Microorganisms with enhanced phosphotransacetylase and acetate kinase operon activity and uses thereof

Abstract

The present disclosure provides microorganisms having enhanced phosphotransacetylase and acetate kinase operon activity, gene expression cassettes comprising a strong promoter and a pta-ackA operon, methods of producing valine using the microorganisms of the present disclosure, and methods of increasing valine production.

Description

Technical Field

[0001] Cross-references to related applications This disclosure claims priority to Korean Patent Application No. 10-2023-0181938, filed on December 14, 2023. The entire disclosure of the aforementioned Korean patent application is incorporated herein by reference and forms part of this disclosure.

[0002] This disclosure relates to microorganisms in which the activities of phosphotransacetylase and acetate kinase operons are enhanced, and their uses. Background Technology

[0003] L-amino acids, as important components of proteins, are widely used as essential materials in pharmaceuticals, food additives, animal feed, nutritional supplements, pesticides, and disinfectants. In particular, branched-chain amino acids (BCAAs) are a collective term for the essential amino acids L-valine, L-leucine, and L-isoleucine, and these branched-chain amino acids are known to have antioxidant effects and directly promote protein synthesis in muscle cells.

[0004] Meanwhile, the microbial production of BCAAs mainly uses microorganisms of the genera Escherichia or Corynebacterium, and in the case of L-valine, it is known to be biosynthesized from pyruvate through multiple steps using 2-oxoisovalerate as a precursor; however, the microbial production of L-valine is not easy to carry out on an industrial scale.

[0005] Therefore, further research is still needed to effectively increase L-valine production capacity.

[0006] [Existing Technical Documents] [Patent Literature] (Patent Document 1) US Patent Publication No. US 2020-0362374 A1 Summary of the Invention

[0007] Technical issues The purpose of this disclosure is to provide a microorganism in which the activity of the phosphotransacetase (Pta) and acetate kinase (ackA) operons (pta-ackA operon) is enhanced.

[0008] Another object of this disclosure is to provide a gene expression cassette comprising a promoter and a structural gene of a pta-ackA operon operatively linked to said promoter.

[0009] Another object of this disclosure is to provide a method for producing valine, which includes culturing the microorganism in a culture medium.

[0010] Another object of this disclosure is to provide a method for increasing valine production, which includes culturing the microorganism in a culture medium.

[0011] Another object of this disclosure is to provide a composition for producing valine, comprising at least one selected from the group consisting of said microorganisms and culture media in which said microorganisms are cultured.

[0012] Technical solution This can be explained in detail below. Furthermore, the various descriptions and embodiments disclosed in this disclosure can also be applied to other various descriptions and embodiments. That is, all combinations of the various elements disclosed in this disclosure fall within the scope of this disclosure. Moreover, the scope of this disclosure is not limited by the specific descriptions below. In addition, numerous papers and patent documents are cited in this specification, and their citations are indicated. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety, thereby more clearly explaining the level of the technical field to which this disclosure pertains and the content of this disclosure.

[0013] In this disclosure, it has been found that enhanced activity of the phosphotransacetase (pta) and acetate kinase (ackA) operon (pta-ackA operon) (hereinafter referred to as "pta-ackA operon") in valine-producing microorganisms increases valine productivity and yield; therefore, this disclosure provides promoters for enhancing the pta-ackA operon and valine-producing microorganisms wherein the activity of the pta-ackA operon is enhanced.

[0014] In this disclosure, the pta-ackA operon may contain the structural gene of pta-ackA and a promoter operatively linked thereto.

[0015] In this disclosure, the term "promoter" refers to a non-translated polynucleotide sequence upstream of a coding region that includes an RNA polymerase binding site and has initiation activity for transcribing a target gene into mRNA, i.e., a DNA region that allows RNA polymerase to bind and initiate transcription of the target gene. The promoter may be located in the 5' region of the mRNA transcription start site.

[0016] The structural gene of the pta-ackA operon may comprise a gene encoding a pta protein (phosphotransacetase; e.g., the amino acid sequence of SEQ ID NO: 1) (e.g., the nucleic acid sequence of SEQ ID NO: 2) and a gene encoding an ackA protein (acetylkinase; e.g., the amino acid sequence of SEQ ID NO: 3) (e.g., the nucleic acid sequence of SEQ ID NO: 4). The gene encoding the pta protein and the gene encoding the ackA protein may be linked regardless of their order. In one example, the structural gene of the pta-ackA operon may comprise the nucleic acid sequence of SEQ ID NO: 31.

[0017] The PTA protein may be derived from Corynebacterium microorganisms, such as Corynebacterium glutamicum (Sequence ID: WP_003862872.1). The PTA protein may possess phosphotransacetylase activity. The sequence of the PTA protein or the gene encoding it can be obtained from known databases (NCBI, etc.). The pta protein may have 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 98.2% or more, 98.4% or more, 98.6% or more, 98.8% or more, 98.9% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more homology or identity with the amino acid sequence of SEQ ID NO: 1, or may contain or be composed of the amino acid sequence. The gene encoding the pta protein may have 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 98.2% or more, 98.4% or more, 98.6% or more, 98.8% or more, 98.9% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more homology or identity with the nucleic acid sequence of SEQ ID NO: 2, or may contain or be composed of the nucleic acid sequence.

[0018] The ackA protein may originate from Corynebacterium species, such as Corynebacterium glutamicum (Sequence ID: WP_003862874.1). The ackA protein may possess acetate kinase activity. The ackA protein may possess phosphoryltransferase activity. The sequence of the ackA protein or the gene encoding it can be obtained from known databases (NCBI, etc.). The ackA protein may have 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 98.2% or more, 98.4% or more, 98.6% or more, 98.8% or more, 98.9% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more homology or identity with the amino acid sequence of SEQ ID NO: 3, or may contain or be composed of the amino acid sequence. The gene encoding the ackA protein may have 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 98.2% or more, 98.4% or more, 98.6% or more, 98.8% or more, 98.9% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more homology or identity with the nucleic acid sequence of SEQ ID NO: 4, or may contain or be composed of the nucleic acid sequence.

[0019] Furthermore, variants of aromatic amino acid transporters, where portions of the amino acid sequence are deleted, modified, substituted, conservedly substituted, or added, can also be included in the aromatic amino acid transporter category, provided that the amino acid sequence exhibits the activity corresponding to an aromatic amino acid transporter. For example, this includes cases where there are sequence additions or deletions, naturally occurring mutations, silent mutations, or conserved substitutions within the N-terminus, C-terminus, and / or amino acid sequence that do not alter the activity of the aromatic amino acid transporter.

[0020] The term "conservative substitution" refers to the replacement of one amino acid with another amino acid having similar structure and / or chemical properties. Such amino acid substitutions can typically occur based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity of the residues. Generally, conservative substitutions have little or no effect on the activity of the protein or peptide.

[0021] In this disclosure, the expression that a polynucleotide or polypeptide “has a specific nucleic acid sequence (nucleotide sequence) or amino acid sequence, contains a specific nucleic acid sequence (nucleotide sequence) or amino acid sequence, is composed of a specific nucleic acid sequence (nucleotide sequence) or amino acid sequence, or is substantially composed of a specific nucleic acid sequence (nucleotide sequence) or amino acid sequence” can mean that the polynucleotide or polypeptide substantially contains the specific nucleic acid sequence or amino acid sequence, and can be interpreted as including (or not excluding mutations) “substantially equivalent sequences”, wherein a mutation (deletion, substitution, modification, and / or addition) is applied to the specific nucleic acid sequence or amino acid sequence to the extent that the original function and / or desired function of the polynucleotide or polypeptide is maintained. In one instance, the statement that a polynucleotide or polypeptide “has a specific nucleic acid sequence (nucleotide sequence) or amino acid sequence, comprises a specific nucleic acid sequence (nucleotide sequence) or amino acid sequence, is composed of a specific nucleic acid sequence (nucleotide sequence) or amino acid sequence, or is substantially composed of a specific nucleic acid sequence (nucleotide sequence) or amino acid sequence” can mean that the polynucleotide or polypeptide (i) substantially comprises the specific nucleic acid sequence or amino acid sequence, or (ii) is composed of or substantially comprises a nucleic acid sequence or amino acid sequence having 70% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 98% or more, 99% or more, 99.5% or more, or 99.9% or more homology or identity with the specific nucleic acid sequence or amino acid sequence, and retains the original function and / or desired function.

[0022] In this disclosure, “homology” or “identity” refers 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 generally used interchangeably.

[0023] Sequence homology or identity of conserved polynucleotides or polypeptides is determined by standard alignment algorithms and can be achieved using default gap penalties established by the procedure employed. Essentially, homologous or identical sequences can typically hybridize with all or part of the sequence under moderately or highly stringent conditions. Clearly, hybridization also includes hybridization with polynucleotides containing codons that take into account universal codons or codon degeneracy within the polynucleotide.

[0024] Whether any two polynucleotide or polypeptide sequences are homologous, similar, or identical can be determined using known computer algorithms, such as the “FASTA” program with default parameters, as described in, for example, 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) can be used to determine, as in the Needleman program (version 5.0.0 or later) in the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, TrendsGenet. 16: 276-277) (which includes the GCG package (Devereux, J. et al., Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, [S.] [F.,] [ et al., J MOLEC BIOL 215]: 403 (1990); Guide to HugeComputers, Martin J. Bishop, [ED.,] Academic Press, San Diego, 1994, and [CARILLO] This is performed as described in ETA / .](1988) SIAM J Applied Math 48: 1073). For example, BLAST or ClustalW from the National Center for Biotechnology Information can be used to determine homology, similarity, or identity.

[0025] Homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using the GAP computer program, as known for example in Needleman et al. (1970), J Mol Biol. 48:443, and in Smith and Waterman, Adv. Appl. Math (1981) 2:482. In summary, the GAP program can be defined as the number of similarly arranged symbols (i.e., nucleotides or amino acids) divided by the total number of symbols in the shorter of the two sequences. The default parameters of the GAP procedure may include: (1) a binary comparison matrix (containing a value of 1 for identity and a value of 0 for non-identity), and the weighted comparison matrix (or the EDNAFULL (EMBOSS version of NCBINUC4.4) permutation matrix) disclosed by Gribskov et al. (1986) Nucl. Acids Res. 14: 6745, as disclosed in Atlas Of Protein Sequence And Structure, edited by Schwartz and Dayhoff, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each vacancy, and an additional penalty of 0.10 for each symbol in each vacancy (or a penalty of 10 for vacancy opening and 0.5 for vacancy extension); and (3) no penalty for terminal vacancy.

[0026] One aspect provides a microorganism in which the activity of the pta-ackA operon is enhanced. The microorganism with enhanced pta-ackA operon activity may possess one or more characteristics selected from the group consisting of: (i) increased valine production capacity; and (ii) increased sugar consumption rate compared to a parental strain or wild-type microorganism with unenhanced activity.

[0027] Enhanced activity of the pta-ackA operon can mean an increase in operon activity compared to its inherent activity. This enhancement can be used interchangeably with terms such as activation, upregulation, overexpression, and increase. In this context, activation, enhancement, upregulation, overexpression, and increase can include exhibiting activity not originally present, or exhibiting increased activity compared to inherent activity or activity before modification. "Inherent activity" refers to the activity of a specific operon originally possessed by the parental strain or unmodified microorganism before transformation, in cases where the trait has been altered due to genetic variation caused by natural or artificial factors. This can be used interchangeably with "activity before modification." An "enhanced," "upregulated," "overexpressed," or "increased" operon activity compared to inherent activity can mean an increase in the activity of a specific operon originally possessed by the parental strain or unmodified microorganism before transformation, for example, an increase in the expression of the operon's structural gene. Whether the activity of the operon is enhanced can be determined by methods well known in the art, such as the degree of increase in the amount of mRNA transcription of the structural gene contained in the operon (e.g., the mRNA level of the pta gene and / or the ackA gene), the expression level of the polypeptide encoded by the structural gene (e.g., the pta protein and / or the ackA protein level), the activity of the polypeptide, and the amount of product produced by the polypeptide (e.g., the amount of valine produced).

[0028] Various methods well-known in the art can be used to enhance operon activity, and there are no limitations, as long as the activity of the target operon is enhanced compared to the unmodified microorganism. Specifically, genetic engineering and / or protein engineering well-known to those skilled in the art can be used, which are routine methods in molecular biology, but are 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.).

[0029] Specifically, the enhancement of the activity of the pta-ackA operon can be a combination of one or more selected from 1) to 8) below, but is not limited thereto.

[0030] 1) Replace the gene expression regulatory region of the pta-ackA operon with a sequence that has strong activity; 2) Increase the intracellular copy number of the pta-ackA operon; 3) Modify the nucleotide sequence encoding the start codon or the 5'-UTR region of the pta-ackA operon structural gene; 4) Modify the amino acid sequence to enhance the activity of PTA and / or ACKA proteins; 5) Modify the polynucleotide sequence of the gene encoding the PTA protein and / or the gene encoding the ackA protein to enhance the activity of the PTA protein and / or the ackA protein (e.g., modify the polynucleotide sequence of the gene to encode a modified polypeptide to enhance the activity of the PTA protein and / or the ackA protein). 6) Introduce exogenous peptides exhibiting PTA protein and / or ACKA protein activity and / or exogenous polynucleotides encoding such exogenous peptides; 7) Codon optimization of genes encoding pta and / or ackA proteins; and 8) Select exposure sites by analyzing the tertiary structure of pta protein and / or ackA protein, and modify or chemically modify the exposure sites.

[0031] The substitution of the gene expression regulatory region of the pta-ackA operon with a highly active sequence as described in item 1) can, for example, involve generating variations in the sequence through deletion, insertion, non-conservative substitution, or conservative substitution, or a combination thereof, to further enhance the activity of the expression regulatory region, or by replacing it with a sequence of stronger activity. The expression regulatory region may include a promoter, an operon sequence, a sequence encoding a ribosome binding site, and sequences regulating transcription and translation termination. For example, replacing the gene expression regulatory region of the pta-ackA operon with a highly active sequence can be achieved by replacing the original promoter with a strong promoter.

[0032] Microorganisms with enhanced pta-ackA operon activity can be those that enhance pta-ackA operon activity by operably linking a strong promoter to the pta-ackA structural gene. As mentioned above, operably linking a strong promoter to the pta-ackA structural gene can refer to replacing (replacing) the original pta-ackA operon promoter with a strong promoter, or positioning a strong promoter before the pta-ackA structural gene so that the expression of the pta-ackA structural gene can be regulated by the strong promoter. The original promoter of the pta-ackA operon can refer to a promoter operably linked to the pta-ackA operon in the chromosome of a wild-type microorganism. In one example, the original promoter can be a promoter linked to a pta gene.

[0033] Furthermore, enhanced activity of the pta-ackA operon can include enhancing the expression of either the pta gene or the ackA gene alone. In one instance, a microorganism with enhanced pta-ackA operon activity can be a microorganism in which a strong promoter is operatively linked to the pta gene and / or a strong promoter is linked to the ackA gene.

[0034] In this disclosure, the term "operably ligated" refers to the functional ligation of the promoter of this disclosure to a target gene sequence (e.g., the pta-ackA structural gene, the pta gene, or the ackA gene) to initiate and mediate the transcription of the target gene. Operable ligations can be prepared using genetic recombination techniques known in the art of this disclosure, and site-specific DNA cutting and ligation can be prepared using cutting and ligases known in the art of this disclosure.

[0035] A strong promoter can be a promoter of an endogenous or exogenous gene from a microorganism. In one instance, a strong promoter can be derived from a Corynebacterium genus. In a specific implementation, a strong promoter can be derived from Corynebacterium glutamicum.

[0036] In one instance, a strong promoter can be PctaE (promoter of the ctaE gene), Ppyk (promoter of the pyk gene), PpfkA (promoter of the pfkA gene), Pald (promoter of the ald gene), CJ1 to CJ7 promoters (US Patent No. US7662943 B2), lac promoter, trp promoter, trc promoter, tac promoter, λ 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., but is not limited to these.

[0037] The ctaE gene may be a gene encoding cytochrome c oxidase subunit 3. In one instance, the promoter of the ctaE gene may contain or consist of the nucleic acid sequence of SEQ ID NO: 5.

[0038] The pyk gene may be a gene encoding pyruvate kinase. In one instance, the promoter of the pyk gene may contain or consist of the nucleic acid sequence of SEQ ID NO: 6. The pfkA gene may be a gene encoding ATP-dependent phosphofructokinase isoenzyme 1. In one instance, the promoter of the pfkA gene may contain or consist of the nucleic acid sequence of SEQ ID NO: 7.

[0039] The ald gene can be a gene encoding acetaldehyde dehydrogenase. In one instance, the promoter of the ald gene can contain or consist of the nucleic acid sequence of SEQ ID NO: 32.

[0040] The increase in intracellular copy number of the pta-ackA operon described in item 2) can be achieved by introducing a vector in which the pta-ackA operon is operatively linked into a microorganism (host cell), and the pta-ackA operon can replicate and function independently of the host. Alternatively, this can be achieved by introducing one or two or more copies of the pta-ackA operon into the chromosome of the microorganism (host cell). Introducing the pta-ackA operon into the chromosome can be done by introducing a vector capable of inserting a gene into the host cell's chromosome, but is not limited thereto.

[0041] Increasing the intracellular copy number of the pta-ackA operon can be achieved by introducing a vector operatively linked to the pta or ackA gene into a host cell, allowing the gene to replicate and function independently of the host. Alternatively, this can be achieved by introducing one or more copies of the pta or ackA gene into the host cell's chromosome. The introduction of the pta or ackA gene into the chromosome can be done, but is not limited to, by introducing a vector capable of inserting the gene into the host cell's chromosome.

[0042] The pta-ackA operon, pta gene, or ackA gene can be operatively linked to a promoter.

[0043] The promoter can be selected from at least one of the groups consisting of the original pta-ackA operon promoter and strong promoters different from the original promoter.

[0044] The original pta-ackA operon promoter and strong promoter are as described above. The pta-ackA operon, pta gene, or ackA gene can be inserted into a location that does not affect the expression of other genes in the host cell, such as a genomic safe harbor site. In one example, a safe harbor gene site could be the site between the NCgl2195 and NCgl2196 genes of *Corynebacterium glutamicum*, or the site between the NCgl0866 and NCgl0867 genes of *Corynebacterium glutamicum*.

[0045] The nucleotide sequence or 5'-UTR region encoding the start codon of the modified polypeptide transcript described in item 3) may, for example, be replaced with a nucleotide sequence encoding another start codon that has a higher polypeptide expression rate compared to the endogenous start codon, but is not limited thereto.

[0046] The modified amino acid sequences or polynucleotide sequences described in items 4) and 5) can be variations introduced into the sequence through deletions, insertions, non-conservative substitutions, or conserved substitutions or combinations thereof in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide, thereby enhancing the activity of the polypeptide, or replacements with amino acid sequences or polynucleotide sequences modified to have stronger activity, or replacements with amino acid sequences or polynucleotide sequences modified to enhance activity, but are not limited thereto. Specifically, the replacements can be made by inserting polynucleotides into chromosomes through homologous recombination, but are not limited thereto. The vectors used herein may also contain selection markers for confirming whether insertion into chromosomes has occurred.

[0047] The introduction of a foreign polynucleotide exhibiting peptide activity as described in item 6) can be the introduction of a foreign polynucleotide encoding a peptide exhibiting the same / similar activity as the peptide into a host cell. There are no restrictions on the source or sequence of the foreign polynucleotide, as long as it exhibits the same / similar activity as the peptide. The method used for introduction can be performed by a known transformation method appropriately selected by someone skilled in the art, and when the introduced polynucleotide is expressed in the host cell, the peptide is produced and its activity can be increased.

[0048] The codon optimization of polynucleotides encoding polypeptides described in item 7) can 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.

[0049] The analysis of the tertiary structure of the polypeptide described in item 8) to select and modify or chemically modify the exposure site may, for example, be done by comparing the sequence information of the polypeptide to be analyzed with a database storing known protein sequence information, determining template protein candidates based on the degree of sequence similarity, identifying the structure based on this, and selecting and modifying or chemically modifying the exposure site.

[0050] This enhancement of peptide activity can be an increase in the activity or expression level or concentration of the corresponding peptide relative to the activity or concentration of the peptide expressed in the wild-type or unmodified microbial strain, or an increase in the amount of product produced by the peptide, but is not limited thereto.

[0051] In one specific implementation, the enhancement may be (i) replacing the promoter of the pta-ackA operon with a strong promoter, (ii) increasing the copy number of the pta-ackA operon, or (iii) a combination thereof.

[0052] In the microorganisms disclosed herein, partial or complete gene modification to enhance pta-ackA operon activity can be induced by: (a) homologous recombination in the microorganism using a chromosomal insertion vector or genome editing using an engineered nuclease (e.g., CRISPR-Cas9), and / or (b) treatment with light such as ultraviolet light and radiation and / or chemicals, but not limited thereto. Methods for modifying partial or complete genes can include methods using DNA recombination techniques. For example, deletion of a partial or complete gene can be achieved by introducing a nucleotide sequence or a vector containing a nucleotide sequence homologous to the target gene into the microorganism to induce homologous recombination. The introduced nucleotide sequence or vector can include, but is not limited to, dominant selection markers.

[0053] The microorganisms disclosed herein may be microorganisms in which the activity of the pta-ackA operon is enhanced, or microorganisms whose activity of the pta-ackA operon is enhanced through genetic modification of a vector (e.g., recombinant microorganisms), but are not limited thereto. The microorganisms (or strains, recombinant cells) disclosed herein may be microorganisms with valine production capacity or with increased valine production capacity (or yield) by enhancing the activity of the pta-ackA operon.

[0054] The microorganisms disclosed herein may be naturally valine-producing microorganisms, or microorganisms that have been conferred or enhanced with valine-producing capacity in parental strains that do not possess valine-producing capacity, but are not limited thereto. The microorganisms may be those with valine-producing capacity or those with increased valine-producing capacity. The microorganisms may be those conferred or enhanced with valine-producing capacity by introducing an enhanced pta-ackA operon into a microorganism that does not possess valine-producing capacity or a microorganism that does possess valine-producing capacity, but are not limited thereto.

[0055] The microorganism having valine production capacity or having enhanced valine production capacity can mean being endowed with valine production capacity, which is different from unmodified microorganisms, pre-recombination cells, parental strains and / or wild-type strains without valine production capacity, or has increased valine production capacity compared to unmodified microorganisms, pre-recombination cells, parental strains and / or wild-type strains.

[0056] According to one embodiment, a microorganism with enhanced pta-ackA operon activity may have increased valine production capacity compared to the original microorganism (i.e., an unmodified microorganism of the same species). In this disclosure, "unmodified microorganism" does not exclude strains with naturally occurring mutations in the microorganism and may refer to the wild-type strain or the natural strain itself, or a strain before its traits were altered due to genetic variation caused by natural or artificial factors. For example, unmodified microorganism may refer to a microorganism in which the pta-ackA operon activity is not enhanced, or a microorganism before the pta-ackA operon activity was enhanced. "Unmodified microorganism" may be used interchangeably with "original strain," "unmodified microorganism," "unmutated strain," "unmodified microorganism," or "reference microorganism."

[0057] Microorganisms (or strains, recombinant cells) may additionally contain mutations that increase valine production, and the location of the mutation and / or the type of gene and / or protein to be mutated may be included without limitation, as long as it increases valine production. Recombinant cells may be used without restriction, as long as they are transformable cells.

[0058] In one instance, the microorganism disclosed herein may also contain an A42V variant of the acetolactate synthase isoenzyme 1 small subunit (IlvN) protein or the nucleotide encoding it (see Biotechnology and Bioprocess Engineering, June 2014, Vol. 19, No. 3, pp. 456-467).

[0059] In one instance, the unmodified microorganism (which is the strain used to compare whether valine production capacity is increased) can be *Corynebacterium glutamicum* strain ATCC13032, *Corynebacterium glutamicum* KCCM11201P (US 8465962 B), or a strain with increased valine production capacity by introducing the A42V mutation into the acetolactate synthase isoenzyme 1 small subunit (IlvN) protein of *Corynebacterium glutamicum* ATCC14067 [ilvN (A42V); Biotechnology and Bioprocess Engineering, June 2014, Vol. 19, No. 3, pp. 456-467; US 11180784 B2], but is not limited thereto.

[0060] As an example, the valine production capacity (or yield, productivity) of a microorganism (or strain, recombinant cell) with enhanced valine production capacity (or productivity, productivity) can be increased by 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 compared to the valine production capacity of the parental strain before mutation or the unmodified microorganism. 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, 34% or more, or 35% or more (the upper limit is not specifically limited, for example it can be 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 not limited to these.

[0061] In another instance, the valine production capacity (or yield, productivity) of a microorganism (or strain, recombinant cell) with enhanced valine production capacity (or productivity, productivity) may be increased by approximately 1.05 times or more, approximately 1.1 times or more, approximately 1.15 times or more, approximately 1.2 times or more, approximately 1.25 times or more, or approximately 1.3 times or more, but is not limited thereto, compared to the valine production capacity of the parental strain before mutation or the unmodified microorganism. The term "approximately" encompasses all ranges including ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all values ​​within a range equal to or similar to the values ​​following the term "approximately," but is not limited thereto.

[0062] The microorganism may be a Corynebacterium.

[0063] Corynebacterium species can be selected from one or more of the following groups: Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium desertti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris, and Corynebacterium acetoacetate. Corynebacterium acetoacidophilum, Corynebacterium acetoglutamicum, Corynebacterium alkanolyticum, Corynebacterium lilium, Corynebacterium melassecola, Corynebacterium thermoaminogenes, Corynebacterium herculis, and Corynebacterium flavescens, but not limited to these.

[0064] On the other hand, a gene expression cassette is provided, which contains a promoter and a structural gene of the pta-ackA operon operatively linked to the promoter.

[0065] In this disclosure, the term "gene expression cassette" can refer to a unit cassette containing a promoter and a target gene and capable of expressing the target gene operatively linked downstream of the promoter. Various factors that can help the target gene be effectively expressed can be contained inside or outside such a gene expression cassette. In addition to the promoter operatively linked to the target gene, a gene expression cassette may typically contain, but is not limited to, transcription termination signals, ribosome binding sites, and translation termination signals.

[0066] "Target gene" refers to a gene whose expression is regulated by the promoter sequence of this disclosure for the purposes of this disclosure. The protein encoded by the target gene can be expressed as "target protein", and the gene encoding the "target protein" can be expressed as "target gene".

[0067] In one instance, the target gene could refer to the structural gene of the pta-ackA operon expressed via a promoter.

[0068] The structural gene of the pta-ackA operon may include a gene encoding the pta protein (SEQ ID NO: 2) and a gene encoding the ackA protein (SEQ ID NO: 4). The structural gene of the pta-ackA operon may include the nucleic acid sequence of SEQ ID NO: 31.

[0069] The promoter may be one or more selected from the group consisting of the promoters of the ctaE gene, the pyk gene, and the pfkA gene, but is not limited thereto.

[0070] The promoter may be derived from the aforementioned Corynebacterium species, such as Corynebacterium glutamicum.

[0071] In one instance, the promoter may be one or more selected from the group consisting of the promoter of the ctaE gene (SEQ ID NO: 5), the promoter of the pyk gene (SEQ ID NO: 6), and the promoter of the pfkA gene (SEQ ID NO: 7), but is not limited thereto.

[0072] In this disclosure, the term "operably ligated" refers to the functional ligation of the promoter of this disclosure to a target gene sequence (e.g., the structural gene of the pta-ackA operon) to initiate and mediate the transcription of the target gene. Operable ligations can be prepared using gene recombination techniques known in the art of this disclosure, and site-specific DNA cutting and ligation can be prepared using cutting and ligases known in the art of this disclosure.

[0073] In one instance, the gene expression cassette may contain at least one promoter selected from the group consisting of the promoter of the pyk gene, the promoter of the ctaE gene, and the promoter of the pfkA gene; and The structural gene of the pta-ackA operon, which is operatively linked to the promoter.

[0074] On the other hand, a composition for producing valine is provided, comprising at least one selected from the group consisting of said microorganisms and culture media for culturing said microorganisms. The compositions disclosed herein may also comprise any suitable excipients commonly used in compositions for producing valine, and such excipients may be, for example, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, or isotonic agents, but are not limited thereto.

[0075] In the compositions disclosed herein, the microorganisms (strains), culture medium, valine, etc., are as described in the other aspects above.

[0076] On the other hand, it provides the use of producing valine by using at least one selected from the group consisting of the microorganism, the microorganism and a culture medium for culturing the microorganism for the production of valine.

[0077] On the other hand, a method for producing valine is provided, which includes the step of culturing the microorganism in a culture medium.

[0078] On the other hand, a method for increasing valine production is provided, which includes the step of culturing the microorganism in a culture medium.

[0079] The microorganisms and valine are as described above.

[0080] In this disclosure, "culture" can refer to the growth of the microorganisms of this disclosure (e.g., Corynebacterium glutamicum strains) under suitable controlled environmental conditions. The culture process of this disclosure can be carried out according to suitable culture media and culture conditions known in the art. Such a culture process can be easily adapted and used by those skilled in the art according to the selected strain. Specifically, the culture can be batch, continuous, and / or fed-batch, but is not limited thereto.

[0081] In this disclosure, "culture medium" refers to a substance in which nutrients required for culturing the microorganisms of this disclosure (e.g., Corynebacterium glutamicum strain) are mixed as the main components, and which provide nutrients, growth factors, etc., including water necessary for survival and growth. Specifically, the culture medium and other culture conditions used to culture the microorganisms of this disclosure can be any and without particular limitation, as long as it is a culture medium for culturing common microorganisms. However, the microorganisms of this disclosure can be cultured under aerobic conditions in a common culture medium containing suitable carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids and / or vitamins, while adjusting temperature, pH, and other conditions.

[0082] In one instance, the culture medium used for Corynebacterium strains can be found in the literature [(American Society for Bacteriology, “Manual of Methods for General Bacteriology” (Washington DC, USA, 1981)].

[0083] In this disclosure, carbon sources 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.; and amino acids such as glutamic acid, methionine, lysine, etc. In addition, natural organic nutrient sources such as starch hydrolysate, molasses, brown molasses, rice bran, cassava, bagasse, and corn steep liquor may be used. Specifically, carbohydrates such as glucose and aseptically pretreated molasses (i.e., molasses converted to reducing sugars) may be used, and other suitable 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.

[0084] As nitrogen sources, inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate can be used; and organic nitrogen sources such as amino acids, such as glutamic acid, methionine, and glutamine, peptone, NZ-amine, meat extracts, yeast extracts, malt extracts, corn steep liquor, casein hydrolysate, fish or its decomposition products, defatted soybean meal or its decomposition products, etc. These nitrogen sources can be used alone or in combination of two or more, but are not limited to these.

[0085] The phosphorus source may include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or their corresponding sodium-containing salts. As inorganic compounds, sodium chloride, calcium chloride, ferric chloride, magnesium sulfate, ferric sulfate, manganese sulfate, calcium carbonate, etc., may be used. In addition, amino acids, vitamins, and / or suitable precursors may be included. These components or precursors may be added to the culture medium in batches or continuously. However, it is not limited to these methods.

[0086] Furthermore, during the cultivation of the microorganisms disclosed herein, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid can be added to the culture medium in an appropriate manner to adjust the pH of the medium. Additionally, during cultivation, foam formation can be suppressed by using antifoaming agents such as polyethylene glycol fatty acids. Furthermore, to maintain an aerobic state in the culture medium, oxygen or oxygen-containing gas can be injected into the medium; or to maintain an anaerobic or microaerobic state, nitrogen, hydrogen, or carbon dioxide gas can be injected, or no gas may be injected, but these are not limited to these methods.

[0087] In the cultivation process disclosed herein, the cultivation temperature can be maintained between 20 and 45°C, specifically between 25 and 40°C, and the cultivation can be carried out for approximately 10 to 160 hours, but is not limited thereto.

[0088] The valine produced by the culture method disclosed herein can be secreted into the culture medium or retained in the cells.

[0089] The method for producing valine or increasing valine yield disclosed herein may further include, for example, a step of preparing the microorganisms (strains) of the present disclosure prior to the culturing step, a step of preparing a culture medium for culturing the microorganisms, or a combination thereof (in any order).

[0090] The method for producing valine or increasing valine yield disclosed herein may further include a step of recovering valine from a culture medium (in which culturing has taken place) or a microorganism (Corynebacterium strain) based on cultivation. A recovery step may also be included after the cultivation step.

[0091] Recovery can be performed using suitable methods known in the art to collect the desired valine according to the microbial culture methods disclosed herein, such as batch, continuous, or fed-batch culture methods. For example, centrifugation, filtration, treatment with a crystalline protein precipitant (salting out), extraction, ultrasonic disruption, ultrafiltration, dialysis, various chromatography methods (such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, etc.), HPLC, or combinations of these methods can be used to recover the desired valine from the culture medium or microorganisms using suitable methods known in the art.

[0092] Furthermore, the method for producing valine or increasing valine yield of this disclosure may additionally include a purification step. Purification can be performed using suitable methods known in the art. In one example, when the method for producing valine or increasing valine yield of this disclosure includes a recovery step and a purification step, the recovery step and the purification step may be performed continuously or discontinuously without regard to the order, or they may be performed simultaneously or integrated into a single step, but are not limited thereto.

[0093] Beneficial effects The microorganisms disclosed herein, which possess enhanced phosphotransacetase and acetate kinase operon activities, exhibit excellent valine production capacity and can therefore be effectively used for the large-scale production of valine. Detailed Implementation

[0094] Example 1. Constructing a plasmid for enhancing pta-ackA.

[0095] Based on the previously disclosed strain Corynebacterium glutamicum KCCM11201P (US Patent Publication No. US 8465962 B), which produces valine, a plasmid was constructed to enhance the activity of the phosphotransacetase and acetate kinase operon (pta-ackA operon), which was enhanced by promoter substitution and additional gene insertion into the chromosome.

[0096] Example 1-1. Constructing a plasmid for promoter substitution.

[0097] To enhance the activity of the phosphotransacetylase and acetate kinase operon (pta-ackA operon), the aim was to select and enhance promoters that are stronger than the endogenous pta-ackA operon. To this end, plasmids that enhance pta-ackA activity were constructed by replacing Ppta (the wild-type promoter of the pta gene) located before the operon with Ppyk (the promoter of the pyk gene), PctaE (the promoter of the ctaE gene), or PpfkA (the promoter of the pfkA gene).

[0098] The amino acid sequences of phosphoryltransferase (Pta) and acetate kinase (ackA), the nucleic acid sequences of the genes encoding them, and the nucleic acid sequences of the various promoters (PctaE, Ppyk, PpfkA) are shown in Table 1 below.

[0099] Specifically, to construct pta-ackA operon-enhanced strains with PctaE, Ppyk, or PpfkA promoters, the chromosome of the valine-producing strain *Corynebacterium glutamicum* KCCM11201P (US 8465962 B) was used as a template, and fragments were obtained by PCR. PfuUltra was used... TM High-fidelity DNA polymerase (Stratagene) was used as the polymerase for the PCR reaction, and the PCR conditions were denaturation at 95°C for 30 seconds, denaturation at 55°C for 30 seconds, and polymerization at 72°C for 1 minute; and the denaturation, annealing and polymerization steps were repeated for 28 cycles.

[0100] As a result, primers SEQ ID NO: 8 and SEQ ID NO: 9 yielded a 606 bp DNA fragment from the 5' upstream region of natural Ppta, and primers SEQ ID NO: 10 and SEQ ID NO: 11 yielded a 600 bp DNA fragment from the 3' downstream region of natural Ppta. Furthermore, primers SEQ ID NO: 12 and SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, and SEQ ID NO: 16 and SEQ ID NO: 17 yielded a 369 bp fragment of PctaE (SEQ ID NO: 5), a 490 bp fragment of Ppyk (SEQ ID NO: 6), and a 500 bp fragment of PpfkA (SEQ ID NO: 7), respectively. The primer sequences used for each PCR using the amplified promoter and the DNA fragments from the upstream and downstream regions of Ppta as templates are shown in Table 2 below.

[0101] [Table 1] [Table 2] PCR was performed using the amplified promoter fragment and upstream and downstream DNA fragments of Ppta as templates, along with primers of SEQ ID NO:8 and SEQ ID NO:11. PCR conditions included 28 cycles of denaturation at 95°C for 5 minutes, followed by denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and polymerization at 72°C for 2 minutes, followed by a final polymerization reaction at 72°C for 5 minutes.

[0102] As a result, DNA fragments containing the target promoter sequences between the upstream and downstream sequences of the Ppta promoter were amplified to replace the natural Ppta promoter with the respective promoters. The amplified products were purified using a PCR purification kit (QUIAGEN) and used as insert DNA fragments for vector construction.

[0103] Vectors for replacing the natural Ppta of Corynebacterium glutamicum KCCM11201P were constructed using the In-Fusion Cloning Kit from TaKaRa, following the provided instructions to clone amplified DNA fragments and the pDC24 vector (SEQ ID NO: 33, Table 3) treated with BamHI and XbaI (New England Biolabs, Beverly, MA). These vectors are pDC24_△Pn_pta::PctaE_pta, pDC24_△Pn_pta::Ppyk_pta, and pDC24_△Pn_pta::PpfkA_pta.

[0104] [Table 3] Examples 1-2. Construction of plasmids for gene insertion To enhance the target gene by additionally inserting the pta-ackA operon into the chromosome of Corynebacterium glutamicum KCCM11201P, non-coding intergenic loci in the KCCM11201P genome were used as insertion sites to eliminate the effects of gene deletion, and the non-coding region between NCgl2195 and NCgl2196 (hereinafter referred to as NCgl2195down) was selected. The PctaE promoter, which is a stronger promoter than the natural promoter or the endogenous pta-ackA operon, was used, and the KCCM11201P genome was used as the template chromosome for PCR in all experiments. PfuUltra™ high-fidelity DNA polymerase (Stratagene) was used as the polymerase for the PCR reaction, and the PCR conditions were: denaturation at 95°C for 30 seconds; denaturation at 55°C for 30 seconds; and polymerization at 72°C for 1 minute; and these denaturation, annealing, and polymerization steps were repeated 28 times.

[0105] The 1200 bp fragment of the 5' upstream region under NCgl2195 was obtained using the primers of SEQ ID NO: 18 and SEQ ID NO: 19, the 1157 bp fragment of the 3' downstream region under NCgl2195 was obtained using the primers of SEQ ID NO: 20 and SEQ ID NO: 21, the 3079 bp fragment of Pn_pta-ackA, which is the complete operon from the native promoter, was obtained using the primers of SEQ ID NO: 22 and SEQ ID NO: 23, the 369 bp fragment of the PctaE promoter region was obtained using the primers of SEQ ID NO: 24 and SEQ ID NO: 25, and the 2579 bp fragment of the ORF part of the pta-ackA operon was obtained using the primers of SEQ ID NO: 26 and SEQ ID NO: 23. The primer sequences used for each PCR are shown in Table 4 below.

[0106] [Table 4] To prepare gene insertion fragments using the native promoter, the upstream and downstream fragments under NCgl2195 and the 3079 bp fragment of Pn_pta-ackA (which covers from the native promoter to the entire operon) were used as templates, and PCR was performed using the primers of SEQ ID NO: 18 and SEQ ID NO: 21. And to prepare gene insertion fragments using the ctaE promoter, the upstream and downstream fragments under NCgl2195, the PctaE promoter fragment, and the 2579 bp fragment of the pta-ackA operon ORF region were used as templates, and PCR was performed using the primers of SEQ ID NO: 18 and SEQ ID NO: 21. The PCR conditions included denaturation at 95°C for 5 minutes, followed by denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and polymerization at 72°C for 2 minutes for 28 cycles, and then a final polymerization reaction at 72°C for 5 minutes.

[0107] Vectors pDC24_△NCgl2195 down::Pn_pta-ackA and pDC24_△NCgl2195 down::PctaE_pta-ackA for the additional insertion of the pta-ackA operon into Corynebacterium glutamicum KCCM11201P were constructed by using the In-fusion cloning kit from TaKaRa to clone the amplified DNA fragments and the pDC24 vector treated with BamHI and XbaI (New England Biolabs, Beverly, MA) according to the provided instructions.

[0108] Example 2. Construction of strains with enhanced activities of phosphotransacetylase and acetate kinase operons (pta-ackA operon) and evaluation of L-valine production capacity. Example 2-1. Construction of strains with promoter substitution The pDC24_△Pn_pta::PctaE_pta, pDC24_△Pn_pta::Ppyk_pta, and pDC24_△Pn_pta::PpfkA_pta vectors constructed in Examples 1-1 were transformed into *Corynebacterium glutamicum* KCCM11201P via homologous recombination on the chromosome (van der Rest et al., Appl Microbiol Biotechnol 52:541-545, 1999). Strains that had inserted the vectors into the chromosome via homologous sequence recombination were selected on a medium containing 25 mg / L kanamycin. Subsequently, for *Corynebacterium glutamicum* transformants that had completed the second recombination, PCR was performed using the primer pair of SEQ ID NO: 8 and SEQ ID NO: 11 to confirm the nucleotide sequence and verify whether the promoter had been replaced. The strains in which the promoter of the pta gene of the parent strain KCCM11201P on the chromosome was replaced were named Corynebacterium glutamicum KCCM11201P_△Pn_pta::PctaE_pta, KCCM11201P_△Pn_pta::Ppyk_pta, and KCCM11201P_△Pn_pta::PpfkA_pta, respectively.

[0109] Example 2-2. Evaluation of L-valine production capacity of promoter-substituted strains Flask evaluation was performed to compare the valine production capacity of the valine-producing strain *Corynebacterium glutamicum* KCCM11201P with that of the four strains constructed in Example 2-1 (i.e., KCCM11201P_△Pn_pta::PctaE_pta, KCCM11201P_△Pn_pta::Ppyk_pta, and KCCM11201P_△Pn_pta::PpfkA_pta). 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 with shaking at 200 rpm for 72 hours. The final OD, valine yield, and relative sugar consumption rate of each strain were measured and are shown in Table 5 below.

[0110] [Nutritional medium (pH 7.2)] Glucose 10 g, beef extract 5 g, polypeptone 10 g, sodium chloride 2.5 g, yeast extract 5 g, agar 20 g, urea 2 g (per 1 liter of distilled water) [Production medium (pH 7.0)] Glucose 100 g, ammonium sulfate 40 g, soy protein 2.5 g, corn steep liquor solids 5 g, urea 3 g, dipotassium hydrogen phosphate 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 (per 1 liter of distilled water) [Table 5] As shown in Table 5, when the native promoter was replaced to enhance the expression of the pta-ackA operon, all strains were superior to the parental strain (KCCM11201P) in terms of relative sugar consumption rate, and the yield was not reduced, thus confirming the improvement of valine productivity by enhancing the expression of the pta-ackA operon.

[0111] Example 2-3. Construction of strains with additional gene insertions To evaluate the effect of improving valine productivity when the expression of the pta-ackA operon was enhanced by promoter replacement and the enhancing effect by additional insertion of the pta-ackA operon into the chromosome, additional gene insertions were performed using the most effective ctaE promoter and the native promoter in Example 2-2. The pDC24_△NCgl2195 down::Pn_pta-ackA and pDC24_△NCgl2195 down::PctaE_pta-ackA vectors constructed in Example 1-2 were transformed into Corynebacterium glutamicum KCCM11201P by homologous recombination on the chromosome (van der Rest et al., Appl Microbiol Biotechnol 52:541-545, 1999). Strains in which the vector was inserted into the chromosome by homologous sequence recombination were selected on a medium containing 25 mg / L kanamycin. Subsequently, PCR was performed on the Corynebacterium glutamicum transformants with the second recombination using the primer pairs of SEQ ID NO: 18 and SEQ ID NO: 21, and the nucleotide sequence was confirmed to identify the promoter replacement. Strains with the pta-ackA operon additionally inserted into the chromosome of the parental strain KCCM11201P were named Corynebacterium glutamicum KCCM11201P_△NCgl2195 down::Pn_pta-ackA and KCCM11201P_△NCgl2195 down::PctaE_pta-ackA, respectively.

[0112] Example 2-4. Evaluation of L-valine production ability of strains with additional gene insertions To compare the valine production abilities of the valine-producing strain Corynebacterium glutamicum KCCM11201P and the two strains KCCM11201P_△NCgl2195::Pn_pta-ackA and KCCM11201P_△NCgl2195::PctaE_pta-ackA constructed in Example 2-3, flask evaluations were performed as in Example 2-2. The final OD, valine productivity, and relative sugar consumption rate of each strain were measured and shown in Table 6 below.

[0113] [Table 6] As shown in Table 6, as a result of additionally inserting the pta-ackA gene to enhance the expression of the pta-ackA operon, an increase in the relative sugar consumption rate and valine productivity was confirmed compared to the parental strain (KCCM11201P).

[0114] Example 2-5. Evaluation of valine production ability of Corynebacterium glutamicum CJ7V strain To evaluate whether enhancing the effect of the pta-ackA operon increased the valine production ability of other valine-producing strains belonging to Corynebacterium glutamicum, a strain with improved valine production ability was prepared by introducing a mutation [ilvN (A42V); Biotechnology and Bioprocess Engineering, June 2014, Vol. 19, No. 3, pp. 456-467] into the acetolactate synthase isozyme 1 small subunit (IlvN) protein of wild-type Corynebacterium glutamicum ATCC14067.

[0115] Specifically, to construct a vector for introducing the A42V mutation into the ilvN gene, genomic DNA was extracted from wild-type Corynebacterium glutamicum strain ATCC14067 using the G-spin Total DNA Extraction Mini Kit (Intron Co., Cat. No. 17045) according to the manufacturer's protocol. Using this genomic DNA as a template, PCR was performed with primer pairs SEQ ID NO: 27 and SEQ ID NO: 28, and primer pairs SEQ ID NO: 29 and SEQ ID NO: 30, to obtain gene fragments A and B, respectively. PCR conditions included 28 cycles of denaturation at 94°C for 5 minutes, followed by 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 a final polymerization at 72°C for 7 minutes. As a result, gene fragment A (528 bp) and gene fragment B (509 bp) were obtained. Overlap PCR was performed using the obtained gene fragments A and B as templates and primer pairs SEQ ID NO: 27 and SEQ ID NO: 30. As a result, a PCR product of 1010 bp was obtained (hereinafter referred to as "mutation-introduced fragment 2").

[0116] The mutant-introduced fragment 2 obtained above was treated with the restriction enzyme SmaI and then ligated to the pDC24 vector treated with the same restriction enzyme. This ligation was then performed by electroporation into *Escherichia coli* DH5α strains (INVITROGEN, DH5α competent cells) to induce homologous recombination on the chromosome. Strains that inserted the vector into the chromosome via homologous sequence recombination were selected on LB medium containing kanamycin. DNA was obtained from the selected *E. coli* transformants using a DNA-spin plasmid DNA purification kit according to the manufacturer's protocol, and the pDC24-ilvN(A42V) vector containing mutant-introduced fragment 2 was constructed to introduce the A42V mutation into the ilvN gene.

[0117] The primer sequences used in this paper are shown in Table 7 below.

[0118] [Table 7] The pDC24-ilvN(A42V) vector prepared above was transformed into wild-type Corynebacterium glutamicum ATCC14067 by homologous recombination on the chromosome. Strains in which the vector was inserted into the chromosome by homologous sequence recombination were selected on a medium containing 25 mg mg / L mg / L kanamycin. Thereafter, using the primer pair of SEQ ID NO: 27 and SEQ ID NO: 30, PCR was performed on the Corynebacterium glutamicum transformant that had completed the second recombination to amplify the gene fragment, and then strains in which the A42V mutation was introduced into the ilvN gene were identified by gene sequence analysis. The recombinant strain was named Corynebacterium glutamicum CJ7V.

[0119] Finally, the pDC24-△Pn_pta::PctaE_pta and pDC24-△NCgl2195 down::PctaE_pta-ackA vectors were transformed into Corynebacterium glutamicum CJ7V in the same manner as in Examples 2-1 and 2-3. The recombinant strains were named Corynebacterium glutamicum CJ7V-△Pn_pta::PctaE_pta and CJ7V-△NCgl2195 down::PctaE_pta-ackA.

[0120] The L-valine production capabilities of the parental strain CJ7V and the strains CJ7V-△Pn_pta::PctaE_pta and CJ7V-△NCgl2195 down::PctaE_pta-ackA were evaluated in the same manner as in Example 2-2 and are shown in Table 8 below.

[0121] [Table 8] As a result, it was again confirmed that when the pta-ackA operon of Corynebacterium glutamicum producing valine was enhanced, the sugar consumption rate and valine productivity increased.

[0122] Based on the foregoing description, those skilled in the art to which the present disclosure pertains will be able to understand that the present disclosure can be implemented in other specific forms without changing the technical spirit or essential features. In this regard, it should be understood that the above embodiments are illustrative in all respects and not restrictive. The scope of the present disclosure should be construed such that all modifications or variations derived from the meaning and scope of the claims to be described later and their equivalent concepts are included within the scope of the present disclosure, rather than being limited by the above detailed description.

Claims

1. A microorganism in which the activities of phosphotransacetase and acetate kinase operons (pta-ackA operon) are enhanced.

2. The microorganism according to claim 1, wherein the enhancement is (i) Replace the promoter of the pta-ackA operator with a strong promoter; (ii) Increase the copy number of the pta-ackA operator; or (iii) Its combination.

3. The microorganism according to claim 2, wherein the strong promoter is selected from at least one of the group consisting of the promoter of the pyk gene, the promoter of the ctaE gene, and the promoter of the pfkA gene.

4. The microorganism according to claim 2, wherein the promoter of the pta-ackA operon is replaced with a promoter comprising at least one nucleic acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO:

7.

5. The microorganism according to claim 1, wherein the pta-ackA operon comprises the nucleic acid sequence of SEQ ID NO: 2 and the nucleic acid sequence of SEQ ID NO:

4.

6. The microorganism according to claim 1, wherein the valine production capacity is increased.

7. The microorganism according to claim 1, wherein the microorganism is a Corynebacterium genus.

8. The microorganism according to claim 7, wherein the Corynebacterium genus microorganism is Corynebacterium glutamicum.

9. A gene expression cassette comprising: (a) Select at least one promoter chosen from the group consisting of the promoters of the pyk gene, the ctaE gene, and the pfkA gene; and (b) The structural gene of the pta-ackA operon operatively linked to the promoter.

10. A method for producing valine, comprising the step of culturing the microorganism according to claim 1 in a culture medium.

11. The method of claim 10, further comprising the step of recovering valine from the culture medium or microorganism after culturing.

12. A method for increasing valine production, comprising the step of culturing the microorganism according to claim 1 in a culture medium.

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