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 containing the same and a Corynebacterium genus microorganism, and a method for producing a target substance using the above microorganism.

Description

[Technical Field]

[0001] 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 containing the same and a Corynebacterium microorganism, a method for producing a target substance using the microorganism, and use of the promoter. [Background technology]

[0002] The production of target substances (e.g., amino acids) using microorganisms has been the subject of extensive research into environmentally friendly and safe production methods, with continuous research being conducted into the mass production of target substances using microorganisms of the genus Corynebacterium. Microorganisms of the genus Corynebacterium, particularly Corynebacterium glutamicum, are Gram-positive microorganisms that are widely used to produce L-amino acids and other useful substances. To produce these L-amino acids and other useful substances, extensive research is being conducted to develop highly efficient production microorganisms and fermentation process technologies.

[0003] L-lysine, a representative substance produced by Corynebacterium microorganisms, is used in animal feed, human pharmaceuticals, and cosmetics, and is produced by fermentation using Corynebacterium strains. Microorganisms with enhanced genes related to L-lysine biosynthesis and a method for producing L-lysine using the same are known (KR 10-0924065B1).

[0004] L-threonine, an essential amino acid, is widely used as a feed and food additive and is also used as a pharmaceutical ingredient in infusions and pharmaceutical synthesis. L-threonine is found in low amounts in plant proteins and is often deficient in animals with a vegetarian diet, making it particularly useful as an animal feed additive. L-threonine is primarily produced by fermentation using Escherichia coli or Corynebacterium microorganisms developed through artificial mutation or genetic engineering. A representative example is the use of genetically engineered strains to produce L-threonine by introducing the threonine operon from E. coli into the threonine-producing strain Brevibacterium flavum (TURBA E. et al., Agric. Biol. Chem. 53:2269-2271, 1989).

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

[0006] Isoleucine is an essential amino acid that cannot be synthesized in the body and is known to have effects such as promoting growth, enhancing nerve function, strengthening liver function, and strengthening muscles. It is usually produced by fermentation using microorganisms.

[0007] There is still a need for a system that can show high expression efficiency in various microorganisms, such as Escherichia, Corynebacterium, and Bacillus, and the need for the development of a versatile promoter continues to grow. Furthermore, if a versatile promoter that is not limited to a specific target substance is developed, it is expected to be useful for the production of various substances. [Prior art documents] [Patent documents]

[0008] [License 1] KR 10-0924065B1 [License 2] WO2008 / 013432 [License 3] Republic of Korea Registration Permit No. 10-09240675 [License 4] Republic of Korea Registration License No. 10-2011994 [Patent Document 5] Republic of Korea Registration License No. 10-1947959 [License 6] Republic of Korea Registration License No. 10-1996769 [License 7] Republic of Korea Public Number 10-2020-0136813 [Non-licensed literature]

[0009] [Non-licensed Document 1] TURBA E. et al, Agric.Biol.Chem.53:2269~2271,1989 [Non-licensed Document 2] Pearson et al (1988)[Proc.Natl.Acad.Sci.USA 85]:2444 [Non-licensed Document 3] Rice et al., 2000, Trends Genet.16:276-277 [Non-licensed Document 4] Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453 [Non-licensed Document 5] Devereux,J.,et al,Nucleic Acids Research 12:387 (1984) [Non-licensed Document 6] Atschul,[S.] [F.,] [ET AL,J MOLEC BIOL 215]:403 (1990) [Non-licensed Document 7] Guide to Huge Computers,Martin J.Bishop,[ED.,] Academic Press,San Diego, 1994

Non-licensed Document 8

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

Non-licensed Document 12

Non-licensed Document 13

Non-licensed Document 14

Non-licensed Document 15

[0010] The present inventors have developed a novel synthetic promoter that, when present in the positive direction, has higher expression activity for downstream genes than known promoters, and have confirmed that this allows for the production of a variety of target substances. [Means for solving the problem]

[0011] One object of the present application is to provide a polynucleotide having promoter activity.

[0012] Another object of the present application is to provide a vector or expression cassette comprising the above polynucleotide; and a gene encoding a protein of interest operably linked to the above polynucleotide.

[0013] Another object of the present application is to provide a Corynebacterium microorganism comprising the above polynucleotide; or the above polynucleotide and a gene encoding a target protein operably linked to the above polynucleotide.

[0014] Another object of the present application is to provide a method for producing a target substance, which includes the steps of culturing the Corynebacterium microorganism in a medium; and recovering the target substance from the medium.

[0015] Another object of the present application is to provide use as a promoter of a polynucleotide having promoter activity in which the nucleotides at positions 27, 28, 31, 32, and 36 in the polynucleotide sequence of SEQ ID NO: 1 are substituted with other nucleotides. [Effects of the Invention]

[0016] The novel polynucleotide having promoter activity of the present application can be introduced into a microorganism that produces a target substance to increase the production amount of the target substance. The improved production yield is expected to be advantageous in industrial terms, such as increasing production convenience and reducing production costs. DETAILED DESCRIPTION OF THE INVENTION

[0017] This will be explained in more detail as follows. Meanwhile, each description and embodiment disclosed in this application may also be applied to different descriptions and embodiments. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the following specific description is not intended to limit the scope of this application.

[0018] Additionally, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific aspects of the present application described herein, and such equivalents are intended to be encompassed by this application.

[0019] One aspect of the present application provides a polynucleotide having promoter activity. Specifically, the polynucleotide having promoter activity of the present application may be a polynucleotide having promoter activity that contains one or more nucleotide substitutions in the polynucleotide sequence of SEQ ID NO:1.

[0020] In this application, the term "polynucleotide" refers to a DNA chain of a certain length or more, which is a polymer of nucleotides in which nucleotide units are linked in a long chain by covalent bonds.

[0021] In this application, the term "polynucleotide having promoter activity" means a DNA region located near the site where transcription of a gene to be expressed, i.e., a gene of interest, occurs, including a site to which RNA polymerase or an enhancer binds for expression of the gene of interest.

[0022] The polynucleotide having promoter activity of the present application can be used as a general-purpose enhanced promoter. For example, it can be used as a promoter that enhances the expression of a polypeptide having glutamate dehydrogenase (GDH) activity. Furthermore, the polynucleotide may be a polynucleotide involved in the production or increased production of a target substance, specifically, lysine, threonine, O-acetylhomoserine, or isoleucine.

[0023] The polynucleotide of the present application may include, without limitation, any polynucleotide sequence having promoter activity. Specifically, in the present application, the polynucleotide having promoter activity may be a polynucleotide having promoter activity that contains one or more, two or more, three or more, four or more, five or more, six or more, or seven or more nucleotide substitutions in the polynucleotide sequence of SEQ ID NO: 1.

[0024] In the present application, the polynucleotide sequence of SEQ ID NO: 1 is an example of a polynucleotide having glutamate dehydrogenase promoter activity. Furthermore, as long as it has promoter activity, a polynucleotide in which a specific nucleotide is substituted in the polynucleotide sequence of SEQ ID NO: 1 may also be a polynucleotide having glutamate dehydrogenase promoter activity. The polynucleotide sequence of SEQ ID NO: 1 may be a representative polynucleotide sequence for indicating the mutation position, and other corresponding polynucleotide sequences having promoter activity are also included in the sequences into which mutations can be introduced. For example, any polynucleotide sequence that can function as a promoter for glutamate dehydrogenase (GDH) or a polypeptide having activity corresponding thereto may be included, without limitation, in the scope of sequences into which mutations can be introduced in the present application.

[0025] The nucleotide sequence of SEQ ID NO: 1 can be confirmed in the publicly known database NCBI Genbank. As a sequence capable of acting as a promoter of glutamate dehydrogenase, the sequence corresponding to SEQ ID NO: 1 may be derived from Corynebacterium sp., specifically, may be a sequence of Corynebacterium glutamicum. However, sequences having activity equivalent to or greater than that of the above polynucleotide may be included, without limitation, in the promoter of the present application.

[0026] The polynucleotides having promoter activity provided in the present application may be those in which nucleotides at specific positions in an existing polynucleotide sequence having promoter activity have been substituted to enhance promoter activity.

[0027] As a specific example, the polynucleotide having promoter activity of the present application may include a polynucleotide having promoter activity in which one or more nucleotides in the nucleotide sequence of SEQ ID NO: 1 are substituted with other nucleotides. Specifically, it may consist of a polynucleotide having promoter activity in which one or more nucleotides in the nucleotide sequence of SEQ ID NO: 1 are substituted with other nucleotides. The above-mentioned polynucleotide having promoter activity may be referred to as a "mutant promoter" in the present application.

[0028] As a specific example, the mutant promoter may be a polynucleotide having promoter activity, which contains a substitution of one or more nucleotides selected from the group consisting of nucleotides 27, 28, 31, 32, and 36 of SEQ ID NO: 1 with other nucleotides. Specifically, the mutant promoter may be substituted with other nucleotides at one or more, two or more, three or more, four or more, or all five of the above positions, or at the corresponding positions. Alternatively, the mutant promoter may be substituted with an additional nucleotide at positions 66 and / or 261.

[0029] The "other nucleotide" is not limited as long as it is different from the nucleotide before substitution. Taking adenine (A), which is the 27th nucleotide in SEQ ID NO: 1, as an example, when it is stated that "the 27th nucleotide in SEQ ID NO: 1 is substituted with another nucleotide," it means that the nucleotide is substituted with cytosine (C), thymine (T), or guanine (G) excluding adenine. Furthermore, even if not otherwise specified, when it is stated in this application that a certain nucleotide is "substituted," it means that the nucleotide before substitution is replaced with another nucleotide.

[0030] Meanwhile, a person skilled in the art can identify the nucleotides at positions corresponding to nucleotides 27, 28, 31, 32, 36, 66, and 261 of SEQ ID NO: 1 in any polynucleotide sequence through sequence alignment known in the art, and it is obvious that the term "nucleotide at a specific position in a specific SEQ ID NO" used in this application includes "nucleotides at corresponding positions" in any polynucleotide sequence, even if not otherwise specified. Therefore, polynucleotide sequences in which one or more nucleotides selected from the group consisting of nucleotides at positions corresponding to nucleotides 27, 28, 31, 32, 36, 66, and 261 of the polynucleotide sequence of SEQ ID NO: 1 in any polynucleotide sequence having promoter activity are substituted with other nucleotides are also included within the scope of this application.

[0031] As a specific example, a polynucleotide having promoter activity in the present application may be one in which one or more nucleotides selected from the group consisting of nucleotides 27, 28, 31, 32, 36, 66, and 261 in the polynucleotide sequence of SEQ ID NO: 1 are substituted with other nucleotides.

[0032] Specifically, in the present application, a polynucleotide having promoter activity may be one in which the nucleotides at positions 27, 28, 31, 32, and 36 in the polynucleotide sequence of SEQ ID NO: 1 are substituted with other nucleotides, or the nucleotides at positions 27, 28, 31, 32, 36, 66, and 261 are substituted with other nucleotides, or the nucleotides at positions 27, 28, 31, 32, 36, and 66 are substituted with other nucleotides, but is not limited thereto.

[0033] For example, substituting one or more, two or more, three or more, four or more, five or more, six or more, or seven nucleotides at positions corresponding to 27, 28, 31, 32, 36, 66, and 261 of the polynucleotide of SEQ ID NO: 1 with other nucleotides can provide a promoter with higher activity than the unsubstituted (unaltered) promoter sequence. Specifically, the polynucleotide having promoter activity of the present application may be one 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. Furthermore, the polynucleotide may also have promoter activity in which the nucleotides at positions 66 and 261 are substituted with other nucleotides, or in which the nucleotide at position 66 is substituted with another nucleotide.

[0034] As a specific example, the polynucleotide having promoter activity of the present application may be one in which the 27th nucleotide adenine (A) is substituted with thymine (T), the 28th nucleotide cytosine (C) is substituted with guanine (G), the 31st nucleotide cytosine (C) is substituted with guanine (G), the 32nd nucleotide cytosine (C) is substituted with thymine (T), and the 36th nucleotide adenine (A) is substituted with cytosine (C) in the polynucleotide sequence of SEQ ID NO: 1; The polynucleotide sequence may be one in which the cytosine (C) at position 36 is substituted with thymine (T), the adenine (A) at position 36 is substituted with cytosine (C), the cytosine (C) at position 66 is substituted with thymine (T), and the adenine (A) at position 261 is substituted with guanine (G); or a polynucleotide in which the adenine (A) at position 27 is substituted with thymine (T), the cytosine (C) at position 28 is substituted with guanine (G), the cytosine (C) at position 31 is substituted with guanine (G), the cytosine (C) at position 32 is substituted with thymine (T), the adenine (A) at position 36 is substituted with cytosine (C), and the cytosine (C) at position 66 is substituted with thymine (T).

[0035] As a more specific example, it may be a polynucleotide represented by any one of the polynucleotide sequences of SEQ ID NOs: 2 to 4. Specifically, in the present application, a polynucleotide having promoter activity may comprise or (essentially) consist of the polynucleotide sequence of SEQ ID NO: 2, 3, or 4.

[0036] Furthermore, the present invention is not limited to the above-mentioned examples, and various modifications can be made to the polynucleotide sequence as long as the promoter activity is not significantly reduced.

[0037] In the present application, a polynucleotide having promoter activity may be a polynucleotide sequence having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity to SEQ ID NO: 2, 3, or 4. Nucleotide sequences having homology or identity within the above category exclude sequences having 100% identity, and may also include sequences having less than 100% identity.

[0038] On the other hand, even if the present application describes a "polynucleotide having a nucleotide sequence described in a specific SEQ ID NO" or a "polynucleotide comprising a nucleotide sequence described in a specific SEQ ID NO," it is obvious that a polynucleotide having a nucleotide sequence in which part of the sequence has been deleted, modified, substituted or added can also be used in the present application, as long as it has the same or corresponding activity as a polynucleotide composed of the nucleotide sequence of the SEQ ID NO.

[0039] For example, if a polynucleotide has the same or corresponding activity as the above-mentioned polynucleotide, it is obvious that a polynucleotide having a meaningless sequence added to the interior or end of the nucleotide sequence of the SEQ ID NO, or a polynucleotide having a partial sequence deleted from the interior or end of the nucleotide sequence of the SEQ ID NO, also falls within the scope of the present application.

[0040] Homology and identity refer to the degree of relatedness between two given base sequences and can be expressed as a percentage.

[0041] The terms homology and identity are often used interchangeably.

[0042] Sequence homology or identity of conserved polynucleotides can be determined using standard sequence algorithms, with default gap penalties established by the program used. Substantially homologous or identical sequences generally hybridize under moderately or highly stringent conditions over the entire sequence or at least about 50%, 60%, 70%, 80%, or 90% of the entire length. Hybridization also takes into account polynucleotides containing degenerate codons in place of codons in the polynucleotide.

[0043] Whether any two polynucleotide sequences have homology, similarity, or identity can be determined using known computer algorithms, such as the "FASTA" program using default parameters, as in, for example, Pearson et al. (1988) [Proc. Natl. Acad. Sci. USA 85]:2444, or using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), as implemented in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) (version 5.0.0 or later). (The GCG program package (Devereux, J., et al., Nucleic Acids Research 12:387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]:403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego, 1994, and [CARILLO ETA / .] (1988) SIAM J Applied Math 48:1073) can be used to determine homology, similarity, or identity, for example, using BLAST from the National Center for Biotechnology Information, or ClustalW.

[0044] Polynucleotide homology, similarity, or identity can be determined by comparing sequence information using the GAP computer program, e.g., as known in Smith and Waterman, Adv. Appl. Math (1981) 2:482, or as described in, e.g., Needleman et al. (1970), J. Mol. Biol. 48:443. Briefly, the GAP program defines a sequence as the total number of symbols in the shorter of the two sequences divided by the number of similar aligned symbols (i.e., nucleotides or amino acids). Default parameters for the GAP program include: (1) a binary comparison matrix (containing a value of 1 for identity and 0 for non-identity) and the weighted comparison matrix of Gribskov et al. (1986) Nucl. Acids Res. 14:6745 (or the EDNAFULL (the EMBOSS version of NCBI NUC4.4) substitution matrix) as disclosed by Schwartz and Dayhoff, eds., Atlas of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap (or a gap open penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for end gaps. Thus, as used herein, the terms "homology" or "identity" indicate the relatedness between sequences.

[0045] Furthermore, probes prepared from known gene sequences, such as polynucleotide sequences that hybridize under stringent conditions with a complementary sequence to all or part of the aforementioned polynucleotide sequences and have the same activity, can be included without limitation. The term "stringent conditions" refers to conditions that allow specific hybridization between polynucleotides. Such 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.A. Usubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York). Examples of conditions include conditions under which genes with high homology or identity of 40% or more, specifically 70% or more, 80% or more, 85% or more, 90% or more, more specifically 95% or more, even more specifically 97% or more, and particularly specifically 99% or more, hybridize with each other, but genes with lower homology or identity do not hybridize with each other; and conditions under which washing is performed once, specifically two to three times, at a salt concentration and temperature equivalent to the washing conditions for conventional Southern hybridization: 60°C, 1X SSC, 0.1% SDS, specifically 60°C, 0.1X SSC, 0.1% SDS, more specifically 68°C, 0.1X SSC, 0.1% SDS.

[0046] Hybridization requires that two nucleic acids have complementary sequences, even though mismatches between bases are possible depending on the stringency of the hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that are capable of hybridizing to one another. For example, with respect to DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Thus, the present application also encompasses isolated nucleic acid fragments that are complementary to entire sequences, as well as substantially similar nucleic acid sequences.

[0047] Specifically, polynucleotides having homology or identity can be detected using the hybridization conditions described above, including a hybridization step at a Tm value of 55° C. The Tm value may be, but is not limited to, 60° C., 63° C., or 65° C., and can be appropriately adjusted by those skilled in the art depending on the purpose.

[0048] The appropriate stringency for hybridizing polynucleotides depends on the length of the polynucleotides and the degree of complementation, variables well known in the art (see Sambrook et al., supra, 9.50-9.51, 11.7-11.8).

[0049] The polynucleotide of the present application having promoter activity can be used as a promoter.

[0050] The promoter can be located 5' to the start site of transcription into mRNA.

[0051] The promoter may have increased promoter activity compared to conventional promoters. That is, it may increase not only the expression of a gene of interest in a host cell but also the expression and / or activity of a protein encoded by the gene of interest. For purposes of this application, the gene of interest for enhanced expression can be changed depending on the product to be produced, and the promoter may be used as a general-purpose promoter for enhancing a gene of interest.

[0052] For the purposes of this application, the "gene of interest" refers to a gene whose expression is to be regulated by the promoter sequence of this application. The protein encoded by the gene of interest can be referred to as the "protein of interest," and the gene encoding the protein of interest can be referred to as the "gene of interest."

[0053] Furthermore, the polynucleotide encoding the target protein may be modified in various ways in its coding region, taking into consideration codon degeneracy or the codons preferred in the organism in which the polynucleotide is to be expressed, without altering the polypeptide sequence. The polynucleotide sequence has been described above.

[0054] As a specific example, the target protein may be a polypeptide having glutamate dehydrogenase (GDH) activity, i.e., the target gene of the promoter may be a gene encoding a polypeptide having glutamate dehydrogenase (GDH) activity.

[0055] In the present application, "glutamate dehydrogenase (GDH)" is also referred to as "glutamate dehydrogenase", etc. The glutamate dehydrogenase is involved in the metabolism of glutamate to 2-oxoglutarate, and by regulating its activity, it is possible to obtain the effect of improving the productivity of useful substances such as lysine, threonine, O-acetylhomoserine, and isoleucine.

[0056] An example of a gene encoding glutamate dehydrogenase is, but is not limited to, the gdh gene (NCgl1999) of Corynebacterium glutamicum ATCC 13032. Those skilled in the art can easily obtain information on genes encoding glutamate dehydrogenase from publicly known databases (e.g., GenBank).

[0057] The amino acid sequence of the glutamate dehydrogenase can be obtained from the publicly known database, GenBank, of NCBI. For example, it may be derived from Corynebacterium glutamicum.

[0058] Furthermore, the "polypeptide having glutamate dehydrogenase activity" in the present application includes not only the wild-type, non-mutated, or natural form of the glutamate dehydrogenase, but also mutants having the same activity or enhanced activity.

[0059] In this application, the term "mutant polypeptide" is used synonymously with "variant" and refers to a protein that differs from the recited sequence in one or more amino acids by conservative substitutions and / or modifications, but that maintains the functions or properties of the protein.

[0060] A variant differs from the identified sequence by the substitution, deletion, or addition of a few amino acids. Such variants can generally be identified by modifying one or more amino acids in the amino acid sequence of the protein and evaluating the properties of the modified protein. That is, the ability of a variant may be increased compared to the native protein. Some variants also include variants in which one or more portions, such as the N-terminal leader sequence or transmembrane domain, have been removed.

[0061] The term "mutant" may refer to a mutant type, a variant, a mutated protein, a mutation, etc. (in English, modification, modified protein, modified polypeptide, mutant, mutein, divergent, variant, etc.), and is not limited thereto as long as it is a term used in the sense of mutation. For purposes of this application, the mutant may be, but is not limited to, a mutated protein with increased activity compared to a native wild-type or non-mutated protein.

[0062] As used herein, the term "conservative substitution" refers to the substitution of one amino acid with another amino acid having similar structural and / or chemical properties. Such variants can have, for example, one or more conservative substitutions while still retaining one or more biological activities. Such amino acid substitutions can generally be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues.

[0063] Variants can also include deletions or additions of amino acids that have minimal effect on the properties and secondary structure of the polypeptide. For example, the polypeptide can be conjugated to an N-terminal signal (or leader) sequence of a protein involved in co-translational or post-translational protein transfer. The polypeptide can also be conjugated to other sequences or linkers that allow the polypeptide to be identified, purified, or synthesized.

[0064] The gene encoding a polypeptide having glutamate dehydrogenase activity of the present application can be referred to as a "gdh gene."

[0065] The gene may be derived from a microorganism of the genus Corynebacterium, specifically Corynebacterium glutamicum.

[0066] In the present application, the "gdh gene," i.e., a polynucleotide encoding a polypeptide having glutamate dehydrogenase activity, may undergo various modifications in the coding region, taking into consideration codon degeneracy or the codons preferred in the organism in which the polypeptide is to be expressed, as long as the amino acid sequence of the polypeptide is not changed.

[0067] The polypeptides having glutamate dehydrogenase activity of the present application can also include mutant sequences, and in particular protein variants that have been mutated to exhibit enhanced glutamate dehydrogenase activity.

[0068] Another aspect of the present application provides a composition for gene expression, comprising the polynucleotide of the present application having promoter activity.

[0069] The above-mentioned composition for gene expression means a composition capable of expressing a gene that can be expressed by a polynucleotide having promoter activity of the present application.

[0070] For example, the gene expression composition may include a polynucleotide having promoter activity of the present application, and may further include, without limitation, a configuration in which the polynucleotide is driven by a promoter.

[0071] In the gene expression composition of the present application, the polynucleotide may be in a form contained within a vector so as to express an operably linked gene in a host cell into which it has been introduced.

[0072] Another aspect of the present application includes an expression cassette comprising the polynucleotide having the promoter activity, the polynucleotide, and a gene encoding a target protein.

[0073] In the present application, the term "expression cassette" refers to a unit cassette that includes the polynucleotide having promoter activity and a gene encoding a target protein, and that can express the target gene operably linked downstream of the promoter. Specifically, the expression cassette may be one in which the polynucleotide having promoter activity and the gene encoding the target protein are operably linked. In the present application, the term "operably linked" means that the gene sequence is functionally linked to a polynucleotide having promoter activity that initiates and mediates transcription of the gene encoding the target protein of the present application.

[0074] Such a gene expression cassette may further contain various factors inside or outside thereof that contribute to efficient expression of the gene of interest. The gene expression cassette may generally contain a transcription termination signal, a ribosome binding site, and a translation termination signal in addition to a promoter operably linked to the gene of interest.

[0075] As a specific example, the target protein may be a polypeptide having glutamate dehydrogenase activity.

[0076] Another aspect of the present application includes a vector comprising the polynucleotide having the above promoter activity, or the above polynucleotide and a gene encoding a target protein.

[0077] As a specific example, the target protein may be a polypeptide having glutamate dehydrogenase activity.

[0078] The term "vector" as used in this application means a DNA construct containing a polynucleotide sequence encoding a protein of interest operably linked to a suitable regulatory sequence so as to express said protein of interest in a suitable host.

[0079] For the purposes of this application, the regulatory sequence may include a polynucleotide having promoter activity of the present application.

[0080] Meanwhile, the regulatory sequence may include components such as a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence regulating the termination of transcription and translation. After being transformed into a suitable host cell, the vector can replicate and function independently of the host genome, or can be integrated into the genome itself. The vector used in this application is not particularly limited as long as it can be expressed in the host cell, and any vector known in the art can be used to transform the host cell. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages, either in their natural or recombinant states.

[0081] For example, pWE15, M13, λLB3, λBL4, λIXII, λASHII, λAPII, λt10, λt11, Charon4A, and Charon21A can be used as phage vectors or cosmid vectors, and pBR series, pUC series, pBluescriptII series, pGEM series, pTZ series, pCL series, and pET series can be used as plasmid vectors.

[0082] Alternatively, the endogenous promoter in the chromosome of a host cell can be replaced with the polynucleotide having promoter activity of the present application using a vector for chromosomal insertion. For example, but not limited to, vectors such as pECCG117, pDZ, pACYC177, pACYC184, pCL, pUC19, pBR322, pMW118, pCC1BAC, pCES208, and pXMJ19 may be used. Alternatively, vectors based on publicly known technology can be used (Korean Patent Registration No. 10-09240675).

[0083] The polynucleotide may be inserted into a chromosome by any method known in the art, including, but not limited to, homologous recombination. A selection marker for confirming the presence or absence of the chromosomal insertion may be further included. The selection marker is used to select cells transformed with the vector, i.e., to confirm the presence or absence of the insertion of the target nucleic acid molecule. Markers that confer a selectable phenotype, such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of a surface protein, may be used. In an environment treated with a selective agent, only cells expressing the selection marker survive or exhibit other phenotypes, allowing the selection of transformed cells. For example, a wild-type polynucleotide can be replaced with a mutated polynucleotide in a chromosome via a vector for intracellular chromosomal insertion.

[0084] In the present application, the term "transformation" may refer to the introduction of a vector containing a polynucleotide encoding a protein of interest into a host cell, so that the protein of interest can be expressed in the host cell.

[0085] Transformed polynucleotides can include any polynucleotide that can be expressed in a host cell, regardless of whether it is located within the host cell's chromosome or extrachromosomally. Furthermore, the polynucleotide encoding the target protein can include DNA and RNA encoding the target protein. The polynucleotide encoding the target protein may be introduced in any form as long as it can be introduced into the host cell and expressed. 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 genetic construct containing all the elements necessary for autonomous expression.

[0086] The expression cassette may typically include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the polynucleotide encoding the target protein. The polynucleotide encoding the target protein may be introduced into a host cell in its own form and operably linked to a sequence required for expression in the host cell, but is not limited thereto.

[0087] In addition, the term "operably linked" as used above means that the gene sequence is functionally linked to a promoter sequence that initiates and mediates transcription of the polynucleotide encoding the target protein of the present application.

[0088] For the purposes of this application, the promoter may be a polynucleotide having promoter activity of the present application.

[0089] Methods for transforming the vectors of the present application include any method for introducing nucleic acids into cells, and can be performed by selecting standard techniques suitable for the host cell, as known in the art, including, but not limited to, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and lithium acetate-DMSO method.

[0090] Another aspect of the present application provides a microorganism comprising: a polynucleotide having promoter activity of the present application; 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.

[0091] In the present application, the term "microorganism" includes all wild-type microorganisms and microorganisms that have undergone natural or artificial genetic modification, and is a concept that includes all microorganisms in which a specific mechanism has been weakened or strengthened by inserting an exogenous gene, strengthening or weakening the activity of an endogenous gene, etc. Specifically, the microorganism may be a microorganism containing a polynucleotide having promoter activity of the present application and a target protein.

[0092] The target protein may be a polypeptide having glutamate dehydrogenase (GDH) activity. The polynucleotide having promoter activity, the target protein, the polypeptide having glutamate dehydrogenase (GDH) activity, the vector, and the expression cassette of the present application are as described above.

[0093] The microorganism may be a microorganism of the genus Corynebacterium, and specifically, may be Corynebacterium glutamicum.

[0094] The microorganism may be, but is not limited to, a microorganism that expresses glutamate dehydrogenase, a microorganism that expresses a polypeptide having glutamate dehydrogenase activity, or a microorganism into which a polypeptide having glutamate dehydrogenase activity has been introduced.

[0095] In the present application, the microorganism may include a polynucleotide having promoter activity of the present application, specifically, the polynucleotide and / or a gene operably linked to the polynucleotide and encoding a target protein. Alternatively, the microorganism may include, but is not limited to, a vector or expression cassette containing the polynucleotide or gene expression regulatory sequence and a gene encoding a target protein. Furthermore, the polynucleotide, the gene encoding the target protein, the vector, and the expression cassette may be introduced into the microorganism by transformation, but is not limited to this. Furthermore, as long as the microorganism can express the gene, it does not matter whether the polynucleotide and the gene encoding the target protein are located on or off the chromosome.

[0096] In the present application, the term "protein is expressed" refers to a state in which a target protein, such as glutamate dehydrogenase or a mutant thereof, is introduced into a microorganism or modified so as to be expressed in the microorganism. When the target protein is a protein present in the microorganism, this may refer to a state in which its activity is enhanced compared to that of the endogenous protein or before modification.

[0097] Specifically, "protein introduction" may mean exhibiting the activity of a specific protein that the microorganism does not originally possess, or exhibiting improved activity compared to the endogenous activity of the protein or the activity of the protein before modification. For example, it may mean introducing a polynucleotide encoding a specific protein into a chromosome in the microorganism, or introducing a vector or expression cassette containing a polynucleotide encoding a specific protein into the microorganism, and exhibiting the activity.

[0098] Furthermore, "enhanced activity" may refer to an improvement in activity compared to the intrinsic activity of a specific protein possessed by a microorganism or the activity before the transformation. When the traits of a microorganism are changed by genetic mutation due to natural or artificial factors, "intrinsic activity" may refer to the activity of a specific protein that was originally possessed by the parent strain before the trait change.

[0099] For purposes of the present application, the enhancement of the activity may be achieved by using the polynucleotide sequence having promoter activity of the present application as an expression regulatory sequence for a target protein. As the target protein may be a natural or mutant form, as described above, the expression regulatory sequence may be an expression regulatory sequence for a gene encoding a protein mutant, or may be an expression regulatory sequence for a gene encoding a natural protein on a chromosome.

[0100] Other methods for enhancing activity may also be used in combination. For example, in addition to using the polynucleotide sequence having promoter activity of the present application as an expression regulatory sequence for a target protein, one or more methods selected from the group consisting of increasing the intracellular copy number of a gene encoding the target protein, replacing a gene encoding a natural protein on a chromosome with a gene encoding the protein variant, introducing an additional mutation into a gene encoding the protein so as to enhance the activity of the protein variant, and introducing the protein variant into a microorganism may be used, but is not limited to these.

[0101] The activity of a target protein may be enhanced by using the polynucleotide of the present application having promoter activity to regulate the expression of the target protein in a microorganism.

[0102] For example, but not limited to, the activity or concentration of the corresponding protein may generally be increased by at least 1%, 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, or 500%, up to 1000% or 2000%, relative to the activity or concentration of the protein in a wild-type or untransformed microbial strain.

[0103] In the present application, the term "non-modified microorganism" does not exclude strains containing mutations that may occur naturally in microorganisms, but also includes naturally occurring strains themselves, microorganisms that do not contain a polynucleotide having the promoter activity of the present application, or microorganisms that have not been transformed with a vector containing a polynucleotide having the promoter activity of the present application.

[0104] In the present application, the term "microorganism capable of producing a target substance" includes all microorganisms that have undergone natural or artificial genetic modification, and may be a microorganism in which a specific mechanism has been weakened or strengthened by inserting an exogenous gene or by enhancing or inactivating the activity of an endogenous gene, or may be a microorganism in which a genetic mutation for producing the target substance has occurred or the activity has been enhanced. For the purposes of the present application, the term "microorganism capable of producing the target substance" may refer to a microorganism that contains a polynucleotide having the promoter activity of the present application and is capable of producing an excess amount of the target substance compared to a wild-type or non-modified microorganism.

[0105] The above-mentioned "microorganism that produces a target substance" can be used interchangeably with terms such as "target substance-producing microorganism," "microorganism having the ability to produce a target substance," "target substance-producing strain," and "strain having the ability to produce a target substance."

[0106] The target substance may be an amino acid, specifically, lysine, threonine, O-acetylhomoserine, or isoleucine. More specifically, the target substance may be L-lysine, the target substance may be L-threonine, and the target substance may be L-isoleucine, but is not limited thereto.

[0107] For the purposes of this application, the microorganism that produces the target substance may have an improved ability to produce the target substance, specifically, lysine, threonine, O-acetylhomoserine, or isoleucine.

[0108] Meanwhile, the target substance-producing microorganism may be a wild-type microorganism or a recombinant microorganism. The recombinant microorganism is as described above. The microorganism may further include mutations such as strengthening a biosynthetic pathway to increase target substance production ability, removing feedback inhibition, or inactivating genes that weaken a degradation pathway or biosynthetic pathway. Such mutations may be induced artificially, for example, by UV irradiation, but this does not exclude natural mutations.

[0109] Specifically, the target substance-producing microorganism may be one that has been mutated to be able to produce the target substance. For example, a microorganism that does not have the ability to produce the target substance may be mutated to have the ability to produce the target substance, or a microorganism whose production ability has been enhanced. For example, a microorganism may be one in which a protein involved in a biosynthetic pathway or a mutant thereof has been introduced into a wild-type microorganism so that the target substance can be produced (KR 10-2011994, KR 10-1947959, KR 10-1996769).

[0110] As a specific example, the target substance-producing microorganism of the present application may be a Corynebacterium microorganism containing aspartokinase (lysC), homoserine dehydrogenase (hom), pyruvate carboxylase (pyc), L-threonine dehydratase (ilvA), or a combination thereof. The aspartokinase, homoserine dehydrogenase, pyruvate carboxylase, or L-threonine dehydratase may be a wild-type protein or a protein mutant whose activity has been weakened or enhanced to be beneficial for the production of the target substance.

[0111] The microorganism of the present application may have an ability to produce a target substance that is at least 1%, 5%, 10%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 22%, 25%, 29%, 33%, 38%, 44%, 45%, or 48% or more of that of a microorganism that does not contain a polynucleotide having promoter activity.

[0112] Another aspect of the present application provides a method for producing a target substance, which comprises culturing the above-mentioned microorganism in a culture medium. The above-mentioned microorganism and target substance are as described above.

[0113] In the present application, the method for producing a target substance using a microorganism containing the polynucleotide can be carried out using methods widely known in the art. Specifically, the culture can be continuous culture using a batch process, fed batch, or repeated fed batch process, but is not limited thereto. The culture medium used for culture should be appropriately adapted to meet the requirements of the specific strain. Culture media for Corynebacterium strains are known (e.g., Manual of Methods for General Bacteriology by the American Society for Bacteriology, Washington DC, USA, 1981).

[0114] The culture medium and other culture conditions used for culturing the strain of the present application can be any medium used for culturing a typical Corynebacterium microorganism without any particular limitations. Specifically, the strain of the present application can be cultured in a typical medium containing an appropriate carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids, and / or vitamins under aerobic or anaerobic conditions, with temperature, pH, etc., being adjusted.

[0115] In the present application, the carbon source may include, but is not limited to, carbohydrates such as glucose, 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 glutamate, methionine, lysine, etc. In addition, natural organic nutrient sources such as starch hydrolysate, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn steeping liquid can be used. Carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted into reducing sugars) can also be used, and various other carbon sources can be used in appropriate amounts without limitation. These carbon sources can be used alone or in combination of two or more.

[0116] Examples of the nitrogen source 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-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolysate, fish or its hydrolyzed products, defatted soybean cake or its hydrolyzed products, etc. These nitrogen sources may be used alone or in combination of two or more, but are not limited thereto.

[0117] The phosphorus source may include monopotassium phosphate, dipotassium phosphate, or the corresponding sodium-containing salts, etc. The inorganic compound may include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc.

[0118] In addition, the medium may contain amino acids, vitamins, and / or appropriate precursors. Specifically, the culture medium for the strain may be supplemented with L-amino acids. Specifically, glycine, glutamate, and / or cysteine ​​may be added, and if necessary, L-amino acids such as lysine may also be added, but the supplement is not limited thereto.

[0119] The medium or precursor may be added to the culture in a batch or continuous manner, but is not limited thereto.

[0120] In the present application, during the cultivation of the bacterial strain, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. can be added to the culture in an appropriate manner to adjust the pH of the culture. Furthermore, during the cultivation, antifoaming agents such as fatty acid polyglycol esters can be used to suppress foam formation. Furthermore, to maintain an aerobic state in the culture, oxygen or an oxygen-containing gas can be injected into the culture, and to maintain anaerobic and microaerobic states, nitrogen, hydrogen, or carbon dioxide gas can be injected without gas injection or to maintain anaerobic and microaerobic states.

[0121] The temperature of the culture water may be, but is not limited to, 25° C. to 40° C., more specifically, 28° C. to 37° C. The culture period can be continued until a desired amount of useful substance is produced, and specifically, may be, but is not limited to, 1 hour to 160 hours, or 10 hours to 100 hours.

[0122] The method for producing the target substance may further include an additional step after the culturing step, which may be appropriately selected depending on the intended use of the target substance.

[0123] Specifically, the method for producing the target substance may include, after the culturing step, recovering the target substance from one or more substances selected from the microorganism, the culture medium, a dried product of the microorganism, an extract of the microorganism, a culture of the microorganism, a supernatant of the culture, and a disrupted product of the microorganism.

[0124] The above method may further include a step of lysing the microorganism (strain) before or simultaneously with the recovery step. Lysis of the strain may be carried out by a method commonly used in the art, such as the use of a lysis buffer, a sonicator, heat treatment, or a French press. The lysis step may include, but is not limited to, an enzymatic reaction using a cell wall-degrading enzyme, a nuclease, a nucleosidase, a protease, or the like.

[0125] In the present application, the term "dried microorganism" is used interchangeably with the term "dried strain," etc. The dried microorganism can be produced by drying the cells that have accumulated the target substance, and specifically, may be contained in a feed composition, a food composition, etc., but is not limited thereto.

[0126] In this application, the term "microbial extract" is used interchangeably with the term "strain extract." The term "strain extract" may refer to the material remaining after separating the cell walls from the bacterial cells of the strain. Specifically, the term "strain extract" may refer to the material remaining after removing the cell walls from the components obtained by lysing the bacterial cells. The strain extract contains the target substance, and other components may include, but are not limited to, one or more of proteins, carbohydrates, nucleic acids, and fibers.

[0127] In the recovery step, the target substance can be recovered using any suitable method known in the art.

[0128] The recovery step may include a purification step, which may involve isolating and purifying only the target substance from the strain. Through this purification step, a purified target substance may be produced.

[0129] If necessary, the method for producing the target substance may further include, after the culturing step, a step of mixing an excipient with a substance selected from the obtained strain, its dried product, extract, culture product, or disrupted product, and the target substance recovered therefrom.

[0130] The excipient may be appropriately selected and used depending on the intended use and form, and may be, for example, one 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, hydroxypropylmethylcellulose, propylene glycol, casein, calcium lactate, Primogel, and gum arabic. Specifically, the excipient may be one or more components selected from starch, glucose, cellulose, lactose, dextrin, glycogen, D-mannitol, and maltodextrin, but is not limited thereto.

[0131] The excipients may include, for example, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers, or isotonic agents, but are not limited thereto.

[0132] Another aspect of the present application provides use of a polynucleotide having promoter activity, in which the nucleotides at positions 27, 28, 31, 32, and 36 in the polynucleotide sequence of SEQ ID NO: 1 are substituted with other nucleotides, as a promoter. The polynucleotide is as described above. [Example]

[0133] The present application will be described in more detail below through examples. However, these examples are for illustrative purposes only and the scope of the present application is not limited to these examples.

[0134] Example 1. Confirmation of the target gene expression induction activity of a novel promoter Example 1-1. gdh promoter mutation library created using random mutation method First, a nucleotide sequence (SEQ ID NO: 1) containing the promoter region of the wild-type Corynebacterium glutamicum ATCC13032 gdh gene (NCBI accession number NCgl1999) was obtained from the National Institutes of Health (NIH) GenBank. Random mutagenesis was performed using the gdh gene promoter (SEQ ID NO: 1) as a template with the Diversify PCR Random Mutagenesis Kit (Takara) and primers SEQ ID NO: 5 and SEQ ID NO: 6 according to the manufacturer's instructions, yielding gdh promoter mutant PCR products (Pmgdh) with different sequences. The ORF (open reading frame) of the GFP gene was isolated by PCR using the pGFPuv vector (Clontech, USA) as a template and primers of SEQ ID NOs: 7 and 8. The PCR was performed by denaturing at 94°C for 5 minutes, followed by 30 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 1 minute, followed by polymerization at 72°C for 7 minutes, to obtain a gene fragment containing the GFP ORF.

[0135] The amplification product, the gdh promoter-mutated PCR product (Pmgdh), and GFP were digested with BamHI / SalI restriction enzymes and mixed with the E. coli-Corynebacterium shuttle vector pCES208 (J. Microbiol. Biotechnol. 18:639-647, 2008). Using the In-Fusion® HD Cloning Kit (clontech), a recombinant vector library in which Pmgdh was linked to GFP was constructed. The vectors were named pCES_Pm1gdh_gfp through pCES_Pm100gdh_gfp.

[0136] A recombinant vector containing the wild-type gdh gene promoter (SEQ ID NO: 1) linked to GFP was used as a control to confirm the activity of the pmgdh library. The wild-type gdh gene promoter fragment was obtained using wild-type Corynebacterium glutamicum ATCC13032 as a template and primers SEQ ID NO: 5 and SEQ ID NO: 6. The GFP ORF (open reading frame) was extracted by PCR using the pGFPuv vector (Clontech, USA) as a template and primers SEQ ID NO: 7 and SEQ ID NO: 8. The PCR was performed at 94°C for 5 minutes, followed by 30 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 1 minute, followed by polymerization at 72°C for 7 minutes.

[0137] The amplified products, the gdh wild-type promoter PCR product (Pgdh) and GFP, were cleaved with BamHI / SalI restriction enzymes and mixed with the prepared E. coli-Corynebacterium shuttle vector pCES208 (J. Microbiol. Biotechnol. 18:639-647, 2008). A recombinant vector in which Pgdh was linked to GFP was constructed using the In-Fusion® HD Cloning Kit (clontech), and named pCES_Pgdh_gfp.

[0138] Example 1-2. Construction of transformed strains 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 above were transformed into Corynebacterium glutamicum ATCC13032 by electroporation (Appl. Microbiol. Biothcenol. (1999) 52:541-545). After transformation, the transformed strains were selected on a selection medium containing 25 mg / L of kanamycin and designated ATCC13032 / pCES, ATCC13032 / pCES_Pmgdh_gfp (ATCC13032 / pCES_Pm1gdh_gfp to ATCC13032 / pCES_Pm100gdh_gfp), and ATCC13032 / pCES_Pgdh_gfp, respectively.

[0139] Example 1-3. Screening of gdh promoter mutations To confirm the activity of the gdh promoter mutants, the transformed strains of Corynebacterium glutamicum ATCC13032 / pCES, ATCC13032 / pCES_Pgdh_gfp, and ATCC13032 / pCES_Pmgdh_gfp (ATCC13032 / pCES_Pm1gdh_gfp to ATCC13032 / pCES_Pm100gdh_gfp) obtained in Example 1-2 above were cultured as described below, and GFP activity was measured.

[0140] Specifically, the transformed Corynebacterium glutamicum strain was inoculated into a flask containing 25 ml of 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, and 3 mg nicotinamide (based on 1 L of distilled water), pH 7.2) and cultured at 30°C for 20 hours with shaking. The cells were harvested from the culture by centrifugation (5,000 rpm, 15 minutes), washed twice with 50 mM Tris-HCl (pH 8.0) buffer, and then suspended in the same buffer. After adding 1.25 g of glass beads per 1.5 ml of suspension, the cells were disrupted using a bead beater for 6 minutes and then centrifuged (15,000 rpm for 20 minutes) to collect the supernatant, which was then assayed for protein concentration using the Bradford method. The same amount of cell extract was subjected to the method described by Laure Gory et al. (FEMS Microbiology Letters 194, 127-133, 2001) by irradiating the same amount of cell extract with 488 nm excitation light and measuring the emitted light at 511 nm using an LS-50B spectrophotometer (Perkin-Elmer). The GFP gene expression levels were compared with those of the control ATCC13032 / pCES_Pgdh_gfp strain, and the top three strains with the highest GFP gene expression levels were selected (Table 1).

[0141] [Table 1]

[0142] As shown in Table 1 above, the Pm3gdh, Pm16gdh, and Pm78gdh promoters were confirmed to exhibit promoter activity in Corynebacterium glutamicum and to exhibit higher fluorescence sensitivity than the wild-type gdh promoter. 3The gdh promoter mutants were sequenced to confirm the mutations introduced into the gdh promoter of the seed strain. To determine the sequence, PCR was performed using primers set forth in SEQ ID NO: 9 and SEQ ID NO: 10, followed by sequence analysis. The sequence of the mutant gdh promoter was confirmed by comparison with the wild-type gdh promoter sequence set forth in SEQ ID NO: 1. The gdh promoter sequences of the selected strains are shown in Table 2 below (Table 2).

[0143] [Table 2]

[0144] Example 2. Construction of Pm3gdh, Pm16gdh, and Pm78gdh promoter mutation introduction vectors To construct vectors for introducing the Pm3gdh, Pm16gdh, and Pm78gdh promoter mutations, PCR products corresponding to the promoter mutations were obtained using primers SEQ ID NOs: 13 and 14, respectively, with the pCES_Pm3gdh_gfp, pCES_Pm16gdh_gfp, and pCES_Pm78gdh_gfp vectors as templates. The primer sets SEQ ID NOs: 11 and 12, and SEQ ID NOs: 15 and 16 were used to obtain gene fragments containing the gdh promoter upstream region and a portion of the gdh ORF using the chromosome of wild-type Corynebacterium glutamicum ATCC 13032 as a template. PCR was performed by denaturing at 94°C for 5 minutes, followed by 30 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 1 minute, followed by polymerization at 72°C for 5 minutes. The PCR products were obtained. The above three amplification products were mixed with the pDCM2 vector (Korea Publication No. 10-2020-0136813), which had been prepared in advance by cleaving with SmaI restriction enzyme, and recombinant vectors were constructed using the In-Fusion® HD Cloning Kit (clontech). These were named pDCM2_Pm3gdh_gdh, pDCM2_Pm16gdh_gdh, and pDCM2_Pm78gdh_gdh, respectively.

[0145] Example 3. Evaluation of target product productivity 3-1. Evaluation of lysine production ability 3-1-1. Construction of L-lysine-producing strains carrying gdh promoter mutants To prepare strains transformed with the gdh promoter mutants using the pDCM2_Pm3gdh_gdh, pDCM2_Pm16gdh_gdh, and pDCM2_Pm78gdh_gdh vectors constructed in Example 2, the L-lysine-producing Corynebacterium glutamicum CJ3P (Binder et al. Genome Biology 2012, 13:R40) strain was transformed with the vectors to introduce the gdh promoter mutant sequences into the chromosome. The CJ3P strain is a Corynebacterium glutamicum strain that has been made capable of producing L-lysine by introducing three mutations (pyc(Pro458Ser), hom(Val59Ala), and lysC(Thr311Ile)) into a wild-type strain using publicly known techniques.

[0146] Specifically, the vector constructed in Example 2 was introduced into the CJ3P strain by electroporation, and the transformed strain was isolated in a selection medium containing 25 mg / L kanamycin. Strains in which the gdh promoter mutants were introduced by the DNA fragment inserted into the chromosome in the secondary recombination process (crossover) were selected through PCR and sequence analysis using primers of SEQ ID NOs: 9 and 10. The selected strains were named Corynebacterium glutamicum CJ3P::Pm3gdh_gdh, CJ3P::Pm16gdh_gdh, and CJ3P::Pm78gdh_gdh.

[0147] 3-1-2. Evaluation of L-lysine production ability of strains harboring gdh promoter mutants To evaluate the L-lysine producing ability of the parent strain Corynebacterium glutamicum CJ3P and the Corynebacterium glutamicum CJ3P::Pm3gdh_gdh, CJ3P::Pm16gdh_gdh, and CJ3P::Pm78gdh_gdh strains constructed in Example 3-1-1, the strains were cultured and analyzed as follows.

[0148] First, each strain was inoculated into a 250-ml corner-baffled flask containing 25 ml of seed medium and cultured with shaking at 200 rpm at 30°C for 20 hours. Then, 1 ml of the seed culture was inoculated into a 250-ml corner-baffled flask containing 24 ml of production medium and cultured with shaking at 200 rpm at 32°C for 48 hours. The compositions of the seed medium and production medium were as follows:

[0149] <Seed medium (pH 7.0)> Glucose 20g, peptone 10g, yeast extract 5g, urea 1.5g, KH2PO4 4g, K2HPO4 8g, MgSO4·7H2O 0.5g, biotin 100μg, thiamine hydrochloride 1000μg, calcium pantothenate 2000μg, nicotinamide 2000μg (based on 1 liter of distilled water)

[0150] <Production medium (pH 7.0)> Glucose 45g, soy protein 10g, molasses 10g, (NH4)2SO4 15g, KH2PO4 0.55g, MgSO4·7H2O 0.6g, FeSO4·7H2O 9mg, MnSO4·5H2O 9mg, biotin 0.9mg, thiamine hydrochloride 4.5mg, CaCO3 30g, calcium pantothenate 4.5mg, nicotinamide 30mg, ZnSO4 0.45mg, CuSO4 0.45mg (based on 1 liter of distilled water)

[0151] After the cultivation, the amount of L-lysine produced was measured using HPLC. The L-lysine concentrations in the culture medium and the rate of increase in concentration for the Corynebacterium glutamicum strains CJ3P, CJ3P::Pm3gdh_gdh, CJ3P::Pm16gdh_gdh, and CJ3P::Pm78gdh_gdh are shown in Table 3 below.

[0152] [Table 3]

[0153] As shown in Table 3, the three strains into which the gdh promoter mutants were introduced were confirmed to have increased L-lysine concentrations compared to the parent strain CJ3P. The above CJ3P::gdhPm3_gdh was designated CM03-1660 and deposited with the Korea Microorganism Collection, a depository under the Budapest Treaty, on April 5, 2021, and was assigned the accession number KCCM12970P.

[0154] Example 3-2. Evaluation of threonine-producing ability 3-2-1. Construction of threonine-producing strain 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, first, an L-threonine-producing strain was constructed by introducing the lysC (L377K) mutant (Korean Patent Registered No. 10-2011994) and the hom (R398Q) mutant (Korean Patent Registered No. 10-1947959) into the Corynebacterium glutamicum ATCC13032 strain.

[0155] Specifically, to construct an L-threonine-producing strain, we first constructed a vector for introducing lysC(L377K). To construct the vector, PCR was performed using the chromosome of wild-type Corynebacterium glutamicum ATCC13032 as a template and primers set forth in SEQ ID NOS: 17 and 18, and 19 and 20. After denaturation at 95°C for 5 minutes, the following cycles were repeated 30 times: denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 30 seconds. Polymerization was then repeated for 7 minutes at 72°C to obtain the respective PCR products. The amplified product was mixed with pDCM2 vector, which had been previously digested with SmaI restriction enzyme. A recombinant vector was constructed using the In-Fusion® HD Cloning Kit and named pDCM2_lysC(L377K).

[0156] The pDCM2_lysC(L377K) vector constructed above was electroporated into the Corynebacterium glutamicum ATCC13032 strain, and transformed strains were isolated on a selection medium containing 25 mg / L kanamycin. Strains with nucleotide mutations in the lysC gene due to the DNA fragment inserted into the chromosome during the secondary recombination process (crossover) were selected through PCR and nucleotide sequence analysis using primers SEQ ID NO: 25 and SEQ ID NO: 26, and the selected strain was designated ATCC13032::lysC(L377K).

[0157] To construct a vector for introducing hom(R398Q), PCR was performed using the chromosome of Corynebacterium glutamicum ATCC13032 as a template, primers SEQ ID NOs: 21 and 22, and primers SEQ ID NOs: 23 and 24. The PCR conditions were denaturation at 95°C for 5 minutes, followed by 30 cycles of 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 pDCM2 vector, which had been previously digested with SmaI restriction enzyme, and a recombinant vector was constructed using the In-Fusion® HD Cloning Kit. This vector was named pDCM2_hom(R398Q).

[0158] The pDCM2_hom(R398Q) vector was electroporated into the Corynebacterium glutamicum ATCC13032::lysC(L377K) strain, and transformants were isolated on a selection medium containing 25 mg / L kanamycin. Strains carrying nucleotide mutations in the hom gene due to the DNA fragment inserted into the chromosome during the secondary recombination process (crossover) were selected by PCR and sequence analysis using primers SEQ ID NOs: 27 and 28. The selected strain was designated Corynebacterium glutamicum ATCC13032::lysC(L377K)_hom(R398Q).

[0159] 3-2-2. Construction of L-threonine-producing strains carrying gdh promoter mutants The vector prepared in Example 2 was introduced into the Corynebacterium glutamicum ATCC13032::lysC(L377K)_hom(R398Q) strain by electroporation, and the transformed strain was isolated in a selection medium containing 25 mg / L kanamycin. Strains in which the gdh promoter mutants were introduced by the DNA fragment inserted into the chromosome during the secondary recombination process (crossover) were selected through PCR and sequence analysis using primers of SEQ ID NO: 9 and SEQ ID NO: 10, and 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.

[0160] 3-2-3. Evaluation of L-threonine-producing ability of strains incorporating gdh promoter mutants To evaluate the L-threonine-producing ability of the parent strain Corynebacterium glutamicum ATCC13032::lysC(L377K)_hom(R398Q), and the ATCC13032::lysC(L377K)_hom(R398Q)::Pm3gdh_gdh, ATCC13032::lysC(L377K)_hom(R398Q)::Pm16gdh_gdh, and ATCC13032::lysC(L377K)_hom(R398Q)::Pm78gdh_gdh strains constructed in Example 3-2-2, the strains were cultured and analyzed as follows.

[0161] First, each strain was inoculated into a 250-ml corner-baffled flask containing 25 ml of seed medium and cultured with shaking at 200 rpm at 30°C for 20 hours. Then, 1 ml of the seed culture was inoculated into a 250-ml corner-baffled flask containing 24 ml of production medium and cultured with shaking at 200 rpm at 32°C for 48 hours. The compositions of the seed medium and production medium are as follows:

[0162] <Seed medium (pH 7.0)> Glucose 20g, peptone 10g, yeast extract 5g, urea 1.5g, KH2PO4 4g, K2HPO4 8g, MgSO4·7H2O 0.5g, biotin 100μg, thiamine hydrochloride 1000μg, calcium pantothenate 2000μg, nicotinamide 2000μg (based on 1 liter of distilled water)

[0163] <Production medium (pH 7.0)> Glucose 45g, soy protein 10g, molasses 10g, (NH4)2SO4 15g, KH2PO4 0.55g, MgSO4·7H2O 0.6g, FeSO4·7H2O 9mg, MnSO4·5H2O 9mg, biotin 0.9mg, thiamine hydrochloride 4.5mg, CaCO3 30g, calcium pantothenate 4.5mg, nicotinamide 30mg, ZnSO4 0.45mg, CuSO4 0.45mg (based on 1 liter of distilled water)

[0164] After the cultivation, the amount of L-threonine produced was measured using HPLC. The L-threonine concentrations in the culture medium and the rate of increase in concentration for the 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 strains are shown in Table 4.

[0165] [Table 4]

[0166] As shown in Table 4 above, it was confirmed that the three strains into which the gdh promoter mutants were introduced had increased L-threonine concentrations compared to the parent strain ATCC13032::lysC(L377K)_hom(R398Q).

[0167] Example 3-3. Evaluation of O-acetylhomoserine productivity 3-3-1. Construction of O-acetylhomoserine-producing strains carrying gdh promoter mutants The vector constructed in Example 2 was electroporated into the wild-type strain Corynebacterium glutamicum ATCC13032, and transformed strains were isolated in a selection medium containing 25 mg / L kanamycin. Strains carrying the gdh promoter mutants introduced by the DNA fragment inserted into the chromosome during the secondary recombination process (crossover) were selected through PCR and sequence analysis using primers set forth in SEQ ID NOs: 9 and 10. The selected strains were designated Corynebacterium glutamicum ATCC13032::Pm3gdh_gdh, ATCC13032::Pm16gdh_gdh, and ATCC13032::Pm78gdh_gdh.

[0168] 3-3-2. Evaluation of O-acetylhomoserine production ability of strains harboring gdh promoter mutants To evaluate the O-acetylhomoserine producing ability of the parent strain Corynebacterium glutamicum ATCC13032 and the ATCC13032::Pm3gdh_gdh, ATCC13032::Pm16gdh_gdh, and ATCC13032::Pm78gdh_gdh strains constructed in Example 3-3-1, the strains were cultured and analyzed as follows.

[0169] One loopful of the strain was inoculated into a 250 ml corner baffle flask containing 25 ml of the medium described below, and cultured at 33°C for 20 hours with shaking at 200 rpm.

[0170] <Production medium (pH 7.2)> Glucose 30g, KH2PO4 2g, urea 3g, (NH4)2SO4 40g, peptone 2.5g, CSL (Corn steep liquor, Sigma) 5g (10ml), MgSO4 7H2O 0.5g, CaCO3 20g (based on 1 liter of distilled water)

[0171] After the cultivation, the productivity of O-acetylhomoserine was measured by HPLC. The O-acetylhomoserine concentrations in the culture medium and the concentration increase rates for the Corynebacterium glutamicum ATCC13032, ATCC13032::Pm3gdh_gdh, ATCC13032::Pm16gdh_gdh, and ATCC13032::Pm78gdh_gdh strains are shown in Table 5.

[0172] [Table 5]

[0173] As shown in Table 5 above, it was confirmed that the three strains into which the gdh promoter mutants were introduced had increased O-acetylhomoserine concentrations compared to the parent wild-type strain ATCC13032.

[0174] Example 3-4. Evaluation of L-isoleucine productivity 3-4-1. Construction of L-isoleucine-producing strains carrying gdh promoter mutants 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, the Corynebacterium glutamicum CJP1 (Korea Patent Registered No. 10-1996769) strain was first transformed with the vectors to introduce the gdh promoter mutant sequences into the chromosome. Subsequently, a vector containing the ilvA gene encoding the publicly known L-threonine dehydratase, in which the 323rd amino acid, valine, was mutated to alanine (V323A) (Appl. Enviro. Microbiol., December 1996, pp. 4345-4351), was further introduced to construct an L-isoleucine-producing strain (Korean Patent Registration No. 10-1996769).

[0175] Specifically, the vector prepared in Example 2 was introduced into the CJP1 strain by electroporation, and the transformed strain was isolated in a selection medium containing 25 mg / L kanamycin. Strains in which the gdh promoter mutants were introduced by the DNA fragment inserted into the chromosome in the secondary recombination process (crossover) were selected through PCR and sequence analysis using primers of SEQ ID NO: 9 and SEQ ID NO: 10, and the selected strains were named Corynebacterium glutamicum CJP1::Pm3gdh_gdh, CJP1::Pm16gdh_gdh, and CJP1::Pm78gdh_gdh.

[0176] The pECCG117-ilvA(V323A) vector (Korea Patent Registration No. 10-1996769) was electroporated into the constructed strain, and the transformed strains were isolated in a selection medium containing 25 mg / L kanamycin. The selected strains were named Corynebacterium glutamicum CJP1::Pm3gdh_gdh / pECCG117-ilvA(V323A), CJP1::Pm16gdh_gdh / pECCG117-ilvA(V323A), and CJP1::Pm78gdh_gdh / pECCG117-ilvA(V323A), respectively.

[0177] 3-4-2. Evaluation of L-isoleucine production ability of strains harboring gdh promoter mutants To evaluate the L-isoleucine production ability of the parent strain Corynebacterium glutamicum CJP1 / pECCG117-ilvA(V323A), CJP1::Pm3gdh_gdh / pECCG117-ilvA(V323A), CJP1::Pm16gdh_gdh / pECCG117-ilvA(V323A), and CJP1::Pm78gdh_gdh / pECCG117-ilvA(V323A) strains constructed in Example 3-4-1, the strains were cultured and analyzed as follows.

[0178] First, each strain was inoculated into a 250-ml corner-baffled flask containing 25 ml of seed medium and cultured with shaking at 200 rpm at 30°C for 20 hours. Then, 1 ml of the seed culture was inoculated into a 250-ml corner-baffled flask containing 24 ml of production medium and cultured with shaking at 200 rpm at 32°C for 48 hours. The compositions of the seed medium and production medium were as follows:

[0179] <Seed medium (pH 7.0)> Glucose 20g, peptone 10g, yeast extract 5g, urea 1.5g, KH2PO4 4g, K2HPO4 8g, MgSO4·7H2O 0.5g, biotin 100μg, thiamine hydrochloride 1000μg, calcium pantothenate 2000μg, nicotinamide 2000μg (based on 1 liter of distilled water)

[0180] <Production medium (pH 7.0)> Glucose 45g, soy protein 10g, molasses 10g, (NH4)2SO4 15g, KH2PO4 0.55g, MgSO4·7H2O 0.6g, SO4·7H2O 9mg, MnSO4·5H2O 9mg, biotin 0.9mg, thiamine hydrochloride 4.5mg, CaCO3 30g, calcium pantothenate 4.5mg, nicotinamide 30mg, ZnSO4 0.45mg, CuSO4 0.45mg (based on 1 liter of distilled water)

[0181] After the cultivation, the amount of L-isoleucine produced was measured using HPLC. The L-isoleucine concentrations in the culture medium and the rate of increase in concentration for the Corynebacterium glutamicum CJP1 / pECCG117-ilvA(V323A), CJP1::Pm3gdh_gdh / pECCG117-ilvA(V323A), CJP1::Pm16gdh_gdh / pECCG117-ilvA(V323A), and CJP1::Pm78gdh_gdh / pECCG117-ilvA(V323A) strains are shown in Table 6.

[0182] [Table 6]

[0183] As shown in Table 6 above, the three strains into which the gdh promoter mutants were introduced showed increased L-isoleucine concentrations compared to the parent wild-type strain CJP1 / pECCG117-ilvA(V323A). Based on these results, it was confirmed that recombinant microorganisms containing polynucleotides having promoter activity of the present application increase the productivity of industrially useful L-lysine, L-threonine, O-acetylhomoserine, and L-isoleucine.

[0184] From the above description, those skilled in the art to which the present application pertains will understand that the present application may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are merely illustrative and not limiting. The scope of the present application should be interpreted as including all modifications and variations derived from the meaning and scope of the claims below, and equivalent concepts thereof, rather than the above detailed description.

[0185] JPEG0007762226000007.jpg241170

Claims

1. In the polynucleotide sequence of SEQ ID NO: 1, the nucleotides at positions 27, 28, 31, 32 and 36 are replaced with other nucleotides; A polynucleotide having promoter activity, in which the 27th nucleotide, adenine (A), is substituted with thymine (T), the 28th nucleotide, cytosine (C), is substituted with guanine (G), the 31st nucleotide, cytosine (C), is substituted with guanine (G), the 32nd nucleotide, cytosine (C), is thymine (T), and the 36th nucleotide, adenine (A), is substituted with cytosine (C) in the polynucleotide sequence of SEQ ID NO:

1.

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

3. The nucleotide at position 66 is further replaced with another nucleotide, 2. The polynucleotide of claim 1, wherein the 66th nucleotide, cytosine (C), is substituted with thymine (T).

4. The polynucleotide of claim 1, wherein the polynucleotide is set forth in SEQ ID NO:

2.

5. The polynucleotide of claim 2, wherein the polynucleotide is set forth in SEQ ID NO:

3.

6. The polynucleotide of claim 3, wherein the polynucleotide is set forth in SEQ ID NO:

4.

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

8. An expression cassette comprising the polynucleotide according to any one of claims 1 to 6; and a gene encoding a target protein operably linked to the polynucleotide.

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

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

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

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

13. A method for producing a target substance, comprising the steps of culturing the Corynebacterium microorganism according to claim 10 in a medium; and recovering the target substance from the medium.

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

15. Use of the polynucleotide described in any one of claims 1 to 6 as a promoter.

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