WO2008097048A1

WO2008097048A1 - Novel promoter and uses thereof

Info

Publication number: WO2008097048A1
Application number: PCT/KR2008/000754
Authority: WO
Inventors: Hye Won Kim; Hyejin Choi; Ji-Hye Lee; Soo Youn Hwang
Original assignee: Cj Cheiljedang Corporation
Priority date: 2007-02-08
Filing date: 2008-02-05
Publication date: 2008-08-14
Also published as: KR100860932B1; KR20080074286A

Abstract

The present invention relates to a novel promoter capable of overexpressing a target gene, and uses thereof. In particular, the present invention relates to a novel polynucleotide having a promoter activity, which is derived from Corynebacterium glutamicum, a recombinant vector containing the polynucleotide, a transformant transformed with the recombinant vector to overexpress a target gene, and a method for overexpressing the target gene using the transformant. The transformant transformed with the novel promoter of the present invention produces target genes, particularly amino acids useful in industry, in a high yield. Therefore, the transformant can be used in medicinal and pharmaceutical industries.

Description

Description NOVEL PROMOTER AND USES THEREOF

Technical Field

[1] The present invention relates to a novel promoter capable of overexpressing a target gene, and uses thereof. In particular, the present invention relates to a novel polynucleotide having a promoter activity, which is derived from Corynebacterium glutamicum, a recombinant vector containing the polynucleotide, a transformant transformed with the recombinant vector to overexpress a target gene, and a method for overexpressing the target gene using the transformant. The target gene may be preferably a gene encoding a protein involved in the production of amino acids, particularly L-arginine.

[2]

Background Art

[3] Strains belonging to thegenus Corynebacterium are industrial microorganisms that produce chemical substances having various industrial applications such as animal feed, drugs, food, and the like, which contain L-lysine, L-threonine, L-arginine and various nucleic acids. To develop Corynebacterium capable of producing target materials in a high yieldby using genetic or metabolic engineering, the expression of genes involved in several metabolic pathways should be selectively regulated. Thus, a regulatory gene, promoter, which is recognized by RNA polymerase to initiate gene transcription, should have strong activity or generality. It is necessary to develop promoters for the overexpression of target materials including amino acids and nucleic acids.

[4] To express genes in Corynebacterium, the genes are generally expressed by their own promoters (Vasicova, P., et al., J. Bacteriol. 181, 6188-6191, (1999), etc.). However, unlike other industrial microorganisms such as Escherichia coli and Bacillus subtilis, there is no information on the basic structure of promoter sequences for gene expression in Corynebacterium. For this reason, the following method has been suggested to produce promoters for gene expression in Corynebacterium. First, a promoter region is removed from a gene associated with resistance to an antibiotic such as chloramphenicol. Separately, a genomic DNA separated from coryneform bacteria is cleaved using suitable restriction enzymes, and the resulting fragment is introduced to the promoter sites. Then, the obtained gene is used to transform coryneform bacteria and the antibiotic resistance of the transformed strain is assessed (Eikmanns, BJ., et al., Gene, 102, 93-98, (1991); Patek, M., et al., Microbiology, 142, 1297-1309, (1996)). However, previously developed promoter sequences still need to be improved with respect to selectivity and efficiency of gene expression.

[5] L-arginine is a semi-essential amino acid, of which supplementation is required for growing animals. L-arginine has been widely used as an efficient additive in medicaments, food, or the like, and is useful as a drug for improving the hepatic function and brain function and treating male sterility, and as an ingredient of multiple amino acid supplements. Also, L-arginine has been used as a food additive in fish cakes and health beverages, and has recently gained interest as a salt substitute for hypertension patients.

[6] It has been known that in Corynebacterium glutamicum, an argCJBDF gene involved in arginine biosynthesis exists as an operon, and is regulated by feedback- inhibition due to arginine (Vehary Sakanyan, et al, Microbiology, 142:9-108, 1996). Thus, there is a limit in producing L-arginine in a high yield. Accordingly, the present inventors have made an effort to develop strains capable of producing L-arginine in higher yield. As disclosed in Korean Patent application No. 2007-5831, they found that a microorganism transformed with an argD2 gene, which is a putative gene having the same function as an argD gene encodingacetylornithine aminotransferases, over- expresses the argD2 gene to produce L-arginine in higher yield than a parent strain. Therefore, to produce a target material in a high yield, there is a need to overexpress the gene encoding the target material, and develop a more powerful promoter.

[7]

Disclosure of Invention

Technical Problem

[8] It is an object of the present invention to provide a novel polynucleotide having a promoter activity, derived from Corynebacterium glutamicum, or a fragment or variant thereof having the same activity.

[9] It is another object of the present invention to provide a recombinant vector comprising the polynucleotide having a promoter activity.

[10] It is still another object of the present invention to provide a transformant transformed by introducing the recombinant vector into a host microorganism.

[11] It is still another object of the present invention to provide a method for over- expressing a target gene using the transformant.

[12]

Technical Solution

[13] Accordingly, in order to identify a powerful promoter capable of overexpressing a target gene, the present inventors have studied a region having the promoter sequence, derived from Corynebacterium glutamicum. They found that a CJP promoter of the present invention can express a target gene in Corynebacterium glutamicum, and overexpress the argD2 genein the transformant transformed with the argD2 geneto produce L-arginine in higher yield than a parent strain, thereby completing the present invention. [14]

Advantageous Effects

[15] The transformant transformed with the novel promoter of the present invention produces target genes, particularly amino acids useful in industry, in a high yield.

Therefore, the transformant can be used in medicinal and pharmaceutical industries. [16]

Brief Description of the Drawings [17] Fig. 1 is a photograph showing the result of silver staining, after two-dimensional electrophoresis of cell lysate of Corynebacterium glutamicum; [18] Fig. 2 illustrates the construction of a recombinant vector "pHC139T-gfp" for monitoring promoter activity, which is prepared by inserting a rrnB B terminator into a pECCGl 17-CJl vector containing a gfp gene encoding a green fluorescent protein (GFP) and substituting the CJl promoter with the CJP promoter of the present invention; and [19] Fig. 3 illustrates the construction of a recombinant vector "pHC139T-argD2" capable of overexpressing an argD2 gene (Ncgl2355), which is a putative gene of acetylornithine aminotransferase involved in arginine biosynthesis of Corynebacterium glutamicum, by the CJP promoter of the present invention. [20]

Best Mode for Carrying Out the Invention [21] In one embodiment, the present invention provides a novel polynucleotide having a promoter activity, derived from Corynebacterium glutamicum. Preferably, the polynucleotide may be represented by a base sequence of SEQ ID NO. 1, and a polypeptide therefrom is designated herein as "CJP promoter". [22] The polynucleotide of the present invention is an isolated nucleic acid having a promoter activity. The term "promoter" as used herein refers to a DNA region which is recognized by RNA polymerase to initiate gene transcription, and positions at the 5' direction of an mRNA transcription initiation site. [23] In a specific embodiment of the present invention, the activity of the CJP promoter of the present invention was compared to that of the tac promoter. [24] The term "tac promoter" refers to a promoter obtained by fusing a sequence obtained from the - 10 region of a tryptophan operon promoter of E.coli and a sequence obtained from the - 10 region of a lactose operon promoter of E.coli. The tac promoter is known to have a strong promoter activity. [25] The "CJP promoter" represented by SEQ ID NO. 1 of the present invention is a promoter of Ncgll341 gene of Corynebacterium glutamicum ATCC 21831, and has activity in bacteria belonging to the genus Corynebacterium.

[26] From the result of comparing the activities of two promoters, it was found that the expression of target gene by the CJP promoter of the present invention was increased by 26%, when the expression of target gene by the tac promoter was taken as 100%

[27] The polynucleotide having a promoter activity of the present invention may be modified to a certain degree by any one of several recently developed methods, for example, directed evolution or site-directed mutagenesis. Those skilled in the art will readily appreciate that variants, in which one or more base bases of the novel polynucleotide according to the present invention are altered by substitutions, deletions, insertions or combinations thereof, or functional fragments thereof exerting identical activity, are equivalent to the novel promoter of the present invention, as long as they retain a promoter activity of expressing a target gene.

[28] In addition, the present invention includes polynucleotides having a promoter activity, selected from the group consisting of nucleotide sequences complementary to the base sequence of polynucleotide having a promoter activity of the present invention, which are derived from Corynebacterium glutamicum and represented by SEQ ID NO. 1.

[29] The term "complementary" as used herein refers to the hybridization or base-pairing between polynucleotides or nucleic acids, for example, between the two strands of a double- stranded DNA molecule or between an oligonucleotide primer and a primer binding site on a single-stranded nucleic acid to be sequenced or amplified.

[30] The Corynebacterium glutamicum promoter of the present invention is a promoter derived from Coryneform bacterium, and is useful for gene expression preferably in prokaryotic cells, and more preferably in Coryneform bacterium.

[31] Conventional methods for promoter isolation include (1) the use of a promoter probe vector system in the random cloning of genomic DNA fragments at the upstream region of a reporter gene, which is expressed only when a cloned fragment contains promoter activity; (2) gene-specific probe-based hybridization by which a gene and its promoter are isolated from a genomic library; and (3) differential hybridization of an inducible and non-inducible cDNA probe to a gene bank.

[32] The polynucleotide of the CJP promoter of the present invention may be isolated or prepared from microorganism belonging to the genus Corynebacterium using a standard molecular biology technique. For example, it may be isolated by PCR using proper primer sequences. Also, it may be prepared by a standard synthesis technique using an automated DNA synthesizer.

[33] Those skilled in the art will readily appreciate that the promoter according to the present invention maintains its activity in any suitable microorganism as well as in microorganisms belonging to the genus Corynebacterium.

[34] Examples of the microorganisms belonging to the genus Corynebacterium include

Corynebacterium glutamicum ATCC 13032, ATCC 21831, ATCC 21493, and L- amino acid-producing mutants prepared therefrom, such as Corynebacterium glutamicum KCCM-10741, Corynebacterium glutamicum KFCC 10881, Corynebacterium glutamicum KFCC 11001, Corynebacterium ammoniagenes CJHBlOO (KCCM- 10330) and ATCC 6871, but are not limited thereto.

[35] In another embodiment, the present invention provides an expression cassette containing the CJ9 promoter of the present invention, operably linked to a coding sequence. The coding sequence may be, for example, the entire gene or a coding sequence which encodes a predetermined region of the gene. Examples of the coding sequence may include a coding sequence of a gene associated with a metabolic product such as amino acid (e.g., L-lysine, L-threonine, or L-arginine), GMP, and IMP, but are not limited thereto. The expression cassette of the present invention may further include 5' and 3' control sequences operably linked to the coding sequence and novel promoter according to the present invention.

[36] In still another embodiment, the present invention also provides a vector including the expression cassette containing the promoter of the present invention. The term "vector" as used herein refers to a DNA construct that contains a DNA sequence which is operably linked to a suitable control sequence to express a target gene in a suitable host. Such control sequences may include a promoter to initiate transcription, a certain operator sequence to control such transcription, a sequence encoding suitable mRNA ribosomal binding sites, and a sequence to control termination of transcription and translation. The vector may be a plasmid, a phage particle, or simply a potential genomic insert. Once the vector is transformed into a suitable host, it may replicate and function independently of the host genome, in some cases, may integrate into the genome itself.

[37] The term "operably linked" as used herein means that the promoter is functionally linked to the coding sequence to initiate and mediate transcription of the coding sequence. The vector used in the present invention is not specifically limited, and may be any vector known in the related art. Example thereof may include pECCGl 17 (KFCC- 10763), but is not limited thereto.

[38] In a specific embodiment, a pECCGl 17-CJl (Korean Patent Application No.

10-2004-107215) vector containing a gfp gene, which codes for a green fluorescent protein (hereinafter, abbreviated to "GFP"), was used to measure activity of the CJ9 promoter of the present invention. To stably express the vector, an rrnB B terminator was inserted into the vector, and the CJl promoter was substituted with the CJ9 promoter of the present invention to manufacture a pHC139T-gfp vector. Meanwhile, the recombinant vector was transformed into E.coli DH5a, and the transformed microorganism was designated as CA06-0014, which was deposited at the Korean Culture Center of Microorganisms on December 13, 2006 under accession number KCCM10822P.

[39] In a preferred embodiment of the present invention, the gfp gene was removed from the pHC139T-gfp vector, and then ligated with a DNA fragment containing ORF (Open Reading Frame) of the argD2 gene to manufacture an αrgD2-overexpressing pHC139T-argD2 vector.

[40] The argD2 gene(Ncgl2355) is a putative gene of acetylornithine aminotransferase involved in arginine biosynthesis of Corynebacterium glutamicum, and is known to be classified into aminotransferases subgroup II according to genome based analysis (Alice C. McHardy, et al, J. Biotechnology, 104, 229-240, 2003). However, function of the protein encoded by the argD2 genehas not been clearly identified. There is a little sequence homology between argD and argD2 genes, however, both genes have the same motif. Therefore, it is inferred that the protein encoded by the argD2 gene has similar function to acetylornithine aminotransferase that is encoded by the argD gene and required for arginine biosynthesis (Vww.genome.jp/keggA). As disclosed in Korean Patent Application No. 2007-5831, the present inventors first found that an L-arginine producing strain which was transformed with a recombinant vector containing the argD2 genehas higher productivity than the parent strain. The disclosure thereof is incorporated herein by reference in its entirety. In the present invention, the present inventors inferred that the expression vector having the CJ9 promoter instead of CJl promoter overexpresses the argD2 gene to produce L-arginine in much higher yield, compared to the vector disclosed in Korean Patent Application No. 2007-5831. They found that the novel CJ9 promoter according to the present invention has an activity of inducing the overexpression.

[41] In one embodiment, the present invention provides a method for overexpressing target genes by operably linking the target genes encoding various proteins to the polynucleotide of the novel promoter. Examples of the target gene include argB, argC, argF, argF2, and gfp, but are not limited thereto.

[42] In still another embodiment, the present invention provides a transformant, prepared by transforming a host microorganism with the recombinant vector containing a target gene.

[43] Specifically, the host microorganism has high transfection and expression efficiency, and may be any microorganism including prokaryotic or eukaryotic cells, preferably microorganisms belonging to the genus Corynebacterium, and more preferably Corynebacterium glutamicum ATCC21831. In a specific embodiment of the present invention, if the target gene to be overexpressed is the argD2 gene, the host microorganism may be microorganisms capable of producing L-arginine, preferably microorganisms belonging to the genus Corynebacterium, which have a resistance to L-arginine analogues and are capable of producing L-arginine, but is not limited thereto, as long as it overexpressesthe argD2 gene to produce L-arginine in a high yield.

[44] The transformant can be easily prepared by those skilled in the art according to any known method. The term "transformation" as used herein means introducing DNA into a host cell so that DNA is replicable, either as an extrachromosomal element or by chromosomal integration. That is, transformation is artificial genetic alteration by introducing a foreign DNA into a host cell. Examples thereof include a CaCl precipitation, a Hanahan method that is an improved CaCl method by using DMSO (dimethyl sulfoxide) as a reducing material, electroporation, calcium phosphate precipitation, protoplast fusion, agitation using silicone carbide fiber, Agrobacterium- mediated transformation, PEG-, dextran sulfate-, and lipofectamine-mediated transformation.

[45] In the transformant to overexpress argD2 gene, prepared according to a specific embodiment of the present invention, the argD2 gene present in the chromosome of the transformant may be additionally subjected to expression or deletion by a conventional recombinant technique to enhance the overexpression. It is well known in the related art that its base sequence can be analyzed by a sequencing method using fluorescence.

[46] In a specific embodiment of the present invention, PCR was performed to obtain the argD2 gene, which encodes a putative protein having the same function as acety- lornithine aminotransferases, from the chromosome of L-arginine-producing strains, Corynebacterium glutamicum ATCC21831, and the obtained argD2 gene was inserted into the gfp region of pHC139T-gfp vector containing the novel CJ9 promoter according to the present invention to manufacture a pHC139T-argD2 vector. Then, electroporation was performed to introduce the pHC139T-argD2 vector into an L- arginine-producing host microorganism, for example, Corynebacterium glutamicum ATCC21831, which has a resistance to L-arginine analogues and is capable of producing L-arginine, to manufacture a transformant capable of overexpressing the argD2 gene to produce L-arginine in a high yield. In the present invention, the transformant was designated as CA06-0015, and deposited at the Korean Culture Center of Microorganisms (hereinafter, abbreviated to "KCCM") on December 13, 2006 under accession number KCCM10823P.

[47] In the biosynthetic pathway of L-arginine, ornithine is an intermediate of the metabolic pathway of arginine and is an important material in nitrogen metabolism along with the urea cycle. It was found that the CA06-0015 strain transformed by the method of the present invention overexpressed the argD2 geneto produce L-arginine in a high yield.

[48] In a specific embodiment, the present invention provides a method for producing L- arginine, comprising the step of culturing the transformant, preferably the transformant identified by accession number KCCM10823P. In particular, the method for producing L-arginine comprises the steps of (a) culturing the transformant prepared by the present invention; (b) enriching L-arginine in the broth or microorganisms; and (c) separating residual L-arginine and any constituent of the fermentation broth and/or the biomass. In the method for producing L-arginine, the transformant of step (a) may be preferably one identified by accession number KCCM10823P, but the desired product is not limited to L-arginine. In the present invention, various polypeptides and proteins may be produced by culturing the transformant transformed with the expression vector containing a target gene linked to the novel promoter of the present invention.

[49] In the production method of L-arginine according to the present invention, the cultivation of the transformed L-arginine overexpressing microorganisms may be conducted in suitable media and under culture conditions known in the art. The culturing procedures can be readily adjusted by those skilled in the art according to the selected strain. Examples of the culturing procedures include batch type, continuous type and fed-batch type manners, but are not limited thereto. Various culturing procedures are disclosed in literature, for example, "biochemical Engineering" (James M. Lee, Prentice-Hall International Editions, ppl38-176, 1991); Chmiel (Bioprozesstechnik 1. Einfuhrung in die Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991); and Storhas (Bioreaktoren und periphere Einrichtungen (Vieweg Verlag, Braunschweig/Wiesbaden, 1994)).

[50] During cultivation, ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid may be properly added to adjust the pH of the cultures. Defoaming agents such as fatty acid polyglycol ester may be properly added to reduce the formation of foams in cultures. Further, to maintain the cultures in aerobic states, oxygen or oxygen-containing gas (e.g., air) may be injected into the cultures. The cultures are maintained at 20 to 45⁰C and preferably at 25 to 4O⁰C. The cultivation may be continued until a desired amount of L-arginine is obtained, and preferably for 10 to 160 hrs. The isolation of L-arginine from the culture broth may be performed by the conventional method known in the art. Examples thereof may include centrifugation, filtration, ion-exchange chromatography, and crystallization. For example, the cultures may be subjected to low-speed centrifugation to remove the biomass, and the supernatant may be separated by ion-exchange chromatography.

[51] Mode for the Invention

[52] Hereinafter, the present invention will be described in more detail with reference to

Examples. However, these Examples are for illustrative purposes only, and the invention is not intended to be limited by these Examples.

[53]

[54] Example

[55] In Examples of the present invention, Corynebacterium glutamicum ATCC 21831 was cultured in a 5 L fermentor to prepare a cell lysate, and two-dimensional electrophoresis was performed to screen the overexpressed proteins. Then, the selected proteins were cleaved, and their peptide sequence was analyzed. Using the obtained peptide sequence data, the genes of the overexpressed proteins were identified to isolate promoter regions of the genes. Next, a vector containing the obtained promoter region was prepared to assess the promoter activity in Corynebacterium glutamicum ATCC21831.

[56]

[57] Example 1. Extraction of peptide overexpressed in Corynebacterium glutamicum ATCC21831

[58] Example 1-1. Preparation of cell lvsate

[59] Corynebacterium glutamicum ATCC21831 was cultured in a medium containing

7.7% glucose. At this time, the cell concentration was measured. When the culture reached the exponential phase, 10 ml of culture media was collected. The sample was centrifuged (12,000 rpm, 10 min) to remove the supernatant. The obtained cell pellet was resuspended in a lysis buffer (1OmM TrisHCl, pH 8.5) to prepare about 100 D of cell lysate.

[60]

[61] Example 1-2. Two-dimensional electrophoresis analysis

[62] Two-dimensional electrophoresis was performed to screen the overexpressed polypeptides from the cell lysate of Corynebacterium glutamicum ATCC21831, prepared in Example 1-1.

[63] To prepare a sample for Two-dimensional electrophoresis, the cell lysate prepared in Example 1-1 was diluted using 6 M urea, 2 M thiourea, 4% CHAPs (Bio-Rad, Hercules, CA), and 0.4% dithiothreitol (hereinafter, abbreviated to "DTT"), and then 7 D of IPG (Immobilized pH Gradient gel strip) buffer and 3 D of 1% Bromophenol Blue (hereinafter, abbreviated to "BPB") were added thereto to give a final volume of 350 D. The prepared sample was loaded onto a rehydration tray using an immobiline™ pH gradient Drystrip (Amersham Bioscience, USA), and then rehydrated at room temperature for about 2 hrs. [64] The rehydrated strip gel was subjected to isoelectric focusing (IEF) at 25 ⁰C at 0 to

100 V for 1 hr, at 300 V for 1 hr, at 600 V for 1 hr, and at 8000 V for a predetermined time, which was adjusted to perform focusing for about 43-97 kVhr (pH 4-7; 43.4 kVhr, pH 4.5-5.5; 97kVhr, pH 5.5-6.7; 97 kVhr) using a Multiphor II electrophoresis chamber (Amersham Pharmacia Biotech, USA).

[65] After the isoelectric focusing was completed, each strip gel was equilibrated with equilibration buffer containing 20 mM Tris-HCl (pH 8.8), 6 M urea, 2% SDS, 20% glycerol, 2.5% acrylamide and 5 mM TBP (tributylphosphate) for 15 min. Then, each equilibrated strip was loaded onto a two-dimensional gel (9-16% concentration gradient), and then sealed with an SDS solution containing 0.5% agarose having a low boiling point and 0.001% BPB. The electrophoresis was performed at 100 V for about 19 hrs.

[66] After the electrophoresis was completed, the gel was immobilized in a 45% methanol solution and 5% acetic acid solution. The immobilized gel was washed with distilled water for 1 hr to remove acetic acid. The gel was sensitized with 0.02% sodium thiosulfate for 2 min, and then washed with distilled water. Next, the gel was reacted with 0.1 % silver nitrate for 20 min and washed with distilled water. The reaction product was developed using a solution containing 2% (w/v) sodium carbonate (Na CO ) and 0.04% (v/v) formaldehyde. When a spot of the desired strength appeared, the reaction was stopped using 1 % acetic acid. The gel was washed with distilled water to remove acetic acid, sealed in a plastic bag, and stored in a cold lab chamber at 4⁰C.

[67]

[68] Example 1-3. Preparation of peptide sample for mass spectrometry

[69] Peptide samples used for mass spectrometry were prepared from the spots of the gel in Example 1-2. The peptides were separated from the spots using a modified version of a known method (Shevchenko et al., Anal. Chem., 68(5), 850-8, 1996).

[70] First, the protein spots were excised from the gel prepared in Example 1-2, and destained in 120 D of a mixed solution of 30 mM potassium ferricyanide and 100 mM of sodium thiosulfate at a ratio of 1:1. The destained spots were washed with distilled water, and then with 120 D of 50% acetonitrile/25 mM ammonium bicarbonate (pH 7.8) for 10 minutes. The resultants were reacted with 50 D of 100% acetonitrile for about 5 min until the spots changed to white, and then vacuum-dried.

[71] 10 D of trypsin (0.02 D/D) was added to the dried spots and then subjected to reaction on ice for 45 min. Then, 10 D of 50 mM ammonium bicarbonate buffer (pH 7.8) was added to the reaction solution, and subjected to reaction at 37⁰C for 12-14 hrs. The reaction solution was cooled to O⁰C to terminate the reaction, and then sonicated three times at 4⁰C for 10 minutes in 10 D of 0.5% TFA (trifluoreacetic acid) and 50% ace- tonitrile to extract peptides. [72] [73] Example 2. Selection of overexpressed protein by mass spectrometry

[74] HPLC-MS/MS was used for mass spectrometry of the peptides extracted in

Example 1-3. HPLC-MS/MS analysis was performed using 1100 series HPLC system (Agilenmt, USA) and a Finnigan LCQ DECA ion-trap mass spectrometry device (ThermoQuest, USA) equipped with a nanospray ionization source.

[75]

[76] Example 2-1. Peptide separation by HPLC

[77] HPLC was performed using a Cl 8 microprobe reverse phase HPLC column, 0.1 % formic acid (solvent A), and 90% (v/v) acetonitrile and 0.1 % formic acid (solvent B) provided in a linear grade (flow rate=l D/min) to separate the peptides extracted in Example 1-3.

[78]

[79] Example 2-2. Detection of peptide

[80] The peptide detection was performed three times using NSI (nanospray ionization)

(spray voltage: 1.8 kV; capillary temperature: 200⁰C; capillary voltage: 34 V; pipe lens offset: 40 V; and electron multiplier: -60 V). The measurements were obtained in a centroid mode. After the entire MS scan of 400-2000 Da was obtained from the result of NCI, a threshold value was set to 1x105 counts and the strongest ions were separated through a high resolution zoom scan. Then, collision-induced dissociation (hereinafter, abbreviated to "CDI") MS/MS was performed. The sequence of CID spectrum that was not encoded was identified using TurboQuest software (Thermo Finnigan, USA). The results of SEQUEST search were identified through cross- correlation and ΔCn (delta nomalized cross-correlation).

[81]

[82] Example 2-3. Analysis and identification of amino acid sequence of peptide

[83] Q-star Pulsar LC MS/MS (Applied Biosystems, USA) was used to analyze amino acid sequences of the peptides analyzed by the methods of Examples 2-1 and 2-2. As a result, 40 proteins were found, and one overexpressed protein was selected among them.

[84] Fig. 1 is a photograph showing the result of silver staining, after the cell lysate prepared in Example 1-1 was subjected to two-dimensional electrophoresis by the method of Example 1-2. As shown in Fig. 1, the selected CJ9 protein was found to be one of the overexpressed spots. To confirm the function of CJ9 protein, the amino acid sequence of the identified peptide was compared to that in the NCBI gene bank database. As a result, the CJ9 protein identified by mass spectrometry or the like was found to be acetylglutamate semialdehyde dehydrogenase. [85] [86] Table 1

[87] [88] The gene sequence was determined from the amino acid sequence of the over- expressed CJ9 protein, which was selected in Examples of the present invention, and analyzed to select a promoter region. As a result, it was assumed that an oligonucleotide of SEQ ID NO. 1, which was separated from the base sequence of gene encoding the CJ9 protein, has promoter activity.

[89] [90] Example 3. Construction of recombinant vector containing C J9 promoter sequence

[91] To assess the activity of CJ9 promoter, a pECCGl 17-CJl vector (Korean Patent Application No. 10-2004-107215) containing a gfp genewhich encodes a green fluorescent protein (hereinafter, abbreviated to "GFP") was used, and to stably express the vector, an rrnB B terminator was inserted to the vector. Next, to confirm the promoter activity of the base sequence obtained in Example 2-3, which is a putative CJ9 promoter, the CJl promoter was substituted with the putative base sequence of CJ9 promoter (Fig. 2).

[92] [93] Example 3-1. Amplification of putative promoter region [94] 500 D of the genomic DNA (gDNA) was extracted from 25 ml c/Corynebacterium glutamicum ATCC21831, which was cultured for 1 day according to the method of Eikmann, et al (Gene, 102:93-98, 1991). In order to amplify the DNA fragment of putative CJ9 promoter, polymerase chain reaction (hereinafter, abbreviated to "PCR") was performed using the gDNA as a template and a PTC-200 Peltier Thermal Cycler (MJ Research, USA, hereinafter the same). At this time, primers used in the amplification of the DNA fragment of putative CJ9 promoterwere represented by SEQ ID Nos. 2 and 3, which are given in Table 2. PCR conditions included 30 cycles of de- naturation at 94⁰C for 1 min, annealing at 55⁰C for 1 min and extension at 72⁰C for 30 sec. The PCR product was subjected to electrophoresis on a 0.8% agarose gel, and then a band of 0.4 kb was eluted from the gel.

[95] [96] Table 2

[97] [98] Example 3-2. Amplification of rrnB B terminator

L~Z_ [99] The genomic DNA (gDNA) was extracted from E.coli K- 12 W3110 using a

TM

Masterpure Gram positive DNA purification kit (Epicentre, hereinafter the same). To amplify the rrnB B terminator, PCR was performed using the gDNA as a template and a PTC-200 Peltier Thermal Cycler. At this time, primers used in the amplification of rrnB B terminator were represented by SEQ ID Nos. 4 and 5, which are given in Table 3. PCR conditions are the same as in the amplification of DNA fragment of putative CJ9 promoter of Example 3-1. The PCR product was subjected to electrophoresis on a 0.8% agarose gel, and then a band of 411 bp was eluted from the gel.

[100] [101] Table 3

[102] [103] Example 3-3. Construction of recombinant vector pECCG117-CJl-rrnB B

L 2 [104] The pECCG117-CJl vector (Korean Patent Application No. 10-2004-107215) containing a gfp genewhich encodes a green fluorescent protein (hereinafter, abbreviated to "GFP") and the rrnB B terminator amplified in Example 3-2 were treated with Xbal/Notl, respectively and then ligated with each other using a Quick ligation kit (NEB, hereinafter the same) to manufacture a recombinant plasmid pECCG117-CJl-rrnB B (about 7.3 Kb), which was designated as "pHC131T-gfp" in the present invention.

[105] [106] Example 3-4. Construction of vector for monitoring activity of CJ9 promoter [107] The pHC131T-gfp vector prepared in Example 3-3 was cleaved with Kpnl/EcoRV, and then subjected to electrophoresis on a 1% agarose gel to elute a DNA fragment of about 7 Kb, excluding the CJl promoter. The obtained DNA fragment and the DNA fragment of putative CJ9 promoter obtained in Example 3-1 were cleaved with the same restriction enzymes as in the present Example, and then ligated with each other using a Quick ligation kit (NEB, hereinafter the same) to manufacture a recombinant vector pHC131T-gfp-CJ9 (about 7.4 Kb). In the recombinant vector, the gfp geneand the oligonucleotide of putative CJP promoter isolated from Corynebacterium glutamicum ATCC21831 werelinked to each other, and the vector was designated as "pHC139T-gfp" in the present invention. In addition, E.coli DH5a was transformed with the recombinant vector. The transformed microorganism was designated as CA06-0014, and deposited at the Korean Culture Center of Microorganisms on December 13, 2006 under accession number KCCM10822P.

[108]

[109] Example 4. Analysis of CJ9 promoter activity and gfp gene expression

[110] Example 4-1. CJP promoter activity

[111] The recombinant vector "pHC139T-gfp" prepared in Example 3-4 was introduced to Corynebacterium glutamicum ATCC21831 ready for transformation according to a method of van der Rest et al. (Appl. Microbiol. Biotechnol., 52:541-545, 1999). The transformed strain was smeared onto a solid medium #3 (0.5% Pepton, 0.3% Beef extract, 1.5% agar) containing 25 ug/ml of kanamycin and cultured at 3O⁰C. The strains grown in the medium were first screened. Next, the strains showing fluorescence under UV radiation were selected from the first screened strains. The fluorescent radiation indicates thatthe CJP oligonucleotide separated from Corynebacterium glutamicum ATCC21831 substantially exhibits promoter activity.

[112]

[113] Example 4-2. Analysis of sfp gene expression

[114] The CJP promoter and gfp gene were contained in the recombinant vector

"pHC139T-gfp" prepared in Example 3-4. Therefore, the gfp gene expression was assessed to quantify the activity of CJP promoter in the present Example.

[115] First, the transformants first selected in Example 4-1 and ATCC21831 were cultured at about 3O⁰C for 15 hrs as an experimental group and control group, respectively. The culture was centrifuged (12,000 rpm, 10 min) to recover the cells from the culture media. The recovered cells were suspended in a protein extraction buffer (PBS containing 1 mM EDTA, 3% glycerol, and 1% solution of triton-X-100, pH 7.5), and disrupted by ultrasonication. The disrupted cells were subjected to centrifugation (12,000 rpm, 5 min), and the supernatant containing cell lysate was collected. Bradford assay was performed to measure the amount of protein contained in the cell lysate. Subsequently, 400 nm pump laser was radiated on the equal amount of cell lysate using a method introduced by Laure Gory et al. (FEMS Microbiology Letters, 194:127-33, 2001) and the emitted light at 509 nm was detected using a Synergy HT microplate reader (Biotek) to assess the gfp gene expression.

[116] The measured fluorescence intensity is given in Table 4. It was found that the CJP promoter of the present invention more effectively expressed the gfp genethanthe control group.

[117] [118] Table 4

[119] [120] Example 5: Comparison of activities of tac promoter and CJ9 promoter [121] In the present Example, the promoter activities of the promoter of the present invention and the known tαc promoter were compared in Corynebαcterium glutαmicum. To use the recombinant vector pECCG17-tac-gfp (Korean Patent Application No. 10-2004-107215) as a control for the comparison of promoter activity of tαc promoter, the recombinant vector was introduced into Corynebαcterium g lutαmicum ATCC21831 by the method of Example 4-1 to prepare a transformant, and then the expression of gfp gene was assessed by the method of Example 4-2.

[122] When the gfp gene expression by the tαc promoter was taken as 100%, the gfp gene expression by the CJ9 promoter of the present invention was found to be increased by 26%, which is shown in Table 5. Accordingly, it can be seen that the CJ9 promoter of the present invention is a strong promoter capable of expressing the gfp gene in a high yield.

[123] [124] Table 5

[125] [126] Example 6: Measurement of L-arginine production titer of transformant having CJ9 promoter

[127] In the present Example, the L-arginine producing strain CA06-0013 (accession number KCCM10821P) was used as a control to compare the L-arginine production titer of transformant which was transformed with the recombinant vector containing the CJ9 promoter of the present invention.

[128] [129] Example 6-1. Construction of αrgD2- overexpressing vector [130] As described below, constructed was a vector capable of overexpressing an αrgD2 gene (Ncgl2355), which is a putative gene of acetylornithine aminotransferase involved in arginine biosynthesis of Corynebacterium glutamicum (Fig. 3).

[131]

[132] <Amplification of CJ9 promoter>

[133] In order to prepare the CJ9 promoter, PCR was performed using the recombinant vector "pHC139T-gfp" prepared in Example 3-4 as a template and a PTC-200 Peltier Thermal Cycler (MJ Research, USA, hereinafter the same). At this time, primers used in the amplification of the CJ9 promoter region containing Kpnl and BamHIrestriction sites were as follows: SEQ ID NO. 6; 5'-aacaaaagctgggtaccggctacttccgaggaatctt-3', SEQ ID NO. 7; 5'-cccttcaatgccatggatccacaccatacacgttatgcatg-3'. PCR conditions included 24 cycles of denaturation at 94⁰C for 30 sec, annealing at 55⁰C for 30 sec and extension at 68⁰C for 1 min. The PCR product was subjected to electrophoresis on a 0.8% agarose gel, and then a band of 0.4 kb was eluted from the gel.

[134]

[135] <Construction of recombinant vector pHC139T>

[136] In order to construct the recombinant vector "pHC139T" from which the CJl promoter and gfp gene were removed, the recombinant vector "pHC139T-gfp" prepared in Example 3-3 was cleaved with Kpnl and BamHI, and subjected to electrophoresis on a 1% agarose gel. Then, a corresponding band was eluted from the gel, and ligation was performed using a Quick ligation kit (NEB, hereinafter the same).

[137]

[138] < Amplification of DNA fragment including ORF oϊargD2 gene>

[139] The genomic DNA (gDNA) was extracted from the L- arginine producing strain

ATCC21831 using a Genomic-tip system (QIAGEN, hereinafter the same). In order to amplify the DNA fragment (1371 bp) including Open Reading Frame (hereinafter, abbreviated to "ORF") of αrgD2 gene, polymerase chain reaction (hereinafter, abbreviated to "PCR") was performed using the gDNA as a template and a PTC-200 Peltier Thermal Cycler (MJ Research, USA, hereinafter the same). At this time, primers used in the amplification of the ORF region of αrgD2 gene were as follows: SEQ ID NO. 10; 5'-tcccccgggggattggcatgaagggttac-3', SEQ ID NO. 11; 5'-gctctagagcttagaacaacgccccagc-3'. PCR conditions are the same as in the amplification of CJ9 promoter. The PCR product was subjected to electrophoresis on a 1.0% agarose gel, and then a band of 1.3 kb was eluted from the gel.

[140]

[141] <Construction of argD2- overexpressing vector pHC139T-argD2 >

[142] The DNA fragment of recombinant vector "pHC139T" cleaved with BamHI and the

DNA fragment including ORF of αrgD2 geneamplified above were ligated using an Infusion ligation kit (Clontech) to construct pHC139T-argD2. At this time, primers of αrgD2 geneused in the In-fusion ligation were as follows: SEQ ID NO. 8; 5'-taacgtgtatggtgtggatccatggcattgaagggttacacc-3', SEQ ID NO. 9; 5'-catccgccaaaacagggatccaaacttagaacaacgcccca-3'. PCR conditions are the same as in the amplification of CJ9 promoter.

[143] [144] Example 6-2. Preparation of transformant [145] The recombinant vector "pHC139T-argD2" prepared in Example 6-1 was introduced into the L-arginine producing parent strain, Corynebacterium glutamicum ATCC21831 by electroporation to prepare the αrgD2-overexpressingtransformant, which was designated as "CA06-0015" in the present invention, and deposited at the Korean Culture Center of Microorganisms (hereinafter, abbreviated to "KCCM") on December 13, 2006 under accession number KCCM10823P.

[146] The ATCC21831 strain and transformant were smeared on LB solid media #3 containing 25 mg/L of Kanamycin (Nutrient medium, hereinafter abbreviated to "#3", composition: 3 g/L of Beef extract, 5 g/L of peptone, 10 g/L of sodium chloride), and cultured at 3O⁰C for 16 hrs. The selected colonies were subjected to a flask titer test as the following Example 6-3. As a result, it was found that due to the CJ9 promoter according to the present invention, the αrgD2-overexpressing transformant produced L-arginine in a high yield.

[147] [148] Example 6-3. Comparison of L-arginine production titer in Erlenmeyer flask [149] The L-arginine producing strain ATCC21831, the CA06-0013 strain (accession number KCCM 1082 IP) which overexpresses the argD2 gene by the CJl promoter to have higher productivity (disclosed in Korean Patent Application No. 2007-5831), and the transformant CA06-0015 (accession number KCCMl 0823P) having the CJ9 promoter according to the present invention, which was prepared from the ATCC21831 strain in Example 6-2, were smeared on solid media #3 containing 25 mg/L of Kanamycin, respectively and cultured at 3O⁰C overnight. Then, 3 single colonies were selected from each strain. The selected colonies were cultured in L- arginine seed media given in Table 6, and then evaluated for L-arginine productivity in an Erlenmeyer flask using titer media given in Table 6. The mean values of the L- arginine productivity were calculated and compared.

[150] [151] Table 6

[152] [153] The selected colonies were inoculated in the seed media and cultured in an incubator at 3O⁰C for 16 hrs. 1 ml of the seed culture was inoculated in 24 ml of the titer media, and culturing was carried out at 3O⁰C and 220 rpm for 72 hrs. The results of L-arginine production titer test for parent strain and transformants are given in Table 7.

[154] As shown in Table 7, the arginine producing strain Corynebacterium glutamicum ATCC 21831 exhibited L-arginine productivity of 4.4 g/L. The Corynebacterium glutamicum CA06-0013, which overexpresses the argD2 bythe CJl promoter, exhibited L-arginine productivity of 4.9 g/L. Meanwhile, the Corynebacterium glutamicum CA06-0015, which overexpresses the argD2 bythe CJl promoter of the present invention, exhibited L-arginine productivity of 5.7 g/L. It can be seen that the productivity of the recombinant strain CA06-0015 was increased by 18%, as compared to the CA06-0013 strain.

[155] [156] Table 7

[157] [158] It will be apparent to those skilled in the art that various modifications and changes may be made without departing from the scope and spirit of the invention. Therefore, it should be understood that the above Examples and Experimental Examples are not limitative, but illustrative in all aspects. The scope of the invention is defined by the appended claims rather than by the description preceding them, and therefore all changes and modifications that fall within meets and bounds of the claims, or equivalents of such meets and bounds are therefore intended to be embraced by the claims.

[159]

[160]

Industrial Applicability

[161] The present invention provides a novel promoter capable of overexpressing a target gene, and uses thereof. In particular, the present invention provides a novel polynucleotide having a promoter activity, which is derived from Corynebacterium glutamicum, a recombinant vector containing the polynucleotide, a transformant transformed with the recombinant vector to overexpress a target gene, and a method for overexpressing the target gene using the transformant. The transformant transformed with the novel promoter of the present invention produces target genes, particularly amino acids useful in industry, in a high yield. Therefore, the transformant can be used in medicinal and pharmaceutical industries.

[162]

Claims

[I] A polynucleotide having a promoter activity, represented by SEQ ID NO. 1, or a fragment or variant thereof.

[2] A recombinant vector comprising the polynucleotide, or the fragment or variant thereof of claim 1.

[3] The recombinant vector according to claim 2, wherein the recombinant vector is pHC139T-gfp shown in Fig. 2.

[4] A transformant identified by accession number KCCM10922P, which is transformed with the recombinant vector of claim 3.

[5] A recombinant expression vector, comprising

(i) the polynucleotide, or the fragment or variant thereof of claim 1, and

(ii) a coding sequence of a target gene operably linked to the polynucleotide, or the fragment or variant thereof. [6] The recombinant expression vector according to claim 5, wherein the target gene is an argD2 gene. [7] A transformant transformed with the recombinant expression vector of claim 5 or

6. [8] The transformant according to claim 7, wherein the transformant belongs to the genus Corynebacterium. [9] The transformant according to claim 8, wherein the transformant is identified by accession number KCCM10823P. [10] A method for overexpressing a target gene, comprising the step of culturing the transformant of claim 7.

[I I] A method for producing L-arginine, comprising the step of culturing the transformant of claim 9.

BUDAPEST TREATY ON THE INTERNATIONAL

RECOGNITION OF THE DEPOSIT OF MICROORGANISMS

FOR THE PURPOSES OF PATENT PROCEDURE

INTERNATIONAL FORM

To. CJ Corp.

500 5-GA NAMDAEMUN-RO RECEIPT IN THE CASE OF AN ORIGINAL issued pursuant to Rule 7.1 by the CHUNG-KU, SEOUL INTERNATIONAL DEPOSITARY AUTHORITY REPUBLIC OF KOREA identified at the bottom of tills page

the AUTHORITY:

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identified under I above, which was

of person(s) having the power

status of lntenfeSaπitMtøsitiry authority was acquired; where a deposit made outside the Budapest Treaty after the acquisition of the status of international depositary authority is converted into a deposit under the Budapest Treaty, such date is the date on which the microorganism was received by the international depositary authouity. Form BP/1 Sole page

— 2!-^ FJ|^ΛO HHS£! E1

BUDAPEST TREATY ON THE INTERNATIONAL

RECOGNITION OF THE DEPOSIT OF MICROORGANISMS

FOR THE PURPOSES OF PATENT PROCEDURE

INTERNATIONAL FORM

To. CJ Corp.

500 5-GA NAMDAEMUN-RO RECEIPT IN THE CASE OF AN ORIGINAL issued pursuant to Rule 7.1 by the CHUNG-KU, SEOUL INTERNATIONAL DEPOSITARY AUTHORITY REPUBLIC OF KOREA identified at the bottom of this page

was acquired; where a deposit made outside the Budapest Treaty after the acquisition of the status of international depositary authority is converted into a deposit under lhe BudapesL Treaty, such date is the date on which the microorganism was received by the international depositary authouity. Form BP/4 Sole page

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