Microorganism with enhanced activity of aromatic amino acid transporter and uses thereof

Abstract

The present disclosure provides a gene expression cassette, a microorganism with enhanced activity of an aromatic amino acid transporter, a method for producing valine using the microorganism, and a method for increasing valine production.

Description

Microorganisms with enhanced aromatic amino acid transporter activity and their uses

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

[0002] This application claims the benefit of priority from Republic of Korea Patent Application No. 10-2023-0190244, dated December 22, 2023, and all contents of said Republic of Korea patent disclosure document are incorporated herein by reference.

[0003] The present disclosure relates to a microorganism with enhanced aromatic amino acid transporter activity and its use.

[0004]

[0005] Production of branched-chain amino acids using microorganisms is mainly accomplished through microorganisms of the genus Escherichia or the genus Corynebacterium, and in the case of L-valine, it is known that it is biosynthesized from pyruvate through several steps using 2-oxoisovalerate as a precursor, but it is not easy to mass-produce L-valine using microorganisms on an industrial scale.

[0006] Therefore, research is still needed to effectively increase L-valine production.

[0007]

[0008] [Prior Art Literature]

[0009] [Patent Document]

[0010] (Patent Document 1) U.S. Patent Publication No. US 2020-0362374 A1

[0011]

[0012] One object of the present disclosure is to provide a microorganism having enhanced valine production capacity and enhanced aromatic amino acid transporter activity.

[0013] Another object of the present disclosure is to provide a gene expression cassette comprising a promoter and a gene encoding an aromatic amino acid transporter operably linked to the promoter.

[0014] Another object of the present disclosure is to provide a method for producing valine, comprising a step of culturing the microorganism in a medium.

[0015] Another object of the present disclosure is to provide a method for increasing valine production, comprising the step of culturing the microorganism in a medium.

[0016] Another object of the present disclosure is to provide a composition for producing valine, comprising at least one selected from the group consisting of the above microorganism and a medium in which the above microorganism is cultured.

[0017]

[0018] This is specifically explained as follows. Meanwhile, each description and embodiment of the present disclosure can also be applied to each other description and embodiment. That is, all combinations of various elements of the present disclosure fall within the scope of the present disclosure. Furthermore, the scope of the present disclosure is not limited by the specific descriptions described below. Furthermore, numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to more clearly explain the level of the technical field to which the present invention pertains and the content of the present invention.

[0019] In the present disclosure, it was confirmed that the activity of an aromatic amino acid transporter (aroP) was enhanced, resulting in an increase in valine production and yield, and a decrease in the production of by-products other than valine. Accordingly, a promoter for enhancing the activity of an aromatic amino acid transporter and a microorganism producing valine with enhanced activity of an aromatic amino acid transporter are provided.

[0020] In the present disclosure, an aromatic amino acid transporter (aromatic amino acid transporter, or aroP protein) may refer to a protein having aromatic amino acid transport activity. The aromatic amino acid transporter may be derived from a microorganism belonging to the genus Corynebacterium. The aromatic amino acid transporter may be derived from Corynebacterium glutamicum. The sequence of the aromatic amino acid transporter may be obtained from a known database (NCBI) or the like (Sequence ID: WP_003863791.1), and may, for example, include or consist of the amino acid sequence of SEQ ID NO: 1. In one example, the aromatic amino acid transporter may have, but is not limited to, an amino acid sequence of SEQ ID NO: 1 or an amino acid sequence of SEQ ID NO: 1 having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.2%, at least 98.4%, at least 98.6%, at least 98.8%, at least 98.9%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% homology or identity with, or comprising, or consisting of, the amino acid sequence.

[0021] Additionally, if the amino acid sequence exhibits an effect corresponding to the aromatic amino acid transporter having such homology or identity, variants having an amino acid sequence in which some of the sequences are deleted, modified, substituted, conservatively substituted, or added may also be included in the aromatic amino acid transporter. For example, the amino acid sequence may have sequence additions or deletions, naturally occurring mutations, silent mutations, or conservative substitutions that do not alter aromatic amino acid transporter activity at the N-terminus, C-terminus, and / or within the amino acid sequence.

[0022] The term "conservative substitution" refers to the replacement of one amino acid with another amino acid with similar structural and / or chemical properties. Such amino acid substitutions may generally be based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. Typically, conservative substitutions may have little or no effect on the activity of a protein or polypeptide.

[0023] The above aromatic amino acid transporter may be a protein having aromatic amino acid transporter activity, encoded by an aromatic amino acid transporter gene. The aromatic amino acid transporter gene may be derived from a microorganism of the genus Corynebacterium. The aromatic amino acid transporter gene may be derived from Corynebacterium glutamicum. The sequence of the aromatic amino acid transporter gene may be obtained from a known database (NCBI), etc. The aromatic amino acid transporter gene may include the nucleic acid sequence of SEQ ID NO: 2, or may be composed of the nucleic acid sequence of SEQ ID NO: 2. In one example, the aromatic amino acid transporter gene may have, but is not limited to, a homology or identity of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.2%, at least 98.4%, at least 98.6%, at least 98.8%, at least 98.9%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% with the nucleic acid sequence of SEQ ID NO: 2, or may comprise the nucleic acid sequence, or may consist of the nucleic acid sequence.

[0024]

[0025] In the present disclosure, the phrase "a polynucleotide or polypeptide has, includes, consists of, or consists essentially of a specific nucleic acid sequence (base sequence) or amino acid sequence" may mean that the polynucleotide or polypeptide essentially includes the specific nucleic acid sequence or amino acid sequence, and may be interpreted as including (or not excluding) a "substantially equivalent sequence" in which a mutation (deletion, substitution, modification, and / or addition) is added to the specific nucleic acid sequence or amino acid sequence within the scope of maintaining the original function and / or desired function of the polynucleotide or polypeptide. In one example, a polynucleotide or polypeptide "has, comprises, consists of, or consists essentially of a particular nucleic acid sequence (base sequence) or amino acid sequence" means that the polynucleotide or polypeptide (i) comprises the particular nucleic acid sequence or amino acid sequence, or (ii) is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.2%, at least 98.4%, at least 98.6%, at least 98.8%, at least 98.9%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, It may mean that it consists of or includes a nucleic acid sequence or amino acid sequence having a homology or identity of 99.8% or more, or 99.9% or more, and maintains the original function and / or the intended function.

[0026] In this disclosure, 'homology' or 'identity' refers to the degree of similarity between two given amino acid sequences or base sequences, which may be expressed as a percentage. The terms homology and identity are often used interchangeably.

[0027] Sequence homology or identity of conserved polynucleotides or polypeptides is determined by standard alignment algorithms, and may be combined with default gap penalties established by the program being used. In practice, homologous or identical sequences can generally hybridize with all or part of the sequence under moderate or high stringency conditions. It should be appreciated that hybridization also includes hybridization with polynucleotides containing common codons or codons that take codon degeneracy into account.

[0028] Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined using known computer algorithms such as the "FASTA" program using default parameters, for example, as in Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444. Alternatively, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later) can be determined using the GCG program package (Devereux, J., et al, Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego, 1994, and [CARILLO ETA / .](1988) SIAM J Applied Math 48: 1073). For example, homology, similarity, or identity can be determined using BLAST or ClustalW of the National Center for Biotechnology Information Database.

[0029] Homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using a GAP computer program, such as that disclosed, for example, in Smith and Waterman, Adv. Appl. Math (1981) 2:482, or in, for example, Needleman et al. (1970), J Mol Biol. 48:443. In brief, the GAP program can be defined as the total number of symbols in the shorter of the two sequences divided by the number of similarly arranged symbols (i.e., nucleotides or amino acids). Default parameters for the GAP program include (1) a binary comparison matrix (containing values ​​of 1 for identity and 0 for non-identity) and (2) a coding sequence matrix, as disclosed by Gribskov et al. (1986) Nucl. Acids Res. 48:443, and (3) a binary comparison matrix, containing values ​​of 1 for identity and 0 for non-identity, as disclosed by Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979). 14: A weighted comparison matrix of 6745 (or a permutation matrix of EDNAFULL (EMBOSS version of NCBI NUC4.4)); (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 extension penalty of 0.5); and (3) no penalty for terminal gaps.

[0030]

[0031] One aspect provides a microorganism with enhanced aromatic amino acid transporter activity. The microorganism with enhanced aromatic amino acid transporter activity may have one or more characteristics selected from the group consisting of (i) increased valine production; (ii) increased sugar consumption rate; and (iii) decreased byproduct production (e.g., aromatic amino acids, such as one or more amino acids selected from the group consisting of phenylalanine, tyrosine, and tryptophan).

[0032] The above-mentioned enhancement of the activity of the aromatic amino acid transporter may mean that the activity of the aromatic amino acid transporter is increased compared to the intrinsic activity. The above-mentioned enhancement may be used interchangeably with terms such as activation, up-regulation, overexpression, and increase. Here, activation, enhancement, up-regulation, overexpression, and increase may all include exhibiting an activity that was not originally present, or exhibiting an enhanced activity compared to the intrinsic activity or the activity before modification. The above-mentioned “intrinsic activity” refers to the activity of a specific aromatic amino acid transporter that a parent strain or an unmodified microorganism originally had before the trait change when the trait changes due to genetic mutation caused by natural or artificial factors. This may be used interchangeably with “activity before modification.” The phrase “enhancement”, “upregulation”, “overexpression” or “increase” in the activity of an aromatic amino acid transporter compared to its endogenous activity may mean an increase in the activity of a specific aromatic amino acid transporter that the parent strain or unmodified microorganism originally had before the transformation, for example, an increase in the expression of a gene encoding the aromatic amino acid transporter. Whether the activity of the aromatic amino acid transporter is enhanced can be confirmed from the amount of mRNA transcription of the gene encoding the aromatic amino acid transporter, the amount of expression of the aromatic amino acid transporter, the degree of activity of the aromatic amino acid transporter or the amount of a byproduct (e.g., an aromatic amino acid) produced by the microorganism.

[0033] The enhancement of the activity of the above aromatic amino acid transporter can be achieved by applying various methods well known in the art, and is not limited as long as the activity of the aromatic amino acid transporter can be enhanced compared to that of the microorganism before transformation. Specifically, it may be achieved by using genetic engineering and / or protein engineering, which are routine methods of molecular biology and are well known to those skilled in the art, but is not limited thereto (e.g., Sitnicka et al. Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol. 2. 1-16, Sambrook et al. Molecular Cloning 2012, etc.).

[0034] Specifically, the enhancement of the polypeptide (aromatic amino acid transporter) of the present disclosure is

[0035] 1) Increase in the intracellular copy number of a polynucleotide encoding a polypeptide;

[0036] 2) Replacing the gene expression control region encoding the polypeptide with a highly active sequence;

[0037] 3) Modification of the base sequence encoding the initiation codon or 5'-UTR region of a gene transcript encoding a polypeptide;

[0038] 4) Modification of the amino acid sequence of the polypeptide so as to enhance polypeptide activity;

[0039] 5) Modification of the polynucleotide sequence encoding the polypeptide so as to enhance the activity of the polypeptide (e.g., modification of the polynucleotide sequence of the polypeptide gene so as to encode a polypeptide modified so as to enhance the activity of the polypeptide);

[0040] 6) Introduction of a foreign polypeptide exhibiting the activity of the polypeptide or a foreign polynucleotide encoding the same;

[0041] 7) Codon optimization of polynucleotides encoding polypeptides;

[0042] 8) Analyzing the tertiary structure of the polypeptide and selecting the exposed portion to modify or chemically modify; or

[0043] 9) It may be a combination of two or more of the above 1) to 8), but is not particularly limited thereto.

[0044] More specifically,

[0045] The increase in the intracellular copy number of the polynucleotide encoding the polypeptide described above 1) may be achieved by introducing one copy or two or more copies of a vector containing the polynucleotide encoding the polypeptide into a microorganism (host cell). The polynucleotide encoding the polypeptide may be introduced into the microorganism by a vector capable of inserting the polynucleotide into the chromosome of the microorganism, or may be introduced into the microorganism in the form of a vector capable of replicating and functioning independently of the microorganism, but is not limited thereto.

[0046] The polynucleotide encoding the above polypeptide may be operably linked to a promoter.

[0047] The term "operably linked" as used herein means that the promoter of the present disclosure is functionally linked to a gene sequence such that the promoter of the present disclosure initiates and mediates transcription of the gene of interest (e.g., a gene encoding an aromatic amino acid transporter). Operable linkages can be produced using genetic recombination techniques known in the art of the present disclosure, and site-specific DNA cleavage and ligation can be produced using cleavage and ligation enzymes known in the art of the present disclosure.

[0048] The above promoter may be at least one selected from the group consisting of the original promoter of the polynucleotide encoding the polypeptide; and a strong promoter different from the original promoter.

[0049] The above native promoter may refer to a promoter operably linked to a polynucleotide encoding the polypeptide within the chromosome of a wild-type microorganism. In one example, the native promoter may be a promoter linked to the NCgl1063 gene of Corynebacterium glutamicum.

[0050] The strong promoter may be a promoter of a microbial endogenous gene or a foreign gene. The strong promoter may be derived from a microorganism of the genus Corynebacterium. In one specific example, the strong promoter may be derived from Corynebacterium glutamicum.

[0051] In one example, the strong promoter may be, but is not limited to, Ppyk (promoter of the pyk gene), Pald (promoter of the ald gene), PctaE (promoter of the ctaE gene), PpfkA (promoter of the pfkA gene), CJ1 to CJ7 promoters (US Patent No. US 7662943 B2), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13 (sm3) promoter (US Patent No. US 10584338 B2), O2 promoter (US Patent No. US 10273491 B2), tkt promoter, yccA promoter, etc.

[0052] The above pyk gene may be a gene encoding pyruvate kinase. In one example, the promoter of the pyk gene may include the nucleic acid sequence of SEQ ID NO: 3 or may be composed of the nucleic acid sequence of SEQ ID NO: 3.

[0053] The above ald gene may be a gene encoding acetaldehyde dehydrogenase. In one example, the promoter of the ald gene may include the nucleic acid sequence of SEQ ID NO: 4 or may be composed of the nucleic acid sequence of SEQ ID NO: 4.

[0054] The above ctaE gene may be a gene encoding cytochrome c oxidase subunit 3. In one example, the promoter of the ctaE gene may include the nucleic acid sequence of SEQ ID NO: 21 or may be composed of the nucleic acid sequence of SEQ ID NO: 21.

[0055] The above pfkA gene may be a gene encoding ATP-dependent 6-phosphofructokinase isozyme 1. In one example, the promoter of the pfkA gene may include the nucleic acid sequence of SEQ ID NO: 22 or may be composed of the nucleic acid sequence of SEQ ID NO: 22.

[0056] The above microorganism may have enhanced activity of an aromatic amino acid transporter, including a gene encoding an aromatic amino acid transporter operably linked to the strong promoter.

[0057] The polynucleotide encoding the polypeptide may be inserted into a location that does not affect the expression of other genes of the host cell, such as a genomic safe harbor. In one example, the safe harbor gene site may be a site between the NCgl0866 gene and the NCgl0867 gene of Corynebacterium glutamicum, or a site between the NCgl2195 gene and the NCgl2196 gene of Corynebacterium glutamicum.

[0058] 2) Replacing the gene expression control region encoding the polypeptide with a sequence having strong activity may involve, for example, mutations in the sequence, such as deletion, insertion, non-conservative or conservative substitution, or a combination thereof, to further enhance the activity of the expression control region, or replacement with a sequence having stronger activity. The expression control region may include, but is not particularly limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating the termination of transcription and translation. As an example, replacing the gene expression control region of the gene encoding the aromatic amino acid transporter with a sequence having strong activity may involve, but is not limited to, replacing the original promoter with a strong promoter.

[0059] The microorganism having enhanced activity of the aromatic amino acid transporter may have enhanced activity of the aromatic amino acid transporter gene by operably linking a strong promoter to the gene encoding the aromatic amino acid transporter. As described above, the operably linking of the strong promoter to the aromatic amino acid transporter gene may mean replacing (substituting) the promoter of the original aromatic amino acid transporter gene with a strong promoter, or positioning the strong promoter in front of the aromatic amino acid transporter gene so that the expression of the aromatic amino acid transporter gene can be regulated by the strong promoter.

[0060] The above strong promoter may be at least one selected from the group consisting of Ppyk (promoter of the pyk gene), Pald (promoter of the ald gene), PctaE (promoter of the ctaE gene), PpfkA (promoter of the pfkA gene), CJ1 to CJ7 promoters (US Patent No. US 7662943 B2), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13 (sm3) promoter (US Patent No. US 10584338 B2), O2 promoter (US Patent No. US 10273491 B2), tkt promoter, yccA promoter, etc., as described above, but is not limited thereto.

[0061] The above 3) modification of the base sequence encoding the initiation codon or 5'-UTR region of the gene transcript encoding the polypeptide may be, for example, a substitution with a base sequence encoding another initiation codon having a higher polypeptide expression rate than the endogenous initiation codon, but is not limited thereto.

[0062] The modification of the amino acid sequence or polynucleotide sequence of the above 4) and 5) may be, but is not limited to, a mutation in the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide to enhance the activity of the polypeptide, or replacement with an amino acid sequence or polynucleotide sequence improved to have stronger activity, or an amino acid sequence or polynucleotide sequence improved to have increased activity. The replacement may be specifically performed by inserting the polynucleotide into a chromosome by homologous recombination, but is not limited thereto. The vector used at this time may additionally include a selection marker to confirm whether or not chromosomal insertion has occurred.

[0063] The introduction of the foreign polynucleotide exhibiting the activity of the polypeptide as described above 6) may be the introduction into the host cell of a foreign polynucleotide encoding a polypeptide exhibiting the same / similar activity as the polypeptide. The foreign polynucleotide is not limited in its origin or sequence as long as it exhibits the same / similar activity as the polypeptide. The method used for the introduction may be performed by a person skilled in the art appropriately selecting a known transformation method, and the polypeptide may be produced by expressing the introduced polynucleotide in the host cell, thereby increasing its activity.

[0064] The above 7) codon optimization of a polynucleotide encoding a polypeptide may be codon optimization of an endogenous polynucleotide to increase transcription or translation within a host cell, or codon optimization of a foreign polynucleotide to achieve optimized transcription or translation within a host cell.

[0065] The above 8) analyzing the tertiary structure of a polypeptide and selecting an exposed portion to modify or chemically modify may be done by, for example, comparing the sequence information of the polypeptide to be analyzed with a database storing the sequence information of known proteins, determining a template protein candidate based on the degree of sequence similarity, confirming the structure based on this, and selecting an exposed portion to modify or chemically modify, and modifying or modifying it.

[0066] Such enhancement of polypeptide activity may be, but is not limited to, an increase in the activity or concentration of the corresponding polypeptide relative to the activity or concentration of the polypeptide expressed in the wild type or pre-transformed microbial strain, or an increase in the amount of a product produced from the polypeptide.

[0067] Modification of part or all of a gene for enhancing the activity of an aromatic amino acid transporter in a microorganism of the present disclosure may be induced by, but is not limited to, (a) homologous recombination using a vector for chromosome insertion into the microorganism or genome editing using engineered nucleases (e.g., CRISPR-Cas9) and / or (b) treatment with light and / or chemicals such as ultraviolet rays and radiation. The method for modifying part or all of the gene may include a method using DNA recombination technology. For example, a nucleotide sequence or vector containing a nucleotide sequence homologous to a target gene may be injected into the microorganism to cause homologous recombination, thereby causing deletion of part or all of the gene. The injected nucleotide sequence or vector may include, but is not limited to, a dominant selection marker.

[0068]

[0069] The microorganism of the present disclosure may be, but is not limited to, a microorganism with enhanced aromatic amino acid transporter activity, or a microorganism genetically modified via a vector to enhance the activity of the aromatic amino acid transporter (e.g., a recombinant microorganism). The microorganism (or strain, recombinant cell) of the present disclosure may be a microorganism with enhanced aromatic amino acid transporter activity, thereby having valine production ability or enhanced valine production ability (or production amount).

[0070] The microorganism of the present disclosure may have one or more characteristics selected from among (i) increased valine production ability; (ii) increased sugar consumption rate; and (iii) decreased production ability of byproducts (e.g., aromatic amino acids, such as one or more amino acids selected from the group consisting of phenylalanine, tyrosine, and tryptophan).

[0071]

[0072] The microorganism of the present disclosure may be a microorganism that naturally has valine production ability, or a microorganism that has valine production ability conferred or enhanced upon a parent strain that does not have valine production ability, but is not limited thereto. The microorganism may be a microorganism that has valine production ability or has increased valine production ability. The microorganism may be a microorganism that does not have valine production ability, or a microorganism that has valine production ability conferred or has valine production ability enhanced by introducing an enhanced aromatic amino acid transporter into a microorganism that does have valine production ability, but is not limited thereto.

[0073] The fact that the above microorganism has valine production ability or has improved valine production ability may mean that the microorganism is endowed with valine production ability, unlike a non-modified microorganism, a cell before recombination, a parent strain, and / or a wild-type strain that does not have valine production ability, or that the valine production ability is increased compared to a non-modified microorganism, a cell before recombination, a parent strain, and / or a wild-type strain.

[0074] According to an example, a microorganism with enhanced activity of an aromatic amino acid transporter may have increased valine production capacity compared to a microorganism prior to introduction, i.e., an identical, unmodified microorganism. In the present disclosure, the term "unmodified microorganism" does not exclude a strain containing a mutation that may occur naturally in a microorganism, and may refer to a wild-type strain or a natural strain itself, or a strain before its characteristics are changed by genetic mutation due to natural or artificial factors. For example, the unmodified microorganism may refer to a microorganism in which the activity of an aromatic amino acid transporter is not enhanced, or before the activity of the aromatic amino acid transporter is enhanced. The term "unmodified microorganism" may be used interchangeably with "pre-modified strain," "pre-modified microorganism," "unmutated strain," "unmodified microorganism," or "reference microorganism."

[0075] The above microorganism (or strain, recombinant cell) may additionally include a mutation that increases valine production, and the location of the mutation and / or the type of gene and / or protein that is the target of the mutation may be included without limitation as long as it increases valine production. The above recombinant cell may be used without limitation as long as it is a cell capable of transformation.

[0076] In one example, the microorganism of the present disclosure may further comprise an A42V variant of the Acetolactate synthase isozyme 1 small subunit (IlvN) protein or a nucleotide encoding the same (see Biotechnology and Bioprocess Engineering, June 2014, Volume 19, Issue 3, pp 456-467).

[0077] In one example, the non-modified microorganism, which is a target strain for comparing whether the valine production ability is increased, may be, but is not limited to, a strain in which the A42V mutation in the acetolactate synthase isozyme 1 small subunit (IlvN) protein is introduced in Corynebacterium glutamicum ATCC13032 strain, Corynebacterium glutamicum KCCM11201P (US Patent Publication No. US 8465962 B), and Corynebacterium glutamicum ATCC14067, thereby improving the valine production ability [ilvN (A42V); Biotechnology and Bioprocess Engineering, June 2014, Volume 19, Issue 3, pp 456-467; US Patent Publication No. US 11180784 B2].

[0078]

[0079] For example, the microorganism (or strain, recombinant cell) with improved (increased) valine production ability (or production amount, yield) has a valine production ability of 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 15% or more, 16% or more, 17% or more, 18% or more, 19% or more, 20% or more, 21% or more, 22% or more, 23% or more, 24% or more, 25% or more, 26% or more, 27% or more, 28% or more, 29% or more, 30% or more, 31% or more, 32% or more, 33% or more, It may be increased by 34% or more or 35% or more (the upper limit is not particularly limited, and may be, for example, about 200% or less, about 150% or less, about 100% or less, about 50% or less, about 45% or less, about 40% or less, or about 35% or less), but is not limited thereto.

[0080] In another example, the microorganism (or strain, recombinant cell) with improved (increased) valine productivity (or production, yield) may have an increased valine productivity of about 1.05 times or more, about 1.1 times or more, about 1.15 times or more, about 1.2 times or more, about 1.25 times or more, or about 1.3 times or more, compared to the parent strain before mutation or the unmodified microorganism, but is not limited thereto. The term “about” includes all of ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all of the values ​​in the range equivalent to or similar to the value following the term “about,” but is not limited thereto.

[0081] The above microorganism may be a microorganism of the genus Corynebacterium.

[0082] The above-mentioned Corynebacterium genus microorganisms are Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes, and Corynebacterium pollutisoli. It consists of Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris, Corynebacterium acetoacidophilum, Corynebacterium acetoglutamicum, Corynebacterium alkanolyticum, Corynebacterium lilium, Corynebacterium melassecola, Corynebacterium thermoaminogenes, Corynebacterium herculis and Corynebacterium flavescens. There may be one or more types selected from the military, but they are not limited thereto.

[0083]

[0084] Another aspect provides a gene expression cassette comprising the promoter and an aromatic amino acid transporter gene operably linked to the promoter.

[0085] As used herein, the term "gene expression cassette" may refer to a unit cassette that includes a promoter and a target gene, and is capable of expressing the target gene operably linked downstream of the promoter. Various factors that can assist in the efficient expression of the target gene may be included inside or outside of such a gene expression cassette. The gene expression cassette may typically include, but is not limited to, a transcription termination signal, a ribosome binding site, and a translation termination signal in addition to a promoter operably linked to the target gene.

[0086] The above "target gene" refers to a gene whose expression is to be controlled by the promoter sequence of the present disclosure for the purposes of the present disclosure. The protein encoded by the target gene may be referred to as the "target protein," and the gene encoding the "target protein" may be referred to as the "target gene."

[0087] In one example, the target gene may refer to an aromatic amino acid transporter gene to be expressed through the promoter.

[0088]

[0089] The gene encoding the aromatic amino acid transporter may be derived from a microorganism of the genus Corynebacterium, as described above. The gene encoding the aromatic amino acid transporter may be derived from Corynebacterium glutamicum. The gene encoding the aromatic amino acid transporter may comprise the nucleic acid sequence of SEQ ID NO: 2, or may consist of the nucleic acid sequence of SEQ ID NO: 2. In one example, the aromatic amino acid transporter gene may have, but is not limited to, a homology or identity of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.2%, at least 98.4%, at least 98.6%, at least 98.8%, at least 98.9%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% with the nucleic acid sequence of SEQ ID NO: 2, or may comprise the nucleic acid sequence, or may consist of the nucleic acid sequence.

[0090] The above promoter may be derived from a microorganism of the genus Corynebacterium, as described above. In one example, the promoter may be derived from Corynebacterium glutamicum.

[0091] The above promoter may be one or more strong promoters selected from the group consisting of Ppyk (promoter of pyk gene), Pald (promoter of ald gene), PctaE (promoter of ctaE gene), PpfkA (promoter of pfkA gene), CJ1 to CJ7 promoters (US Patent No. US 7662943 B2), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13 (sm3) promoter (US Patent No. US 10584338 B2), O2 promoter (US Patent No. US 10273491 B2), tkt promoter, yccA promoter, etc., as described above, but is not limited thereto. no.

[0092] In one example, the promoter may be at least one selected from the group consisting of a promoter of the pyk gene (SEQ ID NO: 3) and a promoter of the ald gene (SEQ ID NO: 4), but is not limited thereto.

[0093] In one embodiment, the gene expression cassette comprises at least one promoter selected from the group consisting of a promoter of a pyk gene, a promoter of an ald gene; and

[0094] It may include a gene encoding an aromatic amino acid transporter operably linked to the above promoter.

[0095] The above microorganism may be a microorganism comprising the above gene expression cassette.

[0096]

[0097] Another aspect provides a composition for producing valine, comprising at least one selected from the group consisting of the above microorganism and a medium in which the above microorganism is cultured. The composition of the present disclosure may further comprise any suitable excipient commonly used in compositions for producing valine, and such excipients may be, for example, but are not limited to, preservatives, wetting agents, dispersing agents, suspending agents, buffering agents, stabilizers, or isotonic agents.

[0098] In the composition of the present disclosure, the microorganism (strain), medium, and valine, etc. are as described in the other aspects above.

[0099] Another aspect provides a use for producing valine by using at least one selected from the group consisting of the above microorganism, the above microorganism, and a medium in which the above microorganism is cultured.

[0100]

[0101] Another aspect provides a method for producing valine or increasing valine production, comprising a step of culturing the microorganism in a medium. The microorganism and valine are as described above.

[0102] In the present disclosure, "cultivation" may refer to growing a microorganism (e.g., a Corynebacterium glutamicum strain) of the present disclosure under appropriately controlled environmental conditions. The culturing process of the present disclosure may be performed using a suitable medium and culture conditions known in the art. Such a culturing process can be easily adjusted and used by those skilled in the art depending on the selected strain. Specifically, the culturing may be batch, continuous, and / or fed-batch, but is not limited thereto.

[0103] In the present disclosure, "medium" means a substance containing as its main component nutrients necessary for culturing the microorganism of the present disclosure (e.g., Corynebacterium glutamicum strain), and supplies nutrients and growth factors, including water essential for survival and growth. Specifically, the medium and other culture conditions used for culturing the microorganism of the present disclosure may be any medium used for culturing general microorganisms without particular limitation, but the microorganism of the present disclosure may be cultured under aerobic conditions while controlling temperature, pH, etc. in a general medium containing an appropriate carbon source, nitrogen source, phosphorus, inorganic compound, amino acid, and / or vitamin.

[0104] For example, culture media for strains of the spp. Corynebacterium can be found in the "Manual of Methods for General Bacteriology" by the American Society for Bacteriology (Washington DC, USA, 1981).

[0105] In the present disclosure, the carbon source may include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvic acid, lactic acid, citric acid, etc.; amino acids such as glutamic acid, methionine, lysine, etc. In addition, natural organic nutrients such as starch hydrolysate, molasses, blackstrap molasses, rice winter, cassava, sugarcane bagasse, and corn steep liquor may be used, and specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted to reducing sugar) may be used, and other appropriate amounts of carbon sources may be used in various ways without limitation. These carbon sources may be used alone or in combination of two or more, but are not limited thereto.

[0106] The nitrogen source may include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc.; organic nitrogen sources such as amino acids such as glutamic acid, methionine, glutamine, etc.; peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolysate, fish or its decomposition product, defatted soybean cake or its decomposition product, etc. These nitrogen sources may be used alone or in combination of two or more, but are not limited thereto.

[0107] The above-mentioned components may include potassium phosphate monobasic, potassium phosphate dibasic, or their corresponding sodium-containing salts. Inorganic compounds may include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc. In addition, amino acids, vitamins, and / or suitable precursors may be included. These components or precursors may be added to the medium in batch or continuous manner, but are not limited thereto.

[0108] Additionally, during the cultivation of the microorganism of the present disclosure, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. may be added to the medium in an appropriate manner to adjust the pH of the medium. Furthermore, during the cultivation, foaming may be suppressed by using an antifoaming agent such as fatty acid polyglycol ester. Furthermore, to maintain the aerobic state of the medium, oxygen or an oxygen-containing gas may be injected into the medium, or to maintain anaerobic and microaerobic states, nitrogen, hydrogen, or carbon dioxide gas may be injected without gas injection, but is not limited thereto.

[0109] In the culture of the present disclosure, the culture temperature can be maintained at 20 to 45°C, specifically 25 to 40°C, and the culture can be performed for about 10 to 160 hours, but is not limited thereto.

[0110] The valine produced by the culture of the present disclosure may be secreted into the medium or remain within the cells.

[0111]

[0112] The method for producing valine or increasing valine production of the present disclosure may additionally include a step of preparing a microorganism (strain) of the present disclosure, a step of preparing a medium for culturing the microorganism, or a combination thereof (in any order), for example, prior to the culturing step.

[0113] The method for producing valine or increasing valine production of the present disclosure may further include a step of recovering valine from a culture medium (a culture medium in which valine is cultured) or a microorganism (Corynebacterium spp. strain) according to the culturing step. The recovering step may be additionally included after the culturing step.

[0114] The above recovery may be performed by collecting the desired valine using a suitable method known in the art according to the culture method of the microorganism of the present disclosure, such as a batch, continuous or fed-batch culture method. For example, various chromatographies such as centrifugation, filtration, treatment with a crystallizing protein precipitant (salting out method), extraction, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC or a combination thereof may be used, and the desired valine may be recovered from the medium or microorganism using a suitable method known in the art.

[0115] Additionally, the method for producing valine or increasing valine production of the present disclosure may additionally include a purification step. The purification may be performed using any suitable method known in the art. In one example, when the method for producing valine or increasing valine production of the present disclosure includes both a recovery step and a purification step, the recovery step and the purification step may be performed sequentially or discontinuously, regardless of the order, or may be performed simultaneously or integrated into a single step, but is not limited thereto.

[0116]

[0117] The microorganism with enhanced activity of the aromatic amino acid transporter of the present disclosure has excellent valine production ability and can be efficiently utilized for mass production of valine.

[0118]

[0119] The present invention will be described in more detail below with reference to the following examples. However, these examples are provided solely to illustrate the present invention, and the scope of the present invention is not limited by these examples.

[0120]

[0121] Example 1. Construction of a plasmid for enhancing aroP

[0122] A plasmid was constructed to enhance the activity of the aromatic amino acid transporter aroP by additionally inserting the aroP gene into the chromosome of Corynebacterium glutamicum KCCM11201P (US Patent Publication No. US 8465962 B), a previously known valine-producing strain. The additionally inserted aroP was designed to be expressed by two types of promoters.

[0123]

[0124]

[0125]

[0126] Example 1-1. Construction of plasmids for gene insertion

[0127] In order to strengthen the target gene by additionally inserting the aroP gene into the Corynebacterium glutamicum KCCM11201P chromosome, an intergenic site that does not encode genetic information in the KCCM11201P genome was used as an insertion site to eliminate the influence of gene deletion. The non-coding region between NCgl0866 and NCgl0867 (hereinafter referred to as NCgl0867up) was selected. At this time, the Ppyk (SEQ ID NO: 3) and Pald (SEQ ID NO: 4) promoters, which are stronger promoters than the promoter of the endogenous aroP gene, were used. PCR was performed using the KCCM11201P genome as the template chromosome for all experiments. PfuUltraTM high-fidelity DNA polymerase (Stratagene) was used as the polymerase for the PCR reaction, and the PCR conditions were denaturation at 95℃ for 30 sec; denaturation at 55℃ for 30 sec; and 72℃ 1 minute polymerization reaction, and denaturation, annealing, and polymerization reaction under these conditions were repeated 28 times.

[0128] Using the primers of SEQ ID NO: 5 and SEQ ID NO: 6, an 819-bp fragment A from the 5' upper portion of NCgl0867up was obtained, using the primers of SEQ ID NO: 9 and SEQ ID NO: 10, an 832-bp fragment B from the 3' lower portion of NCgl0867up was obtained, and using the primers of SEQ ID NO: 7 and SEQ ID NO: 8, a 1430-bp fragment C including the ORF portion of aroP and a portion of the 3' lower portion of aroP was obtained. Using SEQ ID NO: 11 and SEQ ID NO: 12, a 525-bp fragment E from the Ppyk promoter portion was obtained, and using SEQ ID NO: 13 and SEQ ID NO: 14, a 536-bp fragment F from the Pald promoter portion was obtained, respectively. The primer sequences used to perform each of the above PCRs are shown in Table 2 below. The amplified products were purified using a PCR purification kit (PCR Purification kit, QUIAGEN) and used as insert DNA fragments for vector construction.

[0129] SEQ ID NO NAME Sequence 5PrimergctcggtacccggggatccTCATTCGTTCATAATTGATCCCACAG6PrimerGGGGAGTGAATGCTGGGTG7PrimerATGGCTAAATCTAATGAAGGGCTG8PrimerTGGAGGGTCGGACGTCCAGAATAATTTCTATCGG9PrimerTCTGGACGTCCGACCCTCCACGCTGGA10Primercctgcaggtc gactctagaGGGCTCCTTCGTCATCATC11PrimerCCCAGCATTCACTCCCCCTCTACGTAGCTGGTTACACC12PrimerTTCATTAGATTTAGCCATGCCCATAAGCCTAGTACGTCA13PrimerCCCAGCATTCACTCCCCCGATCATCGAACTCGGCGAA14PrimerTCATTAGATTTAGCCATTGGGTCTCCTTTGGGCCAC

[0130] The vector pDC24_△NCgl0867up::Ppyk_aroP was constructed by cloning the previously obtained fragments A, B, C, E and the pDC24 vector (SEQ ID NO: 23, Table 3) treated with BamHI and XbaI (New England Biolabs, Berverly, MA) using the infusion cloning kit from TaKaRa according to the provided manual, thereby inserting the aroP gene expressed by the Ppyk promoter into the position between NCgl0866 and NCgl0867 of KCCM11201P.

[0131] The vector pDC24_△NCgl0867up::Pald_aroP, which can insert the aroP gene expressed by the Pald promoter into the position between NCgl0866 and NCgl0867 of KCCM11201P, was constructed by cloning the previously obtained fragments A, B, C, F and the pDC24 vector treated with BamHI and XbaI (New England Biolabs, Berverly, MA) using the infusion cloning kit from TaKaRa according to the provided manual.

[0132]

[0133]

[0134]

[0135]

[0136] Example 2. Production of a strain with enhanced aromatic amino acid transporter activity and confirmation of L-valine production ability.

[0137] An experiment was conducted to strengthen the aroP gene by additionally inserting the aroP gene into the chromosome of Corynebacterium glutamicum KCCM11201P (US Patent Publication No. US 8465962 B), a valine-producing strain.

[0138]

[0139] Example 2-1. Production of strains with additional insertion of the aroP gene

[0140] The pDC24_△NCgl0867up::Ppyk _aroP, pDC24_△NCgl0867up::Pald_aroP vectors constructed in Example 1-1 were transformed into Corynebacterium glutamicum KCCM11201P by homologous recombination on the chromosome (van der Rest et al., Appl Microbiol Biotechnol 52:541-545, 1999). The strain in which the vector was inserted into the chromosome by recombination of the homologous sequence was selected on a medium containing 25 mg / l of kanamycin. Afterwards, the above-mentioned Corynebacterium glutamicum transformant that completed the second recombination was subjected to PCR using the primers of SEQ ID NO: 15 and SEQ ID NO: 16, and the base sequence was confirmed to confirm whether additional genes were inserted. The strains in which the Ppyk_aroP and Pald_aroP genes were additionally inserted from the parent strain KCCM11201P on the chromosome were named Corynebacterium glutamicum KCCM11201P_△NCgl0867up::Ppyk_aroP and KCCM11201P_△NCgl0867up::Pald_aroP, respectively.

[0141] Sequence number name Sequence 15PrimerCGACATGGAACTAACTCTCCTAG16PrimerCACTTATGATCGCTGTGATCAGG

[0142] Example 2-2. Confirmation of L-valine production ability of strain with additional insertion of the aroP gene.

[0143] To compare the valine production ability of Corynebacterium glutamicum KCCM11201P, a valine-producing strain, and two strains, KCCM11201P_△NCgl0867up::Ppyk_aroP and KCCM11201P_△NCgl0867up::Pald_aroP, prepared in Example 2-1, a flask assay was performed. After subculturing each strain in nutrient medium, each strain was inoculated into a 250 ml corner-baffle flask containing 25 ml of production medium and cultured at 30°C for 72 hours with shaking at 200 rpm. The final OD, valine production yield, and relative sugar consumption rate of each strain were measured and are shown in Table 5 below.

[0144] [Nutrient medium (pH 7.2)]

[0145] 10g glucose, 5g meat extract, 10g polypeptone, 2.5g sodium chloride, 5g yeast extract, 20g agar, 2g urea (per 1 liter of distilled water)

[0146] [Production medium (pH 7.0)]

[0147] Glucose 100 g, ammonium sulfate 40 g, soy protein 2.5 g, corn steep solids 5 g, urea 3 g, potassium phosphate dibasic 1 g, magnesium sulfate heptahydrate 0.5 g, biotin 100 μg, thiamine-HCl 1 mg, calcium pantothenate 2 mg, nicotinamide 3 mg, calcium carbonate 30 g (based on 1 liter of distilled water)

[0148] Strain FN OD Valine yield Valine yield increase (compared to parent strain) Relative sugar consumption rate Tyrosine concentration Phenylalanine concentration 562 nm % % % mg / L mg / LK CCM11201P (parent strain) 71.8 2.7-100 70 190 KCCM11201P_△NCgl0867up::Ppyk_aroP 68.73.1114.8 111100 KCCM11201P_△NCgl0867up::Pald_aroP 70.52.8 103.7 105 30 10

[0149] As shown in Table 5, when the aroP gene was additionally inserted to enhance the expression of aroP, it was superior to KCCM11201P in terms of both relative sugar consumption rate, the yield did not decrease, and it was confirmed that the aromatic amino acids phenylalanine and tyrosine were reduced. Therefore, it was possible to confirm the effect of improving valine productivity and reducing byproducts through enhanced aroP gene expression. In particular, when the aroP gene operably linked to the Ppyk promoter was additionally inserted, it was confirmed that not only the sugar consumption rate increased but also the valine yield improved by 15%.

[0150]

[0151] Example 2-3. Evaluation of valine production capacity of Corynebacterium glutamicum CJ7V strain.

[0152] To determine whether the effect of aroP enhancement increases valine production in other strains of Corynebacterium glutamicum that produce valine, a strain with enhanced valine production was constructed by introducing a mutation [ilvN(A42V); Biotechnology and Bioprocess Engineering, June 2014, Volume 19, Issue 3, pp 456-467] into the acetolactate synthase isozyme 1 small subunit (IlvN) protein of wild-type Corynebacterium glutamicum ATCC14067.

[0153] First, to construct a vector introducing the A42V mutation into the ilvN gene, genomic DNA of the wild-type Corynebacterium glutamicum ATCC14067 strain was extracted using a G-spin Total DNA Extraction Mini Kit according to the manufacturer's protocol. Using the genomic DNA as a template, PCR was performed using the primer pairs of SEQ ID NOs: 17 and 18 and the primer pairs of SEQ ID NOs: 19 and 20, to obtain gene fragments A and B, respectively. The PCR conditions were denaturation at 94°C for 5 minutes; 28 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 60 seconds; and polymerization was performed at 72°C for 7 minutes. As a result, a 528-bp gene fragment H and a 509-bp gene fragment I were obtained. Overlapping PCR was performed using the above-obtained gene fragments H and I as templates and the primer pairs of SEQ ID NO: 17 and SEQ ID NO: 20. As a result, a 1010 bp PCR product (hereinafter referred to as "mutation-introduced fragment J") was obtained.

[0154] The mutation fragment J obtained above was treated with the restriction enzyme SmaI, ligated with the pDC24 vector treated with the same restriction enzyme, and transformed into E. coli DH5α by electroporation to induce homologous recombination on the chromosome. The strain in which the vector was inserted into the chromosome by recombination of homologous sequences was selected on LB medium containing kanamycin. DNA was obtained from the selected E. coli transformant using a DNA-spin plasmid DNA purification kit according to the manufacturer's protocol, thereby constructing the pDC24-ilvN(A42V) vector for introducing the A42V mutation of the ilvN gene, which includes the mutation fragment J.

[0155] The primer sequences used here are as shown in Table 6 below.

[0156] SEQ ID NO Sequence name Sequence (5' -> 3') 17primercggggatcccccgggAGGACGGTACTCAAATACTAAAACTTC18primerTGCCGAGTGTTTCGGTCTTTACAGACACGAGGGACACG19primerTGTCTGTAAAGACCGAAACACTCGGCATCAA20primercggggatcccccgggGACAACTACATTATTATTATACCACA

[0157] The pDC24-ilvN (A42V) vector constructed above was transformed into wild-type Corynebacterium glutamicum ATCC14067 by homologous recombination on the chromosome. The strain in which the vector was inserted into the chromosome by recombination of homologous sequences was selected on a medium containing 25 mg / l of kanamycin. After that, PCR was performed using the primer pair of SEQ ID NO: 17 and SEQ ID NO: 20 on the above-mentioned Corynebacterium glutamicum transformant in which the second recombination was completed to amplify the gene fragment, and the strain in which the A42V mutation was introduced into the ilvN gene was confirmed through gene sequence analysis. The recombinant strain was named Corynebacterium glutamicum CJ7V.

[0158] Finally, the pDC24-△NCgl0867up::Ppyk_aroP vector was transformed into Corynebacterium glutamicum CJ7V using the same method as in Example 2-1. The recombinant strain was named Corynebacterium glutamicum CJ7V-△NCgl0867up::Ppyk_aroP.

[0159] The valine production ability of the parent strain CJ7V and CJ7V-△NCgl0867up::Ppyk_aroP strain was evaluated using the same method as in Example 2-2, and is shown in Table 7 below.

[0160] Strain FN OD Valine yield Valine yield increase (compared to parent strain) Relative sugar consumption rate Tyrosine concentration Phenylalanine concentration 562 nm % % % mg / L mg / LCJ7V (parent strain) 137.32.2 100 100 80 160 CJ7V-△NCgl0867up::Ppyk_aroP 1312.5 113.6 4 10900

[0161] As a result, it was reconfirmed that when aroP of Corynebacterium glutamicum microorganisms was strengthened, the relative sugar consumption rate increased, the production of by-products (tyrosine, phenylalanine) decreased, and the valine yield increased.

[0162]

[0163] From the above description, those skilled in the art will understand that the present application can be implemented in other specific forms without altering the technical concept or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present disclosure should be interpreted as encompassing all changes or modifications derived from the meaning and scope of the claims described below, and their equivalent concepts, rather than the detailed description above.

Claims

1. (a) at least one promoter selected from the group consisting of a promoter of the pyk (Pyruvate kinase) gene, a promoter of the ald (acetaldehyde dehydrogenase) gene, a promoter of the ctaE (Cytochrome c oxidase subunit 3) gene, and a promoter of the pfkA (ATP-dependent 6-phosphofructokinase isozyme 1) gene; and (b) a gene expression cassette comprising a gene encoding an aromatic amino acid transporter operably linked to said promoter.

2. A gene expression cassette according to claim 1, wherein the promoter of the pyk gene comprises a base sequence of SEQ ID NO: 3, the promoter of the ald gene comprises a base sequence of SEQ ID NO: 4, the promoter of the ctaE gene comprises a base sequence of SEQ ID NO: 21, and the promoter of the pfkA gene comprises a base sequence of SEQ ID NO:

22.

3. A gene expression cassette according to claim 1, wherein the gene encoding the aromatic amino acid transporter is derived from a microorganism of the genus Corynebacterium.

4. Microorganisms with enhanced valine production and enhanced aromatic amino acid transporter activity.

5. A microorganism in claim 4, wherein the aromatic amino acid transporter is derived from a microorganism of the genus Corynebacterium.

6. A microorganism in the fourth paragraph, wherein the enhancement is enhancement by (i) overexpression of a gene encoding the aromatic amino acid transporter, (ii) operably linking a gene encoding the aromatic amino acid transporter to a strong promoter, or (iii) a combination thereof.

7. In paragraph 6, a microorganism in which the overexpression of the gene is achieved by additionally introducing the gene into the microorganism.

8. A microorganism in claim 6, wherein the strong promoter is at least one selected from the group consisting of a promoter of the pyk gene, a promoter of the ald gene, a promoter of the ctaE gene, and a promoter of the pfkA gene.

9. A microorganism in claim 8, wherein the promoter of the pyk gene comprises a base sequence of SEQ ID NO: 3, the promoter of the ald gene comprises a base sequence of SEQ ID NO: 4, the promoter of the ctaE gene comprises a base sequence of SEQ ID NO: 21, and the promoter of the pfkA gene comprises a base sequence of SEQ ID NO:

22.

10. In paragraph 4, the microorganism is a microorganism of the genus Corynebacterium.

11. In paragraph 10, the microorganism of the genus Corynebacterium is Corynebacterium glutamicum.

12. A microorganism having increased valine production ability compared to a parent strain or wild-type microorganism in which the activity of an aromatic amino acid transporter is not enhanced according to any one of claims 4 to 11.

13. A method for producing valine, comprising a step of culturing a microorganism according to any one of claims 4 to 11 in a medium.

14. A method for producing valine, further comprising a step of recovering valine from a medium or microorganism according to the culture in accordance with paragraph 13.

15. A method for increasing valine production, comprising the step of culturing a microorganism according to any one of claims 4 to 11 in a medium.

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