Microorganism with enhanced activity of low-specificity threonine aldolase and use thereof

Abstract

The present disclosure provides a microorganism with enhanced activity of low-specific threonine aldolase, 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 amino acid, the composition comprising the microorganism, wherein the microorganism has excellent amino acid production capability.

Description

Microorganism with enhanced activity of low-specificity threonine aldolase and its uses

[0001] A microorganism having enhanced activity of low-specificity threonine aldolase, 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 are provided.

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

[0003] This application claims the benefit of priority based on Korean Patent Application No. 10-2024-0166464 filed on November 20, 2024, and all contents disclosed in the document of said Korean patent application 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 Literature]

[0010] (Patent Document 1) U.S. Registered Patent Publication (US 11661616 B2)

[0011]

[0012] One example of the present disclosure provides a microorganism of the genus Corynebacterium that produces one or more amino acids selected from the group consisting of glycine, alanine, and valine, with enhanced activity of low-specificity threonine aldolase.

[0013] 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.

[0014] 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.

[0015] Another example of the present disclosure provides a use for the microorganism to produce one or more amino acids selected from the group consisting of glycine, alanine, and valine.

[0016] Another example of the present disclosure 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.

[0017]

[0018] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in this disclosure may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this disclosure fall within the scope of this disclosure. Furthermore, the scope of this disclosure is not to be limited by the specific descriptions provided below. Additionally, a person skilled in the art can recognize or identify numerous equivalents to the specific aspects of this disclosure described herein using only ordinary experiments. Moreover, such equivalents are intended to be included in this disclosure.

[0019]

[0020] One aspect provides a microorganism of the genus Corynebacterium that produces one or more amino acids selected from the group consisting of glycine, alanine, and valine, with enhanced activity of low-specificity threonine aldolase.

[0021] The above low-specificity threonine aldolase may refer to a protein having low-specificity threonine aldolase activity. The above low-specificity threonine aldolase activity may refer to enzymatic activity that decomposes threonine into acetaldehyde and glycine (e.g., EC 4.1.2.48). The above low-specificity threonine aldolase activity means that, unlike enzymes with high substrate selectivity, it can exhibit enzymatic reactions on various similar substrates, and means that a protein having low-specificity threonine aldolase activity 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 above 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.

[0022] A microorganism of the genus Corynebacterium that produces one or more amino acids selected from the group consisting of glycine, alanine, and valine, with enhanced activity of the low-specificity threonine aldolase, may be a microorganism into which the low-specificity threonine aldolase or a polynucleotide encoding it has been introduced. The low-specificity threonine aldolase may be an endogenous protein of the microorganism to be introduced or an exogenous protein.

[0023] In one example, the low-specificity threonine aldolase is,

[0024] (a) Sequence No. 1 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%, Amino acid sequences 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;

[0025] (b) Sequence No. 12 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% Amino acid sequences 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; and / or

[0026] (c) Sequence No. 14 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.

[0027] In addition, if a protein possesses such homology or identity and exhibits low-specificity threonine aldolase activity, variants of the low-specificity threonine aldolase having amino acid sequences in which some sequences are deleted, modified, substituted, conservedly substituted, or added may also be included in the low-specificity threonine aldolase. For example, this includes cases where there are sequence additions or deletions, naturally occurring mutations, silent mutations, or conserved substitutions at the N-terminus, C-terminus, and / or within the amino acid sequence that do not alter the low-specificity threonine aldolase activity.

[0028] 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.

[0029]

[0030] In the present disclosure, the introduction of an exogenous protein and / or a polynucleotide encoding it into a host cell (microorganism) may mean the introduction of a protein and / or a polynucleotide encoding it into the host cell derived from a cell of a different genus, a cell of a different species, or another cell of the same species.

[0031] In one example, the low-specificity threonine aldolase and / or the polynucleotide encoding it may be of origin from microorganisms selected from the group consisting of microorganisms of the genus Escherichia, microorganisms of the genus Corynebacterium, microorganisms of the genus Neomicrococcus, and microorganisms of the genus Bombella.

[0032] In one example, the low-specificity threonine aldolase derived from a microorganism of the genus Escherichia and / or the polynucleotide encoding it may be derived from a microorganism selected from the group consisting of Escherichia coli, Escherichia albertii, Escherichia fergusonii, Escherichia hermannii, Escherichia ruysiae and Escherichia marmotae, but is not limited thereto.

[0033] In one example, the low-specificity threonine aldolase derived from the genus Escherichia and / or the polynucleotide encoding it may be of Escherichia coli. In one example, the low-specificity threonine aldolase derived from Escherichia coli may be an ltaE protein. In one example, the low-specificity threonine aldolase derived from Escherichia coli may have, contain, be composed of the amino acid sequence of SEQ ID NO. 1, or be essentially composed of the amino acid sequence.

[0034] The polynucleotide encoding the low-specificity threonine aldolase may refer to a gene encoding the low-specificity threonine aldolase. The polynucleotide encoding the low-specificity threonine aldolase may be of a microorganism of the genus Escherichia, but is not limited thereto, and may be prepared by referring to a known codon table based on the amino acid sequence of the low-specificity threonine aldolase derived from the microorganism of the genus Escherichia.

[0035] In one example, the polynucleotide encoding the low-specificity threonine aldolase derived from Escherichia coli 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%, and 97% It may have, include, be composed of, or be essentially composed of a nucleic 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.

[0036] In one example, a microorganism of the genus Corynebacterium that produces one or more amino acids selected from the group consisting of glycine, alanine, and valine, with enhanced activity of the low-specificity threonine aldolase, may be a microorganism into which a low-specificity threonine aldolase derived from a microorganism of the genus Escherichia or a polynucleotide encoding it has been introduced.

[0037]

[0038] The low-specificity threonine aldolase derived from microorganisms of the genus Neomicrococcus and / or the polynucleotide encoding it may be derived from Neomicrococcus aestuarii and / or Neomicrococcus lactis, but is not limited thereto.

[0039] In one example, a low-specificity threonine aldolase derived from a microorganism of the genus Neomicrococcus and / or a polynucleotide encoding the same may be derived from Neomicrococcus aestuarie, but is not limited thereto. In one example, the low-specificity threonine aldolase derived from Neomicrococcus aestuarie may have, contain, or be composed of the amino acid sequence of SEQ ID NO. 12, or may be essentially composed of the amino acid sequence.

[0040] The polynucleotide encoding the low-specificity threonine aldolase described above may be derived from microorganisms of the genus Neomicrococcus, but is not limited thereto, and may be prepared by referring to a known codon table based on the amino acid sequence of the low-specificity threonine aldolase derived from microorganisms of the genus Neomicrococcus.

[0041] In one example, the polynucleotide encoding low-specificity threonine aldolase derived from Neomicrococcus aestuarie is the nucleic acid sequence of SEQ ID NO. 56 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%, Having, including, being composed of, or essentially consisting of, said nucleic acid sequence having sequence homology or identity of 96.5% or more, 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, or comprising a nucleic acid sequence having sequence homology or identity of 96.5% or more, 97% or more, 97.5% or more, 99.7% or more, 99.8% or more, or 99.9% or more. can.

[0042] In one example, a microorganism of the genus Corynebacterium that produces one or more amino acids selected from the group consisting of glycine, alanine, and valine, with enhanced activity of the low-specificity threonine aldolase, may be a microorganism into which a low-specificity threonine aldolase derived from a microorganism of the genus Neomicrococcus or a polynucleotide encoding the same has been introduced.

[0043]

[0044] The low-specificity threonine aldolase derived from a microorganism of the genus Bombella and / or the polynucleotide encoding it may be derived from a microorganism selected from the group consisting of Bombella sp. ESL0368, Bombella apis, Bombella favorum, Bombella intestini, and Bombella mellum, but is not limited thereto. In one example, the low-specificity threonine aldolase derived from a microorganism of the genus Bombella and / or the polynucleotide encoding it may be derived from Bombella sp. ESL0368, but is not limited thereto. In one example, the low-specificity threonine aldolase derived from Bombella sp. ESL0368 may have, contain, be composed of the amino acid sequence of SEQ ID NO. 14, or be essentially composed of the amino acid sequence.

[0045] The polynucleotide encoding the low-specificity threonine aldolase above may be derived from microorganisms of the genus Bombella, but is not limited thereto, and may be prepared by referring to a known codon table based on the amino acid sequence of the low-specificity threonine aldolase derived from microorganisms of the genus Bombella.

[0046] In one example, the above Bombella sp. A polynucleotide encoding a low-specificity threonine aldolase derived from ESL0368 is the nucleic acid sequence of SEQ ID NO. 57 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 a nucleic 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.

[0047] In one example, a microorganism of the genus Corynebacterium that produces one or more amino acids selected from the group consisting of glycine, alanine, and valine, with enhanced activity of the low-specificity threonine aldolase, may be a microorganism into which a low-specificity threonine aldolase derived from a microorganism of the genus Bombella or a polynucleotide encoding it has been introduced.

[0048]

[0049] The above low-specificity threonine aldolase can be introduced into a microorganism using a recombinant vector. The recombinant vector can be used as an insertion vector or an expression vector. Expressing the above low-specificity threonine aldolase in a microorganism can be performed by culturing a recombinant cell (e.g., a microorganism) containing a low-specificity threonine aldolase gene, a polynucleotide encoding low-specificity threonine aldolase, or a recombinant vector containing the same.

[0050] The introduction of the polynucleotide or recombinant vector encoding the low-specificity threonine aldolase 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 vector containing the target polynucleotide into a host cell to change the genetic traits of the host cell. The transformed polynucleotide may be inserted into or located outside the chromosome of the host cell. There is no limitation on the form in which the polynucleotide is introduced, provided that it can be introduced into the host cell and expressed. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a 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.

[0051] 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.

[0052] In the present disclosure, the term “vector” refers to a DNA product for delivering a target polynucleotide into a suitable host or host cell. For example, a vector may comprise a sequence of nucleotides of a polynucleotide operably linked to a suitable regulatory sequence to enable the expression of the target polynucleotide within a suitable host. The regulatory sequence may comprise a promoter capable of initiating transcription, any operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and / or a sequence regulating the termination of transcription and / or translation. After being transformed into a suitable host cell, the vector may replicate or function independently of the host cell’s genome, or it may be integrated into the genome itself to replicate or function.

[0053] The vectors available for use in the present disclosure are not particularly limited as long as they are capable of replicating within a host cell, and can be selected from all commonly used vectors. Examples of commonly used vectors include plasmids, cosmids, viruses, bacteriophages, etc., in their natural or recombinant state. For example, as the vectors, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A, etc., can be used as phage vectors or cosmid vectors, and pBR-based, pUC-based, pBluescriptII-based, pGEM-based, pTZ-based, pCL-based, and pET-based vectors, etc., can be used as plasmid vectors. Specifically, examples include, but are not limited to, vectors such as pCES208, pDZ, pDC, pDC24, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC.

[0054] The above vector may additionally include a selection marker to determine whether the vector has been transformed into a host cell or, furthermore, whether it has been inserted into a chromosome within the host cell. The selection marker is intended to select cells transformed by the vector or to confirm whether the target polynucleotide has been inserted into a chromosome, and markers conferring selectable phenotypes, such as drug resistance, nutritional requirements, resistance to cytotoxic agents, or the expression of surface proteins, may be used. Since only cells expressing the selection marker survive or exhibit other phenotypic traits in an environment treated with a selective agent, the transformed cells can be selected.

[0055]

[0056] In the present disclosure, the phrase “having, comprising, being composed of, or essentially being composed of a specific nucleic acid sequence (base sequence) or amino acid sequence” means that the polynucleotide or polypeptide has (i) essentially comprises the specific nucleic acid sequence (base sequence) or amino acid sequence, or (ii) 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% or more, 93% or more, 93.5% or more, 94% or more, 94.5% or more, 95% or more, 95.5% or more, 96% or more, 96.5% or more, 97% or more, 97.5% or more, 97.6% 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, It may mean that it consists of or essentially includes a nucleic acid sequence or amino acid sequence having 99.7% or more, 99.8% or more, or 99.9% or more homology or identity, and maintains its original function and / or intended function. In one example, the intended function may mean a function that increases (enhances) or imparts the ability to produce one or more amino acids selected from the group consisting of glycine, alanine, and valine of the microorganism.

[0057] In this disclosure, 'homology' or 'identity' refers 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.

[0058] 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.

[0059] Whether any two polynucleotide or polypeptide sequences have homology or identity can be determined using a known computer algorithm, such as the “FASTA” program, using default parameters as in, for example, Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), as performed in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later) (GCG program package (Devereux, J., et al, Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] (Including Academic Press, San Diego, 1994, and [CARILLO ETA / .](1988) SIAM J Applied Math 48: 1073). For example, homology, similarity, or identity can be determined using BLAST from the National Biotechnology Information Database Center or ClustalW.

[0060] 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.

[0061]

[0062] In the present disclosure, the term “microorganism (or strain)” may include both wild-type microorganisms and microorganisms that have undergone natural or artificial genetic modification. The microorganism may be a microorganism in which a specific mechanism is enhanced or weakened due to causes such as the insertion of an external gene or the enhancement or weakening of the activity of an endogenous gene, and may be a microorganism that includes genetic modification for the production of a desired polypeptide, protein, or product (e.g., one or more amino acids selected from the group consisting of glycine, alanine, and valine).

[0063] In the present disclosure, the term “enhanced” of polypeptide (protein) activity means that the activity of the polypeptide is increased compared to its intrinsic activity. Such enhancement may be used interchangeably with terms such as activation, up-regulation, overexpression, and increase. Here, activation, enhancement, up-regulation, overexpression, and increase may include exhibiting activity that was not originally possessed, or exhibiting activity that is improved compared to the intrinsic activity or activity prior to modification. The “intrinsic activity” refers to the activity of a specific polypeptide originally possessed by the parent strain prior to modification (e.g., a parent strain in which the activity of low-specificity threonine aldolase is not enhanced) or the non-modified microorganism (e.g., a wild-type microorganism) in cases where the trait is altered due to genetic variation caused by natural or artificial factors. This may be used interchangeably with “activity prior to modification.” The statement that the activity of a polypeptide is “enhanced,” “upregulated,” “overexpressed,” or “increased” relative to its intrinsic activity means that it has been enhanced compared to the activity and / or concentration (expression amount) of a specific polypeptide originally possessed by the parent strain or non-modified microorganism prior to transformation.

[0064] The above enhancement can be achieved by introducing an exogenous polypeptide or by enhancing the activity and / or increasing the concentration (expression amount) of the intrinsic polypeptide. Whether the activity of the polypeptide is enhanced can be confirmed by an increase in the degree of activity, expression amount, or amount of product resulting from the polypeptide activity of said polypeptide.

[0065] The enhancement of the activity of the above polypeptide may be achieved by applying various methods well known in the art, and is not limited to, as long as the activity of the target polypeptide can be enhanced compared to the microorganism before modification. Specifically, it may utilize, but is not limited to, gene engineering and / or protein engineering known to a person skilled in the art, which are routine methods of molecular biology (e.g., Sitnicka et al. Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol. 2. 1-16, Sambrook et al. Molecular Cloning 2012, etc.).

[0066] Specifically, the activity enhancement of the polypeptide (protein) of the present disclosure is

[0067] 1) Increase in the intracellular copy number of polynucleotides encoding polypeptides;

[0068] 2) Replace the chromosomal gene expression regulatory region encoding a polypeptide with a potent sequence;

[0069] 3) A modification of the nucleotide sequence encoding the start codon or the 5'-UTR region of the gene transcript encoding the polypeptide;

[0070] 4) Modification of the amino acid sequence of the polypeptide to enhance polypeptide activity;

[0071] 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);

[0072] 6) Introduction of a foreign polypeptide exhibiting polypeptide activity or a foreign polynucleotide encoding the same;

[0073] 7) Codon optimization of polynucleotides encoding polypeptides;

[0074] 8) Analyze the tertiary structure of the polypeptide to select and modify or chemically modify the exposed sites;

[0075] 9) Regulation of the cellular localization of polypeptides; or

[0076] 10) It may be based on two or more combinations selected from 1) to 9) above, but is not specifically limited thereto.

[0077]

[0078] More specifically,

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] 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.

[0089]

[0090] In this disclosure, the term “weakening” of the activity of a polypeptide is a concept that includes both reduced activity and lack of activity relative to intrinsic activity. Such weakening may be used interchangeably with terms such as inactivation, deficiency, down-regulation, decrease, reduce, and attenuation.

[0091] 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 transformation.

[0092] 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.).

[0093] Specifically, the weakening of polypeptide (protein) activity is

[0094] 1) Deletion of all or part of the gene encoding a polypeptide;

[0095] 2) Modification of the expression regulatory region (or expression regulatory sequence) to reduce the expression of the gene encoding the polypeptide;

[0096] 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);

[0097] 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);

[0098] 5) A modification of the nucleotide sequence encoding the start codon or the 5'-UTR region of the gene transcript encoding the polypeptide;

[0099] 6) Introduction of an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the transcript of the gene encoding the polypeptide;

[0100] 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;

[0101] 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);

[0102] 9) Regulation of the cellular localization of polypeptides; or

[0103] 10) It may be based on two or more combinations selected from 1) to 9) above, but is not specifically limited thereto.

[0104] for example,

[0105] 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.

[0106] 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.

[0107] 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.

[0108] 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.

[0109] 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].

[0110] 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.

[0111] 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.

[0112] 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.

[0113] 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 the microbial strain prior to modification, or an increase in the amount of product produced from said polypeptide, but is not limited thereto.

[0114]

[0115] The microorganism (or strain, recombinant cell) of the present disclosure may be a microorganism having the ability (or production volume) to produce one or more amino acids selected from the group consisting of glycine, alanine, and valine, or having an enhanced (or increased) ability to produce one or more amino acids selected from the group consisting of glycine, alanine, and valine.

[0116] Microorganisms that produce one or more amino acids selected from the group consisting of glycine, alanine, and valine, with enhanced activity of the low-specificity threonine aldolase of the present disclosure, may be, but are not limited to, microorganisms that naturally lack the ability to produce said amino acids, or microorganisms that have the ability to produce said amino acids into which the low-specificity threonine aldolase or a polynucleotide encoding it has been introduced. In one example, the polynucleotide encoding the low-specificity threonine aldolase in said microorganism may be operably linked to a strong promoter.

[0117] The fact that the microorganism with enhanced activity of the low-specificity threonine aldolase has improved production capacity for one or more amino acids selected from the group consisting of glycine, alanine, and valine, or possesses said amino acid production capacity, may mean that the microorganism has improved production capacity for one or more amino acids selected from the group consisting of glycine, alanine, and valine compared to the non-modified microorganism, the cell before recombination, the parent strain, or the wild-type microorganism, or that the said amino acid production capacity is conferred unlike the non-modified microorganism, the cell before recombination, the parent strain, and / or the wild-type microorganism that lacks said amino acid production capacity.

[0118] A microorganism with enhanced low-specificity threonine aldolase activity according to one example may have improved production capacity of one or more amino acids selected from the group consisting of glycine, alanine, and valine compared to a microorganism before enhancement, i.e., a non-modified microorganism of the same type. In the present disclosure, "non-modified microorganism" does not exclude strains containing mutations that may naturally occur in microorganisms, and may refer to a wild-type strain or a natural-type strain itself, or a strain before its traits are changed by genetic variation caused by natural or artificial factors. For example, according to one example, the non-modified microorganism may refer to a strain in which the low-specificity threonine aldolase activity is not enhanced or before its activity is enhanced. The "non-modified microorganism" may be used interchangeably with "stratification before modification," "microorganism before modification," "non-mutation strain," "non-modified strain," "non-mutation microorganism," or "reference microorganism." As previously stated, the activity of the above low-specificity threonine aldolase is enhanced.

[0119] 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.

[0120] In one example, the microorganism may be a microorganism with enhanced threonine production ability.

[0121] In one example, the microorganism with enhanced threonine production capacity may be a microorganism with enhanced activity of aspartokinase, L-threonine efflux protein, and / or homoserine dehydrogenase.

[0122] In one example, the microorganism with enhanced activity of the aspartokinase may be a microorganism in which an endogenous or exogenous aspartokinase is introduced, or a variant in which a mutation for releasing feedback inhibition of lysine and / or threonine is introduced to the aspartokinase.

[0123] In one example, the microorganism may include a polypeptide in which the residue corresponding to the 377th position of the aspartokinase (e.g., NCBI Reference Sequence: WP_003855724.1) is substituted with another amino acid.

[0124] The above 'other amino acids' are not limited to any other amino acids except for L-leucine, the 377th amino acid. Specifically, as a representative example, the 377th amino acid residue may be substituted with lysine or methionine. L-lysine is an example of a basic amino acid, and the basic amino acid may be one of L-lysine, L-arginine, and L-histidine. L-methionine is an example of a nonpolar amino acid, and the nonpolar amino acid may be any one of L-methionine, L-phenylalanine, L-alanine, L-cysteine, L-glycine, L-isoleucine, L-leucine, L-proline, L-tryptophan, and L-valine. However, it is not limited thereto.

[0125] In one example, the microorganism with enhanced activity of the L-threonine efflux protein may be a microorganism in which an endogenous or exogenous L-threonine efflux protein (e.g., an L-threonine efflux protein derived from E. coli) is introduced, or a variant in which a mutation for improving the L-threonine efflux ability of the L-threonine efflux protein is introduced.

[0126] In one example, the microorganism may comprise a polypeptide in which the residue corresponding to the 53rd position and / or the 62nd position of the L-threonine efflux protein (e.g., NCBI Reference Sequence: WP_000928824.1) is substituted with another amino acid.

[0127] In one embodiment, the L-threonine excretion protein may have a residue corresponding to the 53rd position substituted with threonine, and / or a residue corresponding to the 62nd position substituted with serine, arginine, alanine, aspartic acid, lysine, proline, cysteine, glycine, threonine, isoleucine, tyrosine, valine, histidine, phenylalanine, methionine, glutamine, asparagine, glutamic acid, or tryptophan, but is not limited thereto.

[0128] In one example, the microorganism with enhanced homoserine dehydrogenase activity may be a microorganism having an endogenous or exogenous homoserine dehydrogenase introduced, or a variant having a mutation that enhances the activity of the homoserine dehydrogenase and / or a mutation for releasing feedback inhibition of threonine introduced.

[0129] In one example, the microorganism may include a polypeptide in which the residue corresponding to the 285th and / or 398th positions of homoserine dehydrogenase (e.g., NCBI Reference Sequence: WP_003854900.1) is substituted with another amino acid.

[0130] In one embodiment, the homoserine dehydrogenase may be substituted with isoleucine for the residue (threonine) corresponding to the 285th position and / or glutamine for the residue (arginine) corresponding to the 398th position, but is not limited thereto.

[0131] In one example, the microorganism may include a polypeptide in which the residue corresponding to the 378th and / or 398th positions of homoserine dehydrogenase is substituted with another amino acid.

[0132] In one example, the homoserine dehydrogenase may have the 378th residue (glycine) substituted with glutamate and / or the 398th residue (arginine) substituted with glutamine, but is not limited thereto.

[0133]

[0134] 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.

[0135] In one example, the microorganism may be Corynebacterium glutamicum.

[0136] In one example, the microorganism may be Corynebacterium glutamicum ATCC13032.

[0137] In one example, a microorganism that produces one or more amino acids selected from the group consisting of glycine, alanine, and valine, with enhanced activity of the low-specificity threonine aldolase, may be a microorganism with further enhanced activity of an aldehyde dehydrogenase.

[0138] The above aldehyde dehydrogenase may refer to a protein having aldehyde dehydrogenase activity. The above aldehyde dehydrogenase activity may refer to enzymatic activity that catalyzes a reaction converting aldehyde or acetaldehyde into a carboxylic acid. In one example, the above aldehyde dehydrogenase may have acetaldehyde dehydrogenase activity.

[0139] A microorganism of the genus Corynebacterium that produces one or more amino acids selected from the group consisting of glycine, alanine, and valine, with the activity of the aldehyde dehydrogenase further enhanced, may be a microorganism into which the aldehyde dehydrogenase or a polynucleotide encoding it has been introduced. The aldehyde dehydrogenase may be an endogenous protein of the microorganism to be introduced or an exogenous protein.

[0140] In one example, the aldehyde dehydrogenase is,

[0141] (a) Sequence No. 32 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% Amino acid sequences 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;

[0142] (b) Sequence No. 34 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% Amino acid sequences 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;

[0143] (c) Sequence No. 36 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% Amino acid sequences 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;

[0144] (d) Sequence No. 38 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% Amino acid sequences 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; and / or

[0145] (e) Sequence No. 40 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.

[0146] 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.

[0147] 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.

[0148]

[0149] 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.

[0150] In one example, the aldehyde dehydrogenase derived from the genus Escherichia and / or the polynucleotide encoding it may be derived from a microorganism selected from the group consisting of Escherichia coli, Escherichia albertii, Escherichia fergusonii, Escherichia hermannii, Escherichia ruysiae, and Escherichia marmotae, but is not limited thereto.

[0151] In one example, the aldehyde dehydrogenase derived from the genus Escherichia and / or the polynucleotide encoding it may be of Escherichia coli. In one example, the aldehyde dehydrogenase derived from Escherichia coli may be an mhpF protein or an eutE protein. In one example, the aldehyde dehydrogenase derived from Escherichia coli may have, contain, or be composed of the amino acid sequence of SEQ ID NO. 32 or SEQ ID NO. 34, or be essentially composed of the amino acid sequence.

[0152] The polynucleotide encoding the aldehyde dehydrogenase may refer to a gene encoding the aldehyde dehydrogenase. The polynucleotide encoding the aldehyde dehydrogenase may be of a microorganism of the genus Escherichia, 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 derived from the microorganism of the genus Escherichia.

[0153] In one example, the polynucleotide encoding an aldehyde dehydrogenase derived from Escherichia coli is the nucleic acid sequence of SEQ ID NO. 33, the nucleic acid sequence of SEQ ID NO. 35, 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%, Having, including, being composed of, or essentially consisting of, said nucleic acid sequence having sequence homology or identity of 96.5% or more, 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, or comprising a nucleic acid sequence having sequence homology or identity of 96.5% or more, 97% or more, 97.5% or more, 99.7% or more, 99.8% or more, or 99.9% or more. can.

[0154] In one example, a microorganism of the genus Corynebacterium that produces one or more amino acids selected from the group consisting of glycine, alanine, and valine, with the activity of the aldehyde dehydrogenase further enhanced, may be a microorganism into which an aldehyde dehydrogenase derived from a microorganism of the genus Escherichia or a polynucleotide encoding the same has been introduced.

[0155]

[0156] 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.

[0157] 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 may have, contain, or be composed of the amino acid sequence of SEQ ID NO. 36, or be essentially composed of the amino acid sequence.

[0158] 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.

[0159] In one example, the polynucleotide encoding an aldehyde dehydrogenase derived from Saccharomyces cerevisiae is the nucleic acid sequence of SEQ ID NO. 37 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.

[0160] In one example, a microorganism of the genus Corynebacterium that produces one or more amino acids selected from the group consisting of glycine, alanine, and valine, with the activity of the aldehyde dehydrogenase 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.

[0161]

[0162] In one example, the aldehyde dehydrogenase derived from a microorganism of the genus Zymomonas and / or the polynucleotide encoding it may be derived from Zymomonas mobilis, but is not limited thereto. In one example, the aldehyde dehydrogenase derived from Zymomonas mobilis may be an AldB protein. In one example, the aldehyde dehydrogenase derived from Zymomonas mobilis may have, contain, or be composed of the amino acid sequence of SEQ ID NO. 38, or be essentially composed of the amino acid sequence.

[0163] The polynucleotide encoding the above aldehyde dehydrogenase may be of a microorganism of the genus Zeomomonas, 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 Zeomomonas.

[0164] In one example, the polynucleotide encoding an aldehyde dehydrogenase derived from the above-mentioned Xymomonas mobilis is the nucleic acid sequence of SEQ ID NO. 39 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.

[0165] In one example, a microorganism of the genus Corynebacterium that produces one or more amino acids selected from the group consisting of glycine, alanine, and valine, with the activity of the aldehyde dehydrogenase further enhanced, may be a microorganism into which an aldehyde dehydrogenase derived from a microorganism of the genus Zemmomonas or a polynucleotide encoding the same has been introduced.

[0166]

[0167] In one example, the aldehyde dehydrogenase derived from the microorganism of the genus Corynebacterium and / or the polynucleotide encoding the same is Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium polutisoli It may be derived from microorganisms selected from the group consisting of (pollutisoli), (Corynebacterium imitans), (Corynebacterium testudinoris), and (Corynebacterium flavescens), but is not limited thereto.

[0168] In one example, the aldehyde dehydrogenase derived from a microorganism of the genus Corynebacterium and / or the polynucleotide encoding it may be derived from Corynebacterium glutamicum. In one example, the aldehyde dehydrogenase derived from Corynebacterium glutamicum may be an Ald protein. In one example, the aldehyde dehydrogenase derived from Corynebacterium glutamicum may have, contain, be composed of the amino acid sequence of SEQ ID NO. 40, or be essentially composed of the amino acid sequence.

[0169] The polynucleotide encoding the above aldehyde dehydrogenase may be of a microorganism of the genus Corynebacterium, but is not limited thereto, and may be prepared by referring to a known codon table based on the amino acid sequence of the aldehyde dehydrogenase of the microorganism of the genus Corynebacterium.

[0170] In one example, the polynucleotide encoding an aldehyde dehydrogenase derived from Corynebacterium glutamicum is the nucleic acid sequence of SEQ ID NO. 41 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%, Having, including, being composed of, or essentially consisting of, said nucleic acid sequence having sequence homology or identity of 96.5% or more, 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, or comprising a nucleic acid sequence having sequence homology or identity of 96.5% or more, 97% or more, 97.5% or more, 99.7% or more, 99.8% or more, or 99.9% or more. can.

[0171] In one example, a microorganism of the genus Corynebacterium that produces one or more amino acids selected from the group consisting of glycine, alanine, and valine, with the activity of the aldehyde dehydrogenase further enhanced, may be a microorganism into which an aldehyde dehydrogenase derived from a microorganism of the genus Corynebacterium or a polynucleotide encoding the same has been introduced.

[0172]

[0173] In one example, the microorganism with enhanced activity of the low-specificity threonine aldolase, or the microorganism with enhanced activity of the low-specificity threonine aldolase and additionally enhanced activity of the aldehyde dehydrogenase, compared to the parent strain before mutation and the non-mutated microorganism, is newly endowed with the ability to produce one or more amino acids selected from the group consisting of glycine, alanine, and valine, or is 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 25% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 100% or more, about 150% or more, about 200% or more, about 250% or more, about 300% or more, about It may be increased by more than 400%, more than 500%, more than 600%, more than 700%, more than 800%, more than 900%, more than 1,000%, more than 1,500%, more than 2,000%, more than 2,500%, or more than 3,000%, but is not limited thereto.

[0174] As another example, a microorganism with enhanced activity of the low-specificity threonine aldolase, or a microorganism with enhanced activity of the low-specificity threonine aldolase and additionally enhanced activity of aldehyde dehydrogenase, compared to the parent strain before mutation and the non-mutated microorganism, has an ability to produce one or more amino acids selected from the group consisting of glycine, alanine, and valine that is 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, about 1.09 times or more, 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, and about 5 times It may be more than, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 15 times, about 20 times, about 25 times, or about 30 times (there is no special limit on the upper limit, for example, it may be about 1,000 times or less), but is not limited thereto.

[0175] As another example, the microorganism with enhanced activity of the low-specificity threonine aldolase, or the microorganism with enhanced activity of the low-specificity threonine aldolase and additionally enhanced activity of aldehyde dehydrogenase, compared to the parent strain before mutation and the non-mutated microorganism, has a production capacity of one or more amino acids selected from the group consisting of glycine, alanine, and valine of about 0.1 g / L or more, about 0.2 g / L or more, about 0.3 g / L or more, about 0.4 g / L or more, about 0.5 g / L or more, about 0.6 g / L or more, about 0.7 g / L or more, about 0.8 g / L or more, about 0.9 g / L or more, about 1 g / L or more, about 1.1 g / L or more, about 1.2 g / L or more, about 1.3 g / L or more, about 1.4 g / L or more, about 1.5 g / L or more, and about 1.6 g / L. It may be, or, about 1.7 g / L or more, about 1.8 g / L or more, about 1.9 g / L or more, about 2.0 g / L or more, about 2.5 g / L or more, about 3 g / L or more, about 3.5 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 value, for example, it may be about 100 g / L or less), but is not limited thereto.

[0176] 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.”

[0177]

[0178] 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.

[0179] In the present disclosure, "culture" means growing the microorganism under appropriately controlled environmental conditions. The culture process of the present disclosure may be carried out according to suitable media and culture conditions known in the art. Such culture process can be easily adjusted and used by those skilled in the art depending on the strain selected. Specifically, the culture may be batch, continuous, and / or fed-batch, but is not limited thereto.

[0180] In the present disclosure, "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 the present disclosure may be any medium used for culturing microorganisms without special limitations, provided that the microorganism of the present disclosure can be cultured under aerobic conditions while controlling the temperature, pH, etc., in a conventional medium containing a suitable carbon source, nitrogen source, phosphorus, inorganic compounds, amino acids, and / or vitamins.

[0181] Specifically, culture media for the microorganisms of the present disclosure, such as strains of the genus Corynebacterium, can be found in the literature ["Manual of Methods for General Bacteriology" by the American Society for Bacteriology (Washington D. Corynebacterium, USA, 1981)].

[0182] In the present disclosure, the carbon source may include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvate, lactic acid, citric acid, etc.; amino acids such as glutamic acid, methionine, lysine, etc. Additionally, natural organic nutrient sources such as starch hydrolysate, molasses (e.g., blackstrap molasses), rice husk, cassava, sugarcane residue, and corn steeping liquid may be used. Specifically, carbohydrates such as glucose and sterilized pre-treated molasses (i.e., molasses converted into reducing sugars) may be used, and other carbon sources in appropriate amounts may be used without limitation. These carbon sources may be used individually or in combination of two or more types, but are not limited thereto.

[0183] 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.

[0184] 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.

[0185] In addition, during the cultivation of the microorganism of the present disclosure, 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 a 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.

[0186] In the culture of the present disclosure, the culture temperature may be maintained at 20 to 45°C, specifically 25 to 40°C, and culture may be carried out for about 10 to 160 hours, but is not limited thereto.

[0187] One or more amino acids selected from the group consisting of glycine, alanine, and valine produced by the culture of the present disclosure may be secreted into the medium or remain in the cell.

[0188] A method for producing one or more amino acids selected from the group consisting of glycine, alanine, and valine of the present disclosure may additionally include, for example, the step of preparing a microorganism of the present disclosure, the step of preparing a medium for culturing said microorganism, or a combination thereof (in any order), prior to the culturing step.

[0189] The method for producing one or more amino acids selected from the group consisting of glycine, alanine, and valine according to the present disclosure may further include the 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 the culture is performed) or a microorganism (e.g., a strain of the genus Corynebacterium) according to the culture. The recovery step may be additionally included after the culture step.

[0190] 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 disclosure, for example, batch, continuous, or fed-batch culture methods. For example, various chromatographic methods such as centrifugation, filtration, treatment with a crystallizing protein precipitating agent (salting out), extraction, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC, or a combination thereof may be used, and 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.

[0191] In addition, the method for producing one or more amino acids selected from the group consisting of glycine, alanine, and valine of the present disclosure may additionally include a purification step. The purification may be performed using a suitable method known in the art. In one example, where the method for producing one or more amino acids selected from the group consisting of glycine, alanine, and valine of the present disclosure includes both a recovery step and a purification step, the recovery step and the purification step may be performed continuously or discontinuously regardless of the order, or simultaneously or integrated into a single step, but are not limited thereto.

[0192]

[0193] 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.

[0194] 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.

[0195] 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.

[0196] Another aspect provides a use for 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.

[0197] The above microorganisms are as described above.

[0198]

[0199] The present disclosure provides a microorganism with enhanced activity of low-specificity threonine aldolase, 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, wherein the ability of said microorganism to produce said amino acids is excellent.

[0200]

[0201] 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.

[0202]

[0203] Example 1. Evaluation of Glycine Production via Introduction of Low-Specific Threonine Aldolase Gene

[0204] Example 1-1. Construction of a vector containing wild-type low-specificity threonine aldolase

[0205] To introduce a low-specificity threonine aldolase gene into a Corynebacterium glutamicum strain, vectors containing low-specificity threonine aldolase derived from Escherichia coli, Neomicrococcus aestuarii, and Bombella sp. ESL0368 were constructed as follows. To be used as a control for the experiment, a vector containing serine hydroxymethyltransferase (SHMT) derived from Corynebacterium glutamicum, which is known to possess intrinsic low-specificity threonine aldolase activity, was constructed.

[0206] The specific production process is as follows.

[0207] A pCES208 vector containing a gene encoding wild-type low-specificity 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 (Korean Registered Patent Publication KR 10-1673080 B1, J.Microbiol. Biotechnol., 18:639-647, 2008). 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 treatment 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.

[0208]

[0209] We constructed a pCES208 vector containing a gene encoding a low-specificity threonine aldolase derived from Neomicrococcus aestuarii (NCBI Reference Sequence: APF39792.1, amino acid sequence: SEQ NO. 12; nucleic acid sequence: SEQ NO. 56) and a pCES208 vector containing a gene encoding a low-specificity threonine aldolase derived from the ESL0368 strain of the genus Bombella (GenBank: QGT74600.1, amino acid sequence: SEQ NO. 14; nucleic acid sequence: SEQ NO. 57).

[0210]

[0211]

[0212] In Table 1 above, lowercase letters are nucleic acid sequences for universal primers, uppercase letters are genes encoding each low-specificity threonine aldolase, and letters in italics are nucleic acid sequences of the Pcj7 promoter. In Table 1 above, the nucleic acid sequence of SEQ ID NO. 13 comprises a gene encoding a low-specificity threonine aldolase derived from Neomicrococcus aestuarii (NCBI Reference Sequence: APF39792.1, amino acid sequence: SEQ ID NO. 12; nucleic acid sequence: SEQ ID NO. 56), a Pcj7 promoter, and a universal primer, and the nucleic acid sequence of SEQ ID NO. 15 comprises a gene encoding a low-specificity threonine aldolase derived from the ESL0368 strain of the genus Bombella (Bombella sp. ESL0368) (GenBank: QGT74600.1, amino acid sequence: SEQ ID NO. 14; nucleic acid sequence: SEQ ID NO. 57), a Pcj7 promoter, and a universal primer.

[0213] In this embodiment, the gene encoding low-specificity threonine aldolase derived from Neomicrococcus aestuarii (NCBI Reference Sequence: APF39792.1, amino acid sequence: SEQ ID NO. 12) was named TA(Nae), and the gene encoding low-specificity threonine aldolase derived from the ESL0368 strain of the genus Bombella (Bombella sp. ESL0368) was named TA(Bom).

[0214] The DNA fragments of the TA(Nae) gene and the TA(Bom) gene were amplified by PCR using the nucleic acid sequence of SEQ ID NO. 13 or SEQ ID NO. 15 as a template and the primer pair of SEQ ID NO. 16 (TA_Fwd) and SEQ ID NO. 17 (TA_Rev). The pCES208_TA(Nae) and pCES208_TA(Bom) plasmids were obtained by performing a Gibson assembly reaction with the vector linearized by treating pCES208 with EcoRV and the DNA fragments of Pcj7_TA(Nae) and Pcj7_TA(Bom), respectively.

[0215] A pCES208 vector containing the glyA gene encoding serine hydroxymethyltransferase (SHMT (Serine Hydroxymethyltransferase) derived from Corynebacterium glutamicum, GenBank: BAB98389.1, amino acid sequence: SEQ ID NO. 8; gene sequence: SEQ ID NO. 9) was constructed. The SHMT enzyme is known to have intrinsic low-specificity threonine aldolase activity.

[0216] The DNA fragment of the glyA gene (Sequence No. 9) was amplified by PCR using the primer pair of Sequence No. 10 (glyA_Fwd) and Sequence No. 11 (glyA_Rev) with Corynebacterium glutamicum ATCC13032 genomic DNA as a template. The Pcj7 promoter (Sequence No. 5) was amplified by PCR using the primer pair of Sequence No. 6 (Pcj7_Fwd) and Sequence No. 7 (Pcj7_Rev) with the synthesized nucleotide sequence (bionics) as a template. The pCES208_glyA plasmid was obtained by performing a Gibson assembly reaction with the vector linearized by EcoRV treatment, the Pcj7 DNA fragment, and the glyA DNA fragment.

[0217]

[0218] Example 1-2. Evaluation of Glycine Production Capacity of Corynebacterium glutamicum Transformers

[0219] Corynebacterium glutamicum transformants were prepared by introducing the pCES208_ltaE, pCES208_TA(Nae), pCES208_TA(Bom), and pCES208_glyA plasmids prepared in Example 1-1 above into the Corynebacterium glutamicum ATCC13032 strain via electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541-545). As a control, a Corynebacterium glutamicum transformant was prepared by introducing the covector pCES208 vector into the Corynebacterium glutamicum ATCC13032 strain. To confirm the effect of introducing low-specificity threonine aldolase, the glycine production capacity of strains introduced with pCES208_ltaE, pCES208_TA(Nae), and pCES208_TA(Bom) plasmids (Table 2, 13032-ltaE WT, 13032-TA(Nae) (WT), 13032-TA(Bom) (WT) respectively) was evaluated using a strain introduced with pCES208_glyA plasmid (Table 2, 13032-glyA (WT)) as a control.

[0220] The glycine productivity of the above Corynebacterium glutamicum transformant was measured by the following method.

[0221] Specifically, in a 250 ml corner-barfle flask containing 25 ml of seed medium, the parent strain Corynebacterium glutamicum ATCC13032_pCES208 (13032-Null), Corynebacterium glutamicum ATCC13032 (13032-glyA (WT)) into which the wild-type glyA gene derived from Corynebacterium glutamicum ATCC13032 has been introduced, Corynebacterium glutamicum ATCC13032 (13032-ltaE (WT)) into which the wild-type ltaE gene derived from E. coli has been introduced, and Corynebacterium glutamicum ATCC13032 (13032-TA(Nae)) into which the gene encoding low-specificity threonine aldolase derived from Neomicrococcus aestuarii has been introduced (WT)), and Corynebacterium glutamicum ATCC13032 (13032-TA(Bom) (WT)) into which a gene encoding a low-specificity threonine aldolase derived from the ESL0368 strain of the genus Bombella (Bombella sp. ESL0368) was introduced were inoculated, respectively, and then cultured at 30°C for 20 hours with shaking at 200 rpm to obtain the inoculum. Subsequently, 1 ml of the inoculum 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 with 200 rpm. After the culture was completed, 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.

[0222] Strain Glycine Concentration (g / l) Increase from Control (%) 13032-Null 4.48 - 13032-glyA (WT) (Control) 5.52 - 13032-ltaE WT 6.52 18.12% 13032-TA(Nae) (WT) 7.12 28.99% 13032-TA(Bom) (WT) 7.33 32.79%

[0223] As shown in Table 2 above, the Corynebacterium glutamicum ATCC13032 pCES208 strain (13032-Null) to which nothing was introduced had low glycine production capacity.

[0224] A strain of Corynebacterium glutamicum into which a wild-type ltaE gene derived from Escherichia coli, a gene encoding low-specificity threonine aldolase derived from Neomicrococcus aestuarii, or a gene encoding low-specificity threonine aldolase derived from Bombella sp. ESL0368 was introduced showed an increase in glycine production capacity of about 18 to 32% compared to a control strain (Corynebacterium glutamicum into which the glyA gene, an endogenous gene of Corynebacterium glutamicum, was introduced (13032-glyA (WT))).

[0225]

[0226] Example 2. Evaluation of Alanine and Valine Production via Introduction of Low-Specific Threonine Aldolase Gene

[0227] The alanine and valine productivity of the Corynebacterium glutamicum transformants obtained in Examples 1-2 above was measured by the following method.

[0228] Specifically, in a 250 ml corner-barfle flask containing 25 ml of seed medium, the parent strain Corynebacterium glutamicum ATCC13032 pCES208 (13032-Null, Tables 3, 4), Corynebacterium glutamicum ATCC13032 (13032-glyA (WT)) into which a wild-type glyA gene derived from Corynebacterium glutamicum ATCC13032 was introduced, Corynebacterium glutamicum ATCC13032 (13032-TA(Nae) (WT)) into which a gene encoding a low-specificity threonine aldolase derived from Neomicrococcus aestuarii was introduced, and a low-specificity strain derived from the genus Bombella ESL0368 (Bombella sp. ESL0368) Corynebacterium glutamicum ATCC13032 (13032-TA(Bom) (WT)) into which the gene encoding threonine aldolase was introduced was inoculated into each strain, and the strain was cultured at 30°C for 20 hours with shaking at 200 rpm to obtain the inoculum. Then, 1 ml of the inoculum 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 with 200 rpm. After the culture was finished, the concentrations of alanine and valine contained in the culture medium were measured using high-performance liquid chromatography (HPLC) to determine the alanine and valine production of each strain. The results obtained are shown in Tables 3 and 4 below, respectively.

[0229] Strain Alanine Concentration (g / l) Increase relative to control (%) 13032-Null 0.466 - 13032-glyA (WT) (Control) 0.472 - 13032-ltaE WT 0.7636 1.65% 13032-TA(Nae) (WT) 0.7836 5.89% 13032-TA(Bom) (WT) 0.6403 5.59%

[0230] Strain Vaulin Concentration (g / l) Increase relative to control (%) 13032-Null 0.01 13032-glyA (WT) (Control) 0.01 13032-ltaE WT 0.06 500.00% 13032-TA(Nae) (WT) 0.2 00 1800.00% 13032-TA(Bom) (WT) 0.07 0600.36%

[0231] As shown in Tables 3 and 4 above, the Corynebacterium glutamicum ATCC13032 pCES208 strain (13032-Null) to which nothing was introduced had low alanine and valine production capacity.

[0232] A Corynebacterium glutamicum strain into which a wild-type ltaE gene derived from Escherichia coli, a gene encoding low-specificity threonine aldolase derived from Neomicrococcus aestuarii, or a gene encoding low-specificity threonine aldolase derived from Bombella sp. ESL0368 was introduced showed significantly increased alanine and valine production capacity compared to a control strain (Corynebacterium glutamicum into which the glyA gene, an endogenous gene of Corynebacterium glutamicum, was introduced (13032-glyA (WT))).

[0233]

[0234] Example 3. Evaluation of Glycine, Alanine, and Valine Production via Introduction of Low-Specific Threonine Aldolase and Aldehyde Dehydrogenase Genes

[0235] Example 3-1. Preparation of a strain with enhanced threonine production pathway

[0236] Example 3-1-1. Plasmid preparation for introduction of Hom(G378E, R398Q) and lysC(L377K) variants

[0237] A mutation (G378E, R398Q) for unlocking feedback inhibition of homoserine dehydrogenase for threonine was introduced into Corynebacterium glutamicum ATCC13032 (Hom(G378E, R398Q), amino acid sequence: SEQ NO. 18, gene sequence: SEQ NO. 19), and a mutation (L377K) for unlocking feedback inhibition of lysine-sensitive aspartokinase 3 for lysine and threonine was introduced (lysC(L377K), amino acid sequence: SEQ NO. 20; gene sequence: SEQ NO. 21) (see U.S. Patent Publication US 2023-0012923 A1).

[0238] A plasmid for the introduction of the Hom(G378E, R398Q) mutation was constructed. Q5 polymerase PCR reactions were performed using Corynebacterium glutamicum ATCC13032 genomic DNA (GenBank: CP025533.1) as a template and each primer pair (Hom*_Up_Fwd and Hom*_Up_Rev, Hom*_Mut_Fwd and Hom*_Mut_Rev, Hom*_Down_Fwd and Hom*_Down_Rev) (Table 5). 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 ℃ for 30 seconds for 1 cycle; 98 ℃ for 10 seconds, 55 ℃ for 30 seconds, 72 ℃ for 15 seconds, 30 cycles; and 72 ℃ for 2 minutes for 1 cycle. Using the three PCR DNA fragments obtained therefrom and the pDC24 plasmid (SEQ No. 55) linearized by restriction enzyme SmaI reaction, a Gibson assembly reaction was performed, and pDC24 hom (G378E, R398Q) was obtained.

[0239] pDC24 lysC (L377K) was constructed in the manner described above. Q5 polymerase PCR was performed using Corynebacterium glutamicum ATCC13032 genomic DNA as a template and each primer pair (lysC*_Up_Fwd and lysC*_Up_Rev, lysC*_Down_Fwd and lysC*_Down_Rev) (Table 5). Gibson assembly was performed using the two PCR DNA fragments obtained therefrom and the pDC24 plasmid linearized by restriction enzyme SmaI reaction, and pDC24 lysC (L377K) was obtained.

[0240]

[0241] Example 3-1-2. Preparation of strains introduced with Hom(G378E, R398Q) and LysC(L377K) variants

[0242] Transformation of wild-type Corynebacterium glutamicum ATCC13032 was performed using the plasmid constructed in Example 3-1-1 and electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541-545). Using pDC24 lysC (L377K) introduced for transformation as a template, homologous recombination (Eggeling, L., & Bott, M. (Eds.). (2005). Handbook of Corynebacterium glutamicum (1st ed.). CRC Press. https: / doi.org / 10.1201 / 9781420039696) was performed to induce intracellular genomic mutations and to possess the kanamycin resistance gene and Levansucrase (SacB), and primary screening was conducted in a medium containing kanamycin. The SacB counter selection method was used to remove the introduced kanamycin marker. Secondary screening was performed on colonies screened in the first screening on high-concentration raw sugar medium, and finally, a strain with the lysC(L377K) mutation was obtained and named CJ-2378.

[0243] Using CJ-2378 as the base strain, a strain with the Hom(G378E, R398Q) mutation was produced by utilizing pDC24 hom(G378E, R398Q) in the same way, and the obtained strain was named CJ-2385.

[0244]

[0245] Example 3-1-3. Evaluation of Threonine Production Capacity of Strains Introduced with Hom(G378E, R3998Q) and lysC(L377K) Variants

[0246] The threonine production ability of the two transgenic strains CJ-2378 and CJ-2385 obtained in Example 3-1-2 above was evaluated as follows.

[0247] Specifically, the parent strain Corynebacterium glutamicum ATCC13032 and the two transformed strains were inoculated into a 250 ml Corner-Baffle flask containing 25 ml of seed medium, respectively, and then cultured at 30 ℃ for 20 hours with shaking at 200 rpm 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 30 ℃ for 48 hours with 200 rpm. After the culture was completed, the threonine production of each strain was determined by measuring the concentration of threonine contained in the culture medium using high-performance liquid chromatography (HPLC). The results obtained are shown in Table 6 below. Quantitative results confirmed that the threonine concentration increased 1.9-fold (approx. 89% increase) and 2.3-fold (approx. 133% increase) compared to the control group.

[0248] Strain OD600 (48h) L-Threonine (g / L) (48h) Increase compared to control (%) Control (Corynebacterium glutamicum ATCC13032) 11 1.1 0.0 9 -CJ-2378 10 1.4 0.1 78 8.8 9%CJ-2385 10 0.7 0.2 11 33.3 3%

[0249] <Batch>

[0250] 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, CAPA 9 mg / L, NCA 60 mg / L, Magnesium sulfate (MgSO4) 0.5 g / L

[0251] Production Medium

[0252] Calcium carbonate (CaCO3) 30 g / L, Sucrose 50 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

[0253]

[0254] Example 3-2. Construction of a vector for the introduction of the aldehyde dehydrogenase gene

[0255] We intended to introduce a plasmid containing a low-specificity threonine aldolase gene and an aldehyde dehydrogenase gene using the threonine-producing strain obtained in Example 3-1 above as a host.

[0256] First, pCES208_ltaE was constructed by introducing a gene encoding wild-type low-specificity 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 into the pCES208 vector (Korean Registered Patent Publication KR 10-1673080 B1, J.Microbiol. Biotechnol., 18:639-647, 2008) in the same manner as described in Example 1-1.

[0257] In addition, aldehyde dehydrogenase genes include mhpF (acetaldehyde dehydrogenase, WP_000044314.1; protein sequence: SEQ ID 32, gene sequence: SEQ ID 33) derived from Escherichia coli, eutE (aldehyde dehydrogenase family protein, WP_001075716.1; protein sequence: SEQ ID 34, gene sequence: SEQ ID 35) derived from Escherichia coli, ALDH1 (aldehyde dehydrogenase, NP_015264.1; protein sequence: SEQ ID 36, gene sequence: SEQ ID 37) derived from Saccharomyces cerevisiae, and AldB (NADP-dependent aldehyde dehydrogenase, WP_011241495.1; protein sequence: SEQ ID 38, gene sequence: SEQ ID NO. 39), or ald (aldehyde dehydrogenase family protein, WP_011015386.1; protein sequence: SEQ ID NO. 40, gene sequence: SEQ ID NO. 41) derived from Corynebacterium glutamicum was introduced.

[0258] The mhpF gene (sequence number 33) and eutE gene (sequence number 35) were amplified by PCR using primer pairs (mhpF_Fwd, mhpF_Rev, eutE_Fwd, eutE_Rev) with Escherichia coli W3110 genomic DNA (ATCC27325) as a template (Table 7).

[0259] The ALDH1 gene (sequence number 37) was amplified by PCR using primer pairs (ALDH1_Fwd, ALDH1_Rev) with the nucleic acid sequence (bionics) of the synthesized sequence number 37 as a template (Table 7).

[0260] The AldB gene (sequence number 39) was amplified by PCR using primer pairs (aldB_Fwd, aldB_Rev) with the nucleic acid sequence (bionics) of the synthesized sequence number 39 as a template (Table 7).

[0261] The Ald gene (sequence number 41) was amplified by PCR using primer pairs (ald_Fwd, ald_Rev) with Corynebacterium glutamicum ATCC13032 genomic DNA (GenBank: CP025533.1) as a template (Table 7).

[0262] The sequence of the PgapA promoter (sequence number 42) was amplified by PCR using primer pairs (PgapA_Fwd, PgapA_Rev) with Corynebacterium glutamicum ATCC13032 genomic DNA (GenBank: CP025533.1) as a template (Table 7).

[0263] Five types of vectors (pCES208 Pcj7_ltaE-PgapA_mhpF, pCES208 Pcj7_ltaE-PgapA_eutE, pCES208 Pcj7_ltaE-PgapA_ALDH1, pCES208 Pcj7_ltaE-PgapA_aldB, pCES208 Pcj7_ltaE-PgapA_ald) were obtained by combinatorially performing a Gibson assembly reaction with a vector linearized by treating pCES208 with restriction enzyme (EcoRV) and DNA fragments of Pcj7 DNA, ltaE DNA, PgapA DNA, and five types of aldehyde dehydrogenase genes.

[0264]

[0265] Example 3-3. Evaluation of Glycine, Alanine, and Valine Production in Corynebacterium glutamicum Transformers Introduced with Low-Specific Threonine Aldolase Gene and Aldehyde Dehydrogenase Gene

[0266] CJ-2385 was transformed by electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541-545) using the pCES208 Pcj7_ltaE, pCES208 Pcj7_ltaE-PgapA_mhpF, pCES208 Pcj7_ltaE-PgapA_eutE, pCES208 Pcj7_ltaE-PgapA_ALDH1, pCES208 Pcj7_ltaE-PgapA_aldB, and pCES208 Pcj7_ltaE-PgapA_ald vectors produced in Example 3-2 and the control vector (pCES208). As a result, seven types of transformed strains could be produced.

[0267] The glycine, alanine, and valine production capabilities of the seven strains produced were evaluated as follows. Specifically, after inoculating the parental strains Corynebacterium glutamicum—control (CJ-2385 pCES208), CJ-2385 pCES208 Pcj7_ltaE, CJ-2385 pCES208 Pcj7_ltaE-PgapA_mhpF, CJ-2385 pCES208 Pcj7_ltaE-PgapA_eutE, CJ-2385 pCES208 Pcj7_ltaE-PgapA_ALDH1, CJ-2385 pCES208 Pcj7_ltaE-PgapA_aldB, and CJ-2385 pCES208 Pcj7_ltaE-PgapA_ald—into a 250 ml Corner-Baffle flask containing 25 ml of inoculum medium, at 200 rpm for 20 hours at 30°C A seed culture was obtained by shaking culture. Then, 1 ml of the seed culture was inoculated into a 250 ml Corner-Baffle flask containing 24 ml of the above production medium, and cultured at 200 rpm for 48 hours at 30°C. After the culture was finished, the concentrations of glycine, alanine, and valine contained in the culture medium were measured using high-performance liquid chromatography (HPLC). The results obtained are shown in Tables 8 to 10 below.

[0268] <Batch>

[0269] 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, CAPA 9 mg / L, NCA 60 mg / L, Magnesium sulfate (MgSO4) 0.5 g / L

[0270] Production Medium

[0271] Calcium carbonate (CaCO3) 30 g / L, Sucrose 50 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

[0272]

[0273] Strain OD600 (48h) L-Threonine (g / L) (48h) Glycine (g / L) (48h) Increase compared to control (%) Control (CJ-2385 pCES208) 104.6 0.2 00.1 2-CJ-2385 pCES208 Pcj7_ltaE 103.5 0.0 1.8 31 42 5.0 0%CJ-2385 pCES208 Pcj7_ltaE-PgapA_mhpF 103.8 0.0 6 2.2 41 76 6.6 7%CJ-2385 pCES208 Pcj7_ltaE-PgapA_eutE 106.9 0.0 7 2.4 21 91 6.6 7%CJ-2385 pCES208 Pcj7_ltaE-PgapA_ALDH1103.70.062.311825.00%CJ-2385 pCES208 Pcj7_ltaE-PgapA_aldB102.50.062.471958.33%CJ-2385 pCES208 Pcj7_ltaE-PgapA_ald105.20.082.21733.33%

[0274] Strain OD600 (48h) L-Threonine (g / L) (48h) L-Alanine (g / L) (48h) Increase compared to control (%) Control (CJ-2385 pCES208) 104.6 0.2 0.1 2 -CJ-2385 pCES208 Pcj7_ltaE 103.5 0.0 0.1 74 1.6 7% CJ-2385 pCES208 Pcj7_ltaE -PgapA_mhpF 103.8 0.0 6 0.1 95 8.3 3% CJ-2385 pCES208 Pcj7_ltaE -PgapA_eutE 106.9 0.0 7 0.2 41 0.0 0% CJ-2385 pCES208 Pcj7_ltaE-PgapA_ALDH1103.70.060.38216.67%CJ-2385 pCES208 Pcj7_ltaE-PgapA_aldB102.50.060.35191.67%CJ-2385 pCES208 Pcj7_ltaE-PgapA_ald105.20.080.34183.33%

[0275] Strain OD600 (48h) L-Threonine (g / L) (48h) L-Valine (g / L) (48h) Increase compared to control (%) Control (CJ-2385 pCES208) 104.6 0.2 1.8 8 -CJ-2385 pCES208 Pcj7_ltaE 103.5 0.0 3.0 26 0.6 4%CJ-2385 pCES208 Pcj7_ltaE-PgapA_mhpF 103.8 0.0 6 3.4 88 5.1 1%CJ-2385 pCES208 Pcj7_ltaE-PgapA_eutE 106.9 0.0 7 3.7 197.3 4%CJ-2385 pCES208 Pcj7_ltaE-PgapA_ALDH1103.70.064.08117.02%CJ-2385 pCES208 Pcj7_ltaE-PgapA_aldB102.50.064.01113.30%CJ-2385 pCES208 Pcj7_ltaE-PgapA_ald105.20.083.81102.66%

[0276] As shown in Tables 8 to 10 above, microorganisms introduced with wild-type ltaE derived from E. coli showed an increase in glycine production capacity of approximately 1425%, alanine production capacity of approximately 42%, and valine production capacity of approximately 61% compared to the control group. In addition, it was confirmed that microorganisms introduced with a low-specificity wild-type threonine aldolase gene derived from E. coli and a gene encoding aldehyde dehydrogenase showed a significant increase in glycine, alanine, and / or valine production capacity compared to the control group. These results indicate that the introduction of the threonine aldolase gene derived from E. coli increased the glycine, alanine, and / or valine production capacity of the microorganisms, and that the co-introduction of the gene encoding aldehyde dehydrogenase further increased the glycine, alanine, and / or valine production capacity of the microorganisms.

[0277]

[0278] 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. Microorganisms of the genus Corynebacterium that produce one or more amino acids selected from the group consisting of glycine, alanine, and valine, with enhanced activity of low-specificity threonine aldolase.

2. In claim 1, the low-specificity threonine aldolase is, (a) Sequence No. 1 or an amino acid sequence having 90% or more sequence identity with respect to it; (b) SEQ ID NO. 12 or an amino acid sequence having 90% or more sequence identity with respect to it; or (c) A microorganism of the genus Corynebacterium comprising SEQ ID NO. 14 or an amino acid sequence having 90% or more sequence identity with respect to it.

3. In paragraph 1, the microorganism of the genus Corynebacterium is, (a) low-specificity threonine aldolase derived from E. coli or a polynucleotide encoding the same; (b) low-specificity threonine aldolase derived from Neomicrococcus aestuarii or a polynucleotide encoding the same; or (c) A microorganism of the genus Corynebacterium into which a low-specificity threonine aldolase derived from the ESL0368 strain of the genus Bombella (Bombella sp. ESL0368) or a polynucleotide encoding it has been introduced.

4. In paragraph 1, the Corynebacterium microorganism is a Corynebacterium microorganism in which the activity of aldehyde dehydrogenase is enhanced.

5. In paragraph 4, the aldehyde dehydrogenase is, (a) Sequence No. 32 or an amino acid sequence having 90% or more sequence identity with respect to it; (b) Sequence No. 34 or an amino acid sequence having 90% or more sequence identity with respect to it; (c) Sequence No. 36 or an amino acid sequence having 90% or more sequence identity with respect to it; (d) an amino acid sequence having at least 90% sequence identity with SEQ ID NO. 38; and (e) A microorganism of the genus Corynebacterium comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO. 40 or amino acid sequences having 90% or more sequence identity with respect to it.

6. In paragraph 4, the microorganism of the genus Corynebacterium is, (a) Acetaldehyde dehydrogenase derived from E. coli or a polynucleotide encoding the same; (b) an aldehyde dehydrogenase derived from E. coli or a polynucleotide encoding the same; (c) an aldehyde dehydrogenase derived from Saccharomyces cerevisiae or a polynucleotide encoding the same; (d) an aldehyde dehydrogenase derived from Zymomonas mobilis or a polynucleotide encoding the same; and (e) A microorganism of the genus Corynebacterium into which one or more selected from the group consisting of an aldehyde dehydrogenase derived from Corynebacterium glutamicum or a polynucleotide encoding the same have been introduced.

7. In paragraph 1, the microorganism of the genus Corynebacterium is Corynebacterium glutamicum.

8. In any one of claims 1 to 7, the Corynebacterium microorganism is a Corynebacterium microorganism having an increased production capacity of one or more amino acids selected from the group consisting of glycine, alanine, and valine compared to a parent strain in which the activity of low-specificity threonine aldolase is not enhanced.

9. 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 the genus Corynebacterium according to any one of claims 1 to 7 in a culture medium.

10. A method for producing amino acids according to claim 9, further comprising the step of recovering the amino acid from a culture medium or microorganism according to the above culture.

11. A composition for producing one or more amino acids selected from the group consisting of glycine, alanine, and valine, comprising a microorganism of the genus Corynebacterium according to any one of claims 1 to 7.

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