KR100898246B1 - Novel Corynebacterium glutamicum promoter - Google Patents

Abstract

The present invention relates to novel promoter nucleic acid molecules derived from Corynebacterium glutamicum cells, vectors comprising such nucleic acid molecules, host cells transformed with such vectors and using such nucleic acid molecules to induce expression of the gene of interest. It is about a method.

Corynebacterium glutamicum, promoter, nucleic acid, vector, transformation

Description

Novel Corynebacterium Glutacum Promoter Novel Corynebacterium glutamicum promoter

The present invention uses a novel Corynebacterium glutamicum promoter nucleic acid molecule capable of inducing expression of a target gene, a vector comprising such nucleic acid molecule, a host cell transformed with such a vector and a promoter nucleic acid molecule. It relates to a method of inducing expression of a gene.

Coryneform bacteria are traditionally the most widely used industrial microorganisms for the production of amino acids and nucleic acid-related substances, mainly L-lysine, L-threonine, L-arginine, L-threonine, And gram-positive bacteria used to produce chemicals having various uses in fields such as feed, pharmaceuticals, and foods including amino acids such as glutamic acid and various nucleic acids, and require biotin for growth. It has a characteristic of snapping at right angles during cell division, and its low degradation activity on the generated metabolite is one of the advantages of the bacteria. Representative species include Corynebacterium, including Corynebacterium glutamicum, Bribibacterium, including Brevibacterium flavum, Athrobacter sp., And Microbacterium sp.

In order to develop such coryneform bacteria into strains capable of producing high titers of target substances using genetic engineering and metabolic engineering techniques, the expression of genes related to various metabolic processes in the strain should be selectively controlled. For this regulation, one of the regulatory genes must have a strong or universal activity of a promoter, a site where RNA synthase binds to a DNA molecule to initiate transcription, and overexpresses target substances such as amino acids or nucleic acids. In order to do this, the search for these promoters is essential.

In order to express genes in coryneform bacteria, it was common to use promoters originally possessed by their own genes (Vasicova, P., et al., J. Bacteriol., 181, 6188-6191, (1999), etc.). ).

Meanwhile, the promoter sequence for gene expression in coryneform bacteria is Escherichia coli ) or Bacillus Unlike other industrial microorganisms such as subtilis ), the general structure is unknown. Therefore, many researchers have removed the promoter portion of antibiotic resistance genes such as chloramphenicol, introduced genomic DNA isolated from coryneform bacteria with appropriate restriction enzymes, and then transformed the coryneform bacteria. Promoters have been developed by measuring the antibiotic resistance of the resulting strains (Eikmanns, BJ, et. al ., Gene , 102: 93-98, 1991; Patek, M., et al ., Microbiology , 142: 1297-1309, 1996).

Prior patents related to promoters derived from Coryneform bacteria include patents describing promoters derived from Corynebacterium, and are described in US Patent 5,700,661 "Gene expression regulatory DNA", US Patent 5,965,391 "DNA which regulates gene expression in coryneform bacteria", USA Patent No. 7,141,388 "Nucleotide sequences for transcreptional regulation in corynebacterium glutamicum", and Korea Patent No. 10-0653742 "New L-lysine-induced promoter", and the like, and disclosed in Korea disclosed a promoter derived from Corynebacterium ammonia genes Patent 10-2006-0068505 “Novel promoter sequences derived from cells of the genus Corynebacterium, expression cassettes and vectors comprising them, host cells comprising said vectors and methods of expressing genes using the same”.

 However, previously developed promoter sequences still have room for improvement in terms of selectivity and expression efficiency of gene expression such as increased expression in the presence of a specific inducer.

On the other hand, the conventional method of searching for a promoter includes (1) the use of a promoter probe vector for randomly cloning a genomic DNA fragment in front of a reporter gene expressed only when the cloned fragment contains promoter activity, and (2) a gene. Hybridization based on specific probes has been used to isolate genes and their promoters from gene libraries, and (3) differential hybridization of gene banks using inducible and non-inducible cDNA probes.

The present inventors cloned genomic DNA fragments in front of the reporter gene and applied the use of a promoter probe vector that can determine the degree of promoter activity through the reporter gene, thereby applying a DNA microarray of the Corynebacterium glutamicum genomic gene. New corynebacterium glutamime by amplifying the predicted promoter polynucleotide of a gene selected as a gene with high expression rate by a) and then linking it to the front of the GFP reporter gene to confirm the degree of GFP activity. It was possible to provide a Qum-derived promoter nucleic acid molecule to complete the present invention.

It is an object of the present invention to provide a novel Corynebacterium glutamicum promoter nucleic acid molecule.

It is still another object of the present invention to provide a vector comprising the novel promoter nucleic acid molecule described above.

Still another object of the present invention is to provide a transformant transformed with the vector.

Still another object of the present invention is to provide a method for inducing expression of a gene of interest using the novel promoter nucleic acid molecule described above.

In one embodiment, the present invention relates to a novel Corynebacterium glutamicum promoter nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1 or 2.

The Corynebacterium glutamicum promoter nucleic acid molecule of the present invention is a promoter derived from Coryneform bacterium, and is preferably used as a promoter for gene expression in prokaryotic cells, particularly E. coli or Coryneform bacteria.

The term "coryneform bacterium" of the present invention is a concept including microorganisms of the genus Corynebacterium (Corynebacterium) or Brevibacterium (Brevibacterium). This coryneform bacterium is a wild-type strain known as Corynebacterium glutamicum, in addition to Corynebacterium glutamicum ATCC 13032, in particular Corynebacterium glutamicum species, known as Corynebacterium thermoaminogenes ( thermoaminogenes) FERM BP-1539, Brevibacterium flavum ATCC 14067, Brevibacterium lactofermentum ATCC 13869, and L-amino acid producing mutants or strains prepared therefrom, for example , Corynebacterium glutamicum KFCC 10881, Corynebacterium glutamicum KFCC 11001 and the like, but is not limited to these examples.

As used herein, the term "promoter" refers to a non-ready nucleic acid sequence upstream of a coding region, that is, a polymerase, that contains a binding site for a polymerase and has a transcription initiation activity to an mRNA of a promoter subgene. Refers to a DNA region for initiating transcription of a gene and is located at the 5 'region of the mRNA transcription initiation site.

In a specific embodiment, the promoter of the present invention is a promoter of the butA gene isolated from the Corynebacterium glutamicum genome (N1, defined by the nucleotide sequence of SEQ ID NO: 1), or the promoter of the pyk gene (N2, SEQ ID NO: 2). As defined by the nucleotide sequence of).

As a specific embodiment of the present invention, the present inventors use a DNA microarray to measure novel genome expression of about 3000 genes of Corynebacterium glutamicum ATCC 13032, and thus, a novel promoter, specifically, butA and pyk genes. Promoter of was selected.

In the present invention, the 'butA gene' and the 'pyk gene' can obtain the genetic information from the nucleotide sequence of the Corynebacterium glutamicum genomic gene that is already published. For example, the butA and pyk genes used in the present invention can obtain genetic information of proteins from the National Institutes of Health Gene Bank (NIH GenBank), but is not limited thereto. The 'butA gene' used in the present invention is GenBank registration number NCgl2582, which encodes L-2,3-butanediol dehydrogenase (L-2.3-butanediol dehydrogenase), and the 'pyk gene' is GenBank registration number NCgl2008 As an example, pyruvate kinase is encoded.

In addition, the 'butA gene' and the 'pyk gene' according to the present invention not only have NCgl2582 and NCgl2008 sequences derived from the Corynebacterium glutamicum ATCC 13032 strain, but also exist in the microorganisms belonging to the genus Corynebacterium. As a gene means a gene that expresses substantially the same product as the gene product of NCgl2582 or NCgl2008. “Substantially identical” means having the same kind of activity and regulatory mechanisms as each gene product.

SEQ ID NO: 1 or 2 nucleotide sequence contained in the promoter nucleic acid molecule of the present invention through a variety of recent research techniques, such as directed evolution or site-directed mutagenesis, etc. Some deformation is possible. That is, the Corynebacterium glutamicum promoter nucleic acid molecule of the present invention of the present invention includes a functional equivalent thereof that performs a functionally identical function to the Corynebacterium glutamicum promoter nucleic acid molecule described above. Equivalents are those polynucleotides in which some sequences of nucleotides are modified by artificial modification, or variants or the same function in which modifications are made by deletion, substitution, insertion, or combination thereof. And functional fragments of.

Those skilled in the art will appreciate that the present invention is equivalent to the novel promoters and equivalents of the present invention, so long as the sequence that maintains at least 70% homology by such an artificial modification retains the promoter activity for the gene expression desired in the present invention. It is easy to understand that it belongs to the scope of rights.

Thus, as another specific aspect, the promoter nucleic acid molecule of the present invention comprises a nucleotide sequence that exhibits at least 70% homology with the above-described nucleotide sequence and has a promoter activity.

As used herein, the term "homology" is intended to indicate a degree of similarity to a wild type nucleic acid sequence, preferably 70% or more of the nucleic acid sequence of the promoter of the present invention defined by SEQ ID NO: 1 or 2. More preferably at least 80%, even more preferably at least 90%, most preferably at least 95%. This homology comparison can be performed by using a comparison program that is easy to see with the naked eye. Commercially available computer programs can calculate the percent homology between two or more sequences, and the percent homology can be calculated for adjacent sequences.

In another specific aspect, a promoter nucleic acid molecule of the present invention comprises a nucleotide sequence complementary to a nucleotide sequence having at least 70% homology with SEQ ID No. 1, 2 or these and having promoter activity, preferably , They preferably have a promoter activity equivalent to the nucleotides.

As used herein, the term “complementary” refers to a hybridization or base between two strands or oligonucleotide primers of a nucleotide or nucleic acid, eg, a double stranded DNA molecule, and a primer binding site on a single stranded nucleic acid to be sequenced or amplified. It means mating.

Promoter nucleic acid molecules of the invention can be isolated or prepared using standard molecular biology techniques. For example, it can be separated by PCR using the appropriate primer sequence. It can also be prepared using standard synthetic techniques using automated DNA synthesizers.

In another aspect, the present invention relates to a vector comprising the novel Corynebacterium glutamicum promoter nucleic acid molecule described above.

As used herein, the term "vector" refers to a DNA preparation containing a nucleotide sequence of a gene operably linked to a suitable regulatory sequence to allow expression of the gene of interest in a suitable host. Such regulatory sequences include promoters capable of initiating transcription, any operator sequence for regulating such transcription, sequences encoding suitable mRNA ribosomal binding sites, and sequences regulating termination of transcription and translation. The term "operably linked" means that the coding sequence and the promoter are functionally linked such that the promoter sequence initiates and mediates the transcription of the coding sequence. The vector used in the present invention is not particularly limited as long as it is replicable in the host, and any vector known in the art may be used. For example, it may be a plasmid, phage particles, or simply a potential genomic insert, preferably pECCG117 (KFCC-10763) or pCR2.1-TOPO (Invitrogen, USA), but are not limited thereto. The vector can be transformed into a suitable host and then replicated or functioned independently of the host genome, and in some cases integrated into the genome itself.

In addition, the vector of the present invention may include a selection market if necessary. The selection marker is for selecting cells transformed with the vector, and markers may be used that confer a selectable phenotype such as drug resistance, nutritional requirements, resistance to cytotoxic agents or expression of surface proteins. In an environment treated with a selective agent, only cells expressing a selection marker survive, so that transformed cells can be selected.

The vector of the present invention can recombinantly produce various target proteins by operatively linking a regulatory sequence comprising a Corynebacterium glutamicum promoter nucleic acid molecule with a coding sequence comprising a target gene. The coding sequence may be, for example, a sequence encoding an entire gene, or a constant region thereof. Examples of coding sequences may be coding sequences of genes associated with amino acids (specifically, such as L-lysine, L-threonine or L-arginine) or metabolites of nucleotides (specifically, GMP or IMP, etc.). The invention is not limited thereto. Preferred genes for expression using the vectors of the invention include, for example, genes encoding various proteins involved in the biosynthetic pathway of L-lysine; Aspartate kinase (lysC), diaminopimelate dehydrogenase (ddh), aspartate semialdehyde dehydrogenase (asd), dihydrodipicolinate synthase (dapA), dihydrodipicolinate reductase (dapB) , Diaminopimelate epilases (dapF), or diaminopimelate decarboxylase (lysA) and the like can be used, but are not necessarily limited thereto.

In addition, the vector of the present invention may be linked to the downstream of the promoter (expression 'reporter gene') to express a gene encoding a protein that can be quantified to measure the degree of promoter activity. As a reporter gene, it is preferable to use Green Fluorescent Protein (GFP).

In a specific embodiment of the present invention, as a vector for inserting the novel promoter nucleic acid molecules (N1, N2) of SEQ ID NOs: 1 and 2 according to the present invention, a shuttle usable for transformation into Corynebacterium glutamicum The vector pECCG117 (Biotechnology letters vol 13, No. 10, p. 721-726 (1991) or Korean Patent Publication No. 92-7401) was used. In addition, gfp encoding GFP as a reporter gene for measuring promoter activity in the vector. A vector pECCG117-N1-GFP comprising the gene of SEQ ID NO: 1, including the kanamycin resistance gene as a selection marker, and finally comprising the promoter of SEQ ID NO: 1, and a promoter (N2) of SEQ ID NO: 2 N2-GFP was produced (FIG. 1).

In another aspect, the present invention relates to a transformant transformed with the vector described above. The transformant of the present invention can be constructed by introducing a vector into a host cell in an embodiment in which a promoter can act.

The term "transformation" of the present invention means that DNA is introduced into a host so that the DNA is replicable as an extrachromosomal factor or by chromosomal integration.

Methods for transforming a vector of the present invention include any method for introducing nucleic acids into cells, and can be carried out by selecting appropriate standard techniques as known in the art depending on the host cell. For example, electroporation, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and Lithium acetate-DMSO method;

As the host cell, it is preferable to use a host having high DNA introduction efficiency and a high expression efficiency of the introduced DNA, and all microorganisms including prokaryotic and eukaryotic may be used, preferably E. coli or coryneform bacteria, more preferably. For example, Corynebacterium glutamicum ATCC 10881 may be used.

In a specific embodiment of the present invention, the vector pECCG117-N1-GFP comprising the promoter (N1) of SEQ ID NO: 1 according to the present invention is transformed into Corynebacterium glutamicum 10881 to transform the target gene overexpression A converter was prepared. In the present invention, the transformant was named CA01-0086, and deposited on the Korean Culture Center of Microorganisms (hereinafter abbreviated as "KCCM") as accession number KCCM 10875P as of July 23, 2007. . In addition, the vector pECCG116-N2-GFP containing the promoter (N2) of SEQ ID NO: 2 was transformed into Corynebacterium glutamicum 10881 to prepare a transformant capable of overexpressing a gene of interest. The sieve was named CA01-0087 and deposited with accession number KCCM 10876P.

In still another aspect, the present invention relates to a method of expressing a gene of interest comprising culturing a transformant transformed with a vector comprising the aforementioned Corynebacterium glutamicum promoter nucleic acid molecule.

In addition, the method of producing L- lysine comprising the step of culturing the transformant.

In the present invention, "expression of the target gene" may mean to produce the protein encoded by the target gene by expressing the target gene. In the present invention, a method of expressing a target gene is a method of expressing a protein encoded by the target gene by culturing a transformant (host cell) transformed with a vector containing the target gene, thereby inducing the protein. The final product of the biosynthetic pathway can be prepared.

The final product may preferably be L-amino acid, more preferably L-lysine. In the present invention, the gene of interest may be genes encoding various proteins involved in the biosynthetic pathway of L-lysine, as described above, and L-lysine biosynthesis through expression of these genes of interest. By producing proteins containing enzymes necessary for the production of the final product L- lysine can be efficiently produced.

Culture of the transformant in the present invention can be carried out according to well-known methods, conditions such as the culture temperature and time, the pH of the medium can be appropriately adjusted. These known culture methods are described in Chmiel; Bioprozesstechnik 1. Einfuhrung in die Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991), and Storhas; Bioreaktoren und periphere Einrichtungen (Vieweg Verlag, Braunschweig / Wiesbaden, 1994).

The culture medium used should suitably meet the requirements of the particular strain. Culture media for various microorganisms are known (eg, "Manual of Methods for General Bacteriology" from American Society for Bacteriology (Washington D.C., USA, 1981)). Carbon sources in the medium include sugars and carbohydrates (e.g. glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose), fats and fats (e.g. soybean oil, sunflower seed oil, peanut oil and coconut oil). ), Fatty acids such as palmitic acid, stearic acid and linoleic acid, alcohols such as glycerol and ethanol, organic acids such as acetic acid, and the like. These materials can be used individually or as a mixture. Nitrogen sources can be nitrogen-containing organic compounds such as peptone, yeast extract, gravy, malt extract, corn steep liquor, soybean meal and urea, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and nitrate Ammonium) can be used, and these materials can also be used individually or as a mixture. Potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium containing salts can be used as the phosphorus source. In addition, the culture medium may contain metal salts necessary for growth (eg, magnesium sulfate or iron sulfate), and finally, essential growth-promoting substances such as amino acids and vitamins may be used in addition to the substances mentioned above. Suitable precursors may further be added to the culture medium. The feed material may be added to the culture all at once or may be appropriately supplied during the culture.

The pH of the culture can be adjusted by appropriate use of a basic compound (eg sodium hydroxide, potassium hydroxide or ammonia) or an acidic compound (eg phosphoric acid or sulfuric acid). Foaming can be controlled using foaming agents such as fatty acid polyglycol esters. The stability of the plasmid can be maintained by the addition of a suitable substance that selectively acts, for example antibiotics. Oxygen or oxygen-containing gas mixtures, such as air, may be introduced into the culture to maintain aerobic conditions. Incubation temperature is usually 20 to 45 ℃, preferably 25 to 40 ℃.

The invention is explained in greater detail through the following examples. However, the present invention should not be limited by these examples.

The novel promoter N1 or N2 derived from Corynebacterium glutamicum according to the present invention exhibits strong promoter activity without specific conditions, inducing high expression of a target gene in various cells including coryneform bacteria and efficiently There is an advantage that can be obtained.

Example  1: Construction of a Recombinant Vector Containing a Promoter Sequence

(1) Selection of novel promoter target genes derived from Corynebacterium glutamicum

After culturing Corynebacterium glutamicum ATCC 13032 in a 5 liter fermenter, the cells were collected. The cells were examined for mRNA expression levels of about 3000 genes through genomic DNA chips (Genomic Tree, Inc., cDNA chip). Among the genes, genes butA and pyk, which account for 2.8% and 1.11% of total genome expression, were selected as target genes for the novel promoter of the present invention.

(2) amplification of expected promoter region DNA fragments

The base sequence of the Corynebacterium glutamicum genome gene is already clearly identified and published (Appl. Microbiol. Biotechnol., 62 (2-3), 99-109 (2003): GenBank Accession No. NC — 003450). Genetic information (butA; NCgl2582, / pyk; NCgl2008) of proteins was obtained from the National Institutes of Health (NIH GenBank). Based on the reported base sequence to amplify the predicted promoter (SEQ ID NO: 1: butA promoter N1, SEQ ID NO: 2: pyk promoter N2) located at the front of each protein open reading frame (ORF) as shown in Table 1 below. A primer comprising the enzyme EcoRV cleavage site was synthesized.

As a template, chromosomal DNA of Corynebacterium glutamicum ATCC 13032 strain was used as a template, and PCR (PCR) was performed using primers 1 and 2 (SEQ ID NOs: 3 and 4) and primers 3 and 4 (SEQ ID NOs: 5 and 6), respectively. Sambrook et al, Molecular Cloning, a Laboratory Manual (1989), Cold Spring Harbor Laboratories (PCR conditions: denaturation = 94 ° C, 1 min / annealing = 55 ° C, 1 min / polymerization = 72 ° C, 30 seconds, 30 times) was used to amplify the expected promoter region of the butA gene and pyk gene was prepared.

Primer sequence N1 Primer 1 (SEQ ID NO: 3) AAGCTG GGTACC GATAG TTCAA ACCGC Primer 2 (SEQ ID NO: 4) ACTTC CTCTT GCTTC CTCAA N2 Primer 3 (SEQ ID NO: 5) AAGCTG GGTACC GTCTG TTGCA GCAGA Primer 4 (SEQ ID NO: 6) CATAA GCCTA GTACG TCATT Gfp Primer 5 (SEQ ID NO: 7) GATATC ATGAG TAAAG GAGAA GAACT Primer 6 (SEQ ID NO: 8) CTGCAG TTATT TGTAG TGAGC TCATC

(3) Construction of vector for measuring promoter activity

In this example, in order to measure the activity of the predicted promoter regions of the butA and pyk genes prepared above in Corynebacterium, the reporter genes GFP and pECCG117-N1-GFP and pECCG117-N2-GFP vectors inserted with the new promoter were inserted. Each was produced.

DNA fragments and GFP obtained in Example 1- (2) To insert the gene into Corynebacterium, the following process was carried out. First, GFP is based on a pGFuv vector (clontech, USA), amplified using primers SEQ ID No. 7 and 8 (Table 1), and ligated to pECCG117 using restriction enzymes EcoRV and PstI, respectively, to pECCG117-GFP. The vector was obtained (Korean Patent Publication No. 92-7401 and Korean Patent Registration No. 10-0620092), and further processed by restriction enzymes with EcoRV to insert 300bps of new promoters (N1, N2) in front of the GFP gene, respectively, pECCG117 -N1-GFP and pECCG117-N2-GFP vectors were constructed (FIG. 1).

Example  2. New  Of a vector comprising a promoter sequence Corynebacterium  My insert

After the above-described pECCG117-N1-GFP and pECCG117-N2-GFP vectors were transformed into L-lysine producing strain Corynebacterium glutamicum KFCC 10881, respectively, (Appl. Microbiol. Biotechnol. ( 1999) using a transformation method of 52: 541-545), and colonies with susceptibility to the antibiotic kanamycin were selected in a selection medium containing kanamycin 25 mg / L.

The strain transformed with the vector containing the promoter N1 was named CA01-0086, and the strain transformed with the vector containing the promoter N2 was named CA01-0087. As of July 23, 2007, the strain CA01-0086 was transferred to the Korea Microbiological Conservation Center. Was deposited with accession number KCCM 10875P, and CA01-0087 strain was deposited with accession number KCCM 10876P.

Example  3: In the Corynebacterium  Activity of promoter sequence

(1) Predicting promoter expression intensity by measuring activity of GFP gene

The obtained transformed strains were cultured in the following manner to measure the activity of the promoter sequence.

Medium [glucose 20 g, ammonium sulfate 5 g, yeast extract 5 g, urea 1.5 g, KH ₂ PO ₄ 4 g, K ₂ HPO ₄ 8 g, MgSO ₄ 7 H ₂ O 0.5 g, biotin 150 μg, thiamine hydrochloride 1.5 mg 1 mg of Corynebacterium glutamicum strains transformed into a 250 ml corner-baffle flask containing 25 ml of calcium pantothenic acid, 3 mg of nicotinamide (1 liter of process water), pH 7.2]. Each was inoculated at a rate and shaken (200 rpm) at 30 ° C. until the middle stage of the culture (absorbance = 10). After completion of the culture, the cells were recovered by centrifugation, suspended in 100 mM Tris-Hcl buffer (pH 7.0), and the cells were disrupted by ultrasonic disruption, followed by high-speed centrifugation to obtain a supernatant. 1 mg of protein was taken from the supernatant obtained by the above method, and GFP expression intensity was measured to determine the degree of enzyme activity. Table 2 shows the results of measuring the GFP activity of the strains with each promoter replaced. As a result, it was confirmed that the strains each promoter was replaced with GFP activity.

Strain Gfp  Expression intensity ( FU Of mg ) CA01-0086 733.4 CA01-0087 1230.3 KFCC10881 0

(2) Comparison of promoter activity by measuring activity of GFP gene

For comparison of the promoter activity of the promoters N1 and N2 of the butA and pyk genes of the present invention with those of other promoters derived from Corynebacterium glutamicum, the gene derived from Corynebacterium glutamicum is mdh (horse Gene coding for rate dehydrogenase), gap (gene coding for glyceraldehyde-3-phosphate dehydrogenase), fba (fructose-bisphosphate aldo) Genes encoding fructose-bisphosphate aldolase), and rotamase (genes encoding peptidyl-prolyl cis-trans isomerase). Gene information of proteins (mdh; NCgl2297 / gap; NCgl1526 / fba; NCgl2673 / rotamase; NCgl0033) was obtained from the National Institutes of Health (NIH GenBank).

 Promoters for each gene were prepared by the method described in Example 1, followed by the method described in Example 2 to prepare a transformant with each promoter inserted. GFP expression intensity by the method of Example 3- (1) together with the transformants (CA01-0086, CA01-0087) of the present invention to compare the promoter activity of the transformants inserted with the promoter sequences of the different genes Was measured.

Table 3 below shows the results of comparing GFP activity of Corynebacterium glutamicum-derived promoters.

gene NCgl Gfp  Expression intensity ( FU Of mg ) butA NCgl2582 733.38 pyk NCgl2008 1230.30 mdh NCgl2297 43.69 gap NCgl1526 173.42 fba NCgl2673 40.15 rotamase NCgl0033 21.23

As shown in the above results, strains in which the novel promoter N1 or N2 of the present invention (strains in which the promoter of butA or pyk gene is inserted) are inserted into other promoters derived from Corynebacterium glutamicum Compared to the GFP expression intensity was high, which means that the promoter activity is large.

From the above description, those skilled in the art can understand that the present invention can be implemented in other specific forms without changing the technical spirit or essential features. In this regard, it should be understood that the examples and experimental examples described above are exemplary in all respects and not restrictive. The scope of the present invention should be construed to encompass the meaning and scope of the following claims rather than the foregoing detailed description, and all changes or modifications derived from the equivalent concept thereof are included in the scope of the present invention.

The novel promoter N1 or N2 derived from Corynebacterium glutamicum according to the present invention exhibits strong promoter activity without specific conditions, and can be used for the development of high titer strains capable of expressing the target protein with high efficiency, and include amino acids, nucleic acids, And it can increase the production efficiency of industrial microorganisms, including coryneform bacteria that produce proteins and the like.

1 is a schematic diagram showing a method for producing a promoter search vector. Green Fluorescence Protein (GFP) enzyme method was used to measure the activity of the promoter.

<110> CJ corporation <120> Novel Corynebacterium glutamicum promoter <130> PA9707-0484 <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 300 <212> DNA <213> Corynebacterium glutamicum <220> <221> promoter (222) (1) .. (300) <223> promoter of but A gene <400> 1 gatagttcaa accgccaggc aacttcactg agattccacc acgcgaatta atagtcaccg 60 gcccgattcg cttcgatccg gaaacgccag acttagaaac attcagccaa ctgtcttttc 120 cggtcttaat aattttccga taattcagtc ccatgtgaac cagcataatc ttcatcacat 180 ggtgtgtgca gaaatatttt tgctggtcta ttgtggcgac cgagggcctt tgaaggttcg 240 acaaactgta taaggccttg aatcttgaga atttattttg aggaagcaag aggaagtgtc 300                                                                          300 <210> 2 <211> 300 <212> DNA <213> Corynebacterium glutamicum <220> <221> promoter (222) (1) .. (300) <223> promoter of pyk gene <400> 2 gtctgttgca gcagatgctg tagcttcgcc ggatggaaaa cccttgccga aagcagggga 60 gggcattgat ggagaaacgc cctcaacgcg ataggtttca accataggcc tgacctggct 120 gagatgtttt tggtagaaaa accgagtgcc cgaattgttt tgtgggtgcc cggttttttc 180 tgatttaagc acgtcagagg cgtagaacat tgtctgttca cactctgggt cgcaagattc 240 atcgagaatt aatggtagta cctgtggctt gagggggaat gacgtactag gcttatgggc 300                                                                          300 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer 1 for amplifying the promoter of but A gene <400> 3 aagctgggta ccgatagttc aaaccgc 27 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer 2 for amplifying the promoter of but A gene <400> 4 acttcctctt gcttcctcaa 20 <210> 5 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer 3 for amplifying the promoter of pyk gene <400> 5 aagctgggta ccgtctgttg cagcaga 27 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer 4 for amplifying the promoter of pyk gene <400> 6 cataagccta gtacgtcatt 20  

Claims (10)

A promoter nucleic acid molecule consisting of the nucleotide sequence of SEQ ID NO: 1 or 2. A vector comprising the promoter nucleic acid molecule of claim 1. The vector of claim 2, wherein the vector is pECCG117-N1-GFP or pECCG117-N2-GFP shown in FIG. 1. A transformant transformed with the vector of claim 2. The transformant of claim 4, wherein the transformant is a genus Corynebacterium or Bribibacterium. The transformant of claim 5, wherein the transformant of the genus Corynebacterium is Corynebacterium glutamicum. The transformant of claim 6, which is Corynebacterium glutamicum KCCM 10875P transformed with a vector comprising a promoter of SEQ ID NO: 1. The transformant of claim 6, which is Corynebacterium glutamicum KCCM 10876P transformed with a vector comprising a promoter of SEQ ID NO: 2. 8. A method for expressing a gene of interest comprising culturing the transformant according to any one of claims 4 to 8. A method for producing L-lysine comprising culturing the transformant according to any one of claims 4 to 8.

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