Novel promoters and uses thereof

Abstract

The present application relates to a novel promoter and a method for producing a target substance using the same, and more particularly, to a novel polynucleotide having promoter activity, a vector gene carrying the same, and a microorganism of Corynebacterium sp. anchoring the same, and a method for producing a target substance using the same.

Description

Technical Field

[0001] This disclosure relates to a novel promoter and a method for producing a target substance using the promoter, and more specifically, to a novel polynucleotide with promoter activity, a vector containing the promoter and a Corynebacterium microorganism, a method for producing a target substance using the microorganism, and the use of the promoter. Background Technology

[0002] Various studies on the production of target substances (e.g., amino acids) in microorganisms have explored eco-friendly and safe production methods, with ongoing research focusing on the high-volume production of target substances in Corynebacterium species. Corynebacterium species, particularly *Corynebacterium glutamicum*, are Gram-positive microorganisms commonly used to produce L-amino acids and other useful substances. Various studies have been conducted to develop microorganisms capable of achieving efficient production and fermentation processes for L-amino acids and other useful substances.

[0003] L-Lysine is a representative substance produced by microorganisms of the genus Corynebacterium, used in animal feed and the human pharmaceutical and cosmetic industries, and is produced through fermentation using strains of Corynebacterium. Microorganisms with genes associated with enhanced L-lysine biosynthesis and methods for producing L-lysine using these microorganisms are known (KR 10-0924065B1).

[0004] L-Threonine is an essential amino acid widely used as a feed and food additive, and also for pharmaceutical purposes, serving as a raw material for extracts and drug synthesis. Because plant proteins contain relatively little L-Threonine, vegetarian animals are prone to L-Threonine deficiency, making L-Threonine a particularly effective feed additive for animals. L-Threonine is primarily produced through fermentation using *E. coli* or *Corynebacterium* microorganisms developed through artificial mutation or genetic recombination. Typically, the method using recombinant strains is known, involving the introduction of a threonine operon from *E. coli* into a threonine-producing *Brevibacterium flavum* strain to produce L-Threonine (TURBAE et al., Agric. Biol. Chem. 53:2269–2271, 1989).

[0005] O-acetylhomoserine is used as a precursor for the production of methionine and is an intermediate in the methionine biosynthetic pathway (WO2008 / 013432). O-acetyl-L-homoserine is synthesized by homoserine O-acetyltransferase using L-homoserine and acetyl-CoA as substrates.

[0006] Isoleucine is an essential amino acid that is not synthesized in the body. It is known to promote growth, enhance nerve function, improve liver function, and strengthen muscles, and is usually produced through microbial fermentation.

[0007] Because systems demonstrating high expression efficiency in a variety of microorganisms, namely Escherichia, Corynebacterium, or Bacillus, are required, the development of universal promoters remains necessary. It is also desirable that universal promoters, during development, are not limited to specific target substances and can be used to generate a variety of substances.

[0008] Technical issues

[0009] In this disclosure, it was identified that, compared with known promoters, the novel synthetic promoter, when present in the positive direction, exhibits high expression activity of downstream genes, leading to the production of various target substances.

[0010] Technical solution

[0011] One aspect of this disclosure is to provide polynucleotides with promoter activity.

[0012] Another aspect of this disclosure is to provide a vector or expression cassette comprising: a polynucleotide; and a gene encoding a target protein and operatively linked to the polynucleotide.

[0013] Another aspect of this disclosure is to provide a Corynebacterium microorganism comprising: a polynucleotide; or a polynucleotide and a gene encoding a target protein and operatively linked to said polynucleotide.

[0014] Another aspect of this disclosure is to provide a method for generating a target substance, the method comprising: culturing a Corynebacterium genus microorganism in a culture medium; and recovering the target substance from the culture medium.

[0015] Another aspect of this disclosure is the use of a polynucleotide as a promoter, said polynucleotide having promoter activity, wherein in the polynucleotide sequence of SEQ ID NO: 1, the nucleotides at positions 27, 28, 31, 32 and 36 are replaced by other nucleotides.

[0016] Beneficial effects

[0017] The novel promoter-active polynucleotide disclosed herein can be introduced into microorganisms that produce target substances, thereby increasing the yield of the target substances. Due to the increased yield, advantages such as ease of production, reduced manufacturing costs, and other benefits can be expected in industrial applications. Summary of the Invention

[0018] The present disclosure will now be described in detail. Each description and embodiment disclosed herein can also be applied to other descriptions and embodiments. That is, all combinations of the various elements disclosed herein fall within the scope of this disclosure. Furthermore, the scope of this disclosure is not limited to the specific description below.

[0019] Furthermore, those skilled in the art will recognize or be able to determine many equivalents of the specific embodiments of this disclosure described herein using only conventional experiments. These equivalents are intended to be covered by this disclosure.

[0020] One aspect of this disclosure is to provide polynucleotides with promoter activity.

[0021] Specifically, the promoter-active polynucleotide of this disclosure may be a polynucleotide having promoter activity and including at least one polynucleotide substitution in the polynucleotide sequence of SEQ ID NO: 1.

[0022] As used herein, the term "polynucleotide" refers to a polymer of nucleotides covalently linked together to form a long chain, and said polynucleotide is a DNA chain having a predetermined length or longer.

[0023] As used in this article, "promoter-active polynucleotides" refers to DNA regions present near the transcription sites of the gene to be expressed (i.e., target genes), including sites where RNA polymerases or enhancers bind to express the target gene.

[0024] The promoter-active polynucleotides disclosed herein can be used as universal promoter enhancers. For example, the polynucleotides can be used as promoters capable of enhancing the expression of polypeptides with glutamate dehydrogenase (GDH) activity. Alternatively, the polynucleotides can be polynucleotides involved in increasing the yield or production of target substances, particularly lysine, threonine, O-acetylhomoserine, or isoleucine.

[0025] The polynucleotides disclosed herein may include any polynucleotide having promoter activity, without limitation. Specifically, the promoter-active polynucleotides of this disclosure may be promoter-active polynucleotides that include at least one, at least two, at least three, at least four, at least five, at least six, or at least seven nucleotide substitutions in the polynucleotide sequence of SEQ ID NO: 1.

[0026] An example of the polynucleotide sequence of SEQ ID NO: 1 may be a polynucleotide having glutamate dehydrogenase promoter activity. A polynucleotide having a specific nucleotide substitution in the polynucleotide sequence of SEQ ID NO: 1 may also be a polynucleotide having glutamate dehydrogenase promoter activity, provided it has promoter activity. The polynucleotide sequence of SEQ ID NO: 1 may be a representative polynucleotide sequence used to indicate the mutation site, and other polynucleotide sequences with corresponding promoter activity are also included in the sequences to which mutations can be introduced. For example, any polynucleotide sequence that can serve as a promoter for a polypeptide having glutamate dehydrogenase (GDH) activity or a corresponding activity thereof may be included within the range of sequences to which mutations of this disclosure can be introduced, without limitation.

[0027] The nucleotide sequence of SEQ ID NO: 1 can be confirmed in the known database NCBI GenBank, and the sequence corresponding to SEQ ID NO: 1, which can be used as a promoter sequence for glutamate dehydrogenase, can be derived from the genus Corynebacterium, particularly Corynebacterium glutamicum. However, sequences having equivalent or higher activity than the polynucleotide can be included in the promoter of this disclosure without limitation.

[0028] The promoter-active polynucleotides disclosed herein can be polynucleotides whose promoter activity is enhanced by substitution of nucleotides at specific positions in existing promoter-active polynucleotide sequences.

[0029] In one embodiment, the promoter-active polynucleotide of this disclosure may include a promoter-active polynucleotide wherein at least one nucleotide in the nucleotide sequence of SEQ ID NO: 1 is substituted by another nucleotide.

[0030] In one embodiment, the modified promoter may be a polynucleotide having promoter activity and comprising at least one nucleotide selected from positions 27, 28, 31, 32, and 36 of SEQ ID NO: 1 substituted by another nucleotide. Furthermore, the modified promoter may also have additional substitutions of the nucleotide at positions 66 and / or 261.

[0031] The term "another nucleotide" or "other nucleotide" is not limited, as long as the nucleotide or nucleotides are different from the nucleotide or nucleotides before substitution. For example, with adenine (A) as the nucleotide at position 27 in SEQ ID NO: 1, the phrase "the nucleotide at position 27 in SEQ ID NO: 1 is replaced by another nucleotide" means that, in addition to adenine, it is replaced by cytosine (C), thymine (T), or guanine (G). Unless otherwise stated, the term "replace" a nucleotide in this disclosure means substitution with a nucleotide different from the nucleotide before substitution.

[0032] Furthermore, those skilled in the art, through sequence alignment known in the art, can determine the nucleotides at positions corresponding to nucleotides 27, 28, 31, 32, 36, 66, and 261 in SEQ ID NO: 1 of this disclosure within any polynucleotide sequence, and even if not described separately herein, the phrase "nucleotide at a specific position in a specific SEQ ID NO" clearly means "nucleotide at its corresponding position" even in any polynucleotide sequence. Therefore, within the scope of this disclosure, any polynucleotide sequence with promoter activity is also included, wherein one or more nucleotides selected from the nucleotides at positions 27, 28, 31, 32, 36, 66, and 261 in the polynucleotide sequence corresponding to SEQ ID NO: 1 are substituted with other nucleotides.

[0033] In one embodiment, the promoter-active polynucleotide of this disclosure may be a polynucleotide in which one or more nucleotides selected from the polynucleotide sequence of SEQ ID NO: 1 at positions 27, 28, 31, 32, 36, 66 and 261 are replaced by other nucleotides.

[0034] Specifically, the promoter-active polynucleotide of this disclosure can be a polynucleotide in which the nucleotides at positions 27, 28, 31, 32 and 36 of the polynucleotide sequence of SEQ IDNO: 1 are replaced by other nucleotides; the nucleotides at positions 27, 28, 31, 32, 36, 66 and 261 are replaced by other nucleotides; and the nucleotides at positions 27, 28, 31, 32, 36 and 66 are replaced by other nucleotides, but not limited thereto.

[0035] For example, when at least one, at least two, at least three, at least four, at least five, at least six, or at least seven nucleotides at positions 27, 28, 31, 32, 36, 66, and 261 in the polynucleotide corresponding to SEQ ID NO: 1 are substituted with other nucleotides, a promoter with higher activity than the unsubstituted (unmodified) promoter sequence can be provided. Specifically, the promoter-active polynucleotide of this disclosure can be a polynucleotide in which the nucleotides at positions 27, 28, 31, 32, and 36 of the polynucleotide sequence of SEQ ID NO: 1 are substituted with other nucleotides. The polynucleotide of this disclosure can also be a promoter-active polynucleotide in which the nucleotides at positions 66 and 261 are further substituted with other nucleotides, or the nucleotide at position 66 is substituted with other nucleotides.

[0036] As a specific example, the promoter-active polynucleotide disclosed herein can be a polynucleotide in which, in the polynucleotide sequence of SEQ ID NO: 1, the adenine (A) at position 27 is replaced by thymine (T), the cytosine (C) at position 28 is replaced by guanine (G), the cytosine (C) at position 31 is replaced by guanine (G), the cytosine (C) at position 32 is replaced by thymine (T), and the adenine (A) at position 36 is replaced by cytosine (C); in SEQ ID NO: 1 In the polynucleotide sequence of NO:1, adenine (A) at position 27 is replaced by thymine (T), cytosine (C) at position 28 is replaced by guanine (G), cytosine (C) at position 31 is replaced by guanine (G), cytosine (C) at position 32 is replaced by thymine (T), adenine (A) at position 36 is replaced by cytosine (C), adenine (C) at position 66 is replaced by thymine (T), and adenine (A) at position 261 is replaced by guanine (G); and in SEQ ID In the polynucleotide sequence of NO:1, adenine (A) at position 27 is replaced by thymine (T), cytosine (C) at position 28 is replaced by guanine (G), cytosine (C) at position 31 is replaced by guanine (G), cytosine (C) at position 32 is replaced by thymine (T), adenine (A) at position 36 is replaced by cytosine (C), and cytosine (C) at position 66 is replaced by thymine (T).

[0037] As a more specific example, the polynucleotide of this disclosure may be any polynucleotide sequence represented by SEQ ID NOS: 2 to 4. Specifically, the promoter-active polynucleotide of this disclosure may contain or be composed of the polynucleotide sequence of SEQ ID NO: 2, 3 or 4.

[0038] Not limited to the above implementation scheme, various modifications to the polynucleotide sequence may also be included within the scope that do not significantly reduce promoter activity.

[0039] The promoter-active polynucleotide disclosed herein may be a polynucleotide sequence having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology or identity with SEQ ID NO: 2, 3, or 4. The nucleotide sequence having homology or identity may exclude sequences having 100% identity from the above range, or may be a sequence having less than 100% identity.

[0040] Although described herein as “polynucleotide having a nucleotide sequence indicated by a specific sequence number” or “polynucleotide containing a nucleotide sequence indicated by a specific sequence number”, polynucleotides having partially deleted, modified, substituted or added polynucleotide sequences may also be clearly used in this disclosure, provided that the polynucleotide has the same or corresponding activity as the polypeptide composed of the nucleotide sequence of the corresponding sequence number.

[0041] For example, polynucleotides with the same or corresponding activity as polynucleotides, in which a nonsense sequence is added inside or at the end of the nucleotide sequence of the corresponding sequence number, or polynucleotides in which a portion of the sequence inside or at the end of the nucleotide sequence of the corresponding sequence number is deleted, are also included within the scope of this disclosure.

[0042] Homology or identity refers to the degree of correlation between two given nucleotide sequences and can be expressed as a percentage.

[0043] The terms cognate and identical are often used interchangeably.

[0044] Sequence homology or identity of conserved polynucleotides can be determined using standard alignment algorithms and can be used in conjunction with default gap penalties established by the program used. Substantially homologous or identical sequences can typically hybridize with each other along the entire sequence or at least about 50%, 60%, 70%, 80%, or 90% of the full-length sequence under moderate to high stringency conditions. Polynucleotides containing degenerate codons instead of codons are also considered in hybridization.

[0045] Whether any two polynucleotide sequences are homologous, similar, or identical can be determined by known computer algorithms, such as the “FASTA” program, using default parameters, such as those in Pearson et al. (1988) Proc. Natl. Acad. Sci. USA 85:2444. Alternatively, this can be determined by the Needleman–Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443–453), which uses the European Molecular Biology Open Software (EMBOSS) package (Rice et al., 2000, Trends Genet. 16:276–277) (version 5.0.0 or later) (including the GCG package (Devereux, J. et al., Nucleic Acids Research 12:387 (1984)), BLASTP, BLASTN, FASTA (Atschul, SF et al., J MOLEC BIOL. 48:443–453), which uses the European Molecular Biology Open Software (EMBOSS) package (Rice et al., 2000, Trends Genet. 16:276–277) (version 5.0.0 or later) (including the GCG package (Devereux, J. et al., Nucleic Acids Research 12:387 (1984)), BLASTP, BLASTN, FASTA (Atschul, SF et al., J MOLEC BIOL. 48:443–453). 215:403 (1990); Guide to Huge Computers, ed. Martin J. Bishop, Academic Press, San Diego, 1994, and CARILLO et al. (1988) SIAM J Applied Math 48:1073) use the Needleman procedure to perform this. For example, BLAST or ClustalW from the National Center for Biotechnology Information (NCBI) can be used to determine homology, similarity, or identity.

[0046] Homology, similarity, or identity of polynucleotides can be determined by comparing sequence information using the GAP computer program (e.g., as disclosed in Smith and Waterman, Adv. Appl. Math (1981) 2:482, and Needleman et al., (1970), J Mol Biol. 48:44). In short, the GAP program defines homology, similarity, or identity as a value obtained by dividing the number of similarly aligned symbols (i.e., nucleotides or amino acids) 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 a weighted comparison matrix as described in Gribskov et al. (1986) Nucl. Acids Res. 14:6745, as disclosed in Schwartz and Dayhoff, Atlas of Protein Sequence and Structure, National Biomedical Research Foundation (353–358, 1979) (or the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (2) a penalty of 3.0 for each vacancy and an additional penalty of 0.10 for each symbol of each vacancy (or a penalty of 10 for vacancy opening and 0.5 for vacancy extension); and (3) no penalty for terminal vacancy. Therefore, the terms “homology” or “identity” as used herein refer to the correlation between sequences.

[0047] Additionally, this may include, but is not limited to, any polynucleotide sequence capable of hybridizing under stringent conditions with probes that can be prepared from known genes and possess the same activity, such as sequences partially or entirely complementary to the aforementioned polynucleotide sequences. The term "stringent conditions" refers to conditions that enable specific hybridization between polynucleotides. These conditions are specifically described in the literature (e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; F.M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York). For example, the conditions may include that genes with high homology or identity, such as those with at least 40%, specifically at least 70%, at least 80%, at least 85%, or at least 90%, more specifically at least 95%, even more specifically at least 97%, or even more specifically at least 99% homology or identity, hybridize with each other, but genes with lower homology or identity than the above ranges do not hybridize with each other; or typical washing conditions for Southern hybridization, i.e., washing once, specifically twice or three times, at salt concentrations and temperatures corresponding to 60°C, 1×SSC and 0.1% SDS, specifically 60°C, 0.1×SSC and 0.1% SDS, more specifically 68°C, 0.1×SSC and 0.1% SDS.

[0048] Hybridization requires two nucleic acids to have complementary sequences, although mismatches between bases may depend on the strictness of hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that can hybridize with each other. For example, in DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Therefore, this disclosure may include not only substantially similar nucleic acid sequences, but also isolated nucleic acid fragments that are complementary to the whole sequence.

[0049] Specifically, T can be used under the conditions described above. m Hybridization conditions of 55°C were used to detect polynucleotides with homology or identity. Additionally, T... m The value can be 60°C, 63°C or 65°C, but is not limited to these, and can be appropriately adjusted by those skilled in the art for the purpose.

[0050] The appropriate stringency of hybrid polynucleotides depends on the length of the polynucleotide and the degree of its complementarity, the variables of which are well known in the art (see Sambrook et al., ibid., 9.50–9.51 and 11.7–11.8).

[0051] The polynucleotides with promoter activity disclosed herein can be used as promoters.

[0052] The promoter can be located in the 5' region of the transcription start site into mRNA.

[0053] The promoter disclosed herein may have enhanced promoter activity compared to conventional promoters. That is, the promoter can increase the expression of the target gene and the expression and / or activity of the protein encoded by the target gene. For the purposes of this disclosure, the target gene used for enhanced expression can be changed depending on the product to be produced, and the promoter can be used as a universal promoter for enhancing target genes.

[0054] For the purposes of this disclosure, the term "target gene" refers to a gene whose expression is regulated by the promoter sequence of this disclosure. A protein encoded by a target gene may be expressed as a "target protein," and a gene encoding a "target protein" may be expressed as a "target gene."

[0055] Due to codon degeneracy or considering the preferred codons of the organism expressing the polynucleotide, the polynucleotide encoding the target protein can have various modifications in its coding region within a range in which the polynucleotide sequence remains unchanged. The polynucleotide sequence is as described above.

[0056] In one embodiment, the target protein may be a polypeptide with glutamate dehydrogenase (GDH) activity. That is, the target gene of the promoter may be a gene encoding a polypeptide with glutamate dehydrogenase (GDH) activity.

[0057] As used in this article, the term "glutamate dehydrogenase (GDH)" may also be referred to as "glutamate dehydrogenase," etc. Glutamate dehydrogenase participates in the metabolism of glutamate to 2-ketoglutarate, and its activity can be controlled to increase the production of useful substances such as lysine, threonine, O-acetylhomoserine, and isoleucine.

[0058] Examples of genes encoding glutamate dehydrogenase include, but are not limited to, the gdh gene (NCgl1999) of Corynebacterium glutamicum ATCC13032. Those skilled in the art can readily obtain information on genes encoding glutamate dehydrogenase from known databases (GenBank, etc.).

[0059] The amino acid sequences that make up glutamate dehydrogenase can be obtained from known databases such as NCBI GenBank. For example, the amino acid sequences can be derived from Corynebacterium glutamicum.

[0060] Furthermore, the "polypeptide with glutamate dehydrogenase activity" disclosed herein includes not only wild-type, unmodified, or native types of glutamate dehydrogenase, but also variants with the same or enhanced activity relative to them.

[0061] As used herein, the term “modified polypeptide” has the same meaning as “variant”, referring to a protein in which at least one amino acid in a conserved substitution and / or modification differs from the sequence but whose function or properties are retained.

[0062] Variant sequences differ from the identified sequence in that a few amino acid substitutions, deletions, or additions are made. Variant sequences are typically identified by modifying one or more amino acids in the protein's amino acid sequence and by assessing the properties of the modified protein. That is, the ability of a variant can be increased compared to the ability of its native protein. Additionally, some variants may include at least some of these features, such as the removal of the N-terminal leader sequence or transmembrane domain.

[0063] The term "variant" may also be used interchangeably with "modification," "modified protein," "modified polypeptide," "mutant," "mutant protein," "differentiation," "variant," etc., and may be used with any term used in the sense of mutation, but is not limited thereto. For the purposes of this disclosure, a variant may refer to those in which the activity of a mutant protein is increased compared to the activity of a native wild-type or unmodified protein, but variants are not limited thereto.

[0064] As used herein, the term "conservative substitution" refers to the substitution of one amino acid by another amino acid having similar structure and / or chemical properties. Variants may have, for example, one or more conserved substitutions while retaining one or more biological activities. Such amino acid substitutions can generally occur based on the similarity of the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilic properties of the residues.

[0065] Furthermore, variants can include the deletion or addition of amino acids that have minimal impact on the properties and secondary structure of the peptide. For example, a peptide can be conjugated to a signal (or leader) sequence at the N-terminus of a protein, which guides protein transfer during or after translation. Additionally, peptides can be conjugated to another sequence or linker for peptide identification, purification, or synthesis.

[0066] The gene encoding the polypeptide containing glutamate dehydrogenase disclosed herein may be referred to as the "gdh gene".

[0067] This gene can originate from the genus Corynebacterium, particularly Corynebacterium glutamicum.

[0068] In this disclosure, due to codon degeneracy or considering the preferred codons of the organism to which the polypeptide is to be expressed, the "gdh gene," i.e., the polynucleotide encoding a polypeptide with glutamate dehydrogenase activity, can be modified in its coding region within the range in which the amino acid sequence of the polypeptide remains unchanged.

[0069] The disclosed polypeptides containing glutamate dehydrogenase also include variant sequences, and specifically include protein variants modified to exhibit enhanced glutamate dehydrogenase activity.

[0070] According to another aspect of this disclosure, a composition for gene expression is provided, said composition containing a promoter-active polynucleotide of the present disclosure.

[0071] Compositions for gene expression refer to compositions capable of expressing genes, which can be expressed by polynucleotides with promoter activity disclosed herein.

[0072] For example, compositions for gene expression may include the promoter-active polynucleotides disclosed herein, and may also include, but are not limited to, configurations capable of operating the polynucleotides as promoters.

[0073] In the gene expression compositions disclosed herein, the polynucleotide may be in the form contained in a vector to express an operatively linked gene in a host cell into which the polynucleotide has been introduced.

[0074] According to another aspect of this disclosure, an expression cassette is provided, comprising a polynucleotide having promoter activity, or a polynucleotide and a gene encoding a target protein.

[0075] As used herein, the term "expression cassette" refers to a unit box comprising a promoter-active polynucleotide and a gene encoding a target protein, thus enabling the expression of a target gene operatively linked downstream of a promoter. Specifically, in an expression cassette, a promoter-active polynucleotide can be operatively linked to a gene encoding a target protein. As used herein, the term "operatively linked" refers to a functional link between a gene sequence and a polynucleotide with promoter activity to initiate and mediate transcription of a gene encoding a target protein.

[0076] The internal or external components of such gene expression cassettes may additionally include various factors that can help the effective expression of the target gene. In addition to the promoter that is operatively linked to the target gene, the expression cassette typically includes transcription termination signals, ribosome binding sites, and translation termination signals.

[0077] In one embodiment, the target protein may be a polypeptide with glutamate dehydrogenase activity.

[0078] According to another aspect of this disclosure, a vector is provided comprising a polynucleotide having promoter activity, or a polynucleotide and a gene encoding a target protein.

[0079] In one embodiment, the target protein may be a polypeptide with glutamate dehydrogenase activity.

[0080] As used herein, the term "vector" refers to a DNA construct containing a polynucleotide sequence encoding a target protein, said polynucleotide sequence being operatively linked to a suitable control sequence to express the target protein in a suitable host.

[0081] For the purposes of this disclosure, the control sequence may include the promoter-active polynucleotides of this disclosure.

[0082] The control sequences may include a promoter capable of initiating transcription, any operator sequences for controlling such transcription, sequences encoding suitable mRNA ribosome binding sites, and sequences for controlling transcription and translation termination. After transformation into a suitable host cell, the vector may replicate or function independently of the host genome, or it may integrate into its genome. The vectors used in this disclosure are not particularly limited, as long as they can be expressed in host cells, and any vector known in the art can be used to transform host cells. Examples of commonly used vectors may include natural or recombinant plasmids, granules, viruses, and bacteriophages.

[0083] For example, pWE15, M13, λLB3, λBL4, λIXII, λASHII, λAPII, λt10, λt11, Charon4A, and Charon21A can be used as phage vectors or granular vectors, and vectors based on pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, and pET can be used as plasmid vectors.

[0084] Furthermore, endogenous promoters in chromosomes can be replaced by promoter-active polynucleotides disclosed herein using vectors for insertion into chromosomes in host cells. For example, vectors such as pECCG117, pDZ, pACYC177, pACYC184, pCL, pUC19, pBR322, pMW118, pCC1BAC, pCES208, or pXMJ19 can be used, but are not limited thereto. Alternatively, vectors known as those in the prior art (Korean Patent No. 10-09240675) can be used.

[0085] Polynucleotides can be inserted into chromosomes using any method known in the art, such as homologous recombination, but are not limited thereto. The vector may also include selection markers for studying chromosome insertion or non-insertion. Selection markers are used to select cells transformed with the vector, i.e., to study the insertion or non-insertion of target nucleic acid molecules, and may use markers conferring a selective phenotype, such as drug resistance, nutritional deficiencies, resistance to cytotoxic drugs, and expression of surface proteins. In the case of treatment with a selection agent, only cells expressing the selection marker can survive or express other phenotypic traits, thus allowing selection of transformed cells. For example, wild-type polynucleotides can be replaced with modified polynucleotides using a vector for chromosome insertion in cells.

[0086] As used in this article, the term “conversion” can refer to the introduction of a vector containing a polynucleotide encoding a target protein into a host cell to allow the target protein to be expressed in the host cell.

[0087] The transformed polynucleotide can include any polypeptide that can be expressed in the host cell, whether the polypeptide is inserted into and located within the host cell's chromosome or located outside the chromosome. Additionally, the polynucleotide encoding the target protein can contain both DNA and RNA encoding the target protein. The polynucleotide can be introduced in any form, as long as it can be introduced and expressed in the host cell. For example, the polynucleotide encoding the target protein can be introduced into the host cell in the form of an expression cassette, which is a gene construct containing all the elements required for self-expression.

[0088] Expression cassettes typically include a promoter, transcription termination signal, ribosome binding site, and translation termination signal operably linked to a polynucleotide encoding a target protein. Additionally, the polynucleotide containing the target protein can be introduced into the host cell in its native form and operably linked to the sequence required for expression in the host cell, but is not limited thereto.

[0089] Furthermore, the term "operably linked" refers to a functional link between a gene sequence and a promoter sequence that initiates and mediates the transcription of a polynucleotide encoding a target protein disclosed herein.

[0090] For the purposes of this disclosure, the promoter may be a polynucleotide with promoter activity as disclosed herein.

[0091] The transformation methods for the vectors disclosed herein include any method for introducing nucleic acids into cells, and can be performed using appropriate standard techniques known in the art, depending on the host cell. Examples of such methods may include, but are not limited to, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, retroviral infection, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, lithium acetate-DMSO method, etc.

[0092] According to another aspect of this disclosure, a microorganism is provided comprising: a promoter-active polynucleotide of this disclosure; an expression cassette comprising the polynucleotide and a gene encoding a target protein; or a vector comprising the polynucleotide and a gene encoding a target protein.

[0093] As used herein, the term "microorganism" includes all wild-type microorganisms, or microorganisms with natural or artificial genetic modifications, and refers to microorganisms in which a particular mechanism is weakened or enhanced due to the insertion of a foreign gene or the enhancement or weakening of the activity of an endogenous gene. Specifically, said microorganisms may include: polynucleotides with promoter activity disclosed herein; and target proteins.

[0094] The target protein can be a polypeptide with glutamate dehydrogenase (GDH) activity. The promoter-active polynucleotides, target proteins, polypeptides with glutamate dehydrogenase (GDH) activity, vectors, and expression cassettes disclosed herein are as described above.

[0095] The microorganisms can be members of the genus Corynebacterium, especially Corynebacterium glutamicum.

[0096] The microorganism may be a microorganism expressing glutamate dehydrogenase, a microorganism expressing a polypeptide with glutamate dehydrogenase activity, or a microorganism incorporating a polypeptide with glutamate dehydrogenase activity, but is not limited thereto.

[0097] In this disclosure, microorganisms may include the promoter-active polynucleotides of this disclosure, and specifically may include polynucleotides and / or genes operatively linked to polynucleotides and encoding target proteins. Alternatively, microorganisms may include vectors or expression cassettes comprising, but not limited to, polynucleotides or gene expression control sequences and genes encoding target proteins. Furthermore, polynucleotides, genes encoding target proteins, vectors, and expression cassettes may be introduced into microorganisms through transformation, but are not limited to this. Moreover, it is irrelevant whether the polynucleotides and genes encoding target proteins are located on or outside chromosomes, as long as the gene can be expressed in the microorganism.

[0098] As used herein, the term "to be expressed / expressed" for proteins refers to a state in which, for example, glutamate dehydrogenase or a variant thereof is introduced into or modified for expression in microorganisms. When the target protein is a protein present in a microorganism, the term refers to a state in which the protein's activity is enhanced compared to its endogenous activity or activity prior to modification.

[0099] Specifically, the term "protein introduction" can refer to the microorganism exhibiting the activity of a specific protein that it did not originally possess, or exhibiting enhanced activity compared to the endogenous activity of the corresponding protein or its unmodified activity. For example, the term can refer to the introduction of a polynucleotide encoding a specific protein into the chromosome of a microorganism, or the introduction of a vector or expression cassette containing a polynucleotide encoding a specific protein into a microorganism to express the activity of that protein.

[0100] The term "enhanced activity" can refer to an increase in the activity of a specific protein in a microorganism compared to its endogenous activity or its activity before modification. The term "endogenous activity" can refer to the activity of a specific protein originally possessed by the parent strain before transformation when a microorganism is transformed through gene mutations induced by natural or artificial factors.

[0101] For the purposes of this disclosure, enhanced activity can be achieved by using a promoter-active polynucleotide sequence of this disclosure as the expression control sequence for the target protein. Since the target protein can be in its native or variant form as described above, the expression control sequence can be the expression control sequence of a gene encoding the variant protein on a chromosome or the expression control sequence of a gene encoding the native protein.

[0102] In addition, other activity enhancement methods can be used in combination. For example, in addition to using the promoter-active polynucleotide sequence of this disclosure as the expression control sequence of the target protein, at least one method selected from the following can be used: increasing the intracellular copy number of the gene encoding the target protein, replacing the gene encoding the native protein on the chromosome with a gene encoding a protein variant, and further, introducing a mutation into the gene encoding the protein to enhance the activity of the protein variant, and introducing the protein variant into microorganisms, but not limited thereto.

[0103] By using the promoter-active polynucleotides disclosed herein for the expression control of target proteins in microorganisms, the activity of target proteins can be enhanced.

[0104] For example, the activity or concentration of a protein relative to that in a wild-type or unmodified microbial strain may be increased by at least 1%, at least 10%, at least 25%, at least 50%, at least 75%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, or at least 500%, and up to 1,000% or 2,000%, but is not limited thereto.

[0105] As used herein, the term "unmodified microorganism" does not exclude mutant strains that can be naturally present in microorganisms, and refers to the natural strain itself, excluding microorganisms with promoter-active polynucleotides of this disclosure or microorganisms not transformed with vectors containing promoter-active polynucleotides of this disclosure.

[0106] As used herein, the term "target-producing microorganism" includes all microorganisms with naturally or artificially produced genetic modifications, and can refer to microorganisms in which a particular mechanism is weakened or enhanced due to the insertion of a foreign gene or the enhancement or inactivation of an endogenous gene, wherein said microorganisms have genetic mutations or enhanced activity for target-producing. For the purposes of this disclosure, target-producing microorganisms can refer to microorganisms capable of producing an excess of the target substance compared to wild-type or unmodified microorganisms by including the promoter-active polynucleotides of this disclosure.

[0107] The term "microorganism that produces target substances" can be used interchangeably with terms such as "microorganism that produces target substances", "microorganism with the ability to produce target substances", "strain that produces target substances", and "strain with the ability to produce target substances".

[0108] The target substance can be an amino acid, particularly lysine, threonine, O-acetylhomoserine, or isoleucine. As more specific examples, lysine can be L-lysine, threonine can be L-threonine, and isoleucine can be L-isoleucine, but is not limited to these.

[0109] For the purposes of this disclosure, microorganisms that produce target substances may have improved ability to produce target substances, particularly lysine, threonine, O-acetylhomoserine, or isoleucine.

[0110] Furthermore, the microorganisms producing the target substance can be wild-type or recombinant microorganisms. Recombinant microorganisms are as described above. Microorganisms may also include mutations, such as enhancing biosynthetic pathways to increase the ability to produce the target substance, releasing feedback inhibition, or inactivating genes that weaken degradation or biosynthetic pathways. Such mutations can be artificially induced, for example, through UV irradiation, but natural mutations are not excluded.

[0111] Specifically, microorganisms that produce target substances can be modified to produce the target substances. For example, microorganisms that do not have the ability to produce target substances can be modified to have the ability to produce target substances, or the production ability of microorganisms can be enhanced. For example, proteins involved in biosynthetic pathways or their variants can be introduced into wild-type microorganisms to produce target substances (KR 10-2011994, KR 10-1947959 and KR 10-1996769).

[0112] As a specific example, the microorganisms that produce the target substance disclosed herein may be Corynebacterium species containing aspartate kinase (lysC), homoserine dehydrogenase (hom), pyruvate carboxylase (pyc), L-threonine dehydratase (ilvA), or combinations thereof. The aspartate kinase, homoserine dehydrogenase, pyruvate carboxylase, or L-threonine dehydratase may be wild-type proteins or protein variants mutated to benefit the production of the target substance through attenuation or enhancement of activity.

[0113] Compared to microorganisms that do not contain polynucleotides with promoter activity, the microorganisms of this disclosure may have an enhanced ability to produce target substances by at least 1%, 5%, 10%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 22%, 25%, 29%, 33%, 38%, 44%, 45%, or 48%.

[0114] According to another aspect of this disclosure, a method for generating a target substance is provided, the method comprising culturing microorganisms in a culture medium. The microorganisms and the target substance are as described above.

[0115] In this disclosure, the method for producing target substances using microorganisms containing polynucleotides can be performed using methods widely known in the art. Specifically, cultivation can be carried out continuously via batch processes, fed-batch processes, or repeated fed-batch processes, but is not limited thereto. The culture medium used in the cultivation must appropriately meet the requirements of the specific strain. Culture media for Corynebacterium strains have been disclosed (e.g., Manual of Methods for General Bacteriology by the American Society for Bacteriology, Washington DC, USA, 1981).

[0116] As for the culture medium and other culture conditions used to cultivate the strains of this disclosure, any culture medium used for the cultivation of typical microorganisms of the genus Corynebacterium can be used without particular limitation. Specifically, the strains of this disclosure can be cultured under aerobic or anaerobic conditions in a conventional culture medium containing appropriate carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids and / or vitamins, while adjusting the temperature, pH, etc.

[0117] In this disclosure, carbon sources may include, but are not limited to, carbohydrates such as glucose, fructose, sucrose, and maltose; sugar alcohols such as mannitol and sorbitol; organic acids such as pyruvic acid, lactic acid, and citric acid; amino acids such as glutamic acid, methionine, and lysine; and so on. In addition, natural organic nutrient sources may be used, such as starch hydrolysate, molasses, crude molasses, rice bran, cassava, bagasse, and corn steep liquor, and carbohydrates such as glucose and aseptically pretreated molasses (i.e., molasses converted to reducing sugars) may be used, and other carbon sources may be used in appropriate amounts. These carbon sources may be used alone or in combination of two or more of them.

[0118] Examples of nitrogen sources may include: inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; and organic nitrogen sources such as amino acids, peptone, NZ-amines, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolysate, fish or its degradation products, defatted soybean meal or its degradation products. These nitrogen sources may be used alone or in combination of two or more, but are not limited thereto.

[0119] Examples of phosphorus sources may include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and their corresponding sodium-containing salts. Examples of inorganic compounds may include sodium chloride, calcium chloride, ferric chloride, magnesium sulfate, ferric sulfate, manganese sulfate, calcium carbonate, etc.

[0120] In addition, the culture medium may contain amino acids, vitamins, and / or appropriate precursors. Specifically, L-amino acids, such as glycine, glutamic acid, and / or cysteine, may be added to the culture medium of the strain. Specifically, glycine, glutamic acid, and / or cysteine ​​may be added, and if desired, further L-amino acids, such as lysine, may be added, but not limited to these.

[0121] These culture media or precursors can be added to the culture in batches or continuously, but are not limited to this.

[0122] In this disclosure, during strain cultivation, the pH of the culture can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid to the culture in an appropriate manner. Furthermore, antifoaming agents such as fatty acid polyethylene glycol esters can be added to inhibit foam formation during cultivation. To maintain an aerobic state in the culture, oxygen or oxygen-containing gas can be injected into the culture; alternatively, to maintain an anaerobic or non-anaerobic state, no gas may be injected, or nitrogen, hydrogen, or carbon dioxide gas may be injected.

[0123] The culture temperature can be from 25°C to 40°C, and specifically from 28°C to 37°C, but is not limited thereto. The culture time can continue until the desired yield of useful substances can be obtained, and can be from 1 to 160 hours, or from 10 to 100 hours, but is not limited thereto.

[0124] Methods for producing target materials may also include additional processes following the culture step. Other methods may be appropriately selected depending on the intended use of the target material.

[0125] Specifically, the method for manufacturing the target substance may include, after the culturing step, recovering the target substance from at least one of the following: microorganisms, culture medium, dried microorganisms, microbial extracts, microbial cultures, culture supernatants, and microbial lysates.

[0126] The method may also include lysing the microorganism (strain) before or simultaneously with the recovery step. Lysis of the strain can be performed using methods commonly used in the art to which this disclosure pertains, such as lysis buffer, sonication, heat treatment, Freund's crusher, etc. Additionally, the lysis step may include, but is not limited to, enzymatic reactions involving cell wall degrading enzymes, nucleic acid degrading enzymes, nucleotransferases, proteolytic enzymes, etc.

[0127] In this disclosure, the term "dried product of microorganisms" may be used interchangeably with the term "dried product of bacterial strains," etc. Dried products of microorganisms can be prepared by drying cells in which target substances have accumulated, and can specifically include, but are not limited to, feed compositions, food compositions, etc.

[0128] In this disclosure, the term "microbial extract" is used interchangeably with the term "strain extract," etc. A strain extract can refer to the material remaining after the cell wall has been separated from the cells of a strain. Specifically, a strain extract can refer to components other than the cell wall among those obtained through cell lysis. A strain extract contains a target substance and, in addition to the target substance, may contain at least one component selected from, but is not limited to, proteins, carbohydrates, nucleic acids, and fibers.

[0129] In the recovery step, the target material can be recovered using appropriate methods known in the art.

[0130] The recovery step may include a purification process. The purification method may involve isolating the target substance solely from the strain and then purifying it to purity. This purification method can produce a pure, purified target substance.

[0131] As needed, the method for generating the target substance may also include mixing the excipient with a material selected from strains or their dried products, extracts, cultures or lysates obtained after the culture step, and recovering the target substance therefrom.

[0132] Excipients may be used appropriately, depending on their intended use or form, and may be selected from starch, glucose, cellulose, lactose, glycogen, D-mannitol, sorbitol, lactitol, maltodextrin, calcium carbonate, synthetic aluminum silicate, calcium hydrogen phosphate, calcium sulfate, sodium chloride, sodium bicarbonate, purified lanolin, dextrin, sodium alginate, methylcellulose, colloidal silica gel, hydroxypropyl starch, hydroxypropyl methylcellulose, propylene glycol, casein, calcium lactate, sodium carboxymethyl starch (Primojel), and gum arabic. Specifically, excipients may be selected from at least one of the following components: starch, glucose, cellulose, lactose, dextrin, glycogen, D-mannitol, and maltodextrin, but are not limited thereto.

[0133] Examples of excipients may include, but are not limited to, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, isotonic agents, etc.

[0134] According to another aspect of this disclosure, the use of a polynucleotide as a promoter is provided, said polynucleotide having promoter activity, wherein in the polynucleotide sequence of SEQ ID NO: 1, the nucleotides at positions 27, 28, 31, 32 and 36 are replaced by other nucleotides.

[0135] Polynucleotides are as described above. Detailed Implementation

[0136] Methods of implementing the present invention

[0137] The present disclosure will be described in more detail below with reference to exemplary embodiments. However, these exemplary embodiments are given for the purpose of illustrating the present disclosure, and the scope of the disclosure is not limited to these examples.

[0138] Example 1: Identification of the activity of novel promoters that induce target gene expression

[0139] Example 1-1. Using a library of randomly mutagenic gdh promoter mutants

[0140] First, the nucleotide sequence (SEQ ID NO: 1) of the promoter region of the gdh gene (NCBI accession number NCgl1999) from wild-type Corynebacterium glutamicum ATCC13032 was fixed in NIH GenBank. Using the gdh gene promoter composed of the nucleotide sequence of SEQ ID NO: 1 as a template, primers of SEQ ID NO: 5 and SEQ ID NO: 6 were used, and a diverse PCR random mutagenesis kit (TaKaRa) was employed to obtain PCR products (Pmgdh) of gdh promoter mutants with different sequences. For the open reading frame (ORF) of the GFP gene, the pGFPuv vector (Clontech, USA) was used as a template, and PCR was performed using primers of SEQ ID NO: 7 and 8. The PCR reaction was performed as follows: denaturation at 94°C for 5 minutes; denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, polymerization at 72°C for 1 minute, for 30 cycles; followed by polymerization at 72°C for 7 minutes, resulting in a gene fragment containing the GFP ORF.

[0141] The amplified product, the gdh promoter mutant PCR product (Pmgdh), and GFP were mixed with pCES208 (an E. coli-Corynebacterium shuttle vector prepared by digestion with BamHI / SalI restriction enzyme (J. Microbiol. Biotechnol. 18:639–647, 2008) to facilitate the use of... The HD Cloning Kit (Clontech) was used to construct recombinant vector libraries in which Pmgdh was linked to GFP. The vectors were named pCES_Pm1gdh_gfp to pCES_Pm100gdh_gfp.

[0142] As a control for studying the activity of the Pmgdh library, a recombinant vector containing the wild-type gdh gene promoter (SEQ ID NO: 1) linked to GFP was used. The promoter gene fragment of the wild-type gdh gene was obtained using wild-type Corynebacterium glutamicum ATCC13032 as a template and primers SEQ ID NO: 5 and SEQ ID NO: 6. For the open reading frame (ORF) of the GFP gene, the pGFPuv vector (Clontech, USA) was used as a template, and PCR was performed using primers SEQ ID NO: 7 and 8. The PCR reaction was performed as follows: 30 cycles of denaturation at 94°C for 5 minutes, followed by denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 1 minute, and then polymerization at 72°C for 7 minutes to obtain the gene fragment containing the GFP ORF.

[0143] The amplified product, the wild-type promoter PCR product of gdh (Pgdh), and GFP were mixed with pCES208 (an E. coli-Corynebacterium shuttle vector prepared by digestion with BamHI / SalI restriction enzyme (J. Microbiol. Biotechnol. 18:639–647, 2008) to allow for the use of... The HD Cloning Kit (Clontech) was used to construct a recombinant vector in which Pgdh and GFP were linked, and the recombinant vector was named pCES_Pgdh_gfp.

[0144] Examples 1-2. Preparation of Transformed Strains

[0145] The vector pCES208 and the recombinant vector pCES_Pmgdh_gfp library (pCES_Pm1gdh_gfp to pCES_Pm100gdh_gfp) and pCES_Pgdh_gfp constructed in Example 1-1 were transformed into Corynebacterium glutamicum ATCC13032 by electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541–545). Then, the transformed strains were selected in selective medium containing 25 mg / L kanamycin and named ATCC13032 / pCES, ATCC13032 / pCES_Pmgdh_gfp (ATCC13032 / pCES_Pm1gdh_gfp to ATCC13032 / pCES_Pm100gdh_gfp), and ATCC13032 / pCES_Pgdh_gfp, respectively.

[0146] Examples 1-3. Selection of gdh promoter mutants

[0147] To investigate the activity of the gdh promoter mutant, the transformed strains *Corynebacterium glutamicum* ATCC13032 / pCES, ATCC13032 / pCES_Pgdh_gfp, and ATCC13032 / pCES_Pmgdh_gfp (from ATCC13032 / pCES_Pm1gdh_gfp to ATCC13032 / pCES_Pm100gdh_gfp) obtained in Examples 1-2 were cultured using the following method, and GFP activity was measured.

[0148] Specifically, each transformed *Corynebacterium glutamicum* strain was inoculated into flasks containing 25 mL of culture medium (20 g glucose, 5 g ammonium sulfate, 5 g yeast extract, 1.5 g urea, 4 g KH₂PO₄, 8 g K₂HPO₄, 0.5 g MgSO₄·7H₂O, 150 μg biotin, 1.5 mg thiamine hydrochloride, 3 mg calcium pantothenate, 3 mg nicotinamide (based on 1 L distilled water), pH 7.2) and cultured at 30 °C with shaking for 20 h. Cells were recovered from the culture by centrifugation (5,000 rpm, 15 min), washed twice with 50 mM Tris-HCl (pH 8.0) buffer, and then resuspended in the same buffer. Cells were disrupted for 6 min using a bead mill tissue homogenizer after adding 1.25 g glass beads to each 1.5 mL suspension. The supernatant was then recovered by centrifugation (15,000 rpm, 20 min), and protein concentration was quantified using the Bradford method. For equal amounts of cell extracts, the expression level of the GFP gene was measured by irradiating the sample with excitation light at 488 nm using the method described by Laure Gory et al. (FEMS Microbiology Letters, 194, 127–133, 2001), and measuring the emission light at 511 nm using an LS-50B spectrophotometer (Perkin Elmer). The top three strains with the highest GFP gene expression levels were selected by comparing them with the control strain ATCC13032 / pCES_Pgdh_gfp (Table 1).

[0149] Table 1

[0150] ATCC13032 / pCES 0 ATCC13032 / pCES_Pgdh_gfp 583 ATCC13032 / pCES_Pm3gdh_gfp 1217 ATCC13032 / pCES_Pm16gdh_gfp 1205 ATCC13032 / pCES_Pm78gdh_gfp 1198

[0151] As shown in Table 1 above, the Pm3gdh, Pm16gdh, and Pm78gdh promoters exhibited promoter activity in *Corynebacterium glutamicum* and also showed higher fluorescence sensitivity than the wild-type gdh promoter. To investigate mutations introduced into the gdh promoters of the three selected strains, gdh promoter mutants were sequenced. PCR was performed using primers for SEQ ID NO: 9 and SEQ ID NO: 10 to determine the sequences, followed by sequencing. The modified gdh promoters were sequenced by comparing them with the wild-type gdh promoter sequence SEQ ID NO: 1. The gdh promoter sequences of the selected strains are shown in Table 2 below.

[0152] Table 2

[0153]

[0154]

[0155] Example 2: Construction of vectors for introducing Pm3gdh, Pm16gdh, and Pm78gdh promoter mutants

[0156] To construct vectors for introducing promoter mutants of Pm3gdh, Pm16gdh, and Pm78gdh, pCES_Pm3gdh_gfp, pCES_Pm16gdh_gfp, and pCES_Pm78gdh_gfp vectors were used as templates, and PCR products corresponding to the promoter mutants were obtained using primers SEQ ID NO: 13 and SEQ ID NO: 14. Using the chromosome of *Corynebacterium glutamicum* strain ATCC13032 as a template, along with primer sets SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 15, and SEQ ID NO: 16, gene fragments containing the upstream region of the GDH promoter and part of the ORF were obtained. The PCR reaction was performed as follows: 30 cycles of denaturation at 94℃ for 5 minutes, followed by denaturation at 94℃ for 30 seconds, annealing at 55℃ for 30 seconds, and polymerization at 72℃ for 1 minute, and then polymerization at 72℃ for 5 minutes to obtain the PCR products. The three amplification products were mixed with the pDCM2 vector (Korean Patent Publication No. 10-2020-0136813) prepared in advance by digestion with Smal restriction enzyme, and then... Recombinant vectors were constructed using the HD Cloning Kit (Clontech). These vectors were named pDCM2_Pm3gdh_gdh, pDCM2_Pm16gdh_gdh, and pDCM2_Pm78gdh_gdh, respectively.

[0157] Example 3: Evaluation of target material production capability

[0158] 3-1. Evaluation of Lysine Production Capacity

[0159] 3-1-1. Preparation of L-lysine-producing strains with gdh promoter mutant

[0160] To prepare strains transformed with the gdh promoter mutant, the pDCM2_Pm3gdh_gdh, pDCM2_Pm16gdh_gdh, and pDCM2_Pm78gdh_gdh vectors constructed in Example 2 were used to transform the L-lysine-producing strain *Corynebacterium glutamicum* CJ3P (Binder et al., Genome Biology 2012, 13:R40) into the chromosome to introduce the gdh promoter mutant sequence. The CJ3P strain is an *Corynebacterium glutamicum* strain capable of L-lysine production by introducing three types of mutations (pyc (Pro458Ser), hom (Val59Ala), and lysC (Thr311Ile)) into the wild-type strain using known techniques.

[0161] Specifically, the vector constructed in Example 2 was introduced into the CJ3P strain via electroporation, and then transformed strains were obtained from a selection medium containing 25 mg / L kanamycin. Strains were selected by PCR and sequencing using primers of SEQ ID NO: 9 and SEQ ID NO: 10, wherein the gdh promoter mutant was introduced into the strain via a DNA fragment inserted into the chromosome through a second exchange. The selected strains were named *Corynebacterium glutamicum* CJ3P::Pm3gdh_gdh, CJ3P::Pm16gdh_gdh, and CJ3P::Pm78gdh_gdh.

[0162] 3-1-2. Evaluation of L-lysine production capacity in strains infused with the gdh promoter mutant

[0163] To evaluate the L-lysine production capacity of the *Corynebacterium glutamicum* strain CJ3P used as the parent strain and the *Corynebacterium glutamicum* strains CJ3P::Pm3gdh_gdh, CJ3P::Pm16gdh_gdh, and CJ3P::Pm78 gdh_gdh prepared in Example 3-1-1, the strains were cultured using the following method and then analyzed.

[0164] First, each strain was inoculated into a 250mL corner-baffle flask containing 25mL of seed culture medium and cultured at 30°C with shaking at 200rpm for 20 hours. Then, 1mL of seed culture was inoculated into a 250mL corner-baffle flask containing 24mL of production culture medium and cultured at 32°C with shaking at 200rpm for 48 hours. The components of the seed culture medium and production culture medium are shown below:

[0165] Seed culture medium (pH 7.0)

[0166] 20g glucose, 10g peptone, 5g yeast extract, 1.5g urea, 4g KH2PO4, 8g K2HPO4, 0.5g MgSO4·7H2O, 100μg biotin, 1000μg thiamine hydrochloride, 2000μg calcium pantothenate, 2000μg nicotinamide (based on 1L distilled water).

[0167] <Production medium (pH 7.0)>

[0168] 45g glucose, 10g soy protein, 10g molasses, 15g (NH4)2SO4, 0.55g KH2PO4, 0.6g MgSO4·7H2O, 9mg FeSO4·7H2O, 9mg MnSO4·5H2O, 0.9mg biotin, 4.5mg thiamine hydrochloride, 30g CaCO3, 4.5mg calcium pantothenate, 30mg nicotinamide, 0.45mg ZnSO4, 0.45mg CuSO4 (based on 1L distilled water)

[0169] After cultivation, the yield of L-lysine was measured by HPLC. The concentration and concentration growth rate of L-lysine in the cultures of Corynebacterium glutamicum CJ3P, CJ3P::Pm3gdh_gdh, CJ3P::Pm16gdh_gdh, and CJ3P::Pm78gdh_gdh strains are shown in Table 3 below.

[0170] Table 3

[0171] CJ3P 4.12 - CJ3P::gdhPm3_gdh 4.96 20.39％ CJ3P::gdhPm16_gdh 4.84 17.48％ CJ3P::gdhPm78_gdh 4.79 16.26％

[0172] As shown in Table 3, compared with the parental strain CJ3P, the three strains introduced with the gdh promoter mutant showed an increased L-lysine concentration. CJ3P::gdhPm3_gdh was named CM03-1660 and was deposited on April 5, 2021, at the Korean Center for Microbial Conservation, a depositary institution under the Budapest Treaty, with accession number KCCM12970P.

[0173] Example 3-2: Evaluation of threonine production capacity

[0174] 3-2-1. Preparation of Threonine-producing strains

[0175] In order to prepare strains transformed with gdh promoter mutants using the pDCM2_Pm3gdh_gdh, pDCM2_Pm16gdh_gdh, and pDCM2_Pm78gdh_gdh vectors constructed in Example 2, L-threonine-producing strains were first prepared based on Corynebacterium glutamicum ATCC13032 strains, which introduced the lysC(L377K) mutant (Korean Patent No. 10-2011994) and the hom(R398Q) mutant (Korean Patent No. 10-1947959).

[0176] Specifically, to prepare the L-threonine-producing strain, a vector for introducing lysC(L377K) was first constructed. To construct the vector, the chromosome of wild-type Corynebacterium glutamicum ATCC13032 was used as a template, and PCR was performed using primers SEQ ID NOS: 17 and 18 and SEQ ID NOS: 19 and 20. The PCR reaction was performed as follows: 30 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 30 seconds, followed by polymerization at 72°C for 7 minutes, to obtain the PCR products. The amplified products were mixed with the pDCM2 vector, which had been prepared beforehand by digestion with the SmaI restriction enzyme, and then... The HD cloning kit was used to construct the recombinant vector, which was then named pDCM2_lysC(L377K).

[0177] The constructed pDCM2_lysC(L377K) vector was introduced into *Corynebacterium glutamicum* strain ATCC13032 via electroporation, and transformed strains were obtained in selection medium containing 25 mg / L kanamycin. The strains were selected by PCR and sequencing using primers SEQ ID NO: 25 and SEQ ID NO: 26, wherein a nucleotide mutant of the lysC gene was introduced into the strain via a DNA fragment inserted into the chromosome through a second crossover, and the selected strain was named ATCC13032::lysC(L377K).

[0178] To construct the vector introducing hom(R398Q), the chromosome of *Corynebacterium glutamicum* strain ATCC13032 was used as a template, and PCR was performed using primers SEQ ID NOS: 21 and 22 and SEQ ID NOS: 23 and 24. The PCR reaction was performed as follows: 30 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 30 seconds, followed by polymerization at 72°C for 7 minutes. The amplified product was mixed with the pDCM2 vector prepared beforehand by digestion with the SmaI restriction enzyme, and then... The HD cloning kit was used to construct the recombinant vector, which was then named pDCM2_hom(R398Q).

[0179] The constructed pDCM2_hom(R398Q) vector was introduced into the *Corynebacterium glutamicum* strain ATCC13032::lysC(L377K) prepared above via electroporation, and then transformed strains were obtained in selection medium containing 25 mg / L kanamycin. The strains were selected by PCR and sequencing using primers SEQ ID NO: 27 and SEQ ID NO: 28, wherein a nucleotide mutant of the hom gene was introduced into the strain via a DNA fragment inserted into the chromosome through a second crossover, and the selected strains were named *Corynebacterium glutamicum* ATCC13032::lysC(L377K)_hom(R398Q).

[0180] 3-2-2. Preparation of L-threonine-producing strains with gdh promoter mutant

[0181] Specifically, the vector constructed in Example 2 was introduced into the *Corynebacterium glutamicum* strain ATCC13032::lysC(L377K)_hom(R398Q) via electroporation, and then transformed strains were obtained from a selection medium containing 25 mg / L kanamycin. The strains were selected by PCR and sequencing using primers of SEQ ID NO: 9 and SEQ ID NO: 10, wherein the gdh promoter mutant was introduced into the strain via a DNA fragment inserted into the chromosome through a second exchange. The selected strains were named *Corynebacterium glutamicum* ATCC13032::lysC(L377K)_hom(R398Q)::Pm3gdh_gdh, ATCC13032::lysC(L377K)_hom(R398Q)::Pm16gdh_gdh, and ATCC13032::lysC(L377K)_hom(R398Q)::Pm78gdh_gdh.

[0182] 3-2-3. Evaluation of L-threonine production capacity in strains inoculated with the gdh promoter mutant

[0183] To evaluate the L-threonine production capacity of *Corynebacterium glutamicum* ATCC13032::lysC(L377K)_hom(R398Q) used as parent strains, and the *Corynebacterium glutamicum* strains ATCC13032::lysC(L377K)_hom(R398Q)::Pm3gdh_gdh, ATCC13032::lysC(L377K)_hom(R398Q)::Pm16gdh_gdh, and ATCC13032::lysC(L377K)_hom(R398Q)::Pm78gdh_gdh prepared in Example 3-2-2, the strains were cultured using the following method and then analyzed.

[0184] First, each strain was inoculated into a 250mL corner-baffle flask containing 25mL of seed culture medium and cultured at 30°C with shaking at 200rpm for 20 hours. Then, 1mL of seed culture was inoculated into a 250mL corner-baffle flask containing 24mL of production culture medium and cultured at 32°C with shaking at 200rpm for 48 hours.

[0185] Seed culture medium (pH 7.0)

[0186] 20g glucose, 10g peptone, 5g yeast extract, 1.5g urea, 4g KH2PO4, 8g K2HPO4, 0.5g MgSO4·7H2O, 100μg biotin, 1000μg thiamine hydrochloride, 2000μg calcium pantothenate, 2000μg nicotinamide (based on 1L distilled water).

[0187] <Production medium (pH 7.0)>

[0188] 45g glucose, 10g soy protein, 10g molasses, 15g (NH4)2SO4, 0.55g KH2PO4, 0.6g MgSO4·7H2O, 9mg FeSO4·7H2O, 9mg MnSO4·5H2O, 0.9mg biotin, 4.5mg thiamine hydrochloride, 30g CaCO3, 4.5mg calcium pantothenate, 30mg nicotinamide, 0.45mg ZnSO4, 0.45mg CuSO4 (based on 1L distilled water)

[0189] After cultivation, the amount of L-threonine produced was measured by HPLC. The L-threonine concentrations and concentration growth rates in the cultures of the following strains of Corynebacterium glutamicum: ATCC13032::lysC(L377K)_hom(R398Q), ATCC13032::lysC(L377K)_hom(R398Q)::Pm3gdh_gdh, ATCC13032::lysC(L377K)_hom(R398Q)::Pm16gdh_gdh, and ATCC13032::lysC(L377K)_hom(R398Q)::Pm78gdh_gdh are shown in Table 4 below.

[0190] Table 4

[0191]

[0192] As shown in Table 4, the three strains introduced with the gdh promoter mutant showed an increase in L-threonine concentration compared with the parental strain ATCC13032::lysC(L377K)_hom(R398Q).

[0193] Example 3-3: Evaluation of O-acetylhomoserine production capacity

[0194] 3-3-1. Preparation of O-acetylhomoserine-producing strains with introduced gdh promoter mutant

[0195] The vector constructed in Example 2 was introduced into the wild-type strain *Corynebacterium glutamicum* ATCC13032 via electroporation, and then transformed strains were obtained from a selection medium containing 25 mg / L kanamycin. Strains were selected by PCR and sequencing using primers of SEQ ID NO: 9 and SEQ ID NO: 10, wherein the gdh promoter mutant was introduced into the strain via a DNA fragment inserted into the chromosome through a second crossover. The selected strains were named *Corynebacterium glutamicum* ATCC13032::Pm3gdh_gdh, ATCC13032::Pm16gdh_gdh, and ATCC13032::Pm78gdh_gdh.

[0196] 3-3-2. Evaluation of the O-acetylhomoserine production capacity of strains inoculated with the gdh promoter mutant

[0197] To evaluate the O-acetylhomoserine production capacity of *Corynebacterium glutamicum* ATCC13032 used as the parent strain and the strains ATCC13032::Pm3gdh_gdh, ATCC13032::Pm16gdh_gdh, and ATCC13032::Pm78gdh_gdh prepared in Example 3-3-1, the strains were cultured using the following method and then analyzed.

[0198] Each strain was inoculated into a 250 mL corner-baffle flask containing 25 mL of the culture medium described below using an inoculation loop, and then cultured at 33°C with shaking at 200 rpm for 20 hours.

[0199] <Production medium (pH 7.2)>

[0200] 30g glucose, 2g KH2PO4, 3g urea, 40g (NH4)2SO4, 2.5g peptone, 5g (10mL) corn steep liquor (CSL, Sigma), 0.5g MgSO4·7H2O and 20g CaCO3 (based on 1L distilled water).

[0201] After cultivation, the production capacity of O-acetylhomoserine was measured by HPLC. The concentrations and concentration growth rates of O-acetylhomoserine in the cultures of Corynebacterium glutamicum strains ATCC13032, ATCC13032::Pm3gdh_gdh, ATCC13032::Pm16gdh_gdh, and ATCC13032::Pm78gdh_gdh are shown in Table 5 below.

[0202] Table 5

[0203]

[0204] As shown in Table 5, compared with the parental wild-type strain ATCC13032, the three strains introduced with the gdh promoter mutant showed an increased concentration of O-acetylhomoserine.

[0205] Examples 3-4: Evaluation of L-Isoleucine Production Capacity

[0206] 3-4-1. Preparation of L-isoleucine-producing strains with introduced gdh promoter mutant

[0207] To prepare strains transformed with the gdh promoter mutant, the pDCM2_Pm3gdh_gdh, pDCM2_Pm16gdh_gdh, and pDCM2_Pm78gdh_gdh vectors constructed in Example 2 were first transformed into Corynebacterium glutamicum CJP1 (Korean Patent No. 10-1996769) to introduce the gdh promoter mutant sequence into the chromosome. Then, a further transformation was performed using strains containing... ilvA A vector of the gene (V323A) was used to prepare an L-isoleucine-producing strain (Korean Patent No. 10-1996769), in which valine at position 323 of the known ilvA gene encoding L-threonine dehydratase was replaced by alanine (Appl. Enviro. Microbiol., 1996.11, pp. 4345–4351).

[0208] Specifically, the vector constructed in Example 2 was introduced into the CJP1 strain via electroporation, and then transformed strains were obtained from a selection medium containing 25 mg / L kanamycin. Strains were selected by PCR and sequencing using primers of SEQ ID NO: 9 and SEQ ID NO: 10, wherein the gdh promoter mutant was introduced into the strain via a DNA fragment inserted into the chromosome via a second exchange, and the selected strains were named *Corynebacterium glutamicum* CJP1::Pm3gdh_gdh, CJP1::Pm16gdh_gdh, and CJP1::Pm78gdh_gdh.

[0209] The prepared strain was introduced into the pECCG117-ilvA(V323A) vector (Korean Patent No. 10-1996769) via electroporation, and then transformed strains were obtained from a selective medium containing 25 mg / L kanamycin. The selected strains were named CJP1::Pm3gdh_gdh / pECCG117-ilvA(V323A), CJP1::Pm16gdh_gdh / pECCG117-ilvA(V323A), and CJP1::Pm78gdh_gdh / pECCG117-ilvA(V323A), respectively.

[0210] 3-4-2. Evaluation of L-isoleucine production capacity in strains inoculated with the gdh promoter mutant

[0211] To evaluate the L-isoleucine production capacity of the *Corynebacterium glutamicum* CJP1 / pECCG117-ilvA(V323A) used as parent strains and the strains CJP1::Pm3gdh_gdh / pECCG117-ilvA(V323A), CJP1::Pm16gdh_gdh / pECCG117-ilvA(V323A), and CJP1::Pm78gdh_gdh / pECCG117-ilvA(V323A) prepared in Example 3-4-1, the strains were cultured using the following method and then analyzed.

[0212] First, each strain was inoculated into a 250mL corner-baffle flask containing 25mL of seed culture medium and cultured at 30°C with shaking at 200rpm for 20 hours. Then, 1mL of seed culture was inoculated into a 250mL corner-baffle flask containing 24mL of production culture medium and cultured at 32°C with shaking at 200rpm for 48 hours.

[0213] Seed culture medium (pH 7.0)

[0214] 20g glucose, 10g peptone, 5g yeast extract, 1.5g urea, 4g KH2PO4, 8g K2HPO4, 0.5g MgSO4·7H2O, 100μg biotin, 1000μg thiamine hydrochloride, 2000μg calcium pantothenate, 2000μg nicotinamide (based on 1L distilled water).

[0215] <Production medium (pH 7.0)>

[0216] 45g glucose, 10g soy protein, 10g molasses, 15g (NH4)2SO4, 0.55g KH2PO4, 0.6g MgSO4·7H2O, 9mg FeSO4·7H2O, 9mg MnSO4·5H2O, 0.9mg biotin, 4.5mg thiamine hydrochloride, 30g CaCO3, 4.5mg calcium pantothenate, 30mg nicotinamide, 0.45mg ZnSO4, 0.45mg CuSO4 (based on 1L distilled water)

[0217] After cultivation, the amount of L-isoleucine produced was measured using HPLC. The concentrations and concentration growth rates of L-isoleucine in the cultures of the following strains are shown in Table 6 below: CJP1 / pECCG117-ilvA(V323A), CJP1::Pm3gdh_gdh / pECCG117-ilvA(V323A), CJP1::Pm16gdh_gdh / pECCG117-ilvA(V323A), and CJP1::Pm78gdh_gdh / pECCG117-ilvA(V323A).

[0218] Table 6

[0219]

[0220] As shown in Table 6, compared with the parental wild-type strain CJP1 / pECCG117-ilvA(V323A), the three strains introduced with the gdh promoter mutant showed an increase in L-isoleucine concentration.

[0221] The above results identified that recombinant microorganisms containing promoter-active polynucleotides of this disclosure increase the production of industrially usable L-lysine, L-threonine, O-acetylhomoserine, and L-isoleucine.

[0222] From the above description, those skilled in the art will understand that this disclosure can be implemented in other specific forms without departing from the technical spirit or essential characteristics of this disclosure. Therefore, the above embodiments should be interpreted as exemplary and not as limiting this disclosure. The scope of this disclosure should be understood to include all changes or modifications derived from the definitions and scope of the claims and their equivalents.

[0223] <110> CJ Daiichi Sugar Co., Ltd. <120> Novel promoters and their applications <130> OPA22002 <150> KR10-2021-0061306 <151> 2021-05-12 <160> 28 <170> KoPatentIn 3.0 <210> 1 <211> 330 <212> DNA <213> Corynebacterium glutamicum <400> 1 attctttgtg gtcatatctg tgcgacactg ccataattga acgtgagcat ttaccagcct 60 aaatgcccgc agtgagttaa gtctcaaagc aagaagttgc tctttagggc atccgtagtt 120 taaaactatt aaccgttagg tatgacaagc cggttgatgt gaacgcagtt tttaaaagtt 180 tcaggatcag atttttcaca ggcattttgc tccagcaaac gcctaggatg tacatggtgc 240 cctcaatggg aaccaccaac atcactaaat ggcccaggta cacactttaa aatcgtgcgc 300 gcatgcagcc gagatgggaa cgaggaaatc 330 <210> 2 <211> 330 <212> DNA <213> Artificial Sequence <220> <223> Pm3gdh <400> 2 attctttgtg gtcatatctg tgcgactgtg gtatacttga acgtgagcat ttaccagcct 60 aaatgcccgc agtgagttaa gtctcaaagc aagaagttgc tctttagggc atccgtagtt 120 taaaactatt aaccgttagg tatgacaagc cggttgatgt gaacgcagtt tttaaaagtt 180 tcaggatcag atttttcaca ggcattttgc tccagcaaac gcctaggatg tacatggtgc 240 cctcaatggg aaccaccaac atcactaaat ggcccaggta cacactttaa aatcgtgcgc 300 gcatgcagcc gagatgggaa cgaggaaatc 330 <210> 3 <211> 330 <212> DNA <213> Artificial sequence <220> <223> Pm16gdh <400> 3 attctttgtg gtcatatctg tgcgactgtg gtatacttga acgtgagcat ttaccagcct 60 aaatgtccgc agtgagttaa gtctcaaagc aagaagttgc tctttagggc atccgtagtt 120 taaaactatt aaccgttagg tatgacaagc cggttgatgt gaacgcagtt tttaaaagtt 180 tcaggatcag atttttcaca ggcattttgc tccagcaaac gcctaggatg tacatggtgc 240 cctcaatggg aaccaccaac gtcactaaat ggcccaggta cacactttaa aatcgtgcgc 300 gcatgcagcc gagatgggaa cgaggaaatc 330 <210> 4 <211> 330 <212> DNA <213> Artificial sequence <220> <223> Pm78gdh <400> 4 attctttgtg gtcatatctg tgcgactgtg gtatacttga acgtgagcat ttaccagcct 60 aaatgtccgc agtgagttaa gtctcaaagc aagaagttgc tctttagggc atccgtagtt 120 taaaactatt aaccgttagg tatgacaagc cggttgatgt gaacgcagtt tttaaaagtt 180 tcaggatcag atttttcaca ggcattttgc tccagcaaac gcctaggatg tacatggtgc 240 cctcaatggg aaccaccaac atcactaaat ggcccaggta cacactttaa aatcgtgcgc 300 gcatgcagcc gagatgggaa cgaggaaatc 330 <210> 5 <211> 39 <212> DNA <213> Artificial sequence <220> <223> Pgdhm primer <400> 5 ctctagaact agtggatcca ttctttgtgg tcatatctg 39 <210> 6 <211> 40 <212> DNA <213> Artificial sequence <220> <223> Pgdhm primer <400> 6 agttcttctc ctttactcat gatttcctcg ttcccatctc 40 <210> 7 <211> 40 <212> DNA <213> Artificial sequence <220> <223> GFP primers <400> 7 gagatgggaa cgaggaaatc atgagtaaag gagaagaact 40 <210> 8 <211> 37 <212> DNA <213> Artificial sequence <220> <223> GFP primers <400> 8 cccccctcga ggtcgactta tttgtagagc tcatcca 37 <210> 9 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Pgdhm primers <400> 9 attctttgtg gtcatatctg 20 <210> 10 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Pgdhm primers <400> 10 gatttcctcg ttcccatctc 20 <210> 11 <211> 38 <212> DNA <213> Artificial sequence <220> <223> pDZ-Pm3, 4, 6_gdh primers <400> 11 gtgaattcga gctcggtacc ccgtcggtgg gggagttg 38 <210> 12 <211> 36 <212> DNA <213> Artificial sequence <220> <223> pDZ-Pm3, 4, 6_gdh primers <400> 12 gatatgacca caaagaatta aaattgtttg aaaatt 36 <210> 13 <211> 36 <212> DNA <213> Artificial sequence <220> <223> pDZ-Pm3, 4, 6_gdh primers <400> 13 aattttcaaa caattttaat tctttgtggt catatc 36 <210> 14 <211> 38 <212> DNA <213> Artificial sequence <220> <223> pDZ-Pm3, 4, 6_gdh primers <400> 14 cctgctcatc aactgtcatg atttcctcgt tcccatct 38 <210> 15 <211> 38 <212> DNA <213> Artificial sequence <220> <223> pDZ-Pm3, 4, 6_gdh primers <400> 15 agatgggaac gaggaaatca tgacagttga tgagcagg 38 <210> 16 <211> 39 <212> DNA <213> Artificial sequence <220> <223> pDZ-Pm3, 4, 6_gdh primers <400> 16 ggtcgactct agaggatccc ccaactccga tgtcacctg 39 <210> 17 <211> 41 <212> DNA <213> Artificial sequence <220> <223> lysC(L377K) primers <400> 17 gtgaattcga gctcggtacc ctccaagatt ttggtgctgc g 41 <210> 18 <211> 40 <212> DNA <213> Artificial sequence <220> <223> lysC(L377K) primers <400> 18 atctcagagg tggaaatctt ttcgatgttc acgttgacat 40 <210> 19 <211> 40 <212> DNA <213> Artificial sequence <220> <223> lysC(L377K) primers <400> 19 atgtcaacgt gaacatcgaa aagatttcca cctctgagat 40 <210> 20 <211> 41 <212> DNA <213> Artificial sequence <220> <223> lysC(L377K) primers <400> 20 ggtcgactct agaggatccc cgttcacctc agagacgatt a 41 <210> twenty one <211> 41 <212> DNA <213> Artificial sequence <220> <223> hom(R398Q) primers <400> twenty one gtgaattcga gctcggtacc ctttccacac ccgtgttacc g 41 <210> twenty two <211> 40 <212> DNA <213> Artificial sequence <220> <223> hom(R398Q) primers <400> twenty two atcatcgcgc tcttcctgtt ggattgtacg caggggatt 40 <210> twenty three <211> 40 <212> DNA <213> Artificial sequence <220> <223> hom(R398Q) primers <400> twenty three aatctccctg cgtacaatcc aacaggaaga gcgcgatgat 40 <210> twenty four <211> 41 <212> DNA <213> Artificial sequence <220> <223> hom(R398Q) primers <400> twenty four ggtcgactct agaggatccc caaccattag ctgcagcaac a 41 <210> 25 <211> 20 <212> DNA <213> Artificial sequence <220> <223> lysC(L377K) primers <400> 25 tcctaatgca cagaagctgg 20 <210> 26 <211> 20 <212> DNA <213> Artificial sequence <220> <223> lysC(L377K) primers <400> 26 gtggtgcagt tagggttcgc 20 <210> 27 <211> 20 <212> DNA <213> Artificial sequence <220> <223> hom(R398Q) primers <400> 27 atccaactgc agacgtcgaa 20 <210> 28 <211> 20 <212> DNA <213> Artificial sequence <220> <223> hom(R398Q) primers <400> 28 atctgggtgg ccttcaaagg 20

Claims

1. A promoter-active polynucleotide, wherein in the polynucleotide sequence of SEQ ID NO: 1, the nucleotides at positions 27, 28, 31, 32, and 36 are substituted with other nucleotides. In the polynucleotide sequence of SEQ ID NO: 1, the adenine (A) at position 27 is replaced by thymine (T); the cytosine (C) at position 28 is replaced by guanine (G); the cytosine (C) at position 31 is replaced by guanine (G); the cytosine (C) at position 32 is replaced by thymine (T); and the adenine (A) at position 36 is replaced by cytosine (C).

2. The polynucleotide of claim 1, wherein the nucleotides at positions 66 and 261 are further substituted with other nucleotides, and The nucleotide cytosine (C) at position 66 is replaced by thymine (T); and the nucleotide adenine (A) at position 261 is replaced by guanine (G).

3. The polynucleotide of claim 1, wherein the nucleotide at position 66 is further replaced by another nucleotide, and The nucleotide cytosine (C) at position 66 is replaced by thymine (T).

4. The polynucleotide according to claim 1, wherein the polynucleotide is as shown in SEQ ID NO:

2.

5. The polynucleotide according to claim 2, wherein the polynucleotide is as shown in SEQ ID NO:

3.

6. The polynucleotide according to claim 3, wherein the polynucleotide is as shown in SEQ ID NO:

4.

7. The polynucleotide according to any one of claims 1 to 6, wherein the polynucleotide is operatively linked to a gene encoding a target protein.

8. An expression box comprising: The polynucleotide of any one of claims 1 to 6; and A gene that encodes a target protein and is operatively linked to the polynucleotide.

9. The expression cassette according to claim 8, wherein the target protein is glutamate dehydrogenase (GDH).

10. A microorganism of the genus Corynebacterium, comprising: The polynucleotide of any one of claims 1 to 6; or The polynucleotide of any one of claims 1 to 6 and the gene encoding the target protein and operably linked to the polynucleotide.

11. The microorganism according to claim 10, wherein the target protein is glutamate dehydrogenase (GDH).

12. The microorganism according to claim 10, wherein the Corynebacterium genus microorganism is Corynebacterium glutamicum.

13. A method for generating a target material, the method comprising: Cultivate the Corynebacterium genus of claim 10 in a culture medium; and Recover the target substance from the culture medium.

14. The method of claim 13, wherein the target substance is lysine, threonine, O-acetylhomoserine, or isoleucine.

15. Use of a polynucleotide as a promoter, said polynucleotide having promoter activity, wherein in the polynucleotide sequence of SEQ ID NO: 1, the nucleotides at positions 27, 28, 31, 32 and 36 are replaced by other nucleotides. In the polynucleotide sequence of SEQ ID NO: 1, the adenine (A) at position 27 is replaced by thymine (T); the cytosine (C) at position 28 is replaced by guanine (G); the cytosine (C) at position 31 is replaced by guanine (G); the cytosine (C) at position 32 is replaced by thymine (T); and the adenine (A) at position 36 is replaced by cytosine (C).

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