Recombinant amino acid molecules, microorganisms for L-arginine production, and methods for producing L-arginine using the same.

Abstract

This disclosure provides an amino acid molecule, a host cell expressing the amino acid molecule, the use of a host cell for producing L-arginine, a nucleic acid molecule having a sequence encoding the amino acid molecule, a vector comprising the nucleic acid molecule having a sequence encoding the amino acid molecule, a method for producing L-arginine, and a method for increasing L-arginine production by a host cell.

Description

[Technical Field]

[0001] This disclosure relates to improved amino acid molecules, microorganisms expressing the amino acid molecules and their use for producing L-arginine, polynucleotides having sequences encoding the amino acid molecules, vectors comprising polynucleotides having sequences encoding the amino acid molecules, methods for producing L-arginine, and methods for increasing L-arginine production by microorganisms expressing the amino acid molecules. [Background technology]

[0002] L-arginine is an amino acid with widespread applications in medicine, food, animal feed, and industrial fields. L-arginine is produced by the body and, for example, has vasodilatory effects. It can also be used as a feed additive. Currently, various studies are underway to develop host cell and fermentation process technologies for highly efficient L-arginine production (U.S. Patent No. 11345940). [Prior art documents] [Patent Documents]

[0003] [Patent Document 1] U.S. Patent No. 11345940 [Patent Document 2] U.S. Patent Application Publication Number 2023 / 0134555 [Patent Document 3] Korean Patent No. 10-1632642 [Patent Document 4] Korean Patent Application Publication Number 10-2020-0136813 [Patent Document 5] U.S. Patent No. 7662943 [Patent Document 6] U.S. Patent No. 10584338 [Patent Document 7] U.S. Patent No. 10273491 [Non-patent literature]

[0004]

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[0005] Currently, various studies are being conducted to develop microorganisms and fermentation process technologies that can produce L-arginine with high efficiency. [Means for solving the problem]

[0006] This disclosure provides amino acid molecules such as amino acid molecules or proteins.

[0007] This disclosure provides a host cell or microorganism that expresses the amino acid molecule.

[0008] This disclosure provides a nucleic acid molecule comprising a nucleic acid sequence encoding the amino acid molecule.

[0009] This disclosure provides a vector containing a nucleic acid molecule.

[0010] This disclosure provides engineered host cells that express recombinant amino acid molecules, including a recombinant protein that is argininosuccinate synthase.

[0011] This disclosure provides the use of the aforementioned engineered host cells or microorganisms for producing L-arginine.

[0012] This disclosure provides a method for producing L-arginine. [Effects of the Invention]

[0013] Microorganisms expressing the amino acid molecules of this disclosure are effective in producing L-arginine with high efficiency. [Brief explanation of the drawing]

[0014] [Figure 1] This shows the protein sequence alignment comparing the alignment of exemplary argininosuccinate synthase proteins between Escherichia coli and Corynebacterium glutamicum in order to determine the amino acid residues essential for substrate binding of argininosuccinate synthase. [Figure 2a] This shows the sequence alignment performed on exemplary argininosuccinate synthase proteins from various heterologous model microbial species to determine non-conserved sites of substrate binding. [Figure 2b] This shows the sequence alignment performed on exemplary argininosuccinate synthase proteins from various heterologous model microbial species to determine non-conserved sites of substrate binding. [Figure 2c] This shows the sequence alignment performed on exemplary argininosuccinate synthase proteins from various heterologous model microbial species to determine non-conserved sites of substrate binding. [Figure 3]To compare structures and predict the role of amino acid extension in terms of protein function, the tertiary structures of argininosuccinate synthase proteins with amino acid extension (derived from Escherichia coli) and exemplary tertiary structures of argininosuccinate synthase proteins without superimposed amino acid extension (derived from Corynebacterium glutamicum) are shown. [Figure 4a] This lists both identified exemplary motifs and generalized versions of motif sequences in argininosuccinate synthase proteins from heterologous model microorganisms whose sequences have been ordered. [Figure 4b] This lists both identified exemplary motifs and generalized versions of motif sequences in argininosuccinate synthase proteins from heterologous model microorganisms whose sequences have been ordered. [Figure 4c] This lists both identified exemplary motifs and generalized versions of motif sequences in argininosuccinate synthase proteins from heterologous model microorganisms whose sequences have been ordered. [Figure 5a] This shows exemplary DNA sequences of the argG gene, which encodes the argininosuccinate synthase protein, from various heterologous model microbial species. [Figure 5b] This shows exemplary DNA sequences of the argG gene, which encodes the argininosuccinate synthase protein, from various heterologous model microbial species. [Figure 5c] This shows exemplary DNA sequences of the argG gene, which encodes the argininosuccinate synthase protein, from various heterologous model microbial species. [Figure 5d] This shows exemplary DNA sequences of the argG gene, which encodes the argininosuccinate synthase protein, from various heterologous model microbial species. [Figure 5e] This shows exemplary DNA sequences of the argG gene, which encodes the argininosuccinate synthase protein, from various heterologous model microbial species. [Figure 5f] This shows exemplary DNA sequences of the argG gene, which encodes the argininosuccinate synthase protein, from various heterologous model microbial species. [Figure 6] This shows exemplary DNA sequences of argR deletion mutations and argB(M54V) mutations in the argG gene, which encodes the argininosuccinate synthase protein. [Modes for carrying out the invention]

[0015] The amino acid molecules, host cells, nucleic acid molecules, vectors, and methods comprising the argininosuccinate synthase protein of this disclosure utilize, unless otherwise noted, the ordinary techniques and descriptions of molecular biology (including recombinant techniques), cell biology, biochemistry, and microarray and sequence analysis techniques that are part of the art of those skilled in the art. Such ordinary techniques include polymerase chain reaction (PCR), protein sequence alignment, and protein and oligonucleotide sequence analysis. Before describing the compositions, research tools, and methods of this disclosure, it should be understood that this disclosure may vary and is not limited to any particular methods, compositions, purposes, and uses of this disclosure. It should also be understood that the terms used in this disclosure are for the purpose of describing specific aspects and are not intended to limit the scope of this disclosure, which is limited only by the appended claims.

[0016] In some embodiments, the disclosure relates to improved amino acid molecules, including, for example, an improved argininosuccinate synthase protein, which is an enzyme responsible for the rate-limiting reaction step in the L-arginine production process. Argininosuccinate synthase promotes the synthesis of argininosuccinate from its substrates, citrulline and aspartate. Argininosuccinate is then broken down into fumarate and L-arginine by the action of argininosuccinate triase. L-arginine is an amino acid required for the production of nitric oxide, which regulates vasodilation, vascular tone, and blood flow. Therefore, L-arginine has been recognized for its vasodilatory effects and has been studied as an option for the treatment of high blood pressure, angina, and erectile dysfunction.

[0017] This disclosure further relates to recombinant and non-natural amino acid molecules comprising argininosuccinate synthase protein, engineered host cells capable of expressing the amino acid molecules and producing L-arginine, recombinant nucleic acid molecules and vectors for expressing the amino acid molecules, and uses and methods for producing or increasing L-arginine of the protein, host cell, vector and nucleic acid molecule.

[0018] As used in this disclosure, the terms “and / or” are used to indicate any combination of components. Furthermore, the singular forms “a,” “an,” and “the” include a plurality of referents unless the context clearly specifies otherwise. For example, “a nucleotide region” refers to one, one or more, or a mixture of such regions, and “an assay” refers to equivalent steps and methods known to those skilled in the art. When used in relation to a part of a nucleic acid or protein, the term “heterologous” indicates that the nucleic acid or protein contains two or more subsequences not found naturally in an identical relationship to one another. For example, nucleic acids are typically produced recombinantly with two or more unrelated gene sequences, e.g., a promoter from one source and a coding region from another source or coding regions from different sources, sequenced to create a new functional nucleic acid. Similarly, heterologous proteins indicate that a protein contains two or more subsequences not found naturally in an identical relationship to one another.

[0019] The term "conservative amino acid substitutions" refers to amino acid sequence modifications that do not eliminate enzyme function or binding sites. Conservative amino acid substitutions include replacing one type of amino acid with another of the same type, where the type is defined by the typical physicochemical properties of the amino acid residue and the high substitution frequency of naturally occurring homologous proteins, determined, for example, by the standard Dayhoff frequency exchange matrix or BLOSUM matrix. Six common types of amino acid residues are classified, including: Type I (Cys); Type II (Ser, Thr, Pro, Ala, Gly); Type III (Asn, Asp, Gln, Glu); Type IV (His, Arg, Lys); Type V (Ile, Leu, Val, Met); and Type VI (Phe, Tyr, Trp). For example, replacing Asp with another Type III residue such as Asn, Gln, or Glu is a conservative substitution. Therefore, amino acid residues predicted to be non-essential in a protein are preferably replaced with other amino acid residues of the same type. Methods for confirming amino acid conservation substitutions that do not eliminate enzyme activity are well known in this field.

[0020] As used in this disclosure, the term “variant” includes, but is not limited to, enzymes having an amino acid sequence that differs from the amino acid sequence of a reference protein through one or more substitutions, deletions, and / or additions at specific positions within or adjacent to the amino acid sequence of the reference protein. The variant may include one or more conservative substitutions in its amino acid sequence compared to the amino acid sequence of a reference antibody. Conservative substitutions may include, for example, substitutions to similarly charged or uncharged amino acids. The variant maintains, for example, the ability to specifically bind to the substrate of the reference protein. The term “variant” also includes PEGylated proteins.

[0021] Nucleic acid sequences implicitly include not only explicitly defined sequences but also their conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more (or all) selected codons is replaced with a mixture of bases and / or deoxyinosine residues. Batzer, et al., Nucleic Acid Res. 1991, 19, 5081; Ohtsuka, et al., J. Biol. Chem. 1985, 260, 2605-2608; Rossolini, et al., Mol. Cell. Probes 1994, 8, 91-98. The term nucleic acid is used interchangeably with cDNA, mRNA, oligonucleotides, and polynucleotides.

[0022] As used in this disclosure, the terms “homology” and “identity” refer to the degree of similarity between two given amino acid sequences or nucleotide sequences, and may be expressed as a percentage. The terms homology and identity are often used interchangeably.

[0023] As used in this disclosure, the terms “correspond(s) to” and “corresponding to” are intended to refer to the enumerated positions in the reference protein and the positions in the sequence of interest that align with the corresponding positions on the reference protein, in relation to sequence alignment. Thus, when the amino acid sequence of interest aligns with the amino acid sequence of the reference sequence, the amino acids in the target sequence that “correspond to” a particular numbered position in the reference sequence refer to the amino acids that align with the corresponding positions in the reference sequence, but do not necessarily have to be at the same numbered position in the reference sequence. Methods for aligning sequences to determine corresponding amino acids between sequences are well known in the art.

[0024] As used in this disclosure, the terms “amino acid molecule,” “peptide,” “polypeptide,” and “protein” generally refer to amino acid chains linked by peptide bonds (also known as amide bonds).

[0025] As used in this disclosure, "amino acid" refers to an organic molecule that contains both an amino group (i.e., -NH2) and a carboxylic acid group (i.e., -COOH).

[0026] As used in this disclosure, the terms “nucleotide molecule,” “polynucleotide,” or “oligonucleotide” refer to a polymer of nucleotides in which nucleotide monomers are covalently linked into a long chain, more specifically, a DNA or RNA chain of a certain length or longer as a polynucleotide fragment encoding a protein.

[0027] As used in this disclosure, the term “enhancement” of polypeptide activity means that the activity of the polypeptide is increased compared to its endogenous activity.

[0028] Furthermore, the term “operably linked” in relation to a promoter sequence that initiates and mediates the transcription of the polynucleotide encoding the target polypeptide of this disclosure means that the promoter sequence and the polynucleotide sequence are functionally linked to each other.

[0029] In one embodiment, the present disclosure provides an amino acid molecule, such as an amino acid molecule or protein. In some embodiments, the amino acid molecule may include the peptide sequence SEQ ID NO: 1, YKPWLDX1X2FX3X4EL, where X1, X2, and X4 are amino acids, respectively; and X3 is I or V.

[0030] In some instances, X1 in sequence number 1 is S. In some instances, X1 in sequence number 1 is Q. In some instances, X1 in sequence number 1 is T.

[0031] In some instances, X2 in sequence number 1 is D. In some instances, X2 in sequence number 1 is A. In some instances, X2 in sequence number 1 is T. In some instances, X2 in sequence number 1 is Q.

[0032] In some implementations, X3 in sequence number 1 is V. In some implementations, X3 in sequence number 1 is I.

[0033] In the additional embodiment, X4 in sequence number 1 is D.

[0034] In some implementations, sequence number 1 consists of a sequence selected from the group consisting of YKPWLDSAFIDEL (sequence number 2), YKPWLDQTFIDEL (sequence number 3), YKPWLDQQFIDEL (sequence number 4), and YKPWLDTDFIDEL (sequence number 5).

[0035] In some implementations, sequence number 1 consists of sequence YKPWLDSAFIDEL (sequence number 2). In some implementations, sequence number 1 consists of sequence YKPWLDQTFIDEL (sequence number 3). In some implementations, sequence number 1 consists of sequence YKPWLDQQFIDEL (sequence number 4). In some implementations, sequence number 1 consists of sequence YKPWLDTDFIDEL (sequence number 5).

[0036] The amino acid molecules (e.g., amino acid molecular proteins) of this disclosure are functionally "enhanced" and capable of producing larger amounts of L-arginine than naturally occurring enzymes; for example, their catalytic activity is enhanced compared to their naturally occurring counterparts. In some embodiments, the amino acid molecules originate from host cells or microorganisms. Thus, for example, the amino acid molecules originate from microorganisms that have been modified (e.g., artificially and / or specifically genetically modified) from naturally occurring mutants or strains. In some embodiments, the host cells or microorganisms of this disclosure may be Corynebacterium, Escherichia, Bacillus, Streptomyces, Penicillium, Klebsiella, Erwinia, or Pantoea. In some embodiments, the host cell or microorganism may be Acidobacterium capsulatum, Alcaligenes faecalis, Bacillus amyloliquefaciens, Burkholderia pyrrocinia, Corynebacterium ammoniagenes, Corynebacterium glutamicum, Cupriavidus necator, Escherichia coli, Mycobacterium smegmatis, or Neisseria weaveri.The amino acid molecules of this disclosure may, more specifically, be derived from microorganisms of the genus Corynebacterium, and more specifically from, but not limited to, Corynebacterium glutamicum, Corynebacterium Deserti, Corynebacterium crenatum, Corynebacterium efficiens, Corynebacterium suranareeae, and the like.

[0037] The term “enhancement” of polypeptides can be used interchangeably with terms such as activation, up-regulation, overexpression, and increase. Here, activation, enhancement, up-regulation, overexpression, and increase can all include exhibiting activity that was not originally present and exhibiting improved activity compared to the intrinsic activity or original activity before the amino acid sequence of the polypeptide was modified or altered. The “intrinsic activity” refers to the activity of a particular polypeptide that is originally present in the parent strain before transformation or in an unmodified host cell or microorganism when a trait is altered by a genetic mutation due to natural or artificial factors. The term “intrinsic activity” can be used interchangeably with the term “activity before modification.” Therefore, when polypeptide activity is described as "enhanced, up-regulated, overexpressed, or increased" compared to its endogenous activity, it means that the polypeptide activity is improved compared to the activity and / or concentration (expression level) of a particular polypeptide that is originally present in the parental strain before transformation or in the untransformed host cell or microorganism.

[0038] The aforementioned enhancement can be achieved by introducing an exogenous polypeptide or by enhancing the activity and / or concentration (expression level) of an endogenous polypeptide. Whether polypeptide activity is enhanced can be confirmed by an increase in polypeptide activity or expression level, or by the amount of product produced from polypeptide activity.

[0039] A variety of methods, including combinations of well-known processes, can be applied to enhance the activity of polypeptides. The claimed enhancement of peptide and / or activity is not limited insofar as the activity of the target polypeptide is enhanced compared to its activity in the host cell or microorganism before the modification. Specifically, the methods used to enhance the activity of peptides may be, but are not limited to, modifications utilizing genetic engineering and / or protein engineering commonly used in molecular biology and well known to the average technician in the art (e.g., Sitnicka et al., Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol. 2. 1-16, Sambrook et al. Molecular Cloning 2012).

[0040] In some embodiments, the enhancement of the polypeptide activity described herein can be achieved as follows:

[0041] 1) An increase in the intracellular copy number of the polynucleotide encoding the polypeptide. In additional embodiments, the increase in the intracellular copy number of the polynucleotide encoding the polypeptide can be achieved by introducing into the host cell a vector that is operablely linked to the polynucleotide encoding the polypeptide and can replicate and function independently of the host cell. Alternatively, the increase can be achieved by introducing one or more copies of the polynucleotide encoding the polypeptide into the chromosome of the host cell. Such introduction into the chromosome may be, but is not limited to, being carried out by introducing into the host cell a vector that can insert the polynucleotide into the chromosome of the host cell.

[0042] 2) Replacing a gene expression regulatory region on a chromosome encoding a polypeptide with a more potent sequence. The replacement of the gene expression regulatory region (or regulatory sequence) on a chromosome encoding a polypeptide with a more potent sequence may involve, for example, the generation of a sequence mutation by deletion, insertion, non-conservative or conservative substitution or a combination thereof to further enhance the activity of the regulatory region; or replacement with a sequence having stronger activity. The regulatory region may include, but is not limited to, promoters, operator sequences, sequences encoding ribosome binding sites, and sequences that regulate transcription and translation termination. As an example, the replacement may involve replacing the original promoter with a more potent promoter. Examples of well-known strong promoters include, but are not limited to, the CJ1-CJ7 promoters (US 7662943 B2), the lac promoter, the trp promoter, the trc promoter, the tac promoter, the lambda phage PR promoter, the PL promoter, the tet promoter, the gapA promoter, the SPL7 promoter, the SPL13 (sm3) promoter (US 10584338 B2), the O2 promoter (US 10273491 B2), the tkt promoter, and the yccA promoter.

[0043] 3) Modification of the start codon or nucleotide sequence encoding the 5'-UTR region of the polypeptide-encoding gene transcript. The modification of the start codon or nucleotide sequence encoding the 5'-UTR region of the polypeptide-encoding gene transcript may include, for example, substitution with a nucleotide sequence encoding another (i.e., different) start codon that has a higher polypeptide expression rate than the endogenous start codon.

[0044] 4) Modification of the amino acid sequence of a polypeptide or modification of the polynucleotide sequence encoding the polypeptide to enhance polypeptide activity (e.g., modification of the polynucleotide sequence of a polypeptide gene encoding a modified polypeptide to enhance polypeptide activity). The modification of the amino acid sequence or polynucleotide sequence may be achieved by generating mutations 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, so as to enhance polypeptide activity, or by replacing the sequence 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. The replacement may be specifically carried out by inserting a polynucleotide into a chromosome by homologous recombination, but may not be limited thereto. The vector used in this case may further include a selection marker for confirming the presence or absence of chromosomal insertion.

[0045] 5) Introduction of an exogenous polypeptide exhibiting polypeptide activity or an exogenous polynucleotide encoding it. The introduction of the exogenous polynucleotide exhibiting polypeptide activity may be done by introducing an exogenous polynucleotide encoding a polypeptide exhibiting the same or similar activity as the polypeptide into the host cell. The exogenous polynucleotide is not restricted in its origin or sequence, as long as it exhibits the same or similar activity as the polypeptide. A person skilled in the art can appropriately select a known transformation method for the introduction. The introduction of the polynucleotide in the host cell can lead to the production of the polypeptide and an increase in its activity.

[0046] 6) Codon optimization of polynucleotides encoding polypeptides. The codon optimization of polynucleotides encoding polypeptides may be codon optimization of endogenous polynucleotides to increase transcription or translation in host cells, or codon optimization of exogenous polynucleotides to enable optimized transcription and translation in host cells.

[0047] 7) Analyze the tertiary structure of the polypeptide, select exposed sites to deform, or chemically modify. Analyzing the tertiary structure of the polypeptide, selecting exposed sites to deform, or chemically modify may, for example, involve comparing the sequence information of the polypeptide to be analyzed with a database containing sequence information of known proteins to determine candidate template proteins according to the degree of sequence similarity, confirming the structure based on that, and selecting exposed sites to deform or chemically modify.

[0048] 8) A combination of two or more items selected from items 1) to 7) above is also acceptable, but is not limited to this.

[0049] Such enhancement of amino acid molecule activity may be, but is not limited to, an increase in the activity or concentration level of the corresponding amino acid molecule compared to the activity or concentration of the amino acid molecule expressed in wild-type host cells or host cells before deformation, or an increase in the amount of products produced by the amino acid molecule.

[0050] In additional embodiments, the amino acid molecule is, for example, a recombinant protein such as recombinant argininosuccinate synthase.

[0051] In an additional embodiment, the amino acid molecule is sequence number 6, X5PX6X7X8X9GX 10 AFSGGLDTSX 11 AX 12It includes an array having. Here, X5 is I, L or V; X6 is an amino acid; X7 is A, E or Q; X8 is K or R; X9 is I or V; X 10 is I or L; X 11 is A, T or V; X 12 is I, L or V.

[0052] In some embodiments, X5 of SEQ ID NO: 6 is I. In some embodiments, X5 of SEQ ID NO: 6 is L. In some embodiments, X5 of SEQ ID NO: 6 is V.

[0053] In some embodiments, X7 of SEQ ID NO: 6 is A. In some embodiments, X7 of SEQ ID NO: 6 is E. In some embodiments, X7 of SEQ ID NO: 6 is Q.

[0054] In some embodiments, X8 of SEQ ID NO: 6 is K. In some embodiments, X8 of SEQ ID NO: 6 is R.

[0055] In some embodiments, X9 of SEQ ID NO: 6 is I. In some embodiments, X9 of SEQ ID NO: 6 is V.

[0056] In some embodiments, X 10 of SEQ ID NO: 6 is I. In some embodiments, X 10 of SEQ ID NO: 6 is L.

[0057] In some embodiments, X 11 of SEQ ID NO: 6 is A. In some embodiments, X 11 of SEQ ID NO: 6 is T. In some embodiments, X 11 of SEQ ID NO: 6 is V.

[0058] In some embodiments, X 12 of SEQ ID NO: 6 is I. In some embodiments, X 12 of SEQ ID NO: 6 is L. In some embodiments, X 12 of SEQ ID NO: 6 is V.

[0059] In an additional embodiment, the amino acid molecule is represented by Sequence ID No. 7, X 13 GAX a X 14 X 15 X 16 YTA X 17 X 18 GQ X 19 Includes a sequence having DE. Here, X 13 is K or N; X a It is an amino acid; X 14 is C or P; X 15 is C or Y; X 16 is A, S or T; X 17 is D or N; X 18 is I or L; X 19 It is A, P, or Y.

[0060] In some concrete examples, X of sequence number 7 13 is K. In some concrete examples, X of sequence number 7. 13 It is N.

[0061] In some concrete examples, X of sequence number 7 14 is C. In some concrete examples, X of sequence number 7 14 P is P.

[0062] In some concrete examples, X of sequence number 7 15 is C. In some concrete examples, X of sequence number 7 15 It is Y.

[0063] In some concrete examples, X of sequence number 7 16 is A. In some concrete examples, X of sequence number 7 16 is S. In some concrete examples, X of sequence number 7. 16 It is T.

[0064] In some concrete examples, X of sequence number 7 17 It is D. In some concrete examples, X of sequence number 7. 17 It is N.

[0065] In some concrete examples, X of sequence number 7 18 is I. In some concrete examples, X of sequence number 7. 18 It is L.

[0066] In some concrete examples, X of sequence number 7 19 is A. In some concrete examples, X of sequence number 7 19 is P. In some concrete examples, X of sequence number 7 19 It is Y.

[0067] In an additional embodiment, the amino acid molecule is represented by Sequence ID No. 8, X 20 X 21 X 22 X 23 X 24 X 25 X 26 X a LX 27 Includes an array having X. 20 is A or S; X 21 is R or V; X 22 is I or L; X 23 is I or V; X 24 is E or D; X 25 is C or G; X 26 is K or R; X a It is an amino acid; X 27 It is either A or V.

[0068] In some concrete examples, X of sequence number 8 20 is A. In some concrete examples, X of sequence number 8. 20 S is.

[0069] In some concrete examples, X of sequence number 8 21 is R. In some concrete examples, X of sequence number 8 21 V is V.

[0070] In some concrete examples, X of sequence number 8 22 is I. In some concrete examples, X of sequence number 8. 22 It is L.

[0071] In some embodiments, X of SEQ ID NO: 8 23 is I. In some embodiments, X of SEQ ID NO: 8 23 is V.

[0072] In some embodiments, X of SEQ ID NO: 8 24 is E. In some embodiments, X of SEQ ID NO: 8 24 is D.

[0073] In some embodiments, X of SEQ ID NO: 8 25 is C. In some embodiments, X of SEQ ID NO: 8 25 is G.

[0074] In some embodiments, X of SEQ ID NO: 8 26 is K. In some embodiments, X of SEQ ID NO: 8 26 is R.

[0075] In some embodiments, X of SEQ ID NO: 8 27 is A. In some embodiments, X of SEQ ID NO: 8 27 is V.

[0076] In additional embodiments, the amino acid molecule comprises a sequence having SEQ ID NO: 9, X 28 AFX 29 X a X a X 30 X 31 and G. Here, X 28 is G or N; X 29 is H or N; X a is an amino acid; X 30 is S or T; X 31 is A or G.

[0077] In some embodiments, X of SEQ ID NO: 9 28 is G. In some embodiments, X of SEQ ID NO: 9 28 is N.

[0078] In some embodiments, X of SEQ ID NO: 9 29 is H. In some embodiments, X of SEQ ID NO: 9 29 is N.

[0079] In some embodiments, X of SEQ ID NO: 9 30 is S. In some embodiments, X of SEQ ID NO: 9 30 is T.

[0080] In some embodiments, X of SEQ ID NO: 9 31 is A. In some embodiments, X of SEQ ID NO: 9 31 is G.

[0081] In additional embodiments, the amino acid molecule comprises a sequence having SEQ ID NO: 10, YFNTTPX 32 GRAVX 33 X 34 TX 35 LV. Here, X 32 is I or L; X 33 is A or T; X 34 is A or G; X 35 is L or M.

[0082] In some embodiments, X of SEQ ID NO: 10 32 is I. In some embodiments, X of SEQ ID NO: 10 32 is L.

[0083] In some embodiments, X of SEQ ID NO: 10 33 is A. In some embodiments, X of SEQ ID NO: 10 33 is T.

[0084] In some embodiments, X of SEQ ID NO: 10 34 is A. In some embodiments, X of SEQ ID NO: 10 34 is G.

[0085] In some embodiments, X of SEQ ID NO: 10 35 is L. In some embodiments, X of SEQ ID NO: 10 35It is M.

[0086] In an additional embodiment, the amino acid molecule is SEQ ID NO: 11, TX 36 Includes a sequence containing KGNDIERF, where X 36 It is either F or Y.

[0087] In some concrete examples, X of sequence number 11 36 is F. In some concrete examples, X of sequence number 11. 36 It is Y.

[0088] In an additional embodiment, the amino acid molecule is SEQ ID NO: 12, YRYGLX 37 X 38 Includes an array having N, where X 37 is L or V; X 38 It is A, T, or V.

[0089] In some concrete examples, X of sequence number 12 37 is L. In some concrete examples, X of sequence number 12 37 V is V.

[0090] In some concrete examples, X of sequence number 12 38 is A. In some concrete examples, X of sequence number 12 38 is T. In some concrete examples, X of sequence number 12 38 V is V.

[0091] In an additional embodiment, the amino acid molecule is SEQ ID NO: 13, GGRX a EMX 39 X 40 X 41 X 42 Includes an array having X. a It is an amino acid; X 39 is A or S; X 40 is A, E, or Q; X 41 is F, W, or Y; X 42 It is L or M.

[0092] In some concrete examples, X of sequence number 13 39 is A. In some concrete examples, X of sequence number 13 39 S is.

[0093] In some concrete examples, X of sequence number 13 40 is A. In some concrete examples, X of sequence number 13 40 It is E. In some concrete examples, X of sequence number 13. 40 Q is Q.

[0094] In some concrete examples, X of sequence number 13 41 is F. In some concrete examples, X of sequence number 13. 41 is W. In some concrete examples, X of sequence number 13. 41 It is Y.

[0095] In some concrete examples, X of sequence number 13 42 is L. In some concrete examples, X of sequence number 13. 42 It is M.

[0096] In an additional embodiment, the amino acid molecule is SEQ ID NO: 14, EKAYSTDX 43 NX 44 X 45 Includes a sequence containing GATHE. Here, X 43 is A or S; X 44 is I, L, or M; X 45 It is either L or W.

[0097] In some concrete examples, X of sequence number 14 43 is A. In some concrete examples, X of sequence number 14. 43 S is.

[0098] In some concrete examples, X of sequence number 14 44 is I. In some concrete examples, X of sequence number 14 44 is L. In some concrete examples, X of sequence number 14 44 It is M.

[0099] In some concrete examples, X of sequence number 14 45 is L. In some concrete examples, X of sequence number 14 45 It is W.

[0100] In an additional embodiment, the amino acid molecule is SEQ ID NO: 15, VX a PIMGVX a X 46 Includes an array having W, where X a It is an amino acid, X 46 It is F, H, or S.

[0101] In some concrete examples, X of sequence number 15 46 is F. In some concrete examples, X of sequence number 15. 46 is H. In some concrete examples, X of sequence number 15. 46 S is.

[0102] In an additional embodiment, the amino acid molecule is represented by Sequence ID No. 16, X 47 GGRHGX 48 GX 49 X 50 DQIENRX 51 Includes a sequence having IEA. Here, X 47 is I or V; X 48 is L or M; X 49 is M or V; X 50 is A or S; X 51 It is either I or V.

[0103] In some concrete examples, X of sequence number 16 47 is I. In some concrete examples, X of sequence number 16. 47 V is V.

[0104] In some concrete examples, X of sequence number 16 48 is L. In some concrete examples, X of sequence number 16 48 It is M.

[0105] In some concrete examples, X of sequence number 16 49is M. In some concrete examples, X of sequence number 16. 49 V is V.

[0106] In some concrete examples, X of sequence number 16 50 is A. In some concrete examples, X of sequence number 16 50 S is.

[0107] In some concrete examples, X of sequence number 16 51 is I. In some concrete examples, X of sequence number 16. 51 V is V.

[0108] In an additional embodiment, the amino acid molecule includes the sequence having SEQ ID NO: 17, KSRGIYEAPG.

[0109] In an additional embodiment, the amino acid molecule is represented by Sequence ID No. 18, X 52 ALX 53 X 54 X 55 AX 56 ERL X a X 57 X 58 Includes a sequence having IHNEDT. Here, X 52 is L or M; X 53 is F or L; X 54 is F, H, or Y; X 55 is A or I; X 56 is F or Y; X a It is an amino acid; X 57 is N, S, or T; X 58 It is either A or G.

[0110] In some concrete examples, X of sequence number 18 52 is L. In some concrete examples, X of sequence number 18 52 It is M.

[0111] In some concrete examples, X of sequence number 18 53 is F. In some concrete examples, X of sequence number 18. 53 It is L.

[0112] In some concrete examples, X of sequence number 18 54 is F. In some concrete examples, X of sequence number 18. 54 is H. In some concrete examples, X of sequence number 18. 54 It is Y.

[0113] In some concrete examples, X of sequence number 18 55 is A. In some concrete examples, X of sequence number 18. 55 It is I.

[0114] In some concrete examples, X of sequence number 18 56 is F. In some concrete examples, X of sequence number 18. 56 It is Y.

[0115] In some concrete examples, X of sequence number 18 57 It is N. In some concrete examples, X of sequence number 18 57 is S. In some concrete examples, X of sequence number 18. 57 It is T.

[0116] In some concrete examples, X of sequence number 18 58 is A. In some concrete examples, X of sequence number 18. 58 It is G.

[0117] In an additional embodiment, the amino acid molecule is SEQ ID NO: 19, LGX 59 LX 60 YX 61 GRWX 62 DX 63 QX 64 X 65 MX 66 RX 67 Includes an array having X. 59 is K or R; X 60 is L or M; X 61 is A, E, or Q; X 62 is F or L; X 63 is P or S; X 64 is A, S, or G; X65 is I, L, or M; X 66 is I, L, or V; X 67 It is either D or E.

[0118] In some concrete examples, X of sequence number 19 59 is K. In some concrete examples, X of sequence number 19. 59 It is R.

[0119] In some concrete examples, X of sequence number 19 60 is L. In some concrete examples, X of sequence number 19 60 It is M.

[0120] In some concrete examples, X of sequence number 19 61 is A. In some concrete examples, X of sequence number 19 61 It is E. In some concrete examples, X of sequence number 19 61 Q is Q.

[0121] In some concrete examples, X of sequence number 19 62 is F. In some concrete examples, X of sequence number 19 62 It is L.

[0122] In some concrete examples, X of sequence number 19 63 is P. In some concrete examples, X of sequence number 19 63 S is.

[0123] In some concrete examples, X of sequence number 19 64 is A. In some concrete examples, X of sequence number 19 64 is S. In some concrete examples, X of sequence number 19 64 It is G.

[0124] In some concrete examples, X of sequence number 19 65 is I. In some concrete examples, X of sequence number 19 65 is L. In some concrete examples, X of sequence number 19 65 It is M.

[0125] In some concrete examples, X of sequence number 19 66 is I. In some concrete examples, X of sequence number 19 66 is L. In some concrete examples, X of sequence number 19 66 V is V.

[0126] In some concrete examples, X of sequence number 19 67 It is D. In some concrete examples, X of sequence number 19 67 It is E.

[0127] In an additional embodiment, the amino acid molecule is SEQ ID NO: 20, LRRGX a DX 68 X 69 X 70 Includes an array having X. a It is an amino acid; X 68 is F or Y; X 69 is S or T; X 70 It is either I or L.

[0128] In some concrete examples, X of sequence number 20 68 is F. In some concrete examples, X of sequence number 20. 68 It is Y.

[0129] In some concrete examples, X of sequence number 20 69 is S. In some concrete examples, X of sequence number 20. 69 It is T.

[0130] In some concrete examples, X of sequence number 20 70 is I. In some concrete examples, X of sequence number 20. 70 It is L.

[0131] In an additional embodiment, the amino acid molecule is represented by Sequence ID No. 21, X 71 X 72 X 73 X 74 X a X 75 X 76 X77 LX 78 MEX 79 Includes an array having X. 71 is A, D, or N; X 72 is F or L; X 73 is S or T; X 74 is F or Y; X a It is an amino acid; X 75 is A, P, or S; X 76 is A, E, or D; X 77 is K or R; X 78 is S or T; X 79 It is either K or R.

[0132] In some concrete examples, X of sequence number 21 71 is A. In some concrete examples, X of sequence number 21 71 It is D. In some concrete examples, X of sequence number 21 71 It is N.

[0133] In some concrete examples, X of sequence number 21 72 is F. In some concrete examples, X of sequence number 21 72 It is L.

[0134] In some concrete examples, X of sequence number 21 73 is S. In some concrete examples, X of sequence number 21 73 It is T.

[0135] In some concrete examples, X of sequence number 21 74 is F. In some concrete examples, X of sequence number 21 74 It is Y.

[0136] In some concrete examples, X of sequence number 21 75 is A. In some concrete examples, X of sequence number 21 75 is P. In some concrete examples, X of sequence number 21 75 S is.

[0137] In some concrete examples, X of sequence number 2176 is A. In some concrete examples, X of sequence number 21 76 is E. In some concrete examples, X of sequence number 21 76 It is D.

[0138] In some concrete examples, X of sequence number 21 77 is K. In some concrete examples, X of sequence number 21 77 It is R.

[0139] In some concrete examples, X of sequence number 21 78 is S. In some concrete examples, X of sequence number 21 78 It is T.

[0140] In some concrete examples, X of sequence number 21 79 is K. In some concrete examples, X of sequence number 21 79 It is R.

[0141] In an additional embodiment, the amino acid molecule is SEQ ID NO: 22, DRIGQLX 80 MRX 81 X 82 DX 83 X a DX 84 Includes a sequence having R, where X 80 is H or T; X 81 is L, N, or T; X 82 is L or N; X 83 is I, L, or V; X a It is an amino acid; X 84 It is either S or T.

[0142] In some concrete examples, X of sequence number 22 80 is H. In some concrete examples, X of sequence number 22 80 It is T.

[0143] In some concrete examples, X of sequence number 22 81 is L. In some concrete examples, X of sequence number 22 80is N. In some concrete examples, X of sequence number 22 80 It is T.

[0144] In some concrete examples, X of sequence number 22 82 is L. In some concrete examples, X of sequence number 22 82 It is N.

[0145] In some concrete examples, X of sequence number 22 83 is I. In some concrete examples, X of sequence number 22 83 is L. In some concrete examples, X of sequence number 22 83 V is V.

[0146] In some concrete examples, X of sequence number 22 84 is S. In some concrete examples, X of sequence number 22 84 It is T.

[0147] In additional embodiments, the amino acid molecule includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 sequences(et) selected from the group consisting of SEQ ID NOs: 6 to 22. In some embodiments, the amino acid molecule includes all of SEQ ID NOs: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22.

[0148] In additional embodiments, the amino acid molecule of the present disclosure may have a length of at least 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, or 171 amino acids and / or 200, 199, 198, 197, 196, 195, 194, 193, 192, 191, 190, 189, 188, 187, 186, 185, 184, 183, 182, 181, 180, 179, 178, 177, 176, 175, 174, 173, 172, 171, 170, 169, 168, 167, 166, 165, 164, 163, 162, 161, 160, 159, 158, 157, 156, 155, 154, 153, 152, 151 amino acids or less.

[0149] In additional embodiments, the amino acid molecule includes a sequence having at least 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity with SEQ ID NO: 23, which is an A. capsulatum sequence.

[0150] In additional embodiments, the amino acid molecule includes a sequence corresponding to at least 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% of positions 1 to 153 and 167 to 445 of SEQ ID NO: 23. In further additional embodiments, the amino acid molecule includes a sequence corresponding to positions 1 to 153 and 167 to 445 of SEQ ID NO: 23.

[0151] In additional embodiments, the amino acid molecule includes a sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity with the A.faecalis sequence (SEQ ID NO: 24).

[0152] In additional embodiments, the amino acid molecule includes sequences corresponding to positions 1-153 and 167-444 of SEQ ID NO: 24 and at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%. In further additional embodiments, the amino acid molecule includes sequences corresponding to positions 1-153 and 167-444 of SEQ ID NO: 24.

[0153] In additional embodiments, the amino acid molecule includes a sequence having at least 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity with sequence number 26, which is a B. pyrrocinia sequence.

[0154] In additional embodiments, the amino acid molecule includes sequences corresponding to positions 1-153 and 167-445 of SEQ ID NO: 26 and at least 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%. In further additional embodiments, the amino acid molecule includes sequences corresponding to positions 1-153 and 167-445 of SEQ ID NO: 26.

[0155] In additional embodiments, the amino acid molecule includes a sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity with the C. necator sequence (SEQ ID NO: 29).

[0156] In other embodiments, the amino acid molecule includes sequences corresponding to positions 1-153 and 167-441 of SEQ ID NO: 29 at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%. In further additional embodiments, the amino acid molecule includes sequences corresponding to positions 1-153 and 167-441 of SEQ ID NO: 29.

[0157] In additional embodiments, the amino acid molecule includes a sequence having at least 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity with the E. coli sequence (SEQ ID NO: 30).

[0158] In other embodiments, the amino acid molecule includes sequences corresponding to positions 1-153 and 167-447 of SEQ ID NO: 30 and at least 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%. In further additional embodiments, the amino acid molecule includes sequences corresponding to positions 1-153 and 167-447 of SEQ ID NO: 30.

[0159] In additional embodiments, the amino acid molecule includes a sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity with the N. weaveri sequence (SEQ ID NO: 32).

[0160] In additional embodiments, the amino acid molecule includes sequences corresponding to positions 1-157 and 171-448 of SEQ ID NO: 32 and at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%. In further additional embodiments, the amino acid molecule includes sequences corresponding to positions 1-157 and 171-448 of SEQ ID NO: 32.

[0161] In additional embodiments, the amino acid molecule has at least 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity with SEQ ID NO: 23; at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity with SEQ ID NO: 25; Includes sequences that have sequence identity; sequence identity with sequence number 26 of at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%; sequence identity with sequence number 27 of at least 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%; and sequence that has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%.

[0162] Furthermore, the present disclosure provides host cells or microorganisms that express the amino acid molecules of this disclosure in other forms.

[0163] Furthermore, in other forms, the present disclosure provides a nucleic acid molecule comprising a nucleic acid sequence encoding the amino acid molecule of the present disclosure.

[0164] In other ways, the Disclosure relates to sequences having at least 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity with sequence number 34, which is an A. faecalis sequence; and sequences having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity with a B. pyrrocinia sequence. Sequences having at least 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity with sequence number 36, which is a C. necator sequence; Sequences having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity with an E. coli sequence (E. coli The present invention provides a nucleic acid molecule comprising a sequence having at least 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity with sequence number 37, which is an N. weaveri sequence, and a sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity with sequence number 38, which is an N. weaveri sequence, wherein the nucleic acid molecule does not exist in nature.

[0165] In other embodiments, vectors comprising nucleic acid molecules of the present disclosure are also provided. In some embodiments of the present disclosure, “vector” may include a DNA product comprising a nucleotide sequence of polynucleotides encoding the polypeptide of interest, operably linked to a suitable regulatory region (or regulatory sequence) so that the polypeptide of interest can be expressed in a suitable host. The regulatory region may include a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA-ribosome binding site, and sequences regulating the termination of transcription and translation. After transformation in a suitable host cell, the vector can replicate or function independently of the host genome and integrate into the genome itself.

[0166] 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 natural or recombinant plasmids, cosmids, viruses, and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage vectors or cosmid vectors, and the pDZ system, pBR system, pUC system, pBluescript II system, pGEM system, pTZ system, pCL system, and pET system can be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC vectors can be used. Additional information regarding the vector can be found in U.S. Patent Application Publication No. 2023 / 0134555, which is included in its entirety for reference.

[0167] As an example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome via a chromosome insertion vector within the cell. Chromosome insertion of the polynucleotide may be performed by any method known in the art, such as homologous recombination, but is not limited thereto. A selection marker may further be included to confirm chromosome insertion. The selection marker is used to select cells transformed with the vector, i.e., to confirm the presence or absence of insertion of the target nucleic acid molecule, and may confer a selectable phenotype such as drug resistance, nutritional requirements, resistance to cytotoxic agents, or expression of a surface polypeptide. Transformed cells can be selected so that only cells expressing the selection marker survive or exhibit other phenotypes under conditions treated with a selective agent.

[0168] Modifications of some or all of the polynucleotides in the host cell or microorganism, as embodied in some of the embodiments of this disclosure, may be induced by (a) homologous recombination using a vector for chromosome insertion into the host cell or microorganism, or by genome editing using a manipulated nuclease (e.g., CRISPR-Cas9), and / or (b) by light and / or chemical treatment such as ultraviolet light and radiation. Methods for modifying some or all of the gene may include methods using DNA recombination techniques. For example, a nucleotide sequence or vector containing a nucleotide sequence homologous to the target gene may be injected into the host cell or microorganism to induce homologous recombination and thereby delete some or all of the gene. The injected nucleotide sequence or vector may include, but is not limited to, a dominant selection marker.

[0169] In the polynucleotides according to some embodiments of the present disclosure, various modifications may be made to the coding region, taking into account codon degeneracy or the highest-priority codon in an organism expressing the protein of the present disclosure, within the limits that the amino acid sequence of the protein does not change. Another aspect of the present disclosure is to provide a polynucleotide comprising a nucleic acid sequence encoding the protein, which does not exist in nature.

[0170] The homology or identity of conserved polynucleotide or polypeptide sequences is determined by standard alignment algorithms, which may also be used in conjunction with a default gap penalty established by the program used. Substantially homologous or identical sequences can generally hybridize all or part of the sequence under moderate or high stringent conditions. It is obvious that hybridization also includes hybridization with polynucleotides containing common codons or codons considering codon degeneracy.

[0171] Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined using known computer algorithms such as the "FASTA" program with default parameters as in the literature [Pearson et al. (1988), Proc. Natl. Acad. Sci. USA 85]:2444]. Alternatively, homology, similarity or identity can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [et al., J. Mol. Biol. 215]:403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego, 1994, and [CARILLO et al.](1988) SIAM J Applied Math 48:1073) as performed by 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 higher) (including the GCG program package (Devereux, J., et al., Nucleic Acids Research 12:387 (1984))). For example, homology, similarity or identity can be determined using BLAST or ClustalW of the National Center for Biotechnology Information.

[0172] The homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using a GAP computer program such as [Needleman et al. (1970), J Mol Biol. 48:443], as is publicly known in [Smith and Waterman, Adv. Appl. Math. (1981) 2:482]. In summary, a GAP program can be defined as a value obtained by dividing the total number of symbols in the shorter sequence of two sequences by the number of similarly aligned symbols (i.e., nucleotides or amino acids). The default parameters for the GAP program include (1) a binary comparison matrix (containing values ​​of 1 for identity and 0 for non-identity) and a weighted comparison matrix (or EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix) as disclosed in the literature [Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979)], as well as the literature [Gribskov et al. (1986) Nucl. Acids Res. 14:6745]; (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 and a gap expansion penalty of 0.5); and (3) no penalty for end gaps.

[0173] In additional aspects, the Disclosure discloses host cells, microorganisms, or “strains.” As used in the Disclosure, the term “host cell” includes all unmodified wild-type host cells or host cells in which genetic mutations occur naturally or “transformation” occurs artificially. In some embodiments, the host cells in the Disclosure may be microorganisms. The term “microorganism (or strain)” includes all unmodified wild-type microorganisms or microorganisms in which genetic mutations occur naturally or “transformation” occurs artificially. As used in the Disclosure, the terms “unmodified host cell” or “unmodified microorganism” refer to wild-type strains or natural strains themselves, or strains before their traits are altered by genetic mutations due to natural or artificial factors, without excluding strains containing naturally occurring mutations in host cells or microorganisms. For example, an unmodified microorganism refers to a strain in which the activity of the protein in the Disclosure has not been enhanced or has not yet been enhanced. The term "unmodified microorganism" can be used interchangeably with "strain before modification," "microorganism before modification," "unmutated strain," "unmodified strain," "unmutated microorganism," or "reference microorganism."

[0174] As used in this disclosure, the term “transformation” refers to introducing a vector containing a polynucleotide encoding a target polypeptide into a host cell or microorganism so that the polynucleotide-encoded polypeptide can be expressed in the host cell. The transformed polynucleotide may include all transformed polynucleotides located inside the host cell’s chromosomes, and all transformed polynucleotides located outside the chromosomes, as long as they can be expressed in the host cell. The polynucleotide may also include DNA and / or RNA encoding the target polypeptide. The polynucleotide may be introduced into the host cell in any form, as long as it can be introduced into the host cell and expressed. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a gene product containing all the elements necessary for self-expression. The expression cassette may generally include a promoter operably linked to the polynucleotide, a transcription termination signal, a ribosome binding site, and a translation termination signal. The expression cassette may also be in the form of a self-replicating expression vector. The polynucleotide may also be introduced into the host cell in its own form and operably linked to the sequences necessary for expression in the host cell, but is not limited thereto.

[0175] In some embodiments, the manipulated host cell or microorganism of the present disclosure may include one, two, three or more vectors, each containing different nucleotide sequences. In some embodiments, the manipulated host cell or microorganism of the present disclosure may include one, two, three or more vectors, each containing nucleotide sequences encoding different amino acid molecules.

[0176] Artificially modified host cells or microorganisms, i.e., “engineered host cells or microorganisms,” refer to host cells or microorganisms in which specific mechanisms are weakened or strengthened, including through genetic modification to produce a target polypeptide, protein, or product, such as insertion of an exogenous gene or enhancement or inactivation of an endogenous gene. Engineered host cells, microorganisms, or strains as embodied in some embodiments of this disclosure are transformed to express recombinant nucleic acid sequences and / or to produce recombinant amino acid molecules or argininosuccinate synthase proteins.

[0177] In one aspect, the present disclosure provides a host cell, microorganism, or strain expressing argininosuccinate synthase. The host cell or microorganism may be an engineered host cell or microorganism, which has been artificially transformed, modified, or mutated by a method known to those skilled in the art to produce a host cell or microorganism that does not exist in nature.

[0178] In some embodiments of the host cell according to this disclosure, the amino acid sequence for the amino acid molecule or argininosuccinate synthase of this disclosure is exogenous to the host cell. Specifically, in some embodiments, the amino acid molecule or argininosuccinate synthase of this disclosure is exogenous to the host cell. In this disclosure, “extrinsic” sequence means that the sequence does not exist in the host cell without any modification, in particular without any artificial modification. The host cell can be modified and may produce amino acid molecules or argininosuccinate synthase from other organisms, or unnatural recombinant amino acid molecules or argininosuccinate synthase that do not exist naturally in the host cell (i.e., without any modification). In some embodiments of the host cell according to this disclosure, the amino acid sequence for the amino acid molecule or argininosuccinate synthase of this disclosure is an endogenous amino acid sequence of a microorganism. In this disclosure, “intrinsic” sequence means that the sequence exists in a host cell without any modification. The host cell is modified to produce a recombinant amino acid molecule or argininosuccinate synthase having the intrinsic sequence of the host cell.

[0179] In the additional embodiments of the host cell according to this disclosure, the nucleotide sequence encoding the argininosuccinate synthase protein is an exogenous nucleotide sequence of the host cell. In the additional embodiments of the host cell according to this disclosure, the nucleotide sequence encoding the argininosuccinate synthase protein is an endogenous nucleotide sequence of the host cell.

[0180] In some embodiments of the present disclosure, the recombinant or modified host cell may be a host cell having increased L-arginine production capacity compared to a natural wild-type host cell, because the activity of the protein or the polynucleotide encoding it is improved in the recombinant or modified host cell compared to a natural wild-type host cell, but is not limited thereto. The L-arginine-producing recombinant or modified host cell of the present disclosure (e.g., of the genus Corynebacterium) can produce L-arginine in high yield and can therefore be used for the industrial production of L-arginine.

[0181] For example, the L-arginine production capacity of the recombinant or modified host cells increased to approximately 0.3% or more compared to the L-arginine production capacity of the parental strain before mutation or the unmodified host cells. Specifically, this increased to approximately 0.5% or more, approximately 1% or more, approximately 2% or more, approximately 3% or more, approximately 4% or more, approximately 5% or more, approximately 6% or more, approximately 7% or more, approximately 8% or more, approximately 9% or more, approximately 10% or more, approximately 10.7% or more, and approximately 11% or more. The production capacity can be increased to approximately 12% or more, approximately 12.3% or more, approximately 13% or more, approximately 14% or more, approximately 14.4% or more, approximately 15% or more, approximately 15.1% or more, approximately 16% or more, or approximately 16.9% or more (there is no particular upper limit, and production capacity can be increased to, for example, approximately 200% or less, approximately 150% or less, approximately 100% or less, approximately 50% or less, approximately 40% or less, approximately 30% or less, or approximately 20% or less). In some embodiments, L-arginine production by the manipulated host cells of the Disclosure may increase to at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or 50% and / or up to about 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, or 40%. However, the production capacity is not limited to these, as long as it has a positive value of the increase compared to the production capacity of the parental strain before mutation or its unmutated host cells. In some embodiments, the parental strain may be cells before transformation with the amino acid molecules of the Disclosure.

[0182] In other examples, the L-arginine production capacity of the host cells with increased production capacity was approximately 1.005 times, 1.01 times, 1.02 times, 1.03 times, 1.04 times, 1.05 times, 1.06 times, 1.07 times, 1.08 times, 1.09 times, 1.10 times, 1.107 times, and 1.11 times higher than the L-arginine production capacity of the parent strain before mutation or the unmutated host cells. It can be increased by more than double, approximately 1.12 times or more, approximately 1.123 times or more, approximately 1.13 times or more, approximately 1.14 times or more, approximately 1.144 times or more, approximately 1.15 times or more, approximately 1.151 times or more, approximately 1.16 times or more, approximately 1.169 times or more, 1.2 times or more, 1.3 times or more, and 1.4 times or more (there is no particular upper limit, and production capacity can be increased to, for example, approximately 10 times or less, approximately 5 times or less, approximately 3 times or less, or approximately 2 times or less), but it is not limited to these.

[0183] As used in this disclosure, the term “about” is relative to the expressed actual value, as understood by those skilled in the art, and allows for approximations, inaccuracies, and limitations of the measurement under the relevant circumstances. In one or more manners, the terms “about,” “substantially,” and “approximately” may provide industry-acceptable tolerances and / or relatives between items, e.g., tolerances from less than 1% to 10% of the expressed actual value, and other suitable tolerances. The described actual value may be, for example, the length of a nucleotide sequence, the degree of error, the size, the amount, concentration, volume of a component in a composition, process temperature, process time, yield, flow rate, pressure, and / or a range of such actual values. In some cases, such tolerances may arise, for example, through typical measurement and handling procedures used to manufacture a compound, composition, concentrate, or dosage form; due to careless errors in such procedures; through differences in the manufacture, source, or purity of the starting materials or components used to carry out the method; and through similar considerations. The term “about” also includes, for example, amounts that change due to the maturation of a composition, dosage form, or cell culture having a particular initial concentration or mixture, and amounts that change due to the mixing or processing of a composition or dosage form with a particular initial concentration or mixture. The claims of this disclosure include equivalents of these amounts, whether or not they are modified by the term “about.” The term “about” further refers to a range of values ​​similar to the described reference value. In particular embodiments, the term “about” refers to, for example, a range of values ​​that are 50%, 25%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, and 1% or less of the described reference value.

[0184] In one aspect, the Disclosure provides engineered host cells expressing a recombinant amino acid molecule, which comprises a recombinant protein that is an argininosuccinate synthase. The amino acid molecule has an amino acid sequence comprising SEQ ID NO: 1 of the Disclosure.

[0185] In some embodiments, the disclosed manipulated host cell may be a manipulated microorganism. In some embodiments, the manipulated microorganism is Corynebacterium. In some embodiments, the manipulated microorganism is Corynebacterium glutamicum. In some embodiments, the manipulated microorganism is Corynebacterium stationis. In some embodiments, the manipulated microorganism is Corynebacterium crudilactis. In some embodiments, the manipulated microorganism is Corynebacterium Deserti. In some embodiments, the manipulated microorganism is Corynebacterium efficiens. In some examples, the manipulated microorganism is Corynebacterium callunae. In some examples, the manipulated microorganism is Corynebacterium singulare. In some examples, the manipulated microorganism is Corynebacterium halotolerans. In some examples, the manipulated microorganism is Corynebacterium striatum. In some examples, the manipulated microorganism is Corynebacterium ammoniagenes. In some examples, the manipulated microorganism is Corynebacterium pollutisoli. In some specific examples, the manipulated microorganism is Corynebacterium imitans.In some examples, the manipulated microorganism is Corynebacterium testudinoris. In some examples, the manipulated microorganism is Corynebacterium flavescens. In some examples, the manipulated microorganism is Corynebacterium crenatum. In some examples, the manipulated microorganism is Corynebacterium suranareeae. In some examples, the manipulated microorganism is Escherichia. In some examples, the manipulated microorganism is Escherichia coli.

[0186] In some embodiments, the manipulated host cells produce L-arginine. In some embodiments, the manipulated microorganisms produce L-arginine.

[0187] In some embodiments, the manipulated host cells or microorganisms are transformed with the argR gene, which contains a sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 100% sequence identity with SEQ ID NO: 39. In some embodiments, the manipulated host cells or microorganisms are transformed with the argB gene, which contains a sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 100% sequence identity with SEQ ID NO: 40, and which has the M54V mutation.

[0188] In some embodiments, the recombinant protein has an exogenous amino acid sequence of the host cell or microorganism. In other embodiments, the recombinant protein has an endogenous amino acid sequence of the host cell or microorganism.

[0189] In other ways, the Disclosure provides the use of the aforementioned engineered host cells or microorganisms for producing L-arginine.

[0190] In additional embodiments, the present disclosure provides a method for producing L-arginine. In some embodiments, the method includes culturing engineered host cells expressing the recombinant protein of the present disclosure. In some embodiments, the host cells are cultured in a culture medium. In the present disclosure, the term “culture” means growing the host cells of the present disclosure under appropriately controlled environmental conditions. The culture process in some embodiments of the present disclosure may be carried out under suitable culture media and culture conditions known to the art. Such a culture process can be readily adapted and used by those skilled in the art with selected host cells. Specifically, the culture may be batch, continuous, and / or fed-batch culture.

[0191] As used in this disclosure, the term "medium" refers to a substance that contains, as its main component, the nutrients necessary for culturing the host cells of this disclosure, and which also supplies water and other nutrients and growth factors essential for survival and growth. Specifically, the medium and other culture conditions used for culturing the host cells of this disclosure are any medium commonly used for culturing host cells, without any special limitations. However, the host cells of this disclosure are cultured under aerobic conditions in a standard medium containing appropriate carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids, vitamins, etc., while adjusting the temperature, pH, etc. For example, culture media for Corynebacterium host cells can be found in the literature ["Manual of Methods for General Bacteriology" by the American Society for Bacteriology (Washington DC, USA, 1981)].

[0192] During the culture of host cells according to some embodiments of this disclosure, the pH of the culture medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, or sulfuric acid to the medium in an appropriate manner. During culture, an antifoaming agent such as a fatty acid polyglycol ester can be used to suppress the generation of bubbles. Oxygen or an oxygen-containing gas can be injected into the culture medium to maintain an aerobic state; or no gas can be injected to maintain anaerobic and microaerobic conditions; or nitrogen, hydrogen, or carbon dioxide can be injected, but the adjustment of air is not limited thereto.

[0193] In an additional embodiment of the L-arginine production method described above, the culture is carried out using at least one carbon source. Suitable carbon sources include, but are not limited to, carbohydrates, sugar alcohols, organic acids, and amino acids. Carbohydrates may include, in particular, glucose, saccharose, lactose, fructose, sucrose, and maltose. Sugar alcohols may include, in particular, mannitol and sorbitol. Organic acids may include, in particular, pyruvic acid, lactic acid, and citric acid, and amino acids may include, in particular, glutamic acid, methionine, and lysine. Natural organic nutrients such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn maceration can also be used. In specific embodiments, carbohydrates such as glucose and sterilized pre-treated molasses (i.e., molasses converted to reducing sugars) can be used, and a variety of other carbon sources may be used in appropriate amounts without limitation. These carbon sources may be used alone or in combination of two or more, but are not limited to this.

[0194] In an additional embodiment of the L-arginine production method described above, the culture is carried out using at least one nitrogen source. Suitable nitrogen sources include inorganic nitrogen sources, organic nitrogen sources, and combinations thereof. Inorganic nitrogen sources include, but are not limited to, ammonia, ammonium sulfate, ammonium chloride, ammonium phosphate, and ammonium nitrate. Organic nitrogen sources include organic acids, including ammonium acetate and ammonium carbonate; amino acids such as glutamic acid, methionine, and glutamine; and other sources of peptone, NZ-amine, meat extracts, yeast extracts, malt extracts, corn maceration, casein hydrolysates, fish or their decomposition products, and defatted soybean meal or its decomposition products. These nitrogen sources may be used individually or in combination of two or more, but are not limited thereto.

[0195] In an additional embodiment of the L-arginine production method described above, the culture is carried out using at least one of the following: monobasic potassium phosphate, dibasic potassium phosphate, monobasic sodium phosphate, dibasic sodium phosphate, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, magnesium sulfate, calcium carbonate, etc. Furthermore, amino acids, vitamins, appropriate precursors, etc., can be added to the culture medium.

[0196] These carbon sources, nitrogen sources, additives, or their precursors can be added to the culture medium in batches or continuously. However, the method of addition is not limited thereto.

[0197] Another embodiment of the L-arginine production method of this disclosure is to carry out the culture using at least one selected from the group consisting of potassium phosphate, dipotassium phosphate, corresponding sodium-containing salts, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, and calcium carbonate.

[0198] In an additional embodiment of the L-arginine production method, the culture is carried out at a temperature maintained between approximately 20°C and approximately 45°C. In some embodiments, the culture is carried out at a temperature maintained between approximately 25°C and approximately 40°C. In some embodiments, the culture is carried out at a temperature ranging from approximately 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36°C to approximately 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45°C. In an additional embodiment, the culture may be carried out for approximately 10 to approximately 160 hours. In additional embodiments, the culture may be carried out for approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110 to approximately 100, 110, 120, 130, 140, 150, or 160 hours. However, the culture conditions are not limited thereto.

[0199] Another aspect of the present disclosure provides a method for increasing L-arginine production by a host cell. In some embodiments, the method includes transforming a host cell to produce the engineered host cell expressing the recombinant amino acid molecule of the present disclosure.

[0200] In additional embodiments of the methods of the present disclosure, the methods further include, for example, producing the host cells of the present disclosure, producing a culture medium for culturing the host cells, or a combination thereof (in any order) prior to the culture step.

[0201] The L-arginine produced by the culture according to some embodiment of the present disclosure may be secreted into the culture medium or remain in the cells. For example, in an additional embodiment of the method for producing L-arginine, which includes culturing engineered host cells expressing a recombinant amino acid molecule such as recombinant argininosuccinate synthase of the present disclosure, the method further includes recovering the L-arginine from the culture medium (cultured medium) or the cultured host cells after the culture step. The recovery step may further include the recovery step after the culture step.

[0202] In some embodiments of a method for producing L-arginine, which includes culturing engineered host cells expressing the recombinant protein of the Disclosure, the production method further includes recovering L-arginine from the cultured engineered host cells and / or the culture medium in which the engineered host cells are cultured. Suitable methods for recovering the L-arginine include protocols known in the art through the method for culturing host cells of the Disclosure, e.g., batch, continuous, or fed-batch culture methods. For example, centrifugation, filtration, treatment with crystallized protein precipitants (salting out), extraction, sonication, ultrafiltration, dialysis, various types of chromatography, e.g., molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, and affinity chromatography, HPLC, or any combination thereof, can be used to recover the desired L-arginine from the culture medium or host cells using suitable methods known in the art.

[0203] In some embodiments of the L-arginine production method of the present disclosure, the production method further includes a purification step. The purification may be carried out using a suitable method known to the art. In one example, if the L-arginine production method of the present disclosure includes both a recovery step and a purification step, the recovery step and the purification step may be carried out sequentially or discontinuously in any order, or simultaneously or combined into a single step, but the manner of execution is not limited thereto.

[0204] In the methods described in some embodiments, the proteins, polynucleotides, vectors, host cells, etc., are as disclosed in other aspects of this disclosure.

[0205] Another aspect of the present disclosure provides a composition for L-arginine production comprising a microorganism of the genus Corynebacterium; a culture medium on which the microorganism is cultured; or a combination thereof. The composition according to some embodiment of the present disclosure may further comprise any suitable excipients commonly used in compositions for L-arginine production, such excipients may include, but are not limited to, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, and isotonic agents.

[0206] Each description and example in this disclosure applies to all other descriptions and examples. That is, all combinations of the diverse elements of this disclosure fall within the scope of this disclosure. Furthermore, the scope of this disclosure is not limited by the specific descriptions below. Also, a person skilled in the art should be able to recognize or confirm many equivalents to specific aspects of this disclosure through ordinary experimentation alone. These equivalents should also be interpreted as falling within the scope of this disclosure.

[0207] Another mode provides the use of an L-arginine product characterized by one or more of the elements disclosed herein. An additional mode provides the use of a method characterized by one or more of the components of the herein.

[0208] Hereafter, this disclosure is disclosed in more detail with reference to examples. However, the following examples are merely preferred embodiments illustrating this disclosure and are not limiting to the scope of this disclosure. On the other hand, technical matters not disclosed herein can be fully understood and readily implemented by a person with ordinary skill in the art of this disclosure or a similar art.

[0209] Example 1. Comparison of protein sequences and tertiary structures of inter-genus argininosuccinate synthases. First, we confirmed the essential amino acid residues for substrate binding, such as L-citrulline, aspartic acid, and ATP, in Corynebacterium glutamicum by comparing the protein sequences of Corynebacterium glutamicum argininosuccinate synthase (SEQ ID NO: 28) with heterologous argininosuccinate synthases with known structures. Although the tertiary structure of Corynebacterium glutamicum argininosuccinate synthase has not been confirmed, the tertiary crystal structure and its internal active site residues have been reported for Escherichia coli argininosuccinate synthase (SEQ ID NO: 30). Of these, threonine at position 130, tyrosine at position 131, lysine at position 132, glycine at position 133, asparagine at position 134, and aspartic acid at position 135 were reported as essential residues for substrate binding (Lemke, CT, and PL Howell., Structure 9(12):1153-64, 2001). Based on this, the protein sequences of argininosuccinate synthase between Escherichia coli and Corynebacterium glutamicum were aligned (see Figure 1), and it was confirmed that in the case of Corynebacterium glutamicum, threonine at position 119, glycine at position 120, lysine at position 121, glycine at position 122, asparagine at position 123, and aspartic acid at position 124 correspond to the aforementioned residues and are essential for substrate binding.

[0210] Next, to elucidate the non-conserved structure of the substrate binding site, protein sequences of argininosuccinate synthases from various heterologous model microorganisms were aligned in multiple sequences.

[0211] As a result, we confirmed that the argininosuccinate synthases of Acidobacterium capsulatum, Alcaligenes faecalis, Burkholderia pyrosinia, Capriavidus necatol, Escherichia coli, or Neisseria warbergi possess a 5-amino acid extension around the essential residue of the binding site, which is not present in Corynebacterium glutamicum (see, for example, Figure 2 and SEQ ID NOs. 23, 24, 26, 29, 30, and 32, respectively).

[0212] To predict the role of such amino acid extensions in terms of protein function, the tertiary structures of argininosuccinate synthase with and without amino acid extensions were compared. For the tertiary structure without amino acid extensions, a protein from Corynebacterium glutamicum was used, and the Alphafold program was employed to generate the relevant tertiary protein structure (Jumper, J., Evans, R., Pritzel, A. et al. Nature 596, 583-589, 2021). For the tertiary structure with amino acid extensions, a protein from Escherichia coli (PDB ID: 1K97) was used. For comparison, these two were superimposed using the VMD program (Humphrey, W et al., J Mol Graph. 14(1):33-8, 27-8, 1996). See Figure 3.

[0213] As shown in Figure 3, the site where amino acid elongation occurs is located at the entrance to the active site in the protein's tertiary structure, particularly adjacent to the substrate binding site. Furthermore, such amino acid elongation can be involved in the formation of a helical secondary structure. In proteins from Corynebacterium glutamicum that lack such amino acid elongation, the corresponding site exists in a loop form without a special secondary structure, whereas in proteins from Escherichia coli that possess such elongation, the corresponding site exists in the form of an alpha helical structure. In addition, the sequence difference between the elongated portion and the preceding portion may increase the distance between the corresponding region and the substrate in the protein's tertiary structure. Consequently, when the corresponding amino acid elongation is present, the entrance to the active site may be enlarged by the formation of the secondary structure, and the substrate may be able to access the active site more easily, which can contribute to improving the catalytic efficiency of the protein. Not only can the substrate be accessed more easily, but it can also contribute to improving catalytic efficiency by promoting and stabilizing the formation of the protein's quaternary structure. Argininosuccinate synthase from Escherichia coli can function as a tetramer. Since the aforementioned amino acid extension is located near the interface between argininosuccinate synthase monomers, it can influence tetramer formation.

[0214] The identity of the 10 argininosuccinate synthases shown in Table 1 below was analyzed.

[0215] [Table 1]

[0216] Furthermore, the protein sequence identity (Percent Identity) matrices for the 10 types of argininosuccinate synthases were generated using the Clustal 2.1 program (Madeira, Fabio et al., Nucleic Acid Research 50(W1) W276-W279, 2022), and are shown in Table 2 below.

[0217] [Table 2]

[0218] Furthermore, in order to derive motifs that can define structural differences, protein motif analysis was performed on the argininosuccinate synthases having the aforementioned amino acid extensions. Protein motifs are indicated by short sequences that appear in a pattern due to molecular function, and are represented by patterned amino acid sequences. The argininosuccinate synthases having the aforementioned amino acid extensions were included, and the protein sequences of 20 argininosuccinate synthases having 5 amino acid extensions at corresponding positions were further collected and included using the NCBI BLAST program (Altschul, SF et al., Nucleic Acid Research 25 (17) 3389-402, 1997) (SEQ ID NOs: 1 to 16). These sequences were searched for motifs using the MEME program, a protein motif search program (Bailey, TL, and C Elkan. Proceedings. International Conference on Intelligent Systems for Molecular Biology 2, 28-36, 1994).

[0219] As a result, a total of 18 motifs were obtained through the argininosuccinate synthase analysis described above. Protein motifs are usually represented using regular expressions, which are also represented in a similar manner in the diagram (Sigrist, Christian JA et al., Briefings in bioinformatics 3(3) 265-74, 2002). It was shown that motifs exist at the precise positions where amino acid residues are extended, and this is represented by motif 8 (SEQ ID NO: 1). This is an essential motif that explains the amino acid extension of the argininosuccinate synthase described above. Motifs 6 (SEQ ID NO: 11) and 13 (SEQ ID NO: 17) are normally present in the argininosuccinate synthase protein.

[0220] In addition, 15 other motifs were identified within the argininosuccinate synthase protein. These additional 15 motifs are typically found in proteins with amino acid extensions in argininosuccinate synthase. Therefore, these proteins, which always contain motif 8 (SEQ ID NO: 1) and optionally contain some of the 17 motifs (SEQ ID NOs: 6-22), may possess the characteristics of argininosuccinate synthase with amino acid extensions. Table 3 below shows the motifs discovered during the search (shown as SEQ ID NOs: 1 and SEQ ID NOs: 6-22 in Figure 4).

[0221] [Table 3]

[0222] Example 2. Production of a vector into which exogenous argininosuccinate synthase has been introduced. Prior to producing L-arginine-producing strains of Corynebacterium glutamicum, vectors containing the argG gene encoding the argininosuccinate synthase enzyme of this disclosure were prepared. The argG gene was derived from Acidobacterium capsulatum (SEQ ID NO: 33), Alcaligenes faecalis (SEQ ID NO: 34), Burgholderia pyrosinia (SEQ ID NO: 35), Bacillus amyloricephaciens (SEQ ID NO: 41), Corynebacterium ammoniagenes (SEQ ID NO: 42), Corynebacterium glutamicum (SEQ ID NO: 43), Capriavidus necatol (SEQ ID NO: 36), Escherichia coli (SEQ ID NO: 37), Mycobacterium smegmatis (SEQ ID NO: 44), and Neisseria wareberg (SEQ ID NO: 38) (see Figure 5). The vector is typically designed so that each argG gene is inserted into the Corynebacterium glutamicum chromosome via homologous recombination, specifically at the BBD29_RS08210 site, and expressed under a constitutive Po2 promoter (Korean Patent No. 10-1632642).

[0223] Two DNA fragments containing homology regions and promoters targeting BBD29_RS08210 were prepared from the vector components. One DNA fragment pertained to the 5'-homologous region and promoter targeting BBD29_RS08210, while the other DNA fragment pertained to the 3'-homologous region targeting BBD29_RS08210. PCR was performed using Corynebacterium glutamicum ATCC13869 genomic DNA as a template and the primer pairs shown in Table 4 (SEQ ID NOs. 45 and 46 and SEQ ID NOs. 47 and 48) to obtain the DNA fragments, which contain the sequences of the 5'-homologous region and the 3'-homologous region of BBD29_RS08210 with the Po2 promoter (hereinafter referred to as "BBD29_RS08210 5'-DNA fragment" and "BBD29_RS08210 3'-DNA fragment"). Specifically, PCR was performed as follows: denaturation at 95°C for 30 seconds for 30 cycles, annealing at 55°C for 30 seconds, and extension at 72°C for 2 minutes. Subsequently, DNA fragments of the argG gene were produced. Specifically, PCR was performed using the primer pairs shown in Table 4 as templates (Sequence IDs 49 and 50, which have the genomic DNA of Acidobacterium capsulatum strain KACC 14500; Sequence IDs 51 and 52, which have the genomic DNA of Bacillus amyloricephasiensis strain KACC 12067; Sequence IDs 53 and 54, which have the genomic DNA of Burkholderia pyrosinia strain KACC 12018; Sequence IDs 55 and 56, which have the genomic DNA of Corynebacterium ammoniagenes strain ATCC 6872; Sequence IDs 57 and 58, which have the genomic DNA of Corynebacterium glutamicum strain ATCC 13869; and Capriavidus necatol strain KCTC The studies were conducted using SEQ ID NOs. 59 and 60, which contain genomic DNA of 22469; SEQ ID NOs. 61 and 62, which contain genomic DNA of Escherichia coli K-12 strain MG1655; SEQ ID NOs. 63 and 64, which contain genomic DNA of Mycobacterium smegmatis strain MC2 155; and SEQ ID NOs. 65 and 66, which contain genomic DNA of Neisseria wareberg strain KCTC 23363. The genomic DNA used was distributed by KCTC and KACC.Specifically, PCR was performed as follows: denaturation at 95°C for 30 seconds for 30 cycles, annealing at 55°C for 30 seconds, and extension at 72°C for 2 minutes. As a result, DNA fragments of the argG gene (hereinafter referred to as argG(A.capsulatum), argG(B.amyloliquefaciens), argG(B.pyrrocinia), argG(C.ammoniagenes), argG(C.glutamicum), argG(C.necator), argG(E.coli), argG(M.smegmatis), and argG(N.weaveri)) were obtained.

[0224] [Table 4]

[0225] Next, the obtained DNA fragments and linearized vectors were cloned. The linear vector was used after treating pDCM2 (Korean Patent Application Publication No. 10-2020-0136813), which is unreplicable with Corynebacterium glutamicum, with SmaI restriction enzyme. The BBD29_RS08210 5'-DNA fragment, BBD29_RS08210 3'-DNA fragment, and argG gene DNA fragments obtained in this way were cloned by fusion. Fusion cloning was performed using the In-Fusion® HD Cloning Kit (Clontech), and the resulting clones were transformed into E. coli DH5α and spread on LB solid medium containing kanamycin (25 mg / L). After selecting colonies transformed with plasmids into which the target gene was inserted via PCR, DNA was obtained using the DNA-Spin Plasmid DNA Purification Kit (iNtRON), and through this, vectors (recombinant plasmids) containing all of the BBD29_RS08210 5'-DNA fragments, BBD29_RS08210 3'-DNA fragments, and DNA fragments of each argG gene were obtained (i.e., pDCM2-△BBD29_RS08210::Po2_argG(A. capsuleum), pDCM2-△BBD29_RS08210::Po2_argG(B. amyloliquefaciens), pDCM2-△BBD29_RS08210::Po2_argG(B. pyrrocinia), pDCM2-△BBD29_RS08210::Po2_argG( C.ammoniagenes), pDCM2-△BBD29_RS08210::Po2_argG( C.glutamicum), pDCM2-△BBD29_RS08210::Po2_argG( C.necator), pDCM2-△BBD29_RS08210::Po2_argG( E.coli ), pDCM2-△BBD29_RS08210::Po2_argG (M.smegmatis), and pDCM2-△BBD29_RS08210::Po2_argG (N.weaveri) were produced.

[0226] Example 3. Production of an L-arginine-producing Corynebacterium glutamicum strain expressing exogenous argininosuccinate synthase (argG) and evaluation of its L-arginine production capacity. Example 3-1. Production of Corynebacterium glutamicum CJR2 strain We produced the Corynebacterium glutamicum CJR2 strain, which is capable of producing L-arginine. The production method involved sequentially introducing two mutations (ΔargR,argB(M54V)) into the wild-type Corynebacterium glutamicum ATCC13869 (Ikeda, Masato et al., Applied and Environmental Microbiology 75(6)1635-41, 2009).

[0227] First, vectors were prepared to introduce argR deletion (sequence number 39) and argB(M54V) mutation (sequence number 40) (see Figure 6). PCR was performed using Corynebacterium glutamicum ATCC13869 genomic DNA as a template and the primer pairs shown in Table 5 (sequence numbers 67 and 68, and 69 and 70). To obtain homologous recombination fragments containing the argR deletion mutation sequence (sequence number 39), duplicate PCR was performed using the primer pairs of sequence numbers 67 and 70. Similarly, to produce homologous recombination fragments containing the argB(M54V) mutation, PCR was performed using the primer pairs shown in Table 5 (sequence numbers 71 and 72, and 73 and 74). To obtain homologous recombination fragments containing the argB(M54V) mutation sequence (sequence number 40), duplicate PCR was performed using sequence numbers 71 and 74. PCR was performed as follows: denaturation at 95°C for 30 seconds for 30 cycles, annealing at 55°C for 30 seconds, and extension at 72°C for 2 minutes. Using the same method as in Example 2, the linearized pDCM2 vector and its homologous recombinant fragments were cloned by fusion. The vectors (recombinant plasmids) thus produced were named pDCM2-ΔargR and pDCM2-argB(M54V), respectively.

[0228] [Table 5]

[0229] Next, an argR deletion mutation was introduced into wild-type Corynebacterium glutamicum ATCC13869, and the cells were transformed using the pDCM2-ΔargR plasmid prepared above by electrolysis (van der Rest et al., Appl Microbiol Biotechnol 52:541-545, 1999). Subsequently, secondary recombination was performed on solid agar plates containing 4% sucrose, and PCR was performed using primer pairs (SEQ ID NOs. 67 and 70) that targeted the transformed strain after secondary recombination to confirm that the deletion mutation had been introduced into the argR gene on the chromosome. Specifically, PCR was performed as follows: denaturation at 95°C for 30 seconds for 30 cycles, annealing at 55°C for 30 seconds, and extension at 72°C for 2 minutes. The transformed strain was named CJR1.

[0230] Subsequently, the argB(M54V) mutation was introduced into Corynebacterium glutamicum CJR1 using the same method as described above. Using the pDCM2-argB(M54V) plasmid produced above, PCR was performed using a primer pair (sequence numbers 71 and 74) that targeted the transformant after secondary recombination, confirming that the M54V mutation had been introduced into the argB gene on the chromosome. The transformed strain was named CJR2.

[0231] The solid plate culture medium was as follows: compound plate medium (pH 7.0), 10g glucose, 10g peptone, 5g meat extract, 5g yeast extract, 18.5g cerebral and cardiac extract, 2.5g NaCl, 2g urea, 91g sorbitol, and 20g agar (per 1L of distilled water).

[0232] Example 3-2. Production of a strain in which argininosuccinate synthase (argG) has been introduced into the CJR2 strain. To introduce the foreign argG gene into the aforementioned CJR2 strain, mutant strains into which the vectors produced in Example 2 were introduced were prepared in the same manner as in Example 3-1. These mutant strains were named CJR2-argG(A. capsuleum), CJR2-argG(B. amyloliquefaciens), CJR2-argG(B. pyrrocinia), CJR2-argG(C. ammoniagenes), CJR2-argG(C. glutamicum), CJR2-argG(C. necator), CJR2-argG(E. coli), CJR2-argG(M. smegmatis), and CJR2-argG(N. weaveri), respectively. The transformed strains, after secondary recombination was completed, were subjected to PCR using primer pairs (SEQ ID NOs. 45 and 48) to confirm that the Po2 promoter and their respective argG genes had been introduced into the BBD29_RS08210 gene on the chromosome. Specifically, PCR was performed as follows: denaturation at 95°C for 30 seconds for 30 cycles, annealing at 55°C for 30 seconds, and extension at 72°C for 4 minutes.

[0233] Example 3-3. Evaluation of L-arginine production capacity To compare the L-arginine production capacity of the CJR2 strains into which the argG gene was introduced, as produced in Example 3-2 (i.e., CJR2-argG(A. capsuleum), CJR2-argG(B. amyloliquefaciens), CJR2-argG(B. pyrrocinia), CJR2-argG(C. ammoniagenes), CJR2-argG(C. glutamicum), CJR2-argG(C. necator), CJR2-argG(E. coli), CJR2-argG(M. smegmatis), and CJR2-argG(N. weaveri)), the concentration of L-arginine in the culture medium was analyzed by culturing using the method described below.

[0234] Corynebacterium glutamicum CJR2 (parent strain) and the strain produced in Example 3-2 were inoculated into 250 ml corner baffle flasks containing 25 ml of production medium, and then cultured at 30°C for 44 hours with shaking at 200 rpm. The composition of each production medium was as follows:

[0235] <Production medium (pH 7.2)> Sucrose 50g, ammonium sulfate 57g, MgSO4·7H2O 2g, beet molasses 5g, calcium chloride 1mg, cobalt chloride 1mg, KH2PO4 2g, biotin 0.01mg, thiamine-HCl 0.1mg, calcium pantothenate 2mg, nicotinamide 3mg, ferrous sulfate 10mg, manganese sulfate 10mg, zinc sulfate 0.02mg, copper sulfate 0.5mg, calcium carbonate 30g (per 1L of distilled water).

[0236] After the culturing was complete, the L-arginine production capacity (concentration) was analyzed using HPLC (Waters 2478), and the improvements in the analyzed L-arginine and L-citrulline concentrations and L-arginine yield are shown in Table 6 below.

[0237] [Table 6]

[0238] As a result, the Corynebacterium glutamicum strain into which the argininosuccinate synthase with the 5-amino acid extension described in Example 1 was introduced showed higher L-arginine production capacity compared to strains into which other enzymes were introduced. As mentioned in Example 1, the argininosuccinate synthases derived from A. capsulatum, B. pyrrocinia, C. necator, E. coli, and N. weaveri were shown to have the corresponding amino acid extensions (Figures 1-3). The average concentrations of L-arginine and L-citrulline present in the fermentation broth of the CJR2-derived strains were measured to be 6.3 g / L and 0.2 g / L, respectively. The average yield improvement of these strains compared to the parent strain CJR2 was 33.6%. In contrast, the average L-arginine and L-citrulline concentrations in the culture medium of strains into which argininosuccinate synthases derived from B. amyloliquefaciens, C. ammoniagenes, C. glutamicum, and M. smegmatis without 5-amino acid extensions were measured to be 4.9 g / L and 0.7 g / L, respectively; therefore, compared to the former group, the L-arginine concentration was 23% lower on average and the L-citrulline concentration was 350% higher on average. The average yield improvement of these strains compared to the parent strain was 3.3%. This indicates that argininosuccinate synthases with amino acid extensions are relatively superior in terms of catalytic efficiency compared to enzymes without amino acid extensions. Furthermore, these results suggest that in Corynebacterium glutamicum, which produces L-arginine, improving the catalytic efficiency of such argininosuccinate synthase leads to an improvement in its L-arginine production capacity, enabling large-scale production of L-arginine.

Claims

1. YKPWLDX １ X ２ FX ３ X ４ An amino acid molecule containing the amino acid sequence of EL (SEQ ID NO: 1), Each of the aforementioned X １ , X ２ , and X ４ is an amino acid; X ３ is I or V, The aforementioned amino acid molecule is an amino acid molecule that does not exist in nature.

2. The amino acid molecule according to claim 1, wherein the sequence number 1 is a sequence selected from the group consisting of YKPWLDSAFIDEL (sequence number 2), YKPWLDQTFIDEL (sequence number 3), YKPWLDQQFIDEL (sequence number 4), and YKPWLDTDFIDEL (sequence number 5).

3. The aforementioned amino acid sequences are SEQ ID NOs: 6-22. X ５ PX ６ X ７ X ８ X ９ GX １０ AFSGGLDTSX １１ AX １２ (SEQ ID NO: 6), said X ５ is I, L or V; X ６ is an amino acid; X ７ is A, E or Q; X ８ is K or R; X ９ is I or V; X １０ is I or L; X １１ is A, T or V; X １２ is I, L or V, and X １３ GAX ａ X １４ X １５ X １６ YTA X １７ X １８ GQ X １９ DE (Sequence No. 7), the X １３ is K or N; X ａ is an amino acid; X １４ is C or P;X １５ is C or Y; X １６ is A, S or T;X １７ is D or N; X １８ is I or L; X １９ is A, P, or Y, X ２０ X ２１ X ２２ X ２３ X ２４ X ２５ X ２６ X ａ LX ２７ (Sequence No. 8), the X ２０ is A or S; X ２１ is R or V; X ２２ is I or L; X ２３ is I or V; X ２４ is E or D; X ２５ is C or G; X ２６ is K or R; X ａ is an amino acid; X ２７ is A or V, X ２８ AFX ２９ X ａ X ａ X ３０ X ３１ G (Sequence No. 9), the aforementioned X ２８ is G or N; X ２９ is H or N; X ａ is an amino acid; X ３０ is S or T;X ３１ is A or G, YFNTTPX ３２ GRAVX ３３ X ３４ TX ３５ LV (Sequence No. 10), the X ３２ is I or L; X ３３ is A or T;X ３４ is A or G; X ３５ It is L or M, TX ３６ KGNDIERF (Sequence No. 11), the X ３６ is F or Y, YRYGLX ３７ X ３８ N (Sequence No. 12), the aforementioned X ３７ is L or V; X ３８ is A, T, or V, GGRX ａ EMX ３９ X ４０ X ４１ X ４２ (SEQ ID NO: 13), said X ａ is an amino acid; X ３９ is A or S; X ４０ is A, E or Q; X ４１ is F, W or Y; X ４２ is L or M, EKAYSTD X ４３ NX ４４ X ４５ GATHE (SEQ ID NO: 14), said X ４３ is A or S; X ４４ is I, L or M; X ４５ is L or W, and VX ａ PIMGVX ａ X ４６ W (Sequence No. 15), the aforementioned X ａ is an amino acid, X ４６ is F, H, or S, X ４７ GGRHGX ４８ GX ４９ X ５０ DQIENRX ５１ IEA (Sequence No. 16), the X ４７ is I or V; X ４８ is L or M; X ４９ is M or V; X ５０ is A or S; X ５１ is I or V, KSRGIYEAPG (Sequence ID 17), X ５２ ALX ５３ X ５４ X ５５ AX ５６ ERLX ａ X ５７ X ５８ IHNEDT (Sequence No. 18), the X ５２ is L or M; X ５３ is F or L; X ５４ is F, H, or Y; X ５５ is A or I; X ５６ is F or Y; X ａ is an amino acid; X ５７ is N, S or T; X ５８ is A or G, LGX ５９ LX ６０ YX ６１ GRWX ６２ DX ６３ QX ６４ X ６５ MX ６６ RX ６７ (Sequence No. 19), the X ５９ is K or R; X ６０ is L or M; X ６１ is A, E, or Q; X ６２ is F or L; X ６３ is P or S; X ６４ is A, S or G; X ６５ is I, L, or M; X ６６ is I, L, or V; X ６７ is D or E, LRRGX ａ DX ６８ X ６９ X ７０ (Sequence No. 20), the X ａ is an amino acid; X ６８ is F or Y; X ６９ is S or T;X ７０ is I or L, X ７１ X ７２ X ７３ X ７４ X ａ X ７５ X ７６ X ７７ LX ７８ MEX ７９ (Sequence No. 21), the X ７１ is A, D, or N; X ７２ is F or L; X ７３ is S or T;X ７４ is F or Y; X ａ is an amino acid; X ７５ is A, P or S; X ７６ is A, E, or D; X ７７ is K or R; X ７８ is S or T;X ７９ is K or R, DRIGQLX ８０ MRX ８１ X ８２ DX ８３ X ａ DX ８４ R (Sequence No. 22), the above X ８０ is H or T;X ８１ is L, N, or T; X ８２ is L or N; X ８３ is I, L, or V; X ａ is an amino acid; X ８４ is S or T. An amino acid molecule according to claim 1 or 2, comprising at least one sequence selected from the group consisting of the following.

4. The amino acid molecule according to any one of claims 1 to 3, wherein the amino acid molecule comprises all of sequence numbers 6 to 22.

5. The amino acid molecule according to any one of claims 1 to 4, comprising at least one sequence selected from the group consisting of the following: A. A sequence having at least 79% sequence identity with sequence number 23, which is a capsulatum sequence; A. A sequence having at least 80% sequence identity with sequence number 24, which is the faecalis sequence; B. Sequences having at least 78% sequence identity with sequence number 25, which is a pyrocinia sequence; C. Sequences having at least 80% sequence identity with sequence number 26, which is a necator sequence; E. A sequence having at least 78% sequence identity with sequence number 27, which is a coli sequence; and A sequence having at least 80% sequence identity with sequence number 28, which is the N. weaveri sequence.

6. The amino acid molecule according to claim 5, wherein the amino acid molecule comprises a sequence having at least 80% sequence identity with SEQ ID NO:

26.

7. The amino acid molecule according to claim 5, wherein the amino acid molecule comprises a sequence having at least 78% sequence identity with SEQ ID NO:

27.

8. The amino acid molecule according to claim 5, wherein the amino acid molecule comprises a sequence having at least 79% sequence identity with SEQ ID NO: 23; at least 80% sequence identity with SEQ ID NO: 24; at least 78% sequence identity with SEQ ID NO: 25; at least 80% sequence identity with SEQ ID NO: 26; at least 78% sequence identity with SEQ ID NO: 27; and at least 80% sequence identity with SEQ ID NO:

28.

9. The amino acid molecule according to any one of claims 1 to 8, comprising at least one sequence selected from the group consisting of the following: Sequences corresponding to positions 1-153 and 167-445 of sequence number 23 with at least 79% accuracy; Sequences corresponding to positions 1-153 and 167-444 of sequence number 24, with at least 80% of the data; Sequences corresponding to positions 1-153 and 167-445 of sequence number 25, with at least 78% of the data; Sequences corresponding to positions 1-153 and 167-441 of sequence number 26, with at least 80% of the data; Sequences corresponding to positions 1-153 and 167-447 of sequence number 27 and at least 78% of the sequences; and A sequence that corresponds to at least 80% of positions 1-157 and 171-448 of sequence number 28.

10. The amino acid molecule according to claim 9, wherein the amino acid molecule includes a sequence that corresponds at least 80% to positions 1 to 153 and 167 to 441 of Sequence ID No.

26.

11. The amino acid molecule according to claim 9, wherein the amino acid molecule includes a sequence that corresponds to at least 78% of positions 1 to 153 and 167 to 447 of Sequence ID No.

27.

12. The amino acid molecule according to any one of claims 1 to 11 comprises at least one sequence selected from the group consisting of the following: The corresponding sequences for positions 1-153 and 167-445 of sequence number 23; The corresponding sequences for positions 1-153 and 167-444 of sequence number 24; The corresponding sequences for positions 1-153 and 167-445 of sequence number 25; The corresponding sequences for positions 1-153 and 167-441 of sequence number 26; The sequences corresponding to positions 1 to 153 and 167 to 447 of sequence number 27; and The sequence corresponding to positions 1-157 and 171-448 of sequence number 28.

13. The amino acid molecule according to claim 12, wherein the amino acid molecule includes sequences corresponding to positions 1 to 153 and 167 to 441 of SEQ ID NO:

26.

14. The amino acid molecule according to claim 12, wherein the amino acid molecule includes sequences corresponding to positions 1 to 153 and 167 to 447 of Sequence ID No.

27.

15. The aforementioned X １ The argininosuccinate synthase according to any one of claims 1 to 14, wherein is S, Q, or T.

16. The aforementioned X ２ An amino acid molecule according to any one of claims 1 to 15, wherein is A, T, Q, or D.

17. The aforementioned X ３ An amino acid molecule according to any one of claims 1 to 16, wherein is I.

18. The aforementioned X ４ An amino acid molecule according to any one of claims 1 to 17, wherein is D.

19. The amino acid molecule according to any one of claims 1 to 18, wherein the amino acid molecule is a recombinant protein.

20. The amino acid molecule according to any one of claims 1 to 19, wherein the amino acid molecule is argininosuccinate synthase.

21. A host cell expressing an amino acid molecule according to any one of claims 1 to 20.

22. The host cell according to claim 21, wherein the host cell is a microorganism.

23. The host cell according to claim 21 or 22, wherein the host cell is Corynebacterium.

24. The host cells mentioned above include Corynebacterium glutamicum, Corynebacterium stationionis, Corynebacterium crudilactis, Corynebacterium Deserti, Corynebacterium efficiens, Corynebacterium carnae, and Corynebacterium singulare. Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris, Corynebacterium flavecens A host cell according to any one of claims 21 to 23, selected from the group consisting of *Corynebacterium flavescens*, *Corynebacterium crenatum*, and *Corynebacterium suranareeae*.

25. The host cell according to any one of claims 21 to 24, wherein the host cell is Corynebacterium glutamicum.

26. The host cell according to any one of claims 21 to 25, wherein the host cell produces L-arginine.

27. The host cell according to any one of claims 21 to 26, wherein the host cell is transformed with an argR gene containing a sequence having at least 90% sequence identity with sequence number 39, and an argB gene containing a sequence having at least 90% sequence identity with sequence number 40 and having M54V.

28. A nucleic acid molecule comprising a nucleic acid sequence encoding an amino acid molecule according to any one of claims 1 to 20.

29. The nucleic acid molecule according to claim 28, comprising at least one sequence selected from the group consisting of the following: A. A sequence having at least 79% sequence identity with sequence number 33, which is a capsulatum sequence; A. A sequence having at least 80% sequence identity with sequence number 34, which is the faecalis sequence; B. Sequences having at least 78% sequence identity with sequence number 35, which is a pyrocinia sequence; C. A sequence having at least 80% sequence identity with sequence number 36, which is a necator sequence; E. A sequence having at least 78% sequence identity with sequence number 37, which is a coli sequence; and A sequence having at least 80% sequence identity with sequence number 38, which is the N. weaveri sequence.

30. The nucleic acid molecule according to claim 28 or 29, wherein the nucleic acid molecule comprises at least one sequence selected from the group consisting of SEQ ID NOs: 33 to 38.

31. A vector comprising a nucleic acid molecule according to any one of claims 28 to 30.

32. YKPWLDX １ X ２ FX ３ X ４ A manipulated host cell expressing a recombinant amino acid molecule containing the amino acid sequence of EL (SEQ ID NO: 1), wherein each of the X １ , X ２ , and X ４ is an amino acid; X ３ is either I or V.

33. The recombinant amino acid molecules are SEQ ID NOs: 6-22. X ５ PX ６ X ７ X ８ X ９ GX １０ AFSGGLDTSX １１ AX １２ (Sequence No. 6), the above X ５ is I, L, or V; X ６ is an amino acid; X ７ is A, E, or Q; X ８ is K or R; X ９ is I or V; X １０ is I or L; X １１ is A, T, or V; X １２ is I, L, or V, X １３ GAX ａ X １４ X １５ X １６ YTA X １７ X １８ GQ X １９ DE (Sequence No. 7), the X １３ is K or N; X ａ is an amino acid; X １４ is C or P;X １５ is C or Y; X １６ is A, S or T;X １７ is D or N; X １８ is I or L; X １９ is A, P, or Y, X ２０ X ２１ X ２２ X ２３ X ２４ X ２５ X ２６ X ａ LX ２７ (Sequence No. 8), the X ２０ is A or S; X ２１ is R or V; X ２２ is I or L; X ２３ is I or V; X ２４ is E or D; X ２５ is C or G; X ２６ is K or R; X ａ is an amino acid; X ２７ is A or V, X ２８ AFX ２９ X ａ X ａ X ３０ X ３１ G (Sequence No. 9), the aforementioned X ２８ is G or N; X ２９ is H or N; X ａ is an amino acid; X ３０ is S or T;X ３１ is A or G, YFNTTPX ３２ GRAVX ３３ X ３４ TX ３５ LV (Sequence No. 10), the X ３２ is I or L; X ３３ is A or T;X ３４ is A or G; X ３５ It is L or M, TX ３６ KGNDIERF (Sequence No. 11), the X ３６ (is F or Y), YRYGLX ３７ X ３８ N (Sequence No. 12), the aforementioned X ３７ is L or V; X ３８ is A, T, or V, GGRX ａ EMX ３９ X ４０ X ４１ X ４２ (Sequence No. 13), the X ａ is an amino acid; X ３９ is A or S; X ４０ is A, E, or Q; X ４１ is F, W, or Y; X ４２ It is L or M, EKAYSTDX ４３ NX ４４ X ４５ GATE (Sequence No. 14), the X ４３ is A or S; X ４４ is I, L, or M; X ４５ is L or W, VX ａ PIMGVX ａ X ４６ W (Sequence No. 15), the aforementioned X ａ is an amino acid, X ４６ is F, H, or S, X ４７ GGRHGX ４８ GX ４９ X ５０ DQIENRX ５１ IEA (Sequence No. 16), the X ４７ is I or V; X ４８ is L or M; X ４９ is M or V; X ５０ is A or S; X ５１ is I or V, KSRGIYEAPG (Sequence ID 17), X ５２ ALX ５３ X ５４ X ５５ AX ５６ ERLX ａ X ５７ X ５８ IHNEDT (Sequence No. 18), the X ５２ is L or M; X ５３ is F or L; X ５４ is F, H, or Y; X ５５ is A or I; X ５６ is F or Y; X ａ is an amino acid; X ５７ is N, S or T; X ５８ is A or G, LGX ５９ LX ６０ YX ６１ GRWX ６２ DX ６３ QX ６４ X ６５ MX ６６ RX ６７ (Sequence No. 19), the X ５９ is K or R; X ６０ is L or M; X ６１ is A, E, or Q; X ６２ is F or L; X ６３ is P or S; X ６４ is A, S or G; X ６５ is I, L, or M; X ６６ is I, L, or V; X ６７ is D or E, LRRGX ａ DX ６８ X ６９ X ７０ (Sequence No. 20), the X ａ is an amino acid; X ６８ is F or Y; X ６９ is S or T;X ７０ is I or L, X ７１ X ７２ X ７３ X ７４ X ａ X ７５ X ７６ X ７７ LX ７８ MEX ７９ (Sequence No. 21), the X ７１ is A, D, or N; X ７２ is F or L; X ７３ is S or T;X ７４ is F or Y; X ａ is an amino acid; X ７５ is A, P or S; X ７６ is A, E, or D; X ７７ is K or R; X ７８ is S or T;X ７９ is K or R, DRIGQLX ８０ MRX ８１ X ８２ DX ８３ X ａ DX ８４ R (Sequence No. 22), the above X ８０ is H or T;X ８１ is L, N, or T; X ８２ is L or N; X ８３ is I, L, or V; X ａ is an amino acid; X ８４ is S or T. The manipulated host cell according to claim 32, comprising at least one sequence selected from the group consisting of the following.

34. The recombinant amino acid molecule comprises all of SEQ ID NOs: 6 to 22, according to the manipulated host cell according to claim 32 or 33.

35. The recombinant amino acid molecule comprises at least one sequence selected from the group consisting of the following, in the manipulated host cell according to any one of claims 32 to 34: A. A sequence having at least 79% sequence identity with sequence number 23, which is a capsulatum sequence; A. A sequence having at least 80% sequence identity with sequence number 24, which is the faecalis sequence; B. Sequences having at least 78% sequence identity with sequence number 25, which is a pyrocinia sequence; C. Sequences having at least 80% sequence identity with sequence number 26, which is a necator sequence; E. A sequence having at least 78% sequence identity with sequence number 27, which is a coli sequence; and A sequence having at least 80% sequence identity with sequence number 28, which is the N. weaveri sequence.

36. The engineered host cell according to claim 35, wherein the recombinant amino acid molecule comprises a sequence having at least 80% sequence identity with SEQ ID NO:

26.

37. The engineered host cell according to claim 35, wherein the recombinant amino acid molecule comprises a sequence having at least 78% sequence identity with SEQ ID NO:

27.

38. The manipulated host cell according to claim 35, wherein the recombinant amino acid molecule comprises a sequence having at least 79% sequence identity with SEQ ID NO: 23; at least 80% sequence identity with SEQ ID NO: 24; at least 78% sequence identity with SEQ ID NO: 25; at least 80% sequence identity with SEQ ID NO: 26; at least 78% sequence identity with SEQ ID NO: 27; and at least 80% sequence identity with SEQ ID NO:

28.

39. The recombinant amino acid molecule comprises at least one sequence selected from the group consisting of the following, in the manipulated host cell according to any one of claims 32 to 38: Sequences corresponding to positions 1-153 and 167-445 of sequence number 23 with at least 79% accuracy; Sequences corresponding to positions 1-153 and 167-444 of sequence number 24, with at least 80% of the data; Sequences corresponding to positions 1-153 and 167-445 of sequence number 25, with at least 78% of the data; Sequences corresponding to positions 1-153 and 167-441 of sequence number 26, with at least 80% of the data; Sequences corresponding to positions 1-153 and 167-447 of sequence number 27 and at least 78% of the sequences; and A sequence that corresponds to at least 80% of positions 1-157 and 171-448 of sequence number 28.

40. The engineered host cell according to claim 39, wherein the recombinant amino acid molecule comprises a sequence that corresponds at least 80% to positions 1 to 153 and 167 to 441 of SEQ ID NO:

26.

41. The engineered host cell according to claim 39, wherein the recombinant amino acid molecule comprises a sequence that corresponds at least 78% to positions 1 to 153 and 167 to 447 of SEQ ID NO:

27.

42. The recombinant amino acid molecule comprises at least one sequence selected from the group consisting of the following, in the manipulated host cell according to any one of claims 32 to 41: The corresponding sequences for positions 1-153 and 167-445 of sequence number 23; The corresponding sequences for positions 1-153 and 167-444 of sequence number 24; The corresponding sequences for positions 1-153 and 167-445 of sequence number 25; The corresponding sequences for positions 1-153 and 167-441 of sequence number 26; The sequences corresponding to positions 1 to 153 and 167 to 447 of sequence number 27; and The sequence corresponding to positions 1-157 and 171-448 of sequence number 28.

43. The engineered host cell according to claim 42, wherein the recombinant amino acid molecule includes sequences corresponding to positions 1 to 153 and 167 to 441 of SEQ ID NO:

26.

44. The engineered host cell according to claim 42, wherein the recombinant amino acid molecule comprises sequences corresponding to positions 1 to 153 and 167 to 447 of SEQ ID NO:

27.

45. The recombinant amino acid molecule is encoded by a nucleotide molecule comprising at least one sequence selected from the group consisting of the following, in the manipulated host cell according to any one of claims 32 to 44: A. A sequence having at least 79% sequence identity with sequence number 33, which is a capsulatum sequence; A. A sequence having at least 80% sequence identity with sequence number 34, which is the faecalis sequence; B. Sequences having at least 78% sequence identity with sequence number 35, which is a pyrocinia sequence; C. Sequences having at least 80% sequence identity with sequence number 36, which is a necator sequence; E. A sequence having at least 78% sequence identity with sequence number 37, which is a coli sequence; and A sequence having at least 80% sequence identity with sequence number 38, which is the N. weaveri sequence.

46. The engineered host cell according to claim 45, wherein the recombinant amino acid molecule is encoded by a nucleotide molecule having at least 80% sequence identity with sequence number 36, which is a C. necator sequence.

47. The engineered host cell according to claim 45, wherein the recombinant amino acid molecule is encoded by a nucleotide molecule having at least 78% sequence identity with sequence number 37, which is an E. coli sequence.

48. The engineered host cell according to any one of claims 32 to 47, wherein the recombinant amino acid molecule is argininosuccinate synthase.

49. The manipulated host cell according to any one of claims 32 to 48, wherein the manipulated host cell is a microorganism.

50. The manipulated host cell according to any one of claims 32 to 49, wherein the manipulated host cell is Corynebacterium.

51. The manipulated host cells include Corynebacterium glutamicum, Corynebacterium stationis, Corynebacterium crudilactis, Corynebacterium Deserti, Corynebacterium efficiens, Corynebacterium callunae, and Corynebacterium singulare. Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris, Corynebacterium flavecens A manipulated host cell according to any one of claims 32 to 50, selected from the group consisting of *Corynebacterium flavescens*, *Corynebacterium crenatum*, and *Corynebacterium suranareeae*.

52. The manipulated host cell according to any one of claims 32 to 51, wherein the manipulated host cell is Corynebacterium glutamicum.

53. The manipulated host cell according to any one of claims 32 to 52, wherein the manipulated host cell produces L-arginine.

54. The manipulated host cell according to any one of claims 32 to 53, wherein the manipulated host cell is transformed with an argR gene containing a sequence having at least 90% sequence identity with sequence number 39, and an argB gene containing a sequence having at least 90% sequence identity with sequence number 40 and having M54V.

55. The recombinant amino acid molecule has the exogenous amino acid sequence of the host cell, according to any one of claims 32 to 54.

56. Use of an amino acid molecule according to any one of claims 1 to 20, a host cell according to any one of claims 21 to 27, a nucleic acid molecule according to any one of claims 28 to 30, a vector according to claim 31, or an engineered host cell according to any one of claims 32 to 55 for L-arginine production.

57. A method for producing L-arginine, comprising culturing a host cell according to any one of claims 21 to 27 or an engineered host cell according to any one of claims 32 to 55.

58. The method according to claim 57, further comprising recovering L-arginine from the cultured host cells.

59. The method according to claim 57 or 58, further comprising recovering L-arginine from the culture medium in which the host cells were cultured.

60. The method according to any one of claims 57 to 59, wherein the culture is carried out using at least one carbon source selected from the group consisting of carbohydrates, sugar alcohols, organic acids, amino acids, starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn maceration.

61. The method according to any one of claims 57 to 60, wherein the culture is carried out using at least one nitrogen source selected from the group consisting of ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, glutamic acid, methionine, glutamine, peptone, NZ-amine, meat extract, yeast extract, malt extract, corn maceration, casein hydrolysate, fish or its decomposition products, and defatted soybean meal or its decomposition products.

62. The method according to any one of claims 57 to 61, wherein the culture is carried out using at least one selected from the group consisting of potassium phosphate, dipotassium phosphate, corresponding sodium-containing salts, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, and calcium carbonate.

63. A method for increasing L-arginine production by host cells, comprising transforming the host cells to produce the manipulated host cells according to any one of claims 32 to 55.

64. A. A sequence having at least 79% sequence identity with sequence number 33, which is a capsulatum sequence; A. A sequence having at least 80% sequence identity with sequence number 34, which is the faecalis sequence; B. Sequences having at least 78% sequence identity with sequence number 35, which is a pyrocinia sequence; C. Sequences having at least 80% sequence identity with sequence number 36, which is a necator sequence; E. A sequence having at least 78% sequence identity with sequence number 37, which is a coli sequence; and A sequence having at least 80% sequence identity with sequence number 38, which is the N. weaveri sequence. A nucleic acid molecule comprising at least one sequence selected from the group consisting of, The aforementioned nucleic acid molecule is a nucleic acid molecule that does not exist in nature.

65. A nucleic acid molecule according to claim 64, comprising a sequence having at least 80% sequence identity with sequence number 36, which is a necator sequence.

66. E. A nucleic acid molecule according to claim 64 or 65, comprising a sequence having at least 78% sequence identity with sequence number 37, which is a coli sequence.

67. Use of a product or method characterized by one or more elements in this disclosure.

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