aldolase homogeneous variant and its effects

[0210] Example 1-1. Confirmation of Glycine Production Ability of Low-Specific Threonine Aldolase Variant

[0211] Example 1-1-1. Construction of a vector library containing a low-specificity threonine aldolase variant

[0212] Error-prone PCR was used to introduce random mutations into low-specificity L-threonine aldolase derived from Escherichia coli (ltaE, WP_000566376.1, protein sequence: SEQ ID NO. 1, gene sequence: SEQ ID NO. 2). The ORF of low-specificity L-threonine aldolase derived from Escherichia coli W3110 (ATCC 27325) was used as the PCR Template, and reactions were carried out using SEQ ID NO. 3 (ltaE_Fwd) and SEQ ID NO. 4 (ltaE_Rev) (Forward and Reverse, respectively, Table 1). PCR buffer conditions were set to introduce a mutation of 2.0 bp per kb, and the process was conducted in accordance with the supplier's manual (Diversify PCR Random Mutagenesis Kit, TAKARA). Error-prone PCR reaction (based on 50 μl) was carried out with a composition of 40 μl PCR grade water, 5 μl 10X TITANIUM Taq buffer, 1 μl 2 mM dGTP, 1 μl 50X Diversify dNTP Mix, 1 μl Primer mix, 1 μl Template DNA, and 1 μl TITANIUM Taq polymerase, and was reacted in a Thermo cycler (Eppendorf) at 94°C for 30 seconds, 1 cycle; 94°C for 30 seconds, 68°C for 1 minute 30 seconds, 25 cycles; and 68°C for 1 minute, 1 cycle. A pCES208 vector containing a PCR product in which random mutations are expected to have been introduced and a Pcj7 promoter (Sequence No. 5) was constructed (Korean Registered Patent Publication KR 10-1673080 B1, J.Microbiol. Biotechnol., 18:639-647, 2008).The pCES208 vector was linearized by treatment with EcoRV (NEB) restriction enzyme for 4 hours, and DNA fragments were obtained after performing Q5 polymerase PCR (NEB) using Pcj7 promoters SEQ ID NO. 6 (Pcj7_Fwd) and SEQ ID NO. 7 (Pcj7_Rev) (Forward and Reverse, respectively, Table 1). The Q5 polymerase reaction (based on 50 μl) was carried out with a composition of 10 μl of 5X Q5 Reaction Buffer, 1 μl of 10 mM dNTPs, 5 μl of 10 μM Primer mix, 0.5 μl of Template DNA, 0.5 μl of Q5 High-Fidelity DNA Polymerase, and 33 μl of PCR-grade water, and was performed in a Thermocycler (Eppendorf) at 98 °C for 30 seconds for 1 cycle; The reaction was carried out at 98°C for 10 seconds, 55°C for 30 seconds, 72°C for 15 seconds, for 30 cycles; and at 72°C for 2 minutes, for 1 cycle. Transformed plasmids were constructed using the Gibson assembly (NEB) method by mixing the Pcj7 promoter DNA fragment and the ltaE PCR DNA fragment containing random mutations with the linearized pCES208 vector. For the Gibson assembly reaction (based on 20 μl), 1 μl of the linearized pCES208 vector, 3 μl of the Pcj7 DNA fragment, 3 μl of ltaE PCR DNA, 10 μl of Gibson assembly material mix, and 3 μl of PCR-grade water were mixed and the reaction was carried out at 50°C for 30 minutes.

[0213] Primer nucleic acid sequence number ltaE_FwdCGAAAGGAAACACTCATGATTGATTTACGCAGTGA3ltaE_RevATCGATAAGCTTGATTTAACGCGCCAGGAATGCAC4Pcj7_FwdCTGCAGGAATTCGATGAAACATCCCAGCGCTACTAAT6Pcj7_RevGCGTAAATCAATCATGAGTGTTTCCTTTCGTTGGG7

[0214] To be used as a control group for the experiment, a pCES208 vector (Korean Registered Patent Publication KR 10-1673080 B1, J.Microbiol. Biotechnol., 18:639-647, 2008) containing a gene encoding low-specificity L-threonine aldolase (ltaE, NCBI Reference Sequence: WP_000566376.1, amino acid sequence: SEQ ID NO. 1; gene sequence: SEQ ID NO. 2) derived from Escherichia coli was constructed. Specifically, a Q5 polymerase PCR reaction was performed using primer pairs SEQ ID NO. 3 (ltaE_Fwd) and SEQ ID NO. 4 (ltaE_Rev) with Escherichia coli W3110 genomic DNA (ATCC27325) as a template. DNA fragments were obtained from the Pcj7 promoter (SEQ No. 5) after performing Q5 polymerase PCR (NEB) using the primer pair SEQ No. 6 (Pcj7_Fwd) and SEQ No. 7 (Pcj7_Rev). The Q5 polymerase reaction (based on 50 μl) was carried out with a composition of 10 μl of 5X Q5 Reaction Buffer, 1 μl of 10 mM dNTPs, 5 μl of 10 μM Primer mix, 0.5 μl of Template DNA, 0.5 μl of Q5 High-Fidelity DNA Polymerase, and 33 μl of PCR-grade water, and was reacted in a Thermocycler (Eppendorf) at 98 °C for 30 seconds for 1 cycle; 98 °C for 10 seconds, 55 °C for 30 seconds, 72 °C for 30 seconds for 30 cycles; and 72 °C for 2 minutes for 1 cycle. The pCES208 vector was linearized by treating it with EcoRV (NEB) restriction enzyme for 4 hours.A Gibson assembly reaction was performed using the two PCR DNA fragments obtained therefrom and the pCES208 plasmid linearized by the restriction enzyme EcoRV reaction, and pCES208_ltaE was obtained.

[0215]

[0216] Example 1-1-2. Production of Corynebacterium glutamicum containing a vector library and screening of transformants

[0217] Using a vector library containing the pCES208 Pcj7_ltaE variant prepared in Example 1-1-1, the Corynebacterium glutamicum ATCC13032 strain was transformed by electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541-545). First, the pCES208 vector was introduced into Corynebacterium glutamicum ATCC13032 to be used as a control for the experiment. Additionally, a vector containing an ltaE variant with random mutations was introduced into Corynebacterium glutamicum ATCC13032 to obtain a library of transformed strains. A library of Corynebacterium glutamicum ATCC13032 transformants containing the ltaE variant was plated onto Brain-Heart Infusion (BD Difco) solid medium containing kanamycin to obtain single colony forms, and 10,032 colonies were evaluated by inoculating them into 96 Deep Well Plates containing 350 μl of amino acid production medium with a packing rate of 17% per well using a Qpix420 (Colony Picker, Molecular Devices). The composition of the amino acid production medium is specified below.

[0218] 96 Deep Well Plates inoculated with the control strain and the strain containing the ltaE variant, respectively, were sealed using a Gas permeable seal mark2 (Azenta) and cultured in a Multitron (shaking incubator, Infors-HT) at 30°C and 1,000 rpm for 48 hours. After 48 hours of culture, the 96 Deep Well Plates were centrifuged in a Centrifuge 5810R (centrifuge, Eppendorf) at 15°C and 4,000 rpm for 20 minutes. Subsequently, 100 μl of the supernatant from the culture medium from which the cells had been isolated was transferred to a 96-Well Black Polystyrene Microplate (Corning) using a Biomek i5 (Liquid handler, Beckman Coulter) for glycine concentration analysis. Subsequently, individual analysis data for each well was obtained by applying the data to a self-developed NIR Spectrometry at the Analysis & Quality Department of CJ CheilJedang’s BIO Technology Research Institute, and 48 strains were initially selected out of 10,032 variant strains. The 48 selected strains were cultured using the same method as described above, and the top two strains with the highest glycine concentrations were finally selected.

[0219] <Batch>

[0220] Glucose (anhydrous glucose) 20 g / L, Polypeptone 10 g / L, Yeast extract 10 g / L, Ammonium sulfate [(NH4)2SO4] 10 g / L, Urea 1.5 g / L, Monopotassium phosphate (KH2PO4) 5.2 g / L, Disodium phosphate (K2HPO4) 10.7 g / L, d-Biotin 1.8 mg / L, Thiamine-HCl 9 mg / L, Ca-pantothenate 9 mg / L, Nicotinamide 60 mg / L, Magnesium sulfate (MgSO4) 0.5 g / L

[0221] Amino acid production medium

[0222] Calcium carbonate (CaCO3) 30 g / L, Sucrose 50 g / L, L-Threonine 20 g / L, MgSO4 0.6 g / L, (NH4)2SO4 20 g / L, KH2PO4 1 g / L, Yeast extract 3 g / L, d-Biotin 0.05 mg / L, Thiamine-HCl 0.1 mg / L, MnSO4 18 μg / L, FeSO4 18 μg / L, ZnSO4 0.9 μg / L, CuSO4 0.9 μg / L, Kanamycin 50 μg / ml

[0223]

[0224] Example 1-1-3. Evaluation of Glycine Production Capacity of Corynebacterium glutamicum Transformers

[0225] The glycine productivity of the transformants of the two strains into which the ltaE variant selected in Example 1-1-2 was introduced was measured by the following method. To confirm the effect of the low-specificity threonine aldolase variant, the glycine production ability was evaluated using a Corynebacterium glutamicum strain into which the covector pCES208 vector was introduced into the Corynebacterium glutamicum ATCC13032 strain and a Corynebacterium glutamicum strain into which the wild-type low-specificity threonine aldolase derived from E. coli was introduced into the Corynebacterium glutamicum ATCC13032 strain as a control.

[0226] Specifically, the parent strain Corynebacterium glutamicum ATCC13032_pCES208 (13032-Null, Table 2), Corynebacterium glutamicum ATCC13032 (13032-ltaE WT, Table 2) into which wild-type low-specificity threonine aldolase derived from E. coli was introduced, and two strains into which the ltaE mutation was introduced (13032-ltaE(V29G), 13032-ltaE(H126L, A289T), respectively) were inoculated into a 250 ml Corner-Baffle flask containing 25 ml of seed medium, and then the seed culture was obtained by shaking at 200 rpm for 20 hours at 30°C. Subsequently, 1 ml of seed culture was inoculated into a 250 ml corner-baffle flask containing 24 ml of the above production medium, and cultured at 30 °C for 48 hours at 200 rpm. After the culture was finished, the glycine production of each strain was determined by measuring the concentration of glycine contained in the culture medium using high-performance liquid chromatography (HPLC). The results obtained are shown in Table 2 below.

[0227] Strain Glycine Concentration (g / l) Increase from Control (%) 13032-Null 4.48 - 13032-ltaE WT (Control) 6.52 - 13032-ltaE(V29G) 7.42 13.80% 13032-ltaE(H126L, A289T) 7.50 15.03%

[0228] As shown in Table 2 above, the Corynebacterium glutamicum ATCC13032 pCES208 strain (13032-Null) to which nothing was introduced had low glycine production capacity.

[0229] The two types of microorganisms into which the ltaE variant obtained through Example 1-1-2 was introduced showed an increase in glycine production capacity of approximately 13–15% compared to the control group (a Corynebacterium glutamicum strain into which wild-type low-specificity threonine aldolase derived from E. coli was introduced, 13032-ltaE WT). To confirm the sequences of the ltaE variants included in the two types of microorganisms into which the ltaE variants were introduced, plasmid extraction and DNA sequencing analysis were commissioned to Bionics. As a result, it was confirmed that each colony contained one variant (V29G) in which the 29th valine was substituted with glycine compared to the wild-type ltaE protein, and one variant (H126L, A289T) in which the 126th histidine was substituted with leucine and the 289th alanine was substituted with threonine. Accordingly, the two types of microorganisms into which each variant was introduced were named 13032-ltaE(V29G) and 13032-ltaE(H126L, A289T), respectively.

[0230]

[0231] Example 1-2. Confirmation of Glycine, Alanine, and Valine Production Capabilities According to Mutation Sites of Low-Specific Threonine Aldolase Variants

[0232] Based on the results of Example 1-1-3 above, the following experiment was performed to determine the effect of single mutations at the 29th, 126th, and 289th positions of low-specificity threonine aldolase on the production capacity of glycine, valine, and alanine.

[0233]

[0234] Example 1-2-1. Confirmation of Glycine and Alanine Production Capacity According to Variation at Position 29

[0235] First, a vector containing an ltaE variant in which the amino acid at the 29th position of low-specificity threonine aldolase is substituted with glycine was prepared. The sequence of the said ltaE variant was prepared through gene synthesis (Bionics) (amino acid sequence: SEQ No. 8, nucleic acid sequence: SEQ No. 33).

[0236] DNA fragments were obtained by performing Q5 polymerase PCR (NEB) using the primer pair of SEQ ID NO. 3 (ltaE_Fwd) and SEQ ID NO. 4 (ltaE_Rev) (Table 1) with the gene fragment synthesized in the same manner as specified in Example 1-1-1 as a template, and Pcj7 DNA fragments were obtained using the primer pair of SEQ ID NO. 6 (Pcj7_Fwd) and SEQ ID NO. 7 (Pcj7_Rev) (Table 1) in the same manner. After performing a Gibson assembly reaction by mixing with the linearized pCES208 plasmid, a recombinant vector containing the ltaE mutation was obtained. Corynebacterium glutamicum ATCC13032 was transformed using the constructed recombinant vector by electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541-545). The glycine and alanine productivity of the above Corynebacterium glutamicum transformant was measured by the following method.

[0237] Specifically, the parent strain Corynebacterium glutamicum ATCC13032_pCES208 (13032-Null, Tables 3 and 4) and the transformant into which the ltaE variant was introduced were inoculated into a 250 ml Corner-Baffle flask containing 25 ml of seed medium, respectively, and then incubated at 200 rpm for 20 hours at 30°C to obtain the seed culture. Subsequently, 1 ml of the seed culture was inoculated into a 250 ml Corner-Baffle flask containing 24 ml of the production medium and incubated at 200 rpm for 48 hours at 30°C. After the incubation was completed, the glycine and alanine production capacity of each strain was measured by measuring the concentrations of glycine and alanine contained in the culture medium using high-performance liquid chromatography (HPLC). The results obtained are shown in Tables 3 and 4 below.

[0238] Strain Glycine Concentration (g / l) Increase from Control (%) 13032-Null 4.482 - 13032-ltaE WT (Control) 6.520 - 13032-ltaE(V29G) 7.415 13.73%

[0239] Strain Alanine Concentration (g / l) Increase relative to control (%) 13032-Null 0.466 - 13032-ltaE WT (Control) 0.763 - 13032-ltaE(V29G) 0.77 0.92%

[0240] As shown in Tables 3 and 4 above, the Corynebacterium glutamicum ATCC13032 pCES208 strain (13032-Null) into which nothing was introduced had low glycine and alanine production capacity. Microorganisms into which the gene encoding the low-specificity threonine aldolase variant derived from E. coli was introduced showed an increase in glycine production capacity of approximately 14% and alanine production capacity of approximately 1% compared to the control group (Corynebacterium glutamicum strain into which the wild-type low-specificity threonine aldolase derived from E. coli was introduced, 13032-ltaE WT). These results indicate that an amino acid substitution variant at the 29th position of the low-specificity threonine aldolase derived from E. coli further increases the glycine and alanine productivity of the microorganisms.

[0241]

[0242] Example 1-2-2. Confirmation of Glycine, Alanine, and Valine Production Capacity According to Variation at Position 126

[0243] A vector containing an ltaE variant in which the amino acid at the 126th position of low-specificity threonine aldolase is substituted with an amino acid other than histidine was prepared. The sequence of the ltaE variant was prepared through gene synthesis (bionics). The sequence of the ltaE variant is shown in Table 5 below.

[0244] ltaE Amino Acid Sequence Nucleic Acid Sequence ltaE(H126L) Sequence No. 9 Sequence No. 34 ltaE(H126A) Sequence No. 15 Sequence No. 40 ltaE(H126C) Sequence No. 16 Sequence No. 41 ltaE(H126D) Sequence No. 17 Sequence No. 42 ltaE(H126E) Sequence No. 18 Sequence No. 43 ltaE(H126F) Sequence No. 19 Sequence No. 44 ltaE(H126G) Sequence No. 20 Sequence No. 45 ltaE(H126I) Sequence No. 21 Sequence No. 46 ltaE(H126K) Sequence No. 22 Sequence No. 47 ltaE(H126M) Sequence No. 23 Sequence No. 48 ltaE(H126N) Sequence No. 24 Sequence No. 49 ltaE(H126P) Sequence No. 25 Sequence No. 50 ltaE(H126Q) Sequence No. 26 Sequence No. 51 ltaE(H126R) Sequence No. 27 Sequence No. 52 ltaE(H126S) Sequence No. 28 Sequence No. 53 ltaE(H126T) Sequence No. 29 Sequence No. 54 ltaE(H126V) Sequence No. 30 Sequence No. 55 ltaE(H126W) Sequence No. 31 Sequence No. 56 ltaE(H126Y) Sequence No. 32 Sequence No. 57

[0245] DNA fragments were obtained by performing Q5 polymerase PCR (NEB) using the primer pair of SEQ ID NO. 3 (ltaE_Fwd) and SEQ ID NO. 4 (ltaE_Rev) (Table 1) with the gene fragment synthesized in the same manner as specified in Example 1-1-1 as a template, and Pcj7 DNA fragments were obtained using the primer pair of SEQ ID NO. 6 (Pcj7_Fwd) and SEQ ID NO. 7 (Pcj7_Rev) (Table 1) in the same manner. After performing a Gibson assembly reaction by mixing with the linearized pCES208 plasmid, a recombinant vector containing the ltaE mutation was obtained. Corynebacterium glutamicum ATCC13032 was transformed using the constructed recombinant vector by electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541-545).

[0246] The glycine, alanine, and valine productivity of the above Corynebacterium glutamicum transformants were measured using the same method as described in Example 1-2-1. The results obtained are shown in Tables 6, 7, and 8 below.

[0247] Strain Glycine Concentration (g / l) Increase from Control (%) 13032-Null 4.48 2% 13032-ltaE WT (Control) 6.5 2% 13032-ltaE(H126L) 7.5 10 15.18% 13032-ltaE(H126A) 7.0 80 8.59% 13032-ltaE(H126C) 7.1 0 8.90% 13032-ltaE(H126F) 6.9 14 6.04% 13032-ltaE(H126I) 7.5 60 15.95% 13032-ltaE(H126K) 7.07 08.44%13032-ltaE(H126M)7.47014.57%13032-ltaE(H126N)7.35012.73%13032-ltaE(H126T)6.5700 .77%13032-ltaE(H126V)6.5500.46%13032-ltaE(H126W)6.8605.21%13032-ltaE(H126Y)7.0808.59%

[0248] Strain Alanine Concentration (g / l) Increase from Control (%) 13032-Null 0.466 - 13032-ltaE WT (Control) 0.763 - 13032-ltaE(H126L) 0.82 7.47% 13032-ltaE(H126A) 0.92 20.58% 13032-ltaE(H126C) 0.81 6.16% 13032-ltaE(H126D) 0.88 15.33% 13032-ltaE(H126E) 0.87 14.02% 13032-ltaE(H126G) 0.91 7.96% 13032-ltaE(H126I) 1.02 33.68% 13032-lta E(H126N)0.816.16%13032-ltaE(H126P)0.9929.75%13032-ltaE(H126Q)0.9321.89%13032-ltaE(H126R)0.770.92%13032- ltaE(H126S)0.8714.02%13032-ltaE(H126T)0.8714.02%13032-ltaE(H126V)0.8511.40%13032-ltaE(H126W)1.74128.05%

[0249] Strain Vaulin Concentration (g / l) Increase relative to control (%) 13032-Null 0.01 13032-ltaE WT (Control) 0.06 13032-ltaE(H126L) 0.16 166.67% 13032-ltaE(H126A) 0.15 150 0.00% 13032-ltaE(H126C) 0.12 100.00% 13032-ltaE(H126D) 0.17 183.33% 13032- ltaE(H126E)0.130116.67%13032-ltaE(H126G)0.130116.67%13032-ltaE(H12 6I)0.320433.33%13032-ltaE(H126K)0.120100.00%13032-ltaE(H126M)0.150 150.00%13032-ltaE(H126N)0.130116.67%13032-ltaE(H126P)0.200233.33%1 3032-ltaE(H126Q)0.160166.67%13032-ltaE(H126R)0.130116.67%13032-lta E(H126S)0.09050.00%13032-ltaE(H126T)0.09050.00%13032-ltaE(H126V)0. 10066.67%13032-ltaE(H126W)0.380533.33%13032-ltaE(H126Y)0.120100.00%

[0250] As shown in Tables 6, 7, and 8 above, the Corynebacterium glutamicum ATCC13032 pCES208 strain (13032-Null) into which nothing was introduced had low glycine, alanine, and valine production capabilities. Microorganisms into which the gene encoding a low-specificity threonine aldolase variant derived from E. coli was introduced showed an increase in glycine production capability of approximately 1–16%, alanine production capability of approximately 1–128%, and valine production capability of approximately 50–533% compared to the control group (Corynebacterium glutamicum strain into which the wild-type low-specificity threonine aldolase derived from E. coli was introduced). These results indicate that an amino acid substitution variant at the 126th position of the low-specificity threonine aldolase derived from E. coli further increases the glycine, alanine, and valine productivity of the microorganisms.

[0251]

[0252] Example 1-2-3. Confirmation of Glycine and Valine Production Capacity According to Variation at Position 289

[0253] A vector containing an ltaE variant in which the amino acid at the 289th position of low-specificity threonine aldolase is substituted with threonine was constructed. The sequence of the said ltaE variant was prepared via gene synthesis (Bionics) (amino acid sequence: SEQ NO. 10, nucleic acid sequence: SEQ NO. 35).

[0254] DNA fragments were obtained by performing Q5 polymerase PCR (NEB) using the primer pair of SEQ ID NO. 3 (ltaE_Fwd) and SEQ ID NO. 4 (ltaE_Rev) (Table 1) with the gene fragment synthesized in the same manner as specified in Example 1-1-1 as a template, and Pcj7 DNA fragments were obtained using the primer pair of SEQ ID NO. 6 (Pcj7_Fwd) and SEQ ID NO. 7 (Pcj7_Rev) (Table 1) in the same manner. After performing a Gibson assembly reaction by mixing with the linearized pCES208 plasmid, a recombinant vector containing the ltaE mutation was obtained. Corynebacterium glutamicum ATCC13032 was transformed using the constructed recombinant vector by electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541-545).

[0255] The glycine and valine productivity of the above Corynebacterium glutamicum transformants was measured using the same method as described in Example 1-2-1. The results obtained are shown in Tables 9 and 10 below.

[0256] Strain Glycine Concentration (g / l) Increase from Control (%) 13032-Null 4.482 - 13032-ltaE WT (Control) 6.520 - 13032-ltaE (A289T) 7.960 22.09%

[0257] Strain Valin Concentration (g / l) Increase from Control (%) 13032-Null 0.011 -13032-ltaE WT (Control) 0.060 -13032-ltaE(A289T) 0.180 200.00%

[0258] As shown in Tables 9 and 10 above, the Corynebacterium glutamicum ATCC13032 pCES208 strain (13032-Null) into which nothing was introduced had low glycine and valine production capacity. Microorganisms into which the gene encoding the low-specificity threonine aldolase variant derived from E. coli was introduced showed an increase in glycine production capacity of approximately 22% and valine production capacity of approximately 200% compared to the control group (Corynebacterium glutamicum strain into which the wild-type low-specificity threonine aldolase derived from E. coli was introduced, 13032-ltaE WT). These results indicate that an amino acid substitution variant at the 289th position of the low-specificity threonine aldolase derived from E. coli further increases the glycine and valine productivity of the microorganisms.

[0259]

[0260] Example 2. Variants with variant combinations at positions 29, 126, and / or 289 of low-specificity threonine aldolase and effects thereof

[0261] Example 2-1. Construction of a vector containing a low-specificity threonine aldolase variant

[0262] Based on the results of Example 1-1 above, the following experiment was performed to determine the effect of combination variations at the 29th, 126th, and / or 289th positions of low-specificity threonine aldolase on glycine and valine production capacity.

[0263] A vector containing an ltaE variant into which combination variants at the 29th, 126th, and / or 289th positions of low-specificity threonine aldolase were introduced was constructed. The sequence of the ltaE variant was prepared via gene synthesis (Bionics). The sequence of the ltaE variant is shown in Table 11 below.

[0264] ltaE amino acid sequence nucleic acid sequence ltaE(V29G, H126L) Sequence No. 11 Sequence No. 36 ltaE(V29G, A289T) Sequence No. 12 Sequence No. 37 ltaE(H126L, A289T) Sequence No. 13 Sequence No. 38 ltaE(V29G, H126L, A289T) Sequence No. 14 Sequence No. 39

[0265] DNA fragments were obtained by performing Q5 polymerase PCR (NEB) using the primer pair of SEQ ID NO. 3 (ltaE_Fwd) and SEQ ID NO. 4 (ltaE_Rev) (Table 1) with the gene fragment synthesized in the same manner as specified in Example 1-1-1 as a template, and Pcj7 DNA fragments were obtained using the primer pair of SEQ ID NO. 6 (Pcj7_Fwd) and SEQ ID NO. 7 (Pcj7_Rev) (Table 1) in the same manner, and a recombinant vector containing the ltaE mutation was obtained by performing a Gibson assembly reaction after mixing with the linearized pCES208 plasmid.

[0266]

[0267] Example 2-2. Confirmation of Glycine and Valine Production Capacity According to Variations at Positions 29 and 126

[0268] The recombinant vector (ltaE(V29G, H126L)) prepared in Example 2-1 above was introduced into the Corynebacterium glutamicum ATCC13032 strain via electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541-545) to produce a Corynebacterium glutamicum transformant, and the glycine and valine productivity of the transformant were measured using the same method as described in Example 1-2-1. The results obtained are shown in Tables 12 and 13 below.

[0269] Strain Glycine Concentration (g / l) Increase from Control (%) 13032-Null 4.482 -13032-ltaE WT (Control) 6.520 -13032-ltaE(V29G H126L) 7.730 18.56%

[0270] Strain Valin Concentration (g / l) Increase relative to control (%) 13032-Null 0.01 13032-ltaE WT (Control) 0.06 13032-ltaE (V29G H126L) 0.1 301 16.67%

[0271] As shown in Tables 12 and 13 above, the Corynebacterium glutamicum ATCC13032 pCES208 strain (13032-Null) into which nothing was introduced had low glycine and valine production capacity. Microorganisms into which the gene encoding the low-specificity threonine aldolase variant derived from E. coli was introduced showed an increase in glycine production capacity of approximately 19% and valine production capacity of 117% compared to the control group (Corynebacterium glutamicum strain into which the wild-type low-specificity threonine aldolase derived from E. coli was introduced, 13032-ltaE WT). These results indicate that amino acid substitution variants at the 29th and 126th positions of the low-specificity threonine aldolase derived from E. coli further increase the glycine and valine productivity of the microorganisms.

[0272]

[0273] Example 2-3. Confirmation of Glycine and Valine Production Capacity According to Variations at Positions 29 and 289

[0274] The recombinant vector (ltaE(V29G, A289T)) prepared in Example 2-1 above was introduced into the Corynebacterium glutamicum ATCC13032 strain via electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541-545) to produce a Corynebacterium glutamicum transformant, and the glycine and valine productivity of the transformant were measured using the same method as described in Example 1-2-1. The results obtained are shown in Tables 14 and 15 below.

[0275] Strain Glycine Concentration (g / l) Increase from Control (%) 13032-Null 4.48 2% WT (Control) 6.5 2% WT (V29G A289T) 7.5 0% 15.0 3%

[0276] Strain Valin Concentration (g / l) Increase from Control (%) 13032-Null 0.01 13032-ltaE WT (Control) 0.06 13032-ltaE (V29G A289T) 0.1 301 16.67%

[0277] As shown in Tables 14 and 15 above, the Corynebacterium glutamicum ATCC13032 pCES208 strain (13032-Null) into which nothing was introduced had low glycine and valine production capacity. Microorganisms into which the gene encoding the low-specificity threonine aldolase variant derived from E. coli was introduced showed an increase in glycine production capacity of approximately 15% and valine production capacity of approximately 117% compared to the control group (Corynebacterium glutamicum strain into which the wild-type low-specificity threonine aldolase derived from E. coli was introduced, 13032-ltaE WT). These results indicate that amino acid substitution variants at the 29th and 289th positions of the low-specificity threonine aldolase derived from E. coli further increase the glycine and valine productivity of the microorganisms.

[0278]

[0279] Example 2-4. Confirmation of glycine and valine production capacity according to position variations at 126 and 289

[0280] The recombinant vector (ltaE(H126L, A289T)) prepared in Example 2-1 above was introduced into the Corynebacterium glutamicum ATCC13032 strain via electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541-545) to produce a Corynebacterium glutamicum transformant, and the glycine and valine productivity of the transformant were measured using the same method as described in Example 1-2-1. The results obtained are shown in Tables 16 and 17 below.

[0281] Strain Glycine Concentration (g / l) Increase from Control (%) 13032-Null 4.482 -13032-ltaE WT (Control) 6.520 -13032-ltaE(H126L A289T) 8.030 23.16%

[0282] Strain Valin Concentration (g / l) Increase from Control (%) 13032-Null 0.01 13032-ltaE WT (Control) 0.06 03032-ltaE (H126L A289T) 0.16 16 6.67%

[0283] As shown in Tables 16 and 17 above, the Corynebacterium glutamicum ATCC13032 pCES208 strain (13032-Null) into which nothing was introduced had low glycine and valine production capacity. Microorganisms into which the gene encoding the low-specificity threonine aldolase variant derived from E. coli was introduced showed an increase in glycine production capacity of approximately 23% and valine production capacity of approximately 167% compared to the control group (Corynebacterium glutamicum strain into which the wild-type low-specificity threonine aldolase derived from E. coli was introduced, 13032-ltaE WT). These results indicate that amino acid substitution variants at the 126th and 289th positions of the low-specificity threonine aldolase derived from E. coli further increase the glycine and valine productivity of the microorganisms.

[0284]

[0285] Example 2-5. Confirmation of glycine and valine production capacity according to position variations at positions 29, 126, and 289

[0286] The recombinant vector (ltaE(V29G, H126L, A289T)) prepared in Example 2-1 above was introduced into the Corynebacterium glutamicum ATCC13032 strain via electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541-545) to produce a Corynebacterium glutamicum transformant, and the glycine and valine productivity of the transformant were measured using the same method as described in Example 1-2-1. The results obtained are shown in Tables 18 and 19 below.

[0287] Strain Glycine Concentration (g / l) Increase from Control (%) 13032-Null 4.482 -13032-ltaE WT (Control) 6.520 -13032-ltaE(V29G H126L A289T) 8.170 25.31%

[0288] Strain Valin Concentration (g / l) Increase relative to control (%) 13032-Null 0.01 13032-ltaE WT (Control) 0.06 032-ltaE (V29G H126L A289T) 0.16 16 6.67%

[0289] As shown in Tables 18 and 19 above, the Corynebacterium glutamicum ATCC13032 pCES208 strain (13032-Null) into which nothing was introduced had low glycine and valine production capacity. Microorganisms into which the gene encoding the low-specificity threonine aldolase variant derived from E. coli was introduced showed an increase in glycine production capacity of approximately 25% and valine production capacity of 167% compared to the control group (Corynebacterium glutamicum strain into which the wild-type low-specificity threonine aldolase derived from E. coli was introduced, 13032-ltaE WT). These results indicate that amino acid substitution variants at positions 29, 126, and 289 of the low-specificity threonine aldolase derived from E. coli further increase the glycine and valine productivity of the microorganisms.

[0290]

[0291] Example 3. Evaluation of Glycine, Alanine, and Valine Production via Introduction of Low-Specific Threonine Aldolase and Aldehyde Dehydrogenase Genes

[0292] Example 3-1. Construction of a vector for the introduction of the aldehyde dehydrogenase gene

[0293] We intended to introduce a plasmid containing an aldehyde dehydrogenase gene using the above-mentioned wild-type Corynebacterium glutamicum ATCC13032 as a host. The recombinant vector containing wild-type ltaE or ltaE variants constructed in Example 1 was linearized by treatment with restriction enzyme (SalI). For the aldehyde dehydrogenase gene, ALDH1 (aldehyde dehydrogenase, NP_015264.1; protein sequence: SEQ ID NO. 76, gene sequence: SEQ ID NO. 77) derived from Saccharomyces cerevisiae was introduced. The above-mentioned ALDH1 gene was amplified by PCR using primer pairs (ALDH1_Fwd (SEQ ID NO. 100), ALDH1_Rev (SEQ ID NO. 101)) with the nucleic acid sequence (bionics) of the synthesized SEQ ID NO. 77 as a template (Table 20). The sequence of the PgapA promoter (sequence number 82) was amplified by PCR using the Corynebacterium glutamicum ATCC13032 genomic DNA (GenBank: CP025533.1) as a template and primer pairs (PgapA_Fwd (sequence number 102), PgapA_Rev (sequence number 103)) (Table 20). The prepared DNA was mixed and subjected to a Gibson assembly reaction to obtain a recombinant vector containing wild-type ltaE or ltaE variants and the aldehyde dehydrogenase gene.

[0294] Primer Sequence Sequence Number ALDH1_FwdTAGAGGAGACACAACATGACCAAGTTGCACTTCGACA100ALDH1_RevGGCCCCCCCTCGAGGTTACAATTTAATGCGGACCG101PgapA_FwdAGCTTATCGATACCGGAAGAAATTTAGATGATTGAAGCC102PgapA_RevACAATCTTTAGAGGAGACACAACATGACCAAGTTGCAC103

[0295] Example 3-2. Evaluation of Glycine Production in Corynebacterium glutamicum Transformers Introduced with Low-Specific Threonine Aldolase Gene and Aldehyde Dehydrogenase Gene

[0296] The recombinant vector prepared in Example 3-1 above was introduced into the Corynebacterium glutamicum ATCC13032 strain via electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541-545) to produce a Corynebacterium glutamicum transformant, and the glycine productivity of the transformant was measured as follows.

[0297] Specifically, the parent strain Corynebacterium glutamicum ATCC13032_pCES208 (13032-Null, Table 21), a transformant with introduced wild-type ltaE, and a transformant with introduced ltaE variant and aldehyde dehydrogenase gene were inoculated into a 250 ml Corner-Baffle flask containing 25 ml of seed medium, respectively, and then cultured at 200 rpm for 20 hours at 30°C to obtain the seed culture. Subsequently, 1 ml of the seed culture was inoculated into a 250 ml Corner-Baffle flask containing 24 ml of the production medium and cultured at 200 rpm for 48 hours at 30°C. After the culture was completed, the glycine production capacity of each strain was measured by measuring the concentration of glycine contained in the culture medium using high-performance liquid chromatography (HPLC). The results obtained are shown in Table 21 below.

[0298] Strain Glycine Concentration (g / l) Increase from Control (%) 13032-Null 4.482 - 13032-ltaE WT (Control) 6.520 - 13032-ltaE(V29G) - ALDH 18.160 25.15% 13032-ltaE(H126L) - ALDH 18.830 35.43% 13032-ltaE(A289T) - ALDH 19.240 41.72% 13032-ltaE(V29G H126L) - ALDH 19.580 46.93% 13032-ltaE(V29G A289T) - ALDH 19.170 40.64% 13032-ltaE(H126L A289T) - ALDH110.08054.60%13032-ltaE(V29G H126L A289T) - ALDH19.02038.34%13032-ltaE(H126A) - ALDH17.82019.94%13032-ltaE(H126C) - ALDH17.27011.50%13032-ltaE(H126D) - ALDH17.43013.96%13032-ltaE(H126F) - ALDH17.78019.33%13032-ltaE(H126G) - ALDH16.6501.99%13032-ltaE(H126I) - ALDH17.80019.63%13032-ltaE(H126K) - ALDH18.09024.08%13032-ltaE(H126M) - ALDH18.28026.99%13032-ltaE(H126N) - ALDH17.48014.72%13032-ltaE(H126P) - ALDH16.7002.76%13032-ltaE(H126Q) - ALDH16.9105.98%13032-ltaE(H126S) - ALDH16.6301.69%13032-ltaE(H126T) - ALDH16.6702.30%13032-ltaE(H126V) - ALDH16.9606.75%13032-ltaE(H126W) - ALDH18.23026.23%13032-ltaE(H126Y) - ALDH17.21010.58%

[0299] As shown in Table 21 above, the Corynebacterium glutamicum ATCC13032 pCES208 strain (13032-Null) to which nothing was introduced had low glycine production capacity. On the other hand, when a gene encoding a low-specificity threonine aldolase variant derived from E. coli and a gene encoding an aldehyde dehydrogenase derived from Saccharomyces cerevisiae were additionally introduced, it was confirmed that glycine production was further improved compared to the control group (Corynebacterium glutamicum strain to which wild-type low-specificity threonine aldolase derived from E. coli was introduced, 13032-ltaE WT). These results indicate that the introduction of a low-specificity threonine aldolase variant derived from E. coli and the enhancement of aldehyde dehydrogenase activity further increase the glycine productivity of the microorganism.

[0300]

[0301] Example 3-3. Evaluation of Alanine Production in Corynebacterium glutamicum Transformers Introduced with Low-Specific Threonine Aldolase Gene and Aldehyde Dehydrogenase Gene

[0302] The recombinant vector prepared in Example 3-1 above was introduced into the Corynebacterium glutamicum ATCC13032 strain via electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541-545) to produce a Corynebacterium glutamicum transformant, and the alanine productivity of the transformant was measured as follows.

[0303] Specifically, the parent strain Corynebacterium glutamicum ATCC13032 pCES208 (13032-Null, Table 22), a transformant with introduced wild-type ltaE, and a transformant with introduced ltaE variant and aldehyde dehydrogenase gene were inoculated into a 250 ml Corner-Baffle flask containing 25 ml of seed medium, respectively, and then cultured at 200 rpm for 20 hours at 30°C to obtain the seed culture. Subsequently, 1 ml of the seed culture was inoculated into a 250 ml Corner-Baffle flask containing 24 ml of the production medium and cultured at 200 rpm for 48 hours at 30°C. After the culture was completed, the alanine production of each strain was determined by measuring the concentration of alanine contained in the culture medium using high-performance liquid chromatography (HPLC). The results obtained are shown in Table 22 below.

[0304] Strain Alanine Concentration (g / l) Increase relative to control (%) 13032-Null 0.466 - 13032-ltaE WT (Control) 0.763 - 13032-ltaE(V29G) - ALDH 10.89 16.64% 13032-ltaE(H126L) - ALDH 10.96 25.82% 13032-ltaE(V29G H126L) - ALDH 10.81 6.16% 13032-ltaE(H126A) - ALDH 11.03 34.99% 13032-ltaE(H126C) - ALDH 10.89 16.64% 13032-ltaE(H126D) - ALDH10.9828.44%13032-ltaE(H126E) - ALDH11.0132.37%13032-ltaE(H126G) - ALDH10.9524.51%13032-ltaE(H126I) - ALDH11.1348.10%13032-ltaE(H126K) - ALDH10.770.92%13032-ltaE(H126M) - ALDH10.827.47%13032-ltaE(H126N) - ALDH10.9625.82%13032-ltaE(H126P) - ALDH11.1550.72%13032-ltaE(H126Q) - ALDH11.0537.61%13032-ltaE(H126R) - ALDH10.9119.27%13032-ltaE(H126S) - ALDH11.0031.06%13032-ltaE(H126T) - ALDH10.9423.20%13032-ltaE(H126V) - ALDH10.9220.58%13032-ltaE(H126W) - ALDH12.05168.68%

[0305] As shown in Table 22 above, the Corynebacterium glutamicum ATCC13032 pCES208 strain (13032-Null) to which nothing was introduced had low alanine production capacity. On the other hand, when a gene encoding a low-specificity threonine aldolase variant derived from E. coli and a gene encoding an aldehyde dehydrogenase derived from Saccharomyces cerevisiae were additionally introduced, it was confirmed that alanine production was further improved compared to the control group (Corynebacterium glutamicum strain to which wild-type low-specificity threonine aldolase derived from E. coli was introduced, 13032-ltaE WT). These results indicate that the introduction of a low-specificity threonine aldolase variant derived from E. coli and the enhancement of aldehyde dehydrogenase activity further increase the alanine productivity of the microorganism.

[0306]

[0307] Examples 3-4. Evaluation of valine production in Corynebacterium glutamicum transformants into which low-specificity threonine aldolase and aldehyde dehydrogenase genes were introduced

[0308] The recombinant vector prepared in Example 3-1 above was introduced into the Corynebacterium glutamicum ATCC13032 strain via electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541-545) to produce a Corynebacterium glutamicum transformant, and the valine productivity of the transformant was measured as follows.

[0309] Specifically, the parent strain Corynebacterium glutamicum ATCC13032 pCES208 (13032-Null, Table 23), a transformant with introduced wild-type ltaE, and a transformant with introduced ltaE variant and aldehyde dehydrogenase gene were inoculated into a 250 ml Corner-Baffle flask containing 25 ml of seed medium, respectively, and then cultured at 200 rpm for 20 hours at 30°C to obtain the seed culture. Subsequently, 1 ml of the seed culture was inoculated into a 250 ml Corner-Baffle flask containing 24 ml of the production medium and cultured at 200 rpm for 48 hours at 30°C. After the culture was completed, the concentration of valine contained in the culture medium was measured using high-performance liquid chromatography (HPLC) to determine the valine production of each strain. The results obtained are shown in Table 23 below.

[0310] Strain Vaulin Concentration (g / l) Increase from Control (%) 13032-Null 0.01 13032-ltaE WT (Control) 0.06 0 13032-ltaE(H126L) - ALDH 10.2 10 25 0.00% 13032-ltaE(A289T) - ALDH 10.1 90 21 6.67% 13032-ltaE(V29G H126L) - ALDH 10.1 30 11 6.67% 13032-ltaE(V29G A289T) - ALDH 10.1 40 13 3.33% 13032-ltaE(H126L A289T) - ALDH 10.1 10 83.33% 13032-ltaE(V29G H126L A289T) - ALDH10.170183.33%13032-ltaE(H126A) - ALDH10.150150.00%13032-ltaE(H126C) - ALDH10.120100.00%13032-ltaE(H126D) - ALDH10.180200.00%13032-ltaE(H126E) - ALDH10.150150.00%13032-ltaE(H126G) - ALDH10.130116.67%13032-ltaE(H126I) - ALDH10.370516.67%13032-ltaE(H126K) - ALDH10.120100.00%13032-ltaE(H126M) - ALDH10.160166.67%13032-ltaE(H126N) - ALDH10.130116.67%13032-ltaE(H126P) - ALDH10.200233.33%13032-ltaE(H126Q) - ALDH10.180200.00%13032-ltaE(H126R) - ALDH10.140133.33%13032-ltaE(H126S) - ALDH10.09050.00%13032-ltaE(H126T) - ALDH10.10066.67%13032-ltaE(H126V) - ALDH10.11083.33%13032-ltaE(H126W) - ALDH10.440633.33%13032-ltaE(H126Y) - ALDH10.140133.33%

[0311] As shown in Table 23 above, the Corynebacterium glutamicum ATCC13032 pCES208 strain (13032-Null), to which nothing was introduced, had low valine production capacity. On the other hand, when a gene encoding a low-specificity threonine aldolase variant derived from E. coli and a gene encoding an aldehyde dehydrogenase derived from Saccharomyces cerevisiae were additionally introduced, it was confirmed that valine production was further improved compared to the control group (Corynebacterium glutamicum strain with introduced wild-type low-specificity threonine aldolase derived from E. coli, 13032-ltaE WT). These results indicate that the introduction of a low-specificity threonine aldolase variant derived from E. coli and the enhancement of aldehyde dehydrogenase activity further increase the valine productivity of the microorganism.

[0312]

[0313] 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 an amino acid corresponding to one or more residues selected from the group consisting of the 29th, 126th, and 289th residues of the amino acid sequence of SEQ NO. 1 is substituted with another amino acid.

2. A variant polypeptide according to claim 1, wherein the amino acid corresponding to the 29th residue of SEQ ID NO. 1 is substituted with glycine, arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan.

3. A variant polypeptide according to claim 1, wherein the amino acid corresponding to the 126th residue of SEQ ID NO. 1 is substituted with leucine, arginine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, valine, isoleucine, methionine, phenylalanine, tyrosine, or tryptophan.

4. A variant polypeptide according to claim 1, wherein the amino acid corresponding to the 289th residue of SEQ ID NO. 1 is substituted with threonine, arginine, histidine, lysine, aspartic acid, glutamic acid, serine, asparagine, glutamine, cysteine, glycine, proline, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan.

5. In claim 1, the amino acid corresponding to the 29th residue of SEQ ID NO. 1 is glycine; and A variant polypeptide in which the amino acid corresponding to the 126th residue of sequence number 1 is substituted with leucine.

6. In claim 1, the amino acid corresponding to the 29th residue of SEQ ID NO. 1 is glycine; and A variant polypeptide in which the amino acid corresponding to the 289th residue of SEQ ID NO. 1 is substituted with threonine, arginine, histidine, lysine, aspartic acid, glutamic acid, serine, asparagine, glutamine, cysteine, glycine, proline, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan.

7. In claim 1, the amino acid corresponding to the 126th residue of SEQ ID NO. 1 is leucine; and A variant polypeptide in which the amino acid corresponding to the 289th residue of SEQ ID NO. 1 is substituted with threonine, arginine, histidine, lysine, aspartic acid, glutamic acid, serine, asparagine, glutamine, cysteine, glycine, proline, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan.

8. In Paragraph 1, The amino acid corresponding to the 29th residue of SEQ ID NO. 1 is glycine; The amino acid corresponding to the 126th residue of SEQ ID NO. 1 is leucine; and A variant polypeptide in which the amino acid corresponding to the 289th residue of SEQ ID NO. 1 is substituted with threonine, arginine, histidine, lysine, aspartic acid, glutamic acid, serine, asparagine, glutamine, cysteine, glycine, proline, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan.

9. In claim 1, the polypeptide is a variant polypeptide having low specificity L-threonine aldolase activity.

10. In claim 1, the polypeptide is a variant polypeptide derived from a microorganism of the genus Escherichia.

11. A variant polypeptide according to claim 1, wherein the polypeptide comprises any one amino acid sequence selected from the group consisting of SEQ ID NOs 8 to 32.

12. A polynucleotide encoding a variant polypeptide of any one of claims 1 to 11.

13. A microorganism comprising a variant polypeptide in which an amino acid corresponding to one or more residues selected from the group consisting of the 29th, 126th, and 289th residues of sequence number 1 is substituted with another amino acid, or a polynucleotide encoding the same.

14. In paragraph 13, the microorganism is a microorganism in which the activity of aldehyde dehydrogenase is enhanced.

15. In paragraph 14, the above aldehyde dehydrogenase is, A microorganism comprising SEQ ID NO. 76 or an amino acid sequence having 90% or more sequence identity with respect to it.

16. In Paragraph 14, the above microorganism is, A microorganism into which one or more selected from the group consisting of an aldehyde dehydrogenase derived from Saccharomyces cerevisiae or a polynucleotide encoding the same have been introduced.

17. In paragraph 14, the microorganism is a microorganism of the genus Corynebacterium.

18. A method for producing one or more amino acids selected from the group consisting of glycine, alanine, and valine, comprising the step of culturing a microorganism of any one of claims 13 to 17 in a culture medium.

19. A method for producing amino acids according to claim 18, further comprising the step of recovering the amino acid from a culture medium or microorganism according to the above culture.

20. A composition for producing one or more amino acids selected from the group consisting of glycine, alanine, and valine, comprising a microorganism according to any one of claims 13 to 17.