Novel type ii citrate synthase variant and method for producing aspartate-derived l-amino acids by using same

Abstract

The present disclosure relates to a type II citrate synthase variant, a Corynebacterium glutamicum strain comprising the type II citrate synthase variant, and a method for producing aspartate-derived L-amino acids by using the strain.

Description

Novel Type II Citrate Synthase Variant and Method for Producing Aspartate-Derived L-Amino Acid Using the Same

[0001] Cross-citation with related application(s)

[0002] The present disclosure claims the benefit of priority based on Korean Patent Application No. 10-2024-0193105 filed on December 20, 2024, and all contents disclosed in the document of said Korean patent application are incorporated as part of the present disclosure.

[0003] Throughout this disclosure, numerous papers and patent documents are referenced and cited. The disclosures of the cited papers and patent documents are incorporated by reference into this disclosure in their entirety to more clearly explain the state of the art to which the present invention pertains and the content of the present invention.

[0004] The present disclosure relates to a novel type II citrate synthase variant, a Corynebacterium glutamicum strain comprising the type II citrate synthase variant, and a method for producing L-amino acids using the strain.

[0005] Various studies are being conducted to develop high-efficiency production microorganisms and fermentation process technologies for the production of L-amino acids and other useful substances. For example, target substance-specific approaches, such as increasing the expression of genes encoding enzymes involved in L-lysine biosynthesis or removing genes unnecessary for biosynthesis, are mainly used (WO2008-082181 A1).

[0006] However, due to the increasing demand for L-amino acids, research is still needed to effectively increase the production capacity of L-amino acids.

[0007] One objective of the present disclosure is to provide a type II citrate synthase variant.

[0008] Another object of the present disclosure is to provide a polynucleotide encoding the type II citrate synthase variant.

[0009] Another object of the present disclosure is to provide a microorganism of the genus Corynebacterium comprising one or more selected from the group consisting of the type II citrate synthase variant and the polynucleotide encoding it.

[0010] Another object of the present disclosure is to provide a composition for producing aspartate-derived L-amino acids comprising the microorganism of the genus of Corynebacterium.

[0011] Another object of the present disclosure is to provide a method for producing aspartate-derived L-amino acid, comprising the step of culturing the microorganism of the genus of Corynebacterium in a medium.

[0012] Another object of the present disclosure is to provide a use for the production of L-amino acids using microorganisms of the genus of Corynebacterium.

[0013] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in this application may be applied to other descriptions and embodiments. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the scope of this application should not be considered limited by the specific descriptions provided below. Additionally, numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated by reference into this specification in their entirety to more clearly explain the state of the art to which the present invention pertains and the content of the present invention.

[0014]

[0015] One aspect of the present disclosure provides a type II citrate synthase variant in which the amino acid corresponding to the 272nd position from the N-terminus in the amino acid sequence of SEQ ID NO. 1, the amino acid corresponding to the 310th position from the N-terminus, or a combination thereof is substituted with an amino acid different from the original.

[0016] In the present disclosure, the term “type II citrate synthase” refers to an enzyme of the first step of the citric acid cycle that synthesizes citric acid by condensing acetyl-CoA derived from glycolysis or other catabolic reactions with oxaloacetate. Specifically, the type II citrate synthase of the present disclosure may be used interchangeably with type II citrate synthase, GltA protein, or GltA. In the present disclosure, the sequence of the type II citrate synthase can be obtained from GenBank of the NCBI, a known database, and may be, for example, GenBank Accession No. WP_011013914.1. Specifically, it may be, but is not limited to, a polypeptide having type II citrate synthase activity encoded by the NCgl0795 gene (or the gltA gene). In one embodiment, the type II citrate synthase may include the amino acid sequence of SEQ ID NO. 1 or be composed of the amino acid sequence of SEQ ID NO. 1, and a polypeptide having an amino acid sequence in which some sequences of the amino acid sequence of SEQ ID NO. 1 are deleted, modified, substituted, or added may also be included within the scope of the present disclosure if it has the same or corresponding conversion properties as the type II citrate synthase. In addition, regardless of the origin of the microorganism, polypeptides having at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity or homology with the amino acid sequence of SEQ ID NO. 1, and polypeptides having conversion activity identical or corresponding to that of type II citrate synthase, may also be included within the scope of the present disclosure as type II citrate synthase.

[0017] In one embodiment, the type II citrate synthase variant of the present disclosure may have the activity of type II citrate synthase.

[0018] In one embodiment, the type II citrate synthase of the present disclosure may have weakened type II citrate synthase activity.

[0019] In the present disclosure, the sequence of SEQ ID NO. 1 is as follows: MFERDIVATDNNKAVLHYPGGEFEMDIIEASEGNNGVVLGKMLSETGLITFDPGYVSTGSTESKITYIDGDAGILRYRGYDIADLAENATFNEVSYLLINGELPTPDELHKFNDEIRHHTLLDEDFKSQFNVFPRDAHPMATLASSVNILSTYYQDQLNPLDEAQLDKATVRLMAKVPMLAAYAHRARKGAPYMYPDNSLNARENFLRMMFGYPTEPY EIDPIMVKALDKLLILHADHEQNCSTSTVRMIGSAQANMFVSIAGGINALSGPLHGGANQAVLEMLEDIKSNHGGDATEFMNKVKNKEDGVRLMGFGHRVYKNYDPRAA IVKETAHEILEHLGGDDLLDLAIKLEEIALADDYFISRKLYPNVDFYTGLIYRAMGFPTDFFTVLFAIGRLPGWIAHYREQLGAAGNKINRPRQVYTGNESRKLVPREER (SEQ ID NO: 1).

[0020] In one embodiment, the type II citrate synthase variant of the present disclosure described above may comprise an amino acid sequence in which an amino acid corresponding to the 272nd position from the N-terminus in the amino acid sequence of SEQ ID NO. 1, an amino acid corresponding to the 310th position from the N-terminus, or a combination thereof is substituted with an amino acid different from the original.

[0021] In one embodiment, the type II citrate synthase variant of the present disclosure described above may essentially consist of an amino acid corresponding to the 272nd position from the N-terminus in the amino acid sequence of SEQ ID NO. 1, an amino acid corresponding to the 310th position from the N-terminus, or a combination thereof substituted with an amino acid different from the original.

[0022] In one embodiment, the type II citrate synthase variant of the present disclosure described above may consist of an amino acid corresponding to the 272nd position from the N-terminus in the amino acid sequence of SEQ ID NO. 1, an amino acid corresponding to the 310th position from the N-terminus, or a combination thereof substituted with an amino acid different from the original.

[0023] In one embodiment, the amino acid corresponding to the 272nd position from the N-terminus in the amino acid sequence of SEQ ID NO. 1 may be leucine (Leu, L), but is not limited thereto.

[0024] In one embodiment, the amino acid corresponding to the 310th position from the N-terminus in the amino acid sequence of SEQ ID NO. 1 may be arginine (Arg, R), but is not limited thereto.

[0025] In one embodiment, the type II citrate synthase variant of the present disclosure described above is such that the amino acid corresponding to the 272nd position from the N-terminus (e.g., leucine (Leu, L)) is substituted with glutamic acid (E), glycine (G), alanine (A), serine (S), threonine (T), cysteine ​​(C), valine (V), isoleucine (I), methionine (M), proline (P), phenylalanine (F), tyrosine (Y), tryptophan (W), aspartic acid (D), glutamine (Q), histidine (H), lysine (K), arginine (R), or asparagine (N) (e.g., arginine (R));

[0026] The amino acid corresponding to the 310th position from the N-terminus (e.g., arginine (Arg, R)) is substituted with glutamic acid (E), glycine (G), alanine (A), serine (S), threonine (T), cysteine ​​(C), valine (V), leucine (L), isoleucine (I), methionine (M), proline (P), phenylalanine (F), tyrosine (Y), tryptophan (W), aspartic acid (D), glutamine (Q), histidine (H), lysine (K), or asparagine (N) (e.g., tyrosine (Y)); or

[0027] The amino acid corresponding to the 272nd position from the N-terminus (e.g., leucine (Leu, L)) is substituted with glutamic acid (E), glycine (G), alanine (A), serine (S), threonine (T), cysteine ​​(C), valine (V), isoleucine (I), methionine (M), proline (P), phenylalanine (F), tyrosine (Y), tryptophan (W), aspartic acid (D), glutamine (Q), histidine (H), lysine (K), arginine (R), or asparagine (N) (e.g., arginine (R)), and the amino acid corresponding to the 310th position from the N-terminus (e.g., arginine (Arg, R)) is substituted with glutamic acid (E), glycine (G), alanine (A), serine (S), threonine (T), cysteine ​​(C), valine (V), leucine (L), isoleucine (I), It may be substituted with methionine (M), proline (P), phenylalanine (F), tyrosine (Y), tryptophan (W), aspartic acid (D), glutamine (Q), histidine (H), lysine (K), or asparagine (N) (e.g., tyrosine (Y)), but is not limited thereto.

[0028] In one embodiment, the amino acid corresponding to the 272nd position from the N-terminus in the amino acid sequence of SEQ ID NO. 1 may be substituted with arginine (Arg, R), but is not limited thereto.

[0029] In one embodiment, the amino acid corresponding to the 310th position from the N-terminus in the amino acid sequence of SEQ ID NO. 1 may be substituted with tyrosine (Tyr, Y), but is not limited thereto.

[0030] In one embodiment, the type II citrate synthase variant may additionally be an amino acid corresponding to the 169th position from the N-terminus in the amino acid sequence of SEQ ID NO. 1, an amino acid corresponding to the 241st position from the N-terminus, or a combination thereof, substituted with an amino acid different from the original.

[0031] In one embodiment, the amino acid corresponding to the 169th position from the N-terminus in the amino acid sequence of SEQ ID NO. 1 may be alanine (Ala, A), but is not limited thereto.

[0032] In one embodiment, the amino acid corresponding to the 241st position from the N-terminus in the amino acid sequence of SEQ ID NO. 1 may be asparagine (Asn, N), but is not limited thereto.

[0033] In one embodiment, the amino acid corresponding to the 169th position from the N-terminus may be substituted with glutamic acid (E), glycine (G), serine (S), threonine (T), cysteine ​​(C), valine (V), leucine (L), isoleucine (I), methionine (M), proline (P), phenylalanine (F), tyrosine (Y), tryptophan (W), aspartic acid (D), glutamine (Q), histidine (H), lysine (K), arginine (R), or asparagine (N), and more specifically, may be substituted with valine (Val, V), but is not limited thereto.

[0034] In one embodiment, the amino acid corresponding to the 241st position from the N-terminus may be substituted with glutamic acid (E), glycine (G), serine (S), threonine (T), cysteine ​​(C), valine (V), leucine (L), isoleucine (I), methionine (M), proline (P), phenylalanine (F), tyrosine (Y), tryptophan (W), aspartic acid (D), glutamine (Q), histidine (H), lysine (K), arginine (R), or alanine (A), and more specifically may be substituted with threonine (Thr, t), but is not limited thereto.

[0035] In the type II citrate synthase variant according to one example of the present application, the amino acid corresponding to the 238th position from the N-terminus in the amino acid sequence of SEQ ID NO. 1 may be an amino acid other than isoleucine (I, Ile), and more specifically may be arginine (R), histidine (H), lysine (K), aspartic acid (D), glutamic acid (E), serine (S), threonine (T), asparagine (N), glutamine (Q), cysteine ​​(C), glycine (G), proline (P), alanine (A), valine (V), leucine (L), methionine (M), phenylalanine (F), tyrosine (Y), or tryptophan (W), and may be, for example, histidine (His, H), but is not limited thereto.

[0036] In the type II citrate synthase variant according to one example of the present application, the amino acid corresponding to the 393rd position from the N-terminus in the amino acid sequence of SEQ ID NO. 1 may be an amino acid other than tyrosine (Tyr, Y), and more specifically may be arginine (R), histidine (H), lysine (K), aspartic acid (D), glutamic acid (E), serine (S), threonine (T), asparagine (N), glutamine (Q), cysteine ​​(C), glycine (G), proline (P), alanine (A), valine (V), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), or tryptophan (W), for example, phenylalanine (F), but is not limited thereto.

[0037] The type II citrate synthase variant of the present disclosure may comprise an amino acid sequence in which an amino acid corresponding to the 272nd position from the N-terminus in the amino acid sequence of SEQ ID NO. 1, an amino acid corresponding to the 310th position from the N-terminus, or a combination thereof is substituted with an amino acid different from the original; or additionally, an amino acid sequence in which an amino acid corresponding to the 169th position from the N-terminus in the amino acid sequence of SEQ ID NO. 1, an amino acid corresponding to the 241st position from the N-terminus, or a combination thereof is substituted with an amino acid different from the original, or may comprise an amino acid sequence having at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.7% or more homology or identity with said amino acid sequence. Furthermore, it is obvious that variants having amino acid sequences in which some sequences are deleted, modified, substituted, conservatively substituted, or added are included within the scope of the present disclosure, provided that such an amino acid sequence has such homology or identity and exhibits an efficacy corresponding to that of the variants of the present disclosure (efficacy of increasing the production of aspartate-derived L-amino acids (e.g., L-lysine)).

[0038] For example, cases where there are sequence additions or deletions, naturally occurring mutations, silent mutations, or conservative substitutions that do not alter the function of the variant of the present disclosure at the N-terminus, C-terminus, and / or within the amino acid sequence.

[0039] In one embodiment, the type II citrate synthase variant of the present disclosure described above may have at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.7% or more of the amino acid sequence of SEQ ID NO. 1; and less than 100% homology or identity, but is not limited thereto.

[0040] In one embodiment, the type II citrate synthase variant of the present disclosure described above may have at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.7% or more of the amino acid sequence of SEQ ID NO. 1; and less than 100% homology or identity, but is not limited thereto. Furthermore, if an amino acid sequence includes the amino acid substitution described above and possesses the homology or identity described above, and exhibits an efficacy corresponding to that of the variant of the present application (efficacy of increasing the production of aspartate-derived L-amino acid (e.g., L-lysine)), it is obvious that variants having amino acid sequences in which some sequences are deleted, modified, substituted, conservatively substituted, or added are also included within the scope of the present disclosure.

[0041] The type II citrate synthase variant of the present disclosure described above may be an amino acid corresponding to the 272nd position from the N-terminus in the amino acid sequence of SEQ ID NO. 1, an amino acid corresponding to the 310th position from the N-terminus, or a combination thereof, substituted with an amino acid different from the original, and may exhibit the activity of type II citrate synthase having at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.7% or more and less than 100% homology or identity with the amino acid sequence of SEQ ID NO. 1, but is not limited thereto.

[0042] In one embodiment, the type II citrate synthase variant of the present disclosure described above may have at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.7% or more of the amino acid sequence of SEQ ID NO. 1, and less than 100% of homology or identity with the amino acid sequence of SEQ ID NO. 1, but is not limited thereto. Furthermore, if an amino acid sequence includes the amino acid substitution described above and possesses the homology or identity described above, and exhibits an efficacy corresponding to that of the variant of the present application (efficacy of increasing the production of aspartate-derived L-amino acid (e.g., L-lysine)), it is obvious that variants having amino acid sequences in which some sequences are deleted, modified, substituted, conservatively substituted, or added are also included within the scope of the present disclosure.

[0043] The type II citrate synthase variant of the present disclosure described above may be a variant in which the amino acid corresponding to the 272nd position from the N-terminus, the amino acid corresponding to the 310th position from the N-terminus, or a combination thereof in the amino acid sequence of SEQ ID NO. 1 is substituted with an amino acid different from the original, and additionally, the amino acid corresponding to the 169th position from the N-terminus, the amino acid corresponding to the 241st position from the N-terminus, or a combination thereof in the amino acid sequence of SEQ ID NO. 1 is substituted with an amino acid different from the original, and may exhibit type II citrate synthase activity having at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.7% or more and less than 100% homology or identity with the amino acid sequence of SEQ ID NO. 1, but, It is not limited to this.

[0044] In one embodiment, the type II citrate synthase variant of the present disclosure described above may comprise the amino acid sequence of SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 9, or SEQ ID NO. 10. In another embodiment, the type II citrate synthase variant may essentially consist of the amino acid sequence of SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 9, or SEQ ID NO. 10. In yet another embodiment, the type II citrate synthase variant may consist of the amino acid sequence of SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 9, or SEQ ID NO. 10.

[0045] The type II citrate synthase variants of the present disclosure may comprise the amino acid sequence of SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 9, or SEQ ID NO. 10, or may comprise an amino acid sequence having at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% or more homology or identity with said amino acid sequence. Furthermore, it is obvious that variants having amino acid sequences in which some sequences are deleted, modified, substituted, conservatively substituted, or added are also included within the scope of the present disclosure, provided that such amino acid sequences have such homology or identity and exhibit an efficacy corresponding to the variants of the present disclosure (efficacy of increasing the production of aspartate-derived L-amino acids (e.g., L-lysine).

[0046] For example, cases where there are sequence additions or deletions, naturally occurring mutations, silent mutations, or conservative substitutions that do not alter the function of the variant of the present disclosure at the N-terminus, C-terminus, and / or within the amino acid sequence.

[0047] In one embodiment, the type II citrate synthase variant of the present disclosure comprises the amino acid sequence of SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 9, or SEQ ID NO. 10, and may have at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.7% or more of the amino acid sequence of SEQ ID NO. 1; and less than 100% homology or identity, but is not limited thereto. Furthermore, it is obvious that variants having amino acid sequences in which some sequences are deleted, modified, substituted, conservatively substituted, or added are included within the scope of the present disclosure, provided that such an amino acid sequence has such homology or identity and exhibits an efficacy corresponding to that of the variants of the present disclosure (efficacy of increasing the production of aspartate-derived L-amino acids (e.g., L-lysine)).

[0048] The type II citrate synthase variant of the present disclosure described above comprises the amino acid sequence of SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 9, or SEQ ID NO. 10, and has at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.7% or more of the amino acid sequence of SEQ ID NO. 1; and has homology or identity of less than 100%, and may exhibit the activity of a type II citrate synthase, but is not limited thereto.

[0049]

[0050] 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.

[0051] In this disclosure, the term “variant” refers to a polypeptide in which one or more amino acids are conservatively substituted and / or modified, resulting in a sequence of amino acids different from that of the variant prior to modification, while retaining functions or properties. Such variants can generally be identified by modifying one or more amino acids in the amino acid sequence of the polypeptide and evaluating the properties of the modified polypeptide. That is, the capabilities of the variant may be increased, unchanged, or decreased compared to the polypeptide prior to modification. Additionally, some variants may include variants in which one or more parts, such as an N-terminal leader sequence or a transmembrane domain, have been removed. Other variants may include variants in which a portion of the N- and / or C-terminus of a mature protein has been removed. The term "variant" mentioned above may be used interchangeably with terms such as variant, modification, variant polypeptide, mutated protein, mutation, and variant (in English, modification, modified polypeptide, modified protein, mutant, mutein, divergent, etc.), and is not limited to these terms as long as they are used with the meaning of being mutated.

[0052] Additionally, the variant may include deletions or additions of amino acids that have minimal effect on the properties and secondary structure of the polypeptide. For example, a signal (or leader) sequence involved in co-translational or post-translational protein translocation may be conjugated to the N-terminus of the variant. Additionally, the variant may be conjugated with another sequence or linker to enable identification, purification, or synthesis.

[0053]

[0054] In this disclosure, the terms 'homology' or 'identity' refer to the degree of similarity between two given amino acid sequences or base sequences and may be expressed as a percentage. The terms homology and identity may often be used interchangeably.

[0055] 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.

[0056] Whether any two polynucleotide or polypeptide sequences have homology or identity can be determined using a known computer algorithm, such as the “FASTA” program, using default parameters as in, for example, Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), as performed in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later) (GCG program package (Devereux, J., et al, Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] Homology or identity can be determined, for example, using BLAST from the National Biotechnology Information Database Center or ClustalW.

[0057] 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.

[0058] As an example of the present disclosure, a variant of the present disclosure may have activity that increases the production capacity of aspartate-derived L-amino acids (e.g., L-lysine) compared to wild-type type II citrate synthase.

[0059] In this application, the term "aspartate (Aspartic acid)" is an α-amino acid used in the biosynthesis of proteins, which may be used interchangeably with "aspartic acid," abbreviated as Asp or D, and, like all other amino acids, contains an amino group and a carboxylic acid. Generally, aspartate is converted into aspartyl phosphate by aspartokinase (LysC), and then can be converted in vivo into L-lysine, L-threonine, L-methionine, L-homoserine, or L-isoleucine.

[0060] In the present disclosure, the term “aspartate-derived L-amino acid” refers to a substance that can be biosynthesized using aspartate as a precursor, and is not limited to any substance that can be produced through a biosynthetic process using aspartate as a precursor. The aspartate-derived L-amino acid may include not only aspartate-derived L-amino acids but also derivatives thereof. For example, it may be, but is not limited to, L-lysine, L-threonine, L-methionine, L-glycine, homoserine, O-acetylhomoserine, O-succinylhomoserine, O-phosphohomoserine, L-isoleucine, and / or cadaverine. In one embodiment, the aspartate-derived L-amino acid may be one or more selected from the group consisting of L-aspartate, L-lysine, L-threonine, L-methionine, L-homoserine, and L-isoleucine, and more specifically, may be L-lysine, but is not limited thereto.

[0061] In this disclosure, the term “corresponding to” refers to an amino acid residue at a position listed in a polypeptide, or an amino acid residue that is similar, identical, or homologous to a residue listed in a polypeptide. Identifying the amino acid at the corresponding position may involve determining a specific amino acid of a sequence that references a specific sequence. As used in this disclosure, “corresponding region” generally refers to a similar or corresponding position in a related protein or a reference protein.

[0062] For example, any amino acid sequence is an amino acid sequence in which the amino acid corresponding to the 272nd position from the N-terminus in the amino acid sequence of SEQ ID NO. 1, the amino acid corresponding to the 310th position from the N-terminus, or a combination thereof, is substituted with an amino acid different from the original; or additionally, an amino acid sequence in which the amino acid corresponding to the 169th position from the N-terminus in the amino acid sequence of SEQ ID NO. 1, the amino acid corresponding to the 241st position from the N-terminus, or a combination thereof, is substituted with an amino acid different from the original; or is aligned with the amino acid sequence of SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 9, or SEQ ID NO. 10, and based thereon, each amino acid residue of the said amino acid sequence is an amino acid sequence in which the amino acid corresponding to the 272nd position from the N-terminus in the amino acid sequence of SEQ ID NO. 1, the amino acid corresponding to the 310th position from the N-terminus, or a combination thereof, is substituted with an amino acid different from the original; Alternatively, additionally, an amino acid sequence in which the amino acid corresponding to the 169th position from the N-terminus, the amino acid corresponding to the 241st position from the N-terminus, or a combination thereof is substituted with an amino acid different from the original; or numbering may be performed by referring to the numerical positions of amino acid residues corresponding to amino acid residues of the amino acid sequences of SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 9, or SEQ ID NO. 10. For example, a sequence alignment algorithm such as that described in the present disclosure can identify the position of an amino acid, or the position where modifications such as substitution, insertion, or deletion occur, by comparing with a query sequence (also referred to as a “reference sequence”).

[0063] For such alignment, examples such as the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000), Trends Genet. 16: 276-277) may be used, but are not limited thereto, and sequence alignment programs and pairwise sequence comparison algorithms known in the art may be appropriately used.

[0064]

[0065] Another aspect of the present disclosure is to provide a polynucleotide encoding a type II citrate synthase variant of the present disclosure.

[0066] In the present disclosure, the term “polynucleotide” refers to a polymer of nucleotides in which nucleotide monomers are linked together in a long chain by covalent bonds, as a DNA or RNA strand of a certain length or longer, and more specifically, as a polynucleotide fragment encoding the variant.

[0067] In one example, the polynucleotide may be an amino acid sequence in which the amino acid corresponding to the 272nd position from the N-terminus of the amino acid sequence of SEQ ID NO. 1, the amino acid corresponding to the 310th position from the N-terminus, or a combination thereof is substituted with an amino acid different from the original; or additionally, an amino acid sequence in which the amino acid corresponding to the 169th position from the N-terminus of the amino acid sequence of SEQ ID NO. 1, the amino acid corresponding to the 241st position from the N-terminus, or a combination thereof is substituted with an amino acid different from the original; or a polynucleotide encoding a type II citrate synthase variant comprising the amino acid sequence of SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 9, or SEQ ID NO. 10.

[0068] In one example, the polynucleotide may include any one nucleotide sequence selected from the group consisting of the nucleotide sequences of SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 17, and SEQ ID NO. 18.

[0069] The polynucleotide encoding the type II citrate synthase variant of the present disclosure may have or include any one nucleotide sequence selected from the group consisting of the nucleotide sequences of SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 17, and SEQ ID NO. 18. In another example, the polynucleotide may include a nucleotide sequence having at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% or more homology or identity with the nucleotide sequence. In addition, it is obvious that polynucleotides having nucleotide sequences in which some sequences are deleted, modified, substituted, conservatively substituted, or added, are included within the scope of the present disclosure, provided that the sequences have such homology or identity and encode a polypeptide or protein that exhibits efficacy corresponding to the variants of the present disclosure (increased production of aspartate-derived L-amino acids (e.g., L-lysine)).

[0070] In one example, the nucleotide sequence or amino acid sequence provided in this specification may include one that has been modified by conventional mutagenesis, such as direct evolution and / or site-directed mutagenesis, to the extent that their original or intended function is maintained. In one example, the phrase “a polynucleotide or polypeptide comprises a specific nucleotide sequence or amino acid sequence” may mean that the polynucleotide or polypeptide is composed of or essentially comprises the specific nucleotide sequence or amino acid sequence, or (ii) composed of or essentially comprises an amino acid sequence having 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 99.9% or more homology with the specific nucleotide sequence or amino acid sequence, and maintains its original function and / or intended function.

[0071] The polynucleotide of the present disclosure may have various modifications made to its coding region within the scope of not altering the amino acid sequence of the variant of the present disclosure, taking into account the degeneracy of codons or the codons preferred by the organism intended to express the variant of the present disclosure. Specifically, the polynucleotide of the present disclosure comprises an amino acid sequence in which the amino acid corresponding to the 272nd position from the N-terminus, the amino acid corresponding to the 310th position from the N-terminus, or a combination thereof is substituted with an amino acid different from the original; or additionally, an amino acid sequence in which the amino acid corresponding to the 169th position from the N-terminus, the amino acid corresponding to the 241st position from the N-terminus, or a combination thereof is substituted with an amino acid different from the original. or having or including a nucleotide sequence having 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homology or identity with any one nucleotide sequence selected from the group consisting of the nucleotide sequence of a polynucleotide encoding a type II citrate synthase variant comprising the amino acid sequence of SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 9, or SEQ ID NO. 10, and the nucleotide sequence of SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 17, and SEQ ID NO. 18, or having 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, It may consist of or essentially consist of a sequence of 98% or more and less than 100%, but is not limited thereto.

[0072] Additionally, the polynucleotides of the present disclosure may be included without limitation as long as they are probes that can be prepared from known gene sequences, for example, sequences that can be hybridized under stringent conditions with a sequence complementary to all or part of the polynucleotide sequence of the present disclosure. The term "stringent condition" means a condition that enables specific hybridization between polynucleotides. For example, conditions may be listed in which polynucleotides with high homology or identity are hybridized with each other, polynucleotides with homology or identity of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, and polynucleotides with lower homology or identity are not hybridized with each other, or conditions in which washing is performed once, specifically two to three times, at a salt concentration and temperature equivalent to the washing conditions of conventional southern hybridization, such as 60°C, 1×SSC, 0.1% SDS, specifically 60°C, 0.1×SSC, 0.1% SDS, more specifically 68°C, 0.1×SSC, 0.1% SDS.

[0073] Hybridization requires that two nucleic acids have complementary sequences, even though a mismatch between bases may be possible depending on the degree of hybridization. The term “complementary” is used to describe the relationship between nucleotide bases that can hybridize with each other. For example, with respect to DNA, adenine is complementary to thymine and cytosine is complementary to guanine. Accordingly, the polynucleotides of the present disclosure may also include isolated nucleic acid fragments that are complementary to the entire sequence as well as substantially similar nucleotide sequences.

[0074] Specifically, a polynucleotide having homology or identity with the polynucleotide of the present disclosure can be detected using hybridization conditions including a hybridization step at a Tm value of 55°C and using the conditions described above. Additionally, the Tm value may be 60°C, 63°C, or 65°C, but is not limited thereto and can be appropriately adjusted by a person skilled in the art according to the purpose.

[0075] The appropriate strictness for hybridizing the above polynucleotides depends on the length and degree of complementarity of the polynucleotides, and the variables are well known in the relevant technical field.

[0076]

[0077] Another aspect of the present disclosure is to provide a vector comprising the polynucleotide of the present disclosure. The vector may be an expression vector for expressing the polynucleotide in a host cell, but is not limited thereto.

[0078] The vector of the present disclosure may comprise a DNA product comprising a sequence of a polynucleotide encoding said target polypeptide, which is operably linked to a suitable expression control region (or expression control sequence) so as to enable the expression of said target polypeptide within a suitable host. The expression control region may comprise a promoter capable of initiating transcription, any operator sequence for regulating such transcription, a sequence coding for a suitable mRNA ribosome binding site, and a sequence regulating the termination of transcription and translation. After being transformed into a suitable host cell, the vector may replicate or function independently of the host genome and may be incorporated into the genome itself.

[0079] The vectors used in this disclosure are not particularly limited, and any vector known in the art may be used. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages in their natural or recombinant state. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A may be used as phage vectors or cosmid vectors, and pDZ-based, pBR-based, pUC-based, pBluescriptII-based, pGEM-based, pTZ-based, pCL-based, and pET-based vectors may be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC, or pDC24 vectors may be used.

[0080] For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome using a vector for intracellular chromosome insertion. The insertion of the polynucleotide into the chromosome may be achieved by any method known in the art, for example, homologous recombination, but is not limited thereto. A selection marker may be additionally included to confirm whether the chromosome insertion has occurred. The selection marker is intended to select cells transformed by the vector, that is, to confirm whether the target nucleic acid molecule has been inserted, and markers conferring selectable phenotypes such as drug resistance, nutritional requirements, resistance to cytotoxic agents, or expression of a surface polypeptide may be used. Since only cells expressing the selection marker survive or exhibit other phenotypes in an environment treated with a selective agent, the transformed cells can be selected.

[0081] In the present disclosure, the term “transformation” means introducing a vector containing a polynucleotide encoding a target polypeptide into a host cell or microorganism so that the polypeptide encoded by said polynucleotide can be expressed within the host cell. The transformed polynucleotide may include both inserted into and located within the chromosomes of the host cell and extrachromosomally, as long as it can be expressed within the host cell. Additionally, said polynucleotide includes DNA and / or RNA encoding the target polypeptide. said polynucleotide may be introduced in any form that can be introduced into and expressed within the host cell. For example, said polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a genetic structure containing all the elements necessary for self-expression. That expression cassette may typically include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to said polynucleotide. The above expression cassette may be in the form of a self-replicating expression vector. Additionally, 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, but is not limited thereto.

[0082] In addition, the term "operably connected" as above means that a promoter sequence and a polynucleotide sequence are functionally connected to initiate and mediate the transcription of a polynucleotide encoding the target variant of the present disclosure.

[0083]

[0084] Another aspect of the present disclosure provides a microorganism of the genus Corynebacterium comprising one or more selected from the group consisting of the type II citrate synthase variant of the present disclosure and the polynucleotide of the present disclosure.

[0085] The microorganism of the present disclosure may include a type II citrate synthase variant of the present disclosure, a polynucleotide encoding said type II citrate synthase variant, or a vector comprising the polynucleotide of the present disclosure.

[0086] In the microorganism above, the polynucleotide encoding the type II citrate synthase variant may exist or be located in a modified or mutated form of the existing (pre-mutation or wild-type) type II citrate synthase-encoding nucleotide sequence on the chromosome of the microorganism, or may be introduced externally. In the case of introduction externally, the polynucleotide encoding the type II citrate synthase variant may be inserted into a target site through homologous recombination, etc., inserted into a non-target site by a non-homologous mechanism, or exist in the form of a vector such as a plasmid, but is not limited thereto.

[0087] In the present disclosure, the term “microorganism (or strain)” includes both wild-type microorganisms and microorganisms that have undergone natural or artificial genetic modification, and may be microorganisms in which specific mechanisms are weakened or strengthened due to causes such as the insertion of external genes or the enhancement or inactivation of the activity of endogenous genes, and may be microorganisms that include genetic modification for the production of a desired polypeptide, protein, or product.

[0088] The microorganism of the present disclosure may be, but is not limited to, a microorganism comprising one or more of the type II citrate synthase variant of the present disclosure, the polynucleotide of the present disclosure, and a vector comprising the polynucleotide of the present disclosure; a microorganism modified to express a variant of the present disclosure or a polynucleotide of the present disclosure; a microorganism expressing a variant of the present disclosure or a polynucleotide of the present disclosure (e.g., a recombinant microorganism); or a microorganism having variant activity of the present disclosure (e.g., a recombinant microorganism).

[0089] The microorganism of the present disclosure may be a microorganism capable of producing aspartate-derived L-amino acids (e.g., L-lysine).

[0090] The above aspartate-derived L-amino acid is as described above.

[0091] The microorganisms of the present disclosure may be, but are not limited to, microorganisms that naturally possess the ability to produce aspartate-derived L-amino acids (e.g., L-lysine), or microorganisms to which a variant of the present disclosure or a polynucleotide encoding the same (or a vector containing said polynucleotide) is introduced into a parent strain that lacks the ability to produce aspartate-derived L-amino acids (e.g., L-lysine), and / or microorganisms to which the ability to produce aspartate-derived L-amino acids (e.g., L-lysine) is conferred.

[0092] For example, the microorganism of the present disclosure is a cell or microorganism that expresses the type II citrate synthase variant of the present disclosure by being transformed with a vector comprising the polynucleotide of the present disclosure or a polynucleotide encoding a variant of the present disclosure, and for the purposes of the present disclosure, the strain of the present disclosure may include all microorganisms capable of producing aspartate-derived L-amino acids (e.g., L-lysine), including the variant of the present disclosure. For example, the microorganism of the present disclosure may be a recombinant microorganism in which the ability to produce aspartate-derived L-amino acids (e.g., L-lysine) is increased by introducing a polynucleotide encoding a variant of the present disclosure into a natural wild-type microorganism or a microorganism that produces aspartate-derived L-amino acids (e.g., L-lysine).

[0093] In one embodiment, the microorganism of the present disclosure is, compared to a microorganism of the genus Corynebacterium that does not include one or more selected from the group consisting of the type II citrate synthase variant of the present disclosure described above and polynucleotides encoding said type II citrate synthase variant (an amino acid sequence in which an amino acid corresponding to the 272nd position from the N-terminus in the amino acid sequence of SEQ ID NO. 1, an amino acid corresponding to the 310th position from the N-terminus, or a combination thereof, is substituted with an amino acid different from the original; or additionally, an amino acid sequence in which an amino acid corresponding to the 169th position from the N-terminus in the amino acid sequence of SEQ ID NO. 1, an amino acid corresponding to the 241st position from the N-terminus, or a combination thereof, is substituted with an amino acid different from the original; or a microorganism that does not express a type II citrate synthase variant comprising the amino acid sequence of SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 9, or SEQ ID NO. 10, or a microorganism that expresses a wild-type protein corresponding to said type II citrate synthase variant), aspartate derived It may be that the production capacity of L-amino acids (e.g., L-lysine) has increased.

[0094] Microorganisms of the genus Corynebacterium that do not include one or more selected from the group consisting of the type II citrate synthase variant of the present disclosure and the polynucleotide encoding the type II citrate synthase variant may be natural wild-type microorganisms or non-modified microorganisms, and may also be expressed as host cells or parent strains. Microorganisms of the genus Corynebacterium that do not include one or more selected from the group consisting of the type II citrate synthase variant of the present disclosure and the polynucleotide encoding the type II citrate synthase variant may specifically be ATCC13032 strain and / or CJ3P (US 9556463 ​​B2), but are not limited thereto.

[0095] The recombinant microorganism of the present disclosure having increased aspartate-derived L-amino acid (e.g., L-lysine) production capacity may be a microorganism having increased aspartate-derived L-amino acid (e.g., L-lysine) production capacity compared to a natural wild-type microorganism or an unmodified microorganism, but is not limited thereto. For example, the unmodified microorganism that is the target strain for comparing whether the aspartate-derived L-amino acid (e.g., L-lysine) production capacity is increased may be the ATCC13032 strain and / or CJ3P (US 9556463 ​​B2), but is not limited thereto.

[0096] In one embodiment, the microorganism of the present disclosure having increased aspartate-derived L-amino acid (e.g., L-lysine) production capacity (production amount) may be increased by about 1% or more, about 5% or more, about 10% or more, about 15% or more, about 20% or more, or about 21% or more compared to the parent strain before mutation or the non-mutated microorganism (the upper limit is not specifically limited and may be, for example, about 200% or less, about 150% or less, about 100% or less, or about 50% or less, about 30% or less), but is not limited thereto. In another embodiment, the microorganism of the present disclosure having increased aspartate-derived L-amino acid (e.g., L-lysine) production capacity (production amount) may be increased by about 1.01 times, about 1.05 times, about 1.1 times, about 1.15 times, about 1.2 times, or about 1.21 times compared to the parent strain before mutation or the non-mutated microorganism (the upper limit is not specifically limited and may be, for example, about 10 times or less, about 5 times or less, about 3 times or less, or about 2 times or less), but is not limited thereto.

[0097] 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.”

[0098]

[0099] In this disclosure, the term "non-mutated microorganism" does not exclude microorganisms containing mutations that may occur naturally in microorganisms, and may refer to wild-type microorganisms or natural-type microorganisms themselves, or microorganisms prior to genetic mutations caused by natural or artificial factors. For example, the non-mutated microorganism may refer to a microorganism in which the type II citrate synthase variant described herein has not been introduced or is prior to being introduced. The term "non-mutated microorganism" may be used interchangeably with "pre-mutation strain," "pre-mutation microorganism," "non-mutated strain," "non-mutated strain," "non-mutated microorganism," or "reference microorganism."

[0100] In one embodiment, the microorganism of the present disclosure is Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium It may be Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris, or Corynebacterium flavescens, and more specifically, Corynebacterium glutamicum.

[0101]

[0102] In this disclosure, the term "weakening" of a polypeptide is a concept that includes both reduced activity and lack of activity relative to its intrinsic activity. Such weakening may be used interchangeably with terms such as inactivation, deficiency, down-regulation, decrease, reduce, and attenuation.

[0103] The above 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 not active even if the polynucleotide is expressed. The above "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-modified microorganism prior to transformation.

[0104] 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.).

[0105] Specifically, the weakening of the polypeptide of the present disclosure is

[0106] 1) Deletion of all or part of the gene encoding a polypeptide;

[0107] 2) Modification of the expression regulatory region (or expression regulatory sequence) to reduce the expression of the gene encoding the polypeptide;

[0108] 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);

[0109] 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 nucleotides on the nucleotide sequence of the polypeptide gene to code for a polypeptide modified so as to remove or weaken the activity of the polypeptide);

[0110] 5) A modification of the nucleotide sequence encoding the start codon or the 5'-UTR region of the gene transcript encoding the polypeptide;

[0111] 6) Introduction of an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the transcript of the gene encoding the polypeptide;

[0112] 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;

[0113] 8) Addition of a reverse-transcribed promoter to the 3' end of the open reading frame (ORF) of a gene sequence encoding a polypeptide (Reverse transcription engineering, RTE); or

[0114] 9) Regulation of the cellular localization of proteins (polypeptides); or

[0115] 10) It may be based on two or more combinations selected from 1) to 9) above, but is not specifically limited thereto.

[0116] for example,

[0117] 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.

[0118] 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.

[0119] 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.

[0120] In addition, the modification of the amino acid sequence or polynucleotide sequence 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 is 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 is not limited thereto.

[0121] 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].

[0122] 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.

[0123] 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.

[0124] The above 9) regulation of the intracellular localization of the protein (polypeptide) may involve targeting the protein (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 protein (polypeptide), but is not limited thereto.

[0125] Such weakening of polypeptide activity may involve a reduction in the activity or concentration expression of the corresponding polypeptide relative to the activity or concentration of the polypeptide expressed in the wild-type or pre-modification microbial strain, or a decrease in the amount of product produced from said polypeptide, but is not limited thereto.

[0126]

[0127] In the present disclosure, the term “enhancement” of polypeptide activity means that the activity of the polypeptide is increased compared to its intrinsic activity. Such enhancement may be used interchangeably with terms such as activation, up-regulation, overexpression, and increase. Here, activation, enhancement, up-regulation, overexpression, and increase may include exhibiting activity that was not originally possessed, or exhibiting improved activity compared to the intrinsic activity or activity prior to modification. The “intrinsic activity” refers to the activity of a specific polypeptide originally possessed by the parent strain or the non-modified microorganism prior to the change in traits caused by genetic mutations due to natural or artificial factors. This may be used interchangeably with “activity prior to modification.” "Enhancement," "upregulation," "overexpression," or "increase" of polypeptide activity relative to intrinsic activity means that the activity and / or concentration (expression amount) of a specific polypeptide were originally possessed by the parent strain or non-modified microorganism prior to transformation.

[0128] The above enhancement can be achieved by introducing an exogenous polypeptide or by enhancing the activity and / or concentration (expression amount) of an 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 released from the polypeptide.

[0129] 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.).

[0130] Specifically, the reinforcement of the polypeptide of the present disclosure is

[0131] 1) Increase in the intracellular copy number of polynucleotides encoding polypeptides;

[0132] 2) Replace the chromosomal gene expression regulatory region encoding a polypeptide with a potent sequence;

[0133] 3) A modification of the nucleotide sequence encoding the start codon or the 5'-UTR region of the gene transcript encoding the polypeptide;

[0134] 4) Modification of the amino acid sequence of the polypeptide to enhance polypeptide activity;

[0135] 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);

[0136] 6) Introduction of a foreign polypeptide exhibiting polypeptide activity or a foreign polynucleotide encoding the same;

[0137] 7) Codon optimization of polynucleotides encoding polypeptides;

[0138] 8) Analyze the tertiary structure of the polypeptide to select and modify or chemically modify the exposed sites; or

[0139] 9) Regulation of the cellular localization of proteins (polypeptides); or

[0140] 10) It may be based on two or more combinations selected from 1) to 9) above, but is not specifically limited thereto.

[0141] 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.

[0142] 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.

[0143] 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.

[0144] 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.

[0145] The modification of the amino acid sequence or polynucleotide sequence 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 enhance the activity of the polypeptide, or may involve 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, but is not limited thereto. 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 chromosome insertion has occurred. The selection marker is as described above.

[0146] 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.

[0147] The above 7) codon optimization of a polynucleotide encoding a polypeptide may be a codon optimization of the intrinsic polynucleotide such that transcription or translation within the host cell increases, or a codon optimization of the extrinsic polynucleotide such that optimized transcription or translation occurs within the host cell.

[0148] 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.

[0149] The above 9) regulation of the intracellular localization of the protein (polypeptide) may involve targeting the protein (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 protein (polypeptide), but is not limited thereto.

[0150] 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.

[0151]

[0152] Modification of part or all of a polynucleotide in the microorganism of the present disclosure (e.g., modification to code for the type II citrate synthase variant described above) may be induced by (a) homologous recombination using a vector for chromosomal insertion within the microorganism or genome editing using engineered nucleases (e.g., CRISPR-Cas9) and / or (b) treatment by light and / or chemicals such as ultraviolet rays and radiation, but is not limited thereto. The method of modifying part or all of the gene may include methods using DNA recombination technology. For example, deletion of part or all of the gene may be achieved by inducing homologous recombination by injecting a nucleotide sequence or vector containing a nucleotide sequence homologous to the target gene into the microorganism. The injected nucleotide sequence or vector may include a dominant selection marker, but is not limited thereto.

[0153] In the microorganism of the present disclosure, type II citrate synthase variants, polynucleotides, and aspartate-derived L-amino acids (e.g., L-lysine), etc., are as described in the other embodiments above.

[0154]

[0155] Another aspect of the present disclosure provides a method for producing aspartate-derived L-amino acids, comprising the step of culturing a microorganism of the genus Corynebacterium in a medium, the microorganism comprising one or more selected from the group consisting of a type II citrate synthase variant of the present disclosure and a polynucleotide encoding said type II citrate synthase variant.

[0156] The method for producing aspartate-derived L-amino acids of the present disclosure may include the step of culturing a microorganism of the genus Corynebacterium in a medium comprising the type II citrate synthase variant of the present disclosure, the polynucleotide of the present disclosure, or the vector of the present disclosure.

[0157] The above aspartate-derived L-amino acid is as described in a previous embodiment.

[0158] In this disclosure, the term "culture" means growing the microorganisms of the genus Corynebacterium of this disclosure under appropriately controlled environmental conditions. The culture process of this 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.

[0159] In this disclosure, the term "medium" refers to a substance mixed with nutrients as the main component required to culture the microorganisms of the genus Corynebacterium of this disclosure, 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 microorganisms of the genus Corynebacterium of this disclosure may be any medium used for culturing ordinary microorganisms without special limitations; however, the microorganisms of the genus Corynebacterium of this disclosure may be cultured under aerobic conditions while controlling the temperature, pH, etc., in a conventional medium containing a suitable carbon source, nitrogen source, phosphorus, inorganic compounds, amino acids, and / or vitamins.

[0160] Specifically, culture media for strains of the genus Corynebacterium can be found in the literature ["Manual of Methods for General Bacteriology" by the American Society for Bacteriology (Washington DC, USA, 1981)].

[0161] 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.; and amino acids such as glutamic acid, methionine, lysine, etc. Additionally, natural organic nutrient sources such as starch hydrolysate, molasses, blackstrap molasses, rice winter, 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.

[0162] 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.; and organic nitrogen sources such as 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 types, but are not limited thereto.

[0163] 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.

[0164] In addition, during the cultivation of the microorganisms of the genus Corynebacterium disclosed in this 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. In one embodiment, the method for producing aspartate-derived L-amino acid (e.g., L-lysine) may be performed at a pH of 6 to 9, and more specifically at a pH of 6 to 9, 6 to 8.5, 6 to 8, 6 to 7.5, 6 to 7.25, 6 to 7, 6.5 to 9, 6.5 to 8.5, 6.5 to 8, 6.5 to 7.5, 6.5 to 7.25, 6.5 to 7, 6.75 to 9, 6.75 to 8.5, 6.75 to 8, 6.75 to 7.5, 6.75 to 7.25, or 6.75 to 7. no.

[0165] In the culture of the present disclosure, the culture temperature is 20°C to 45°C, specifically 20°C to 45°C, 20°C to 40°C, 20°C to 38°C, 20°C to 37.5°C, 20°C to 37°C, 25°C to 45°C, 25°C to 40°C, 25°C to 38°C, 25°C to 37.5°C, 25°C to 37°C, 30°C to 45°C, 30°C to 40°C, 30°C to 38°C, 30°C to 37.5°C, 30°C to 37°C, 32.5°C to 45°C, 32.5°C to 40°C, 32.5°C to 38°C, 32.5°C to 37.5°C, 32.5°C to 37°C, 35°C to 45°C, 35°C to 40°C, 35°C to 38°C, 35°C to 37.5°C, or 35°C to 37°C may be maintained, but is not limited thereto. In the culture of the present disclosure, the culture time may be about 10 to 160 hours, but is not limited thereto.

[0166] Aspartate-derived L-amino acids (e.g., L-lysine) produced by the culture of the present disclosure may be secreted into the medium or remain in the cell.

[0167] The method for producing an aspartate-derived L-amino acid (e.g., L-lysine) of the present disclosure may additionally include, for example, the step of preparing a microorganism of the genus Corynebacterium of the present disclosure, the step of preparing a medium for culturing said strain, or a combination thereof (in any order), prior to said culturing step.

[0168] The method for producing aspartate-derived L-amino acids (e.g., L-lysine) of the present disclosure may further include a step of recovering aspartate-derived L-amino acids (e.g., L-lysine) from a culture medium (a culture medium in which the culture is performed) or from a microorganism of the genus Corynebacterium. The recovery step may be further included after the culture step.

[0169] The above recovery may involve collecting the desired L-amino acid using a suitable method known in the art according to the culture method of the microorganism disclosed in this disclosure, for example, a batch, continuous, or fed-batch culture method. For example, various chromatographs such as centrifugation, filtration, treatment with a crystallizing protein precipitating agent (salting out method), extraction, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC, or a combination thereof may be used, and the desired L-amino acid may be recovered from the culture medium or microorganism using a suitable method known in the art.

[0170] Additionally, the method for producing aspartate-derived L-amino acids (e.g., L-lysine) of the present disclosure may further include a purification step. The purification may be performed using a suitable method known in the art. In one example, where the method for producing aspartate-derived L-amino acids (e.g., L-lysine) 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.

[0171] In the method of the present disclosure, the variant, polynucleotide, aspartate-derived L-amino acid, vector, strain, etc. are as described in the other embodiments above.

[0172]

[0173] Another aspect of the present disclosure provides a composition for producing aspartate-derived L-amino acids comprising one or more selected from the group consisting of a type II citrate synthase variant of the present disclosure, a polynucleotide encoding said type II citrate synthase variant, a vector containing said polynucleotide, the microorganism of the present disclosure described above, and a culture medium in which said are cultured.

[0174] The composition of the present disclosure may further include any suitable excipients commonly used in compositions for producing amino acids, and such excipients may be, for example, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers or isotonic agents, but are not limited thereto.

[0175] In the composition of the present disclosure, the variant, polynucleotide, vector, strain, medium, and aspartate-derived L-amino acid, etc. are as described in the other embodiments above.

[0176]

[0177] According to another aspect of the present disclosure, the present disclosure may provide a method for increasing the production capacity of aspartate-derived L-amino acids (e.g., L-lysine) of a microorganism, comprising the step of introducing (e.g., transforming) into the microorganism said type II citrate synthase variant of the present disclosure, said type II citrate synthase variant, said polynucleotide encoding said type II citrate synthase variant, and / or said vector containing said polynucleotide; a method for conferring the production capacity of aspartate-derived L-amino acids (e.g., L-lysine) to said microorganism; and / or a method for producing a microorganism with increased production capacity of aspartate-derived L-amino acids (e.g., L-lysine).

[0178] In the method of increasing the production capacity of aspartate-derived L-amino acids (e.g., L-lysine) of a microorganism of the present disclosure, the method of imparting aspartate-derived L-amino acid (e.g., L-lysine) production capacity to a microorganism, and / or the method of producing a microorganism with increased aspartate-derived L-amino acid (e.g., L-lysine) production capacity, the type II citrate synthase variant, polynucleotide, recombinant vector, aspartate-derived L-amino acid, and microorganism, etc. are as described above.

[0179]

[0180] According to another aspect of the present disclosure, the present disclosure provides a use for producing aspartate-derived L-amino acids, producing aspartate-derived L-amino acids from a microorganism selected from the group consisting of: a type II citrate synthase variant of the present disclosure described above; a polynucleotide encoding said type II citrate synthase variant; a recombinant vector comprising said polynucleotide; and one or more microorganisms comprising said type II citrate synthase variant, said polynucleotide encoding said type II citrate synthase variant, and / or said recombinant vector comprising said polynucleotide; and / or imparting and / or increasing the ability of the microorganism to produce aspartate-derived L-amino acids.

[0181] In the use for the production of aspartate-derived L-amino acids; the preparation of aspartate-derived L-amino acids by microorganisms; and / or for imparting and / or increasing the production capacity of aspartate-derived L-amino acids by microorganisms, the type II citrate synthase variant, polynucleotide, recombinant vector, aspartate-derived L-amino acids, and microorganisms, etc. are as described above.

[0182]

[0183] According to another aspect of the present disclosure, the present disclosure provides a composition, method, product, process, or use characterized by one or more elements disclosed in the present disclosure.

[0184] When culturing a Corynebacterium glutamicum strain containing the type II citrate synthase variant of the present disclosure, it is possible to produce L-amino acids derived from L-aspartate in a high yield compared to microorganisms having the existing type II citrate synthase.

[0185] The present application will be described in more detail below through examples. These examples are intended solely to illustrate the present application more specifically, and it will be obvious to those skilled in the art that the scope of the present application is not limited by these examples according to the gist of the application.

[0186]

[0187] Examples

[0188]

[0189] (Throughout this specification, "%" used to indicate the concentration of a particular substance is (weight / weight) % for solid / solid, (weight / volume) % for solid / liquid, and (volume / volume) % for liquid / liquid, unless otherwise noted.)

[0190]

[0191] Example 1: Derivation of sequence variant candidates for type II citrate synthase derived from Corynebacterium glutamicum

[0192] Variant candidates were derived through a method that predicts substrate binding sites and Kcat values ​​based on the corresponding substrate binding site variations.

[0193] To predict substrate binding sites for regulating the activity of type II citrate synthase (GltA), protein structure data such as the Protein Data Bank (PDB) for the amino acid sequence of GltA (Sequence No. 1) was input into the Pyvol program, and a spherification technique was implemented to identify the protein shape as a cluster to predict pocket sites (Ryan HB Smith, et al., 2019). Random mutations were performed on the GltA sequences based on the predicted pocket sites, and the Kcat values ​​of the wild-type and mutant GltA sequences were predicted using the following method.

[0194] The Kcat values ​​were predicted by generating an artificial intelligence model based on the literature of DLKcat to predict variant sequences and Kcat values ​​(Feiran Li, et al., 2022). The process of generating the artificial intelligence model is as follows. Based on papers and experimental data, a dataset of approximately 16,838 protein sequences, substrate structure information (Simplified Molecular-Input Line-Entry System, SMILES), and Kcat was obtained from the BRENDA enzyme database, which provides information on enzymes such as function, structure, and reaction mechanisms, and SABIO-RK, which provides information on biochemical reactions and their reaction kinetics. Non-quantitative information (protein sequences, etc.) was converted into quantitative information through a Convolutional Neural Network (CNN) or Graph Neural Network (GNN), and then the weights of matrices and vectors at each stage were adjusted to generate the artificial intelligence model.

[0195] The wild-type sequence (Sequence No. 1) and the variant sequence predicted as the pocket site were input as the GltA protein sequence, and the KEGG number (C00024) of the substrate acetyl-CoA was input to obtain predicted Kcat values. Among these, the following four variants were obtained that have Kcat values ​​lower than those of the GltA wild-type sequence: a variant in which the leucine (L) at the 272nd position from the N-terminus in the amino acid sequence of Sequence No. 1 is substituted with arginine (R) (L272R, Sequence No. 3); a variant in which the arginine (R) at the 310th position from the N-terminus in the amino acid sequence of Sequence No. 1 is substituted with tyrosine (Y) (R310Y, Sequence No. 4); and a variant in which the histidine (H) at the 238th position from the N-terminus in the amino acid sequence of Sequence No. 1 is substituted with isoleucine (I) (H238I, Sequence No. 7). and a variant in which the phenylalanine (F) at the 393rd position from the N-terminus in the amino acid sequence of SEQ ID NO. 1 is substituted with tyrosine (Y) (F393Y, SEQ ID NO. 8).

[0196]

[0197] Example 2: Construction of a vector containing a type II citrate synthase variant sequence and introduction of the strain

[0198] To construct a vector containing each predicted GltA variant, primers were designed based on the wild-type gltA gene sequence of SEQ ID NO. 2. Template PCR was performed using the chromosome of Corynebacterium glutamicum ATCC13032 as a template, using primers of SEQ ID NO. 19 and 22 and primers of SEQ ID NO. 20 and 21 to construct a vector containing the GltA(H238I) sequence, primers of SEQ ID NO. 19 and 24 and primers of SEQ ID NO. 20 and 23 to construct a vector containing the GltA(L272R) sequence, primers of SEQ ID NO. 19 and 26 and primers of SEQ ID NO. 20 and 25 to construct a vector containing the GltA(R310Y) sequence, and primers of SEQ ID NO. 19 and 27 and primers of SEQ ID NO. 20 and 28 to construct a vector containing the GltA(F393Y) sequence. The PCR conditions were as follows: denaturation at 95°C for 5 minutes, followed by denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 1 minute 30 seconds, repeated 30 times, followed by polymerization at 72°C for 5 minutes. Each template PCR fragment was fusion cloned after treating the plasmid pDC24 (Korean Patent Publication No. 10-2024-0167588, SEQ ID No. 29 of the said patent) for gene insertion and replacement within the Corynebacterium chromosome with restriction enzymes BamHI and XbaI at 37°C for 4 hours. The In-Fusion® HD Cloning Kit (Clontech) was used, and subsequently, the fragments were transformed into E. coli DH5α and plated on LB solid medium containing kanamycin (25 mg / l). A vector with the target gene inserted was identified through PCR using SEQ ID NOs 29 and 30, and the final target plasmid was obtained by confirming the sequence of the target gene. Each plasmid was named pDC_gltA(H238I), pDC_gltA(L272R), pDC_gltA(R310Y), and pDC_gltA(F393Y).The sequences of the primers used in this example are listed in Table 1 below.

[0199] Sequence number sequence (5' -> 3')19GCTCGGTACCCGGGGATCCATGTTTGAAAGGGATATCG20CTGCAGGTCGACTCTAGACATTTCGCTGGCGCGCGAGCT21GCACGCTGACATCGAGCAGAACTGCTCCACCT22CAGTTCTGCTCGATGTCAGCGTGCAGGATGAG23GTCCGGCCCACGGCACGGTGGCGCAAACCAGG24CGCCACCGTGCCGTGGGCCGGACAGAGCGT TG25AAGACGGCGTCTACCTCATGGGCTTCGGACAC26AAGCCCATGAGGTAGACGCCGTCTTCCTTGT27TCACCGTATTGTACGCAATCGGTCGTCTGCCA28GACC GATTGCGTACAATACGGTGAAGAAGTC29AAGTTGGGTAACGCCAGG30CGGCTCGTATGTTGTGTGG31ACTGATAACAACAAGGCTG32GCTCCTCGCGAGGAACCAA

[0200] The vectors pDC_gltA(H238I), pDC_gltA(L272R), pDC_gltA(R310Y), and pDC_gltA(F393Y) prepared as above were transformed into Corynebacterium glutamicum CJ3P (US 9556463 ​​B2) by the electro-pulse method (Van der Rest et al., Appl. Microbiol. Biotechnol. 52:541-545, 1999). After confirming that the target variant was introduced into the gltA gene by homologous chromosome recombination using the primer pair of sequences 31 and 32, the strains were named CJ3P_gltA(H238I), CJ3P_gltA(L272R), CJ3P_gltA(R310Y), and CJ3P_gltA(F393Y), respectively.

[0201]

[0202] Example 3: Comparison of L-lysine production capacity of synthesized gltA variant strains

[0203] Flask fermentation activity was evaluated to confirm L-lysine production ability, with the strain produced in Example 2 as the experimental group and the parent strain CJ3P as the control. In addition, for comparison, strains with the GltA A169V and N241T mutations known in KR10-2277031B1 and KR10-1915433B1 (CJ3P_gltA(A169V), CJ3P_gltA(N241T)) were also evaluated for fermentation activity under the same conditions.

[0204] First, each strain was inoculated into a 250 ml Corner-Baffle flask containing 25 ml of seed medium and cultured at 37 °C for 20 hours with shaking at 200 rpm. 1 ml of seed culture was inoculated into a 250 ml Corner-Baffle flask containing 24 ml of production medium and cultured at 37 °C for 36 hours with shaking at 200 rpm. To accurately determine the point of glycogen consumption, 700 µL of culture medium was taken at the 18-hour culture time to measure the L-lysine concentration, after which the production capacity of each strain was calculated. The above experiment was repeated three times, and the average values ​​of the analysis results are shown in Table 2 below. The production capacity (g / L / h), production capacity growth rate (%), and lysine concentration (g / L) after the completion of culture for each tested strain are as shown in Table 2 below.

[0205]

[0206] <Seed medium (pH 7.0)>

[0207] Glucose 20 g, Peptone 10 g, Yeast extract 5 g, Urea 1.5 g, KH2PO44 g, K2HPO48 g, MgSO4·7H2O 0.5 g, Biotin 0.1 mg, Thiamine HCl 1 mg, Calcium-Pantothenic Acid 2 mg, Nicotinamide 2 mg (based on 1 liter of distilled water)

[0208]

[0209] Production Medium (pH 7.0)

[0210] Glucose 100 g, (NH4)2SO4 40 g, soybean protein 2.5 g, corn stem solids 5 g, urea 3 g, KH2PO4 1 g, MgSO4 7H2O 0.5 g, biotin 0.1 mg, thiamine hydrochloride 1 mg, calcium pantothenic acid 2 mg, nicotinamide 3 mg, CaCO3 30 g (based on 1 liter of distilled water).

[0211]

[0212] Strain Lysine Concentration (g / L) Lysine Production Capacity (g / L / h) Improvement in Lysine Production Capacity (%) 18h 36hat 18hCJ3P (gltA (WT))4.127.450.229100CJ3P_gltA(A169V)4.498.090.249108.7CJ3P_gltA(N241T)4.668.120.259113.1CJ3P_gltA(H238I)2.323. 880.12956.3CJ3P_gltA(L272R)4.968.120.276120.5CJ3P_gltA(R310Y)5.038.210.279121.8CJ3P_gltA(F393Y)3.125.560.17375.5

[0213] As shown in Table 2, among the four newly discovered GltA variants, it was confirmed that two strains, CJ3P_gltA(L272R) and CJ3P_gltA(R310Y), including the GltA(L272R) and gltA(R310Y) variants, showed an increase in L-lysine production capacity of 120.5% and 121.8%, respectively, compared to the control group CJ3P, and also showed increased lysine production capacity compared to previously known variants. The other variants showed a decrease in lysine concentration and lysine production capacity, and in particular, it was confirmed that the growth of the CJ3P_gltA(H238I) strain was completely inhibited.

[0214]

[0215] Example 4: Construction of an effective mutant integrated strain and confirmation of L-lysine production capacity

[0216] In order to construct a strain in which the gltA(N241T) variant identified in the prior art (Republic of Korea Registered Patent No. 10-1915433) and two novel variants whose effects were confirmed in Example 3 were integrated, and to further confirm the effects, the vectors pDC_gltA(L272R) and pDC_gltA(R310Y) constructed in Example 2 were transformed into Glutamicum CJ3P_gltA(N241T) by the electric pulse method (Van der Rest et al., Appl. Microbiol. Biotechnol. 52:541-545, 1999). After confirming strains with mutations in the gltA gene by homologous chromosome recombination using the primer pair of sequence numbers 23 and 24, they were named CJ3P_gltA(N241T / L272R) and CJ3P_gltA(N241T / R310Y).

[0217] To confirm the L-lysine production ability, the strain obtained through this was used as the experimental group and CJ3P_gltA(N241T) as the control, and the flask fermentation activity was evaluated using the same method as in Example 3.

[0218] Strain Lysine Concentration (g / L) Lysine Production Capacity (g / L / h) Improvement in Lysine Production Capacity (%) 18h 36hat 18h CJ3P_gltA(N241T) 4.7 18.15 0.26 2100 CJ3P_gltA(N241T / L272R) 5.35 8.35 0.29 71 13.4 CJ3P_gltA(N241T / R310Y) 5.46 8.43 0.30 31 15.6

[0219] As shown in Table 3, it was confirmed that the L-lysine production capacity of the two strains, CJ3P_gltA(N241T / L272R) and CJ3P_gltA(N241T / R310Y), which were created by additionally introducing the gltA(L272R) and gltA(R310Y) variants based on the CJ3P_gltA(N241T) strain, increased by 113.4% and 115.6%, respectively, compared to the control strain CJ3P_gltA(N241T).

Claims

A type II citrate synthase variant in which the amino acid corresponding to the 272nd position from the N-terminus in the amino acid sequence of SEQ ID NO. 1, the amino acid corresponding to the 310th position from the N-terminus, or a combination thereof is substituted with an amino acid different from the original. In paragraph 1, The amino acid corresponding to the 272nd position from the N-terminus is substituted with glutamic acid (E), glycine (G), alanine (A), serine (S), threonine (T), cysteine ​​(C), valine (V), isoleucine (I), methionine (M), proline (P), phenylalanine (F), tyrosine (Y), tryptophan (W), aspartic acid (D), glutamine (Q), histidine (H), lysine (K), arginine (R), or asparagine (N); The amino acid corresponding to the 310th position from the N-terminus is substituted with glutamic acid (E), glycine (G), alanine (A), serine (S), threonine (T), cysteine ​​(C), valine (V), leucine (L), isoleucine (I), methionine (M), proline (P), phenylalanine (F), tyrosine (Y), tryptophan (W), aspartic acid (D), glutamine (Q), histidine (H), lysine (K), or asparagine (N); or The amino acid corresponding to the 272nd position from the N-terminus is substituted with glutamic acid (E), glycine (G), alanine (A), serine (S), threonine (T), cysteine ​​(C), valine (V), isoleucine (I), methionine (M), proline (P), phenylalanine (F), tyrosine (Y), tryptophan (W), aspartic acid (D), glutamine (Q), histidine (H), lysine (K), arginine (R), or asparagine (N), and the amino acid corresponding to the 310th position from the N-terminus is substituted with glutamic acid (E), glycine (G), alanine (A), serine (S), threonine (T), cysteine ​​(C), valine (V), leucine (L), isoleucine (I), methionine (M), proline (P), phenylalanine (F), tyrosine (Y), tryptophan (W), A type II citrate synthase variant substituted with aspartic acid (D), glutamine (Q), histidine (H), lysine (K), or asparagine (N). In claim 1, the type II citrate synthase variant is additionally a type II citrate synthase variant in which the amino acid corresponding to the 169th position from the N-terminus in the amino acid sequence of SEQ ID NO. 1, the amino acid corresponding to the 241st position from the N-terminus, or a combination thereof is substituted with an amino acid different from the original. A type II citrate synthase variant according to claim 3, wherein the amino acid corresponding to the 169th position from the N-terminus is substituted with glutamic acid (E), glycine (G), serine (S), threonine (T), cysteine ​​(C), valine (V), leucine (L), isoleucine (I), methionine (M), proline (P), phenylalanine (F), tyrosine (Y), tryptophan (W), aspartic acid (D), glutamine (Q), histidine (H), lysine (K), arginine (R), or asparagine (N). A type II citrate synthase variant according to claim 3, wherein the amino acid corresponding to the 241st position from the N-terminus is substituted with glutamic acid (E), glycine (G), serine (S), threonine (T), cysteine ​​(C), valine (V), leucine (L), isoleucine (I), methionine (M), proline (P), phenylalanine (F), tyrosine (Y), tryptophan (W), aspartic acid (D), glutamine (Q), histidine (H), lysine (K), arginine (R), or alanine (A). A type II citrate synthase variant according to claim 1, comprising the amino acid sequence of SEQ ID NO. 3 or SEQ ID NO.

4. In paragraph 3, a type II citrate synthase variant comprising the amino acid sequence of SEQ ID NO. 9 or SEQ ID NO.

10. In claim 1, the type II citrate synthase variant is a type II citrate synthase variant having 80% or more and less than 100% amino acid sequence identity with the amino acid sequence of SEQ ID NO.

1. A polynucleotide encoding a type II citrate synthase variant of any one of claims 1 to 8. A microorganism of the genus Corynebacterium comprising one or more selected from the group consisting of a type II citrate synthase variant of any one of claims 1 to 8 and a polynucleotide encoding said type II citrate synthase variant. In paragraph 10, the microorganism of the genus Corynebacterium is a microorganism that is Corynebacterium glutamicum. In claim 10, the microorganism is a microorganism having increased production capacity of aspartate-derived L-amino acids compared to a microorganism of the genus Corynebacterium that does not contain one or more selected from the group consisting of the type II citrate synthase variant and the polynucleotide encoding the type II citrate synthase variant. In paragraph 12, the microorganism wherein the aspartate-derived L-amino acid is one or more selected from the group consisting of L-aspartate, L-lysine, L-threonine, L-methionine, L-homoserine, and L-isoleucine. A composition for producing aspartate-derived L-amino acid comprising the microorganism of claim 10. A method for producing aspartate-derived L-amino acid, comprising the step of culturing the microorganism of claim 10 in a culture medium. A method for producing aspartate-derived L-amino acids according to claim 15, wherein the method further comprises the step of recovering aspartate-derived L-amino acids from a cultured medium or microorganism. A method for producing aspartate-derived L-amino acids according to claim 15, wherein the aspartate-derived L-amino acid is one or more selected from the group consisting of L-aspartate, L-lysine, L-threonine, L-methionine, L-homoserine, and L-isoleucine.