KR20130130377A - Promoter variant of sod gene derived from coryneform bacteria and method for producing l-lysine using the promoter variant - Google Patents

Abstract

The present invention provides a recombinant vector comprising a promoter mutant of a gene (sod gene) encoding a superoxide dismutase (SOD) derived from coryneform bacteria, a recombinant vector comprising the promoter mutant, a host cell transformed with the recombinant vector, Lysine and a method for producing L-lysine by culturing the same using a host cell, wherein the host cell in which the promoter mutant of the sod gene is inserted is used to enhance the biosynthesis of the amino acid rather than the parent strain, And has the effect of increasing the fermentation production of the same amino acid.

Description

A promoter variant of the sory gene derived from coryneform bacteria and a method for producing L-lysine using the promoter variant,

The present invention relates to a promoter mutant of a sory gene derived from a coryneform bacterium, a method for producing L-lysine using the promoter mutant, and a method for producing L-lysine using the promoter variant, and more particularly, A promoter mutant of a gene encoding an oxidized dismutase (SOD) (hereinafter referred to as "sod gene"), a recombinant vector containing the promoter mutant, a host cell transformed with the recombinant vector, And culturing the L-lysine.

Coryneform bacteria are Gram-positive bacteria and have high GC content. C. glutamicum was isolated from soil in 1957. It is characterized by the production of glutamate by biotin limitation method, has a characteristic of being bent during cell division and does not form spores.

The coryneform bacterium is a widely used industrial bacterium, and since it has an ability to secrete a large amount of metabolites under appropriate culture conditions, it can be used as a microorganism in a variety of amino acids such as L-lysine, L-arginine and L- And the like.

Among them, L-lysine is an essential amino acid which is not synthesized in human or animal body, and is used for food, medicine, and feed additive. Since industrial production of L-lysine is an economically important business process, various methods for increasing L-lysine production efficiency have been studied.

Since the biosynthetic pathway of L-lysine has been studied since the 1950's, most of it has now been produced by fermentation with the genus Corynebacteriun sp. And Brevibacterium sp. And the market for L-lysine is increasing by more than 5% per year worldwide (Kalinowski et al., 2003. J Biotechnol 104: 5-25, Patek et al. 1994. Appl. Environ Microbiol. 60: 133-140).

In order to increase production of L-lysine using coryneform bacterial strains, mutant strains are obtained through classical mutagenesis or fermentation conditions are improved, and amplification of genes related to L-lysine biosynthetic pathway through genetic engineering And other methods of gene deletion have been used (European Patent No. 0737101), and according to Korean Patent Publication Nos. 2010-20140, 2008-8620 and 2008-10073, production of L-lysine The method of modifying the promoters of aspartate aminotransferase, aspartate kinase and diaminopimelate dehydrogenase which are related genes to increase the expression of the gene was used.

SOD (superoxide dismutase, Gene ID: 1020869) is known as an enzyme that catalyzes the dismutation of superoxide radicals with O ₂ and H ₂ O _2. The enzymes are Cu / Zn SOD, Fe SOD, Mn SOD and Ni SOD are known, and SOD found in bacteria is mostly found in the cytoplasm of Mn SOD and Fe SOD (Tainer and Getzoff, 1983. Nature 306: 284-287) .

On the contrary, in the case of pathogenic bacteria, it is known that Cu / Zn SOD enzyme mainly exists in the periplasm in relation to defense and attack against host ROS attack. That is, Cu / Zn SOD is found in eukaryotes and pathogenic and human symbiotic bacteria, whereas Fe / Mn SOD is found in prokaryotic cells, protists, and mitochondria (Borgstahl et al., 1992 Cell 71: 107-118). . In addition, Ni-hooked SOD (Barondeau et al., 2004 Biochem. 43 (25): 8038-8047) and sulfate reducing bacteria Desulfo Non-classical SODs have been reported in vibrio (Silva et al., 1999 Eur. J. Biochem. 259: 235).

Escherichia The SOD of coli is reported to be two isozymes, with Fe SOD in aerobic and anaerobic states and only Mn SOD in aerobic conditions (Hassen and Fridovich. 1977 Fed. Proc. 36: 715). It is known that fur (ferric uptake regulation) is involved in the regulation (Tardat and Touati, 1993 Mol Microbiol. 9 (1) 53-63).

In bacteria, SOD studies related to oxidative stress have been reported in Lactococcus after E. coli lactis (Sanders et al., 1995 J. Bacteriol. 177: 5254-5260), Vibrio species (Kimoto et al., 2001 Microbiol. Immunol. 45: 135-142), Porphyromonas gingivalis (Ohara et al., 2006 Microbiology. 152: 955-966), Corynebacterium melassecola (Merkamm and Guyonvarch, 2001 J. Bacteriol. 183 (4): 1284), Corynebacterium glutamicum (Shafey et al., 2008 Microbiol. Res. 163: 80-86), Staphylococcus aureus (2009 J. Bacteriol. 191 (10): 3301), Helicobacter pylori (2009 J. Agric. Food Chem., 57 (17): 7743).

Among them, studies on regulators for SOD expression are known, but in most species, SOD enzyme purification and enzyme properties are under study. However, studies on the promoters that play an important role in controlling gene expression are still incomplete, and the results of predicting the promoter region of the gene and improving the promoter have not been reported yet.

The inventors of the present invention, while conducting research to solve the above limitations of the prior art, determine the promoter region of the coryneform bacteria-derived sod gene, and measure the activity of the promoter through a promoter variant mutated the promoter region. The inventors have found that they can provide novel sod gene promoter variants that are increased over intrinsic activity, and have completed the present invention.

Accordingly, an object of the present invention is to provide a promoter variant of a novel coryneform bacteria-derived sod gene, and to provide a method for mass production of amino acids such as L-lysine using the increased activity of the promoter variant.

The present invention encodes SOD derived from coryneform bacteria, including any one or more nucleotide sequences selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 to achieve the above object It provides a promoter variant of the gene (sod gene).

The present invention also provides a recombinant vector comprising the promoter variant.

The present invention also provides a host cell transformed with the recombinant vector.

In addition, the present invention provides a method for producing an amino acid comprising the step of culturing using the host cell.

The promoter mutant of the sod gene derived from a coryneform bacterium according to the present invention is a gene encoding a diaminopimelate dehydrogenase (ddh gene), a gene encoding an aspartokinase (ask gene), a transketolase coding (DapA gene) encoding a dihydropicolate synthase, a gene (dapB gene) encoding a dihydropicolinate dehydrogenase, a gene encoding a diaminopimelate decarboxylase (Asd gene) encoding an aspartate transaminase (aspB gene), a gene encoding a glucose 6-phosphate dehydrogenase (zwf gene), a gene coding for an aspartate transaminase A gene coding for gluconate dehydrogenase (gnd gene), a fructose bspspar The activity of an enzyme expressed by being operably linked with a coding sequence of one or more genes of interest selected from the group consisting of an enzyme coding gene (fbp gene) and a pyruvate carboxylase gene (pycA gene) This can be increased.

In addition, the present invention has an effect of increasing the production of amino acids such as L-lysine by overexpressing the biosynthesis of amino acids such as L-lysine than the parent strain by using the host cell into which the promoter variants of the sod gene are inserted.

1 is a diagram showing a region of a promoter of sod gene.
Figure 2 is a diagram showing a vector pK19mobsacB for Corynebacterium chromosome insertion, which is the basic structure of the vector used in the present invention.
3 is a diagram showing a vector for corynebacterium base substitution pCGDH.

The term "promoter" in the present invention refers to a nucleotide sequence that is upstream of the coding region and contains a nucleotide sequence that is complementary to that of the coding region, including a binding site for a polymerase and having a transcription initiation activity into the mRNA of a promoter downstream gene it means.

In addition, in the present invention, the term "operably linked" means that the transcriptional initiation of a gene whose promoter activity encodes a ddh gene and other enzymes, and the functional sequence of the promoter sequence and the gene sequence, that is, a gene requiring expression And its regulatory sequences are functionally linked to each other and linked in such a way as to enable gene expression.

In the present invention, the term "homology" refers to a percentage of identity between two polynucleotides, wherein sequence information between two polypeptide molecules is directly measured using a computer program that aligns sequence information and is readily available. The homology can be determined by aligning.

Furthermore, the term "vector" in the present invention refers to an expression vector capable of expressing a desired protein in a suitable host cell, which gene construct contains an essential regulatory element operatively linked to the expression of the gene insert. As used herein, the term " regulatory element "includes promoters for performing transcription, any operator sequences for regulating transcription, sequences encoding suitable mRNA ribosome binding sites, and sequences controlling transcription and translation termination.

In addition, in the present invention, the term "host cell" refers to a cell in which a vector is transformed into a host cell and thus has various genetic or molecular effects in the host cell.

In addition, the term "transformation" in the present invention means that the DNA is introduced into the host so that the DNA is replicable as an extrachromosomal factor or by insertion into the chromosome.

One aspect of the present invention, Coryneform bacteria-derived superoxide dismutase (SOD) comprising any one or more nucleotide sequences selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 It relates to a promoter variant of the gene encoding (sod gene).

The promoter variant according to the present invention may be a promoter variant of the sod gene consisting of any one of the nucleotide sequences selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO:

The promoter in prokaryotes is defined as the binding site near the transcriptional site to which the RNA polymerase binds. The promoter is composed of two short base sequences spaced apart from the bases at -10 and -35 bases from the start of transcription. The SOD promoter variant of the present invention is improved to have higher activity than a wild-type promoter, and can mutate within the -35 to -10 region to increase the expression of the downstream gene.

The promoter mutant may be used together with a naturally occurring promoter of the gene to increase the expression of a downstream target gene, and the length and type of the contained sequence may be adjusted as needed.

In addition, the promoter variants of the present invention have improved promoter activity compared to the native promoter, thereby improving the function of the promoter to suit the purpose, the mutation can be made by a variety of methods, for example Site-directed mutagenesis, error-prone PCR, DNA shuffling methods (DNA shuffling), and the like.

In a more specific embodiment, the promoter variant according to the present invention may be a DNA fragment comprising any one or more nucleotide sequences selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO:

More preferably, it may be a DNA fragment consisting of any one of the nucleotide sequences selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4

In addition, the promoter variants of the present invention may include substitution, insertion and deletion variants and combinations thereof of one or more nucleotides constituting SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

Any combination of the substitutions, deletions or insertions may be induced so that the function of the components remains unchanged. The promoter variant according to the present invention is a DNA fragment in which the DNA fragment is the DNA represented by SEQ ID NO: 1, 2, 3 or 4 Or a DNA fragment having at least 95% or more homology to the fragment.

Another aspect of the invention relates to a recombinant vector comprising said promoter variant.

The vectors used in the present invention can be transformed into suitable host cells as well as coryneform bacteria, and then can be replicated or sewn into the genome itself irrespective of the genome of the host cell. Wherein said suitable host cell is one in which the vector is replicable and may comprise a replication origin which is a specific nucleic acid sequence at which replication is initiated.

The plasmid used as the vector has a marker that can be selected after insertion into the host. Preferably, the plasmid pK19mobsacB is cleaved using XbaI and EcoRI restriction enzymes, and the vector can be prepared by inserting a promoter mutant sequence.

The vector according to the present invention may be functionally linked to a promoter mutant nucleic acid sequence comprising a portion having different variants of the N-terminal sequence of the structural gene and a nucleotide sequence encoding a protein of interest to perform a general function. Linkage with the vector is well known in the art, using genetic recombination technology, DNA cutting and linkage can be used generally known enzymes and the like.

In addition, the recombinant vector according to the present invention may include a selection marker, which is used for screening a transformant transformed with a vector, wherein a selection marker is selected from the selection marker-treated medium Since only the expressing cells can survive, selection of transformed cells is possible. Representative examples of the selection marker include kanamycin, streptomycin, chloramphenicol, and the like, and kanamycin may be used in the present invention.

The recombinant vector according to the present invention may be such that the promoter mutant is operably linked to the coding sequence of the gene of interest. As the gene of interest, the ddh gene coding for diaminopimelate dehydrogenase, aspartokinase, , The tkt gene encoding transketolase, the dapA gene coding for dihydropicolate synthase, the dapB gene coding for dihydropicolleyridylase, the aminopimelate decarboxylase coding gene an asd gene encoding an aspartate semialdehyde, an aspB gene encoding an aspartate transaminase, a zwf gene encoding a glucose 6-phosphate dehydrogenase, a gnd gene encoding a phosphate gluconate dehydrogenase, Fructose bisphosphatase May contain one or more kinds of genes selected from the group consisting of the fbp gene, the pyruvate kinase-carboxylic La encoding a pycA gene encoding, but is not limited to this.

Wherein the nucleotide sequence of the ddh gene is Gene ID: 3344238, the nucleotide sequence of the ask gene is Gene ID: 3345161, the nucleotide sequence of the tkt gene is Gene ID: 3343601, the nucleotide sequence of the dapA gene is Gene ID: 3345490, The nucleotide sequence of the asd gene is Gene ID: 3345564, the nucleotide sequence of the asdB gene is the Gene ID: 3343262, the nucleotide sequence of the zwf gene is the nucleotide sequence of the asp gene, The nucleotide sequence is Gene ID: 3345621, the nucleotide sequence of the gnd gene is Gene ID: 3344993, the nucleotide sequence of the fbp gene is Gene ID: 3345276, and the nucleotide sequence of pycA is Gene ID: 3344537.

Another aspect of the present invention relates to a host cell transformed with the recombinant vector.

In the present invention, the host cell may be a coryneform bacterium, that is, a strain belonging to the genus Co-rynebacterium or Brevibacterium, in particular Corynebacterium glutamicum, An L-amino acid producing mutant derived from Corynebacterium glutamicum ATCC 13032, preferably Corynebacterium glutamicum ATCC 13032, or the above-mentioned Corynebacterium glutamicum ATCC 13032, preferably Corynebacterium glutamicum KFCC10065.

In addition, the host cell is, for example, Corynebacterium glutamicum and other Corynebacterium strains Corynebacterium thermoaminogenes (Corynebacterium thermoaminogenes), Brevibacterium flavum (Brevibacterium flavum) , Brevibacterium lactofermentum, and L-amino acid producing mutants prepared therefrom, but are not limited thereto.

In the transformed host cell of the present invention, the promoter variant may be inserted by homologous recombination in the chromosome. The homologous recombination method is a genetic technique that knocks out or introduces a gene. The homologous recombination method may be one designed to use a modified promoter region in a host cell. More specifically, the promoter mutant sequence prepared to enhance the promoter activity can be replaced with a promoter region of the target gene of Corynebacterium glutamicum such as ddh gene, ask gene, tkt gene, etc. by a homologous recombination method .

Another aspect of the invention relates to a method for producing an amino acid comprising culturing using the transformed host cell.

Examples of the amino acid include L-lysine, L-threonine, and L-methionine, but L-lysine is preferable.

Cultivation of the transformed host cell (transformant) as described above may be carried out according to conventional methods known in the art. 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 medium used for the culture should meet the requirements of the particular strain in an appropriate manner. Culture media for Corynebacteria strains are known (eg, Manual of Methods for General Bacteriology. American Society for Bacteriology.Washington D.C., USA, 1981).

Sugar sources that may be used include sugars and carbohydrates such as glucose, saccharose, lactose, fructose, maltose, starch and cellulose, oils and fats such as soybean oil, sunflower oil, castor oil, coconut oil, palmitic acid, stearic acid, linoleic acid , Alcohols such as glycerol, ethanol, and organic acids such as acetic acid. These materials may be used individually or as a mixture. Examples of nitrogen sources that may be used include peptone, yeast extract, gravy, malt extract, corn steep liquor, soybean wheat and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. Nitrogen sources can also be used individually or as a mixture.

Potassium which may be used include potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts. The culture medium may also contain metal salts such as magnesium sulfate or iron sulfate required for growth. In addition to the above substances, essential growth substances such as amino acids and vitamins may be used. In addition, precursors suitable for the culture medium may be used. The above-mentioned raw materials can be added to the culture in a batch manner or in a continuous manner by an appropriate method.

Basic compounds such as sodium hydroxide, potassium hydroxide, ammonia, or acid compounds such as phosphoric acid or sulfuric acid can be used in a suitable manner to adjust the pH of the culture. In addition, bubble formation can be suppressed by using a defoaming agent such as a fatty acid polyglycol ester. Oxygen or an oxygen-containing gas (eg, air) may be injected into the culture to maintain aerobic conditions.

The temperature of the culture is usually 20 ° C to 45 ° C, preferably 25 ° C to 40 ° C. Culture continues until the amount of target material desired is maximized. Usually for 10 to 160 hours for this purpose.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

< Example >

In the present embodiment, a promoter prediction program is used to identify regions of promoters of superoxide dismutases (SOD, EC 1.15.1.1), and to identify the activity of various sod promoter variants, A transformant that can be operably linked to a gene involved in L-lysine synthesis was prepared, and an improved strain was obtained by substitution method through homologous recombination to Corynebacterium glutamicum strain.

< Example  1> Coryne type  Bacterial origin SOD  Promoter of gene and nucleotide sequence analysis

One. sod  Promoter designation and determination

In this example, the region between the gene of MsrA (EC.1.8.4.11) and the gene of SodA (EC.1.15.1.1.) Among the genomes of C. glutamicum ATCC 13032 was designated as forward primer (SEQ ID NO: 5: 5'-GGGATCCTC GAAGGATC GTA (Promega Corporation, Madison, Wis. Cat. No. A1360) amplified by PCR using AACAACCTCCAC-3 'and a reverse primer (SEQ ID NO: 6: 5'-GGATCCTCCTTTCGTA GGTTTCCGCACC GAG-3' , DNA sequencing was performed to determine the promoter of sod gene.

The results of performing computer-based analysis of the DNA sequencing corresponding to the SOD promoter using PROBABS and the PROMOSCAN program are shown as regions of the SOD promoter of FIG. 1.

2. sod  Designation of promoter site and transcription start site

Corynebacterium glutamicum (C. glutamicum) the putative -10 and -35 regions were identified the promoter region involved in the SOD gene expression through computer-aided program.

Total RNA was isolated from C. glutamicum ATCC 13032 by Trizol (TM) solution (Invitrogen) according to the manufacturer's method to identify the transcription initiation site of the promoter. However, in the above Trizol (TM) treatment, glass beads (? 100 占 퐉) were added and treated with bead beater 3 times at 4 占 폚 for 3 seconds on / 5 seconds off to induce cell lysis. At this time, the primer used for the 5'-Full RACE PCR and the nucleotide sequence of each primer were used.

Briefly, using the C. glutamicum SOD specific primer (RI) on the isolated bacterial RNA, 1 ^st The cDNA was reacted with reverse transcriptase at 37 ° C for 1 hour and then heat-treated at 75 ° C for 5 minutes.

The template RNA was then digested with RNase treatment. The untranslated region of SOD was amplified by PCR with a primer set prepared as a template, and then cloned and finally confirmed the transcription initiation site of the SOD gene through DNA sequence analysis.

< Example  2> Construction of Recombinant Vector for Mutation Promoter Insertion

The promoter wild type promoter was obtained by digesting the C. glutamicum ATCC13032 strain as a template with a size of about 300 bp recognized as a SOD promoter site. The promoter mutant of SOD was transformed with the plasmid pCGDH PCR was performed with a forward primer (SEQ ID NO: 7) and a pCGDH reverse primer (SEQ ID NO: 8) to obtain a fragment.

A DNA fragment (ddh gene) encoding diaminopimelate dehydrogenase was operatively linked to the promoter, and overlap PCR was performed to obtain a PCR fragment.

For cloning, restriction enzymes were treated with XbaI and EcoRI, and the pK19mobsacB vectorSchafer A, et al. 1994 Gene 145: same XbaI and EcoRI, using a PCR purification kit was digested with restriction enzymes to 69-73) (Qiagen (Qiagen), Hilden, Germany was purified by) T4 DNA Riga cloned using the agent, and E . coli DH5a: was transformed to (... RBC Bioscience, Taiwan Cat No. RH618 Hanahan, D. 1983 J Mol Biol 166.. 557-580).

The selection of plasmid-containing cells was plated on LB (Luria-Bertani) agar plates containing kanamycin (50 μg / ml) and left at 37 ° C. for 16 hours. Plasmid identification of the resulting colonies was performed using a plasmid miniprep kit (Qiagen, Hilden, Germany), and XbaI and EcoRI restriction enzymes used for cloning were used to identify the gene fragments. Respectively.

The resulting plasmid was named "pCGDH" (SEQ ID NO: 1). The pCGDH was used as a template and a mutagen was introduced into a Quick-change site directed mutagenesis kit (Stratagene, La Jolla, Calif.) Using primers for various sod promoter mutants.

As the primers for the sod promoter mutants, SDm1 used SEQ ID NOs: 9 and 10, SDm2 used SEQ ID NOs: 11 and 12, and SDm3 used SEQ ID NOs: 13 and 14, respectively, as shown in Table 1 below.

125 ng of each primer was used and PfuUltra DNA polymerase was added, followed by preheating at 95 ° C for 1 minute and then 18 cycles. Each cycle consisted of denaturation at 95 캜 for 50 seconds, annealing at 60 캜 for 50 seconds, extension at 68 캜 for about 4 minutes, and a final cycle at 68 캜 for 7 minutes.

Sod obtained by PCR and DpnI enzyme treatment, transformed into XL10-Gold Escherichia coli cells, and then plated on LB (Luria-Bertani) agar plate containing kanamycin (50 µg / ml) and standing at 37 ° C for 16 hours. Promoter variant clones were named "SDm1", "SDm2" and "SDm3", respectively. The presence or absence of mutation of the selected colonies was confirmed by the final DNA sequencing.

Primers for sod promoter variants primer Base sequence SEQ ID NO: pCGDH (forward direction) CGT CTA GAC TGC AGC TTT AAG CGC CTC ATCAGC GG 7 pCGDH (reverse direction) CGC GAA TTC TTG CTT CGT CGA TGA AGT TGAC 8 SDm1 (forward direction) TAGGTCGGCTGAAAAATCCCGTTGCAATATCAACAA 9 SDm1 (reverse direction) TTGTTGATATTGCAACGGGATTTTTCAGCCGACCTA 10 SDm2 (forward direction) ATCAACAAAAAGGTGTATAATGGGGAGGTGTCGCA 11 SDm2 (reverse direction) TGCGACACCTCCCCATTATACACCTTTTTGTTGAT 12 SDm3 (forward direction) ATATCAACAAAAAGGTGTACAATTGGGAGGTGTCGC 13 SDm3 (reverse direction) GCGACACCTCCCAATTGTACACCTTTTTGTTGATAT 14

Example 3 Corynebacterium Insertion and Enzyme Activity Measurement into Glutamicum Strain

In this Example, the mutant gene was inserted by transforming Corynebacterium glutamicum with homologous recombination using the above-mentioned group vector in which a promoter mutant of Corynebacterium was inserted.

1. Transformation

C. glutamicum KFCC10065 was transformed into competent cells to transform the clone in which the promoter mutation was confirmed in the pK19mobsacB vector into C. glutamicum .

Incubated overnight at 30 ° C. in 10 ml CM-broth medium [10 g of glucose, 10 g of polypeptone, 5 g of yeast extract, 2.5 g of NaCl, 2 g of Urea (based on 1 liter of distilled water), pH 7.0]. The pre-cultured cells were inoculated in 100 ml of BHIS medium (37 g of BHI, 250 ml of 2 M sorbitol (based on 1 L of distilled water)) to an OD (600 nm) of 0.2-0.3, and the OD (600 nm) And incubated for 6 hours until it reached 0.9.

The culture solution was placed in a prechilled tube and washed repeatedly with 10% glycerol 3-4 times at 5000 rpm at 4 ° C. to recover the cells. The cells were suspended in 10% glycerol and divided into 100 μl. Used while storing at ℃.

The constructs constructed using the water-soluble cells were transformed into a pulsar (2.5 kv, 25 ㎌, 200 Ω) of BIO-RAD using an electroporation 0.2 cm cuvette. Thereafter, 1 ml of CM-broth medium was added, followed by pre-warming at 46 ° C for 6 minutes, shaking culture at 200 rpm and 30 ° C for 2 hours, and then adding kanamycin (25 μg / ml) (Brain Heart Infusion) agar plate, and allowed to stand at 30 DEG C for 32 hours.

The resulting colonies were inoculated into 200 μl of BHIS medium for use in secondary recombination, cultured overnight at 30 ° C. and 200 rpm, diluted to 1: 1000 with 10% sucrose (Jager, et al., 1992 . J Bacteriol. 174: 5462), and incubated at 30 ° C for 72 hours. After the colonies were confirmed to be resistant to kanamycin, they were confirmed by PCR and finally confirmed by DNA sequencing .

2. Enzyme activity measurement

The selected colonies were activated and inoculated one loop to 10 ml of CM medium and incubated at 30 ° C for 16 hours with shaking at 180 rpm. 1 ml of shaken cells was inoculated in 10 ml of CM medium and cultured with shaking at 30 DEG C and 180 rpm for 96 hours.

The cultured cells were harvested by centrifugation (3,660 g, 15 min, 4 ° C), washed three times with 5 ml of 50 mM potassium phosphate buffer (pH 7.5), resuspended to 2.5 ml using the same buffer , And the procedure of placing 1.25 g of glass beads per 1 ml of the suspension and using a mini-beadbeater (Biospec Products, OK, USA) for 30 seconds and allowing to stand for 3 minutes on ice was repeated 10 times to disrupt the cells .

The supernatant was collected through centrifugation (10,000 g, 30 min, 4 ° C) and proteins were quantified using the Bradford method (Anal. Biochem. 1976. 72: 248-254, BIO-RAD LABORATORIES. , Diaminopimelate dehydrogenase (ddh) enzyme activity was used as crude protein solution. The enzyme activity was measured by mixing a reaction solution containing 0.2 M Glycine / NaOH (pH 10.5), 2 mM NADP, and 4 mM mesodiami nopimelate (meso-DAP) with about 0.01 ml of crude protein solution. After the reaction was initiated with a volume of 1 ml, the reaction was carried out at room temperature for 5 minutes and the absorbance was measured at a wavelength of 340 nm. At this time, diaminopimelate dehydrogenase enzyme activity unit was defined as NADPH mmole reduced by 1 mg of protein for 1 minute.

As a result of the measurement, it was confirmed that Corynebacterium glutamicum 10065-SDm2 was increased about 2.5 times as compared with Corynebacterium glutamicum 10065-SDwt (strain in which the wild type sod promoter was inserted) as shown in Table 2 below. In addition, Corynebacterium glutamicum 10065-SDm1 was 1.5-fold more potent than Corynebacterium glutamicum 10065-SDwt and Corynebacterium glutamicum 10065-SDm3 was 1.7-fold more potent than Corynebacterium glutamicum 10065-SDwt .

Enzyme activity measurement result Strain Specific activity (UNIT) P _E SD _mutant / P _E SD _wt 10065-SDwt 10.9 One 10065-SDm1 16.4 1.5 10065-SDm2 27.3 2.5 10065-SDm3 18.5 1.7

 < Example  4> Production of L-lysine using transformed host cells

Corynebacterium glutamicum 10065 strain and the modified strain transformed with the promoter mutant prepared in Example 3 were cultured as follows for the production of L-lysine.

In a 100 ml flask containing 10 ml of CM medium [10 g of glucose, 10 g of polypeptone, 5 g of yeast extract, 2.5 g of NaCl, 2 g of Urea (based on 1 liter of distilled water), pH 7.0], 10 ml of the mother strain Corynebacterium glue Corynebacterium glutamicum SDm1, Corynebacterium glutamicum SDm2, and Corynebacterium glutamicum SDm3 were inoculated, respectively, and cultured at 30 DEG C for 16 hours at 180 rpm in a shaking culture Respectively.

10 ml of the following L-lysine medium was added to a 100 ml flask to inoculate 1 ml of shake-cultured cells in CM medium, and shake culture was performed at 30 ° C, 180 rpm, and 96 hours. The amount of L-lysine produced after the completion of the culture was analyzed by HPLC (LC-20AD Shimazu, Japan) (Hill DW et al., 1979. Anal Chem 51: 1338) using an RI detector. The column was analyzed with an Ionospher 5C column (100 × 3.0 mm; Chrompack, Engstingen, Germany) at 40 ° C. The measurement results are shown in Table 3.

As a result of Table 3, it was found that L-lysine production of Corynebacterium glutamicum SDm2, which exhibited strong ddh activity, was increased by 10.3% compared to the parent strain, and the wild-type sod promoter strain (Corynebacterium glutami) Qum 10065-SDwt) was found to increase 7.2%

In addition, Corynebacterium glutamicum SDm1 and Corynebacterium glutamicum SDm2 were 4.7% higher than 10065-SDwt (strain inserted with wild type sod promoter), and SDm1 and SDm3 were 2.2% higher than 10065-SDwt strains, respectively. And 2.8% increase.

[L-lysine medium composition (pH 7.0)]

Molasses 16g, Raw Sugar 51g, (NH ₄ ) ₂ SO ₄ 30g, KH ₂ PO ₄ 1g, MgSO ₄ -7H ₂ O 1g, FeSO ₄ -7H ₂ O 10mg, MnSO ₄ -5H ₂ O 10mg, Thiamine 1mg, Biotin 1mg , CaCO ₃ 5% (based on 1L of distilled water)

L-lysine production measurement result Bacterium L-lysine (g / l) Corynebacterium glutamicum 10065-SDwt 32.0 Corynebacterium glutamicum 10065-SDm1 32.7 Corynebacterium glutamicum 10065-SDm2 33.5 Corynebacterium glutamicum 10065-SDm3 32.9

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art in light of the above teachings. It will be understood that the invention may be varied and varied without departing from the scope of the invention.

The promoter mutant of the sod gene derived from a coryneform bacterium according to the present invention is a gene encoding a diaminopimelate dehydrogenase (ddh gene), a gene encoding an aspartokinase (ask gene), a transketolase coding (DapA gene) encoding a dihydropicolate synthase, a gene (dapB gene) encoding a dihydropicolinate dehydrogenase, a gene encoding a diaminopimelate decarboxylase (Asd gene) encoding an aspartate transaminase (aspB gene), a gene encoding a glucose 6-phosphate dehydrogenase (zwf gene), a gene coding for an aspartate transaminase A gene coding for gluconate dehydrogenase (gnd gene), a fructose bspspar The activity of an enzyme expressed by being operably linked with a coding sequence of one or more genes of interest selected from the group consisting of an enzyme coding gene (fbp gene) and a pyruvate carboxylase gene (pycA gene) This can be increased.

In addition, the present invention overexpresses biosynthesis of an amino acid such as L-lysine by using a host cell into which a promoter mutant of the sod gene is inserted, thereby being useful for the development of an amino acid fermentation-producing strain such as L-lysine have.

<110> PAIK KWANG INDUSTRIAL CO., Ltd .; Kyungpook National University <120> Promoter variant of sod gene derived from coryneform bacteria and          method for producing L-lysine using the promoter variant <160> 14 <170> Kopatentin 1.71 <210> 1 <211> 300 <212> DNA <213> Artificial Sequence <220> <223> SOD promoter sequence (pCGI) <400> 1 aagcgcctca tcagcggtaa ccatcacggg ttcgggtgcg aaaaaccatg ccataacagg 60 aatgttcctt tcgaaaattg aggaagcctt atgcccttca accctactta gctgccaatt 120 attccgggct tgtgacccgc tacccgataa ataggtcggc tgaaaaattt cgttgcaata 180 tcaacaaaaa ggcctatcat tgggaggtgt cgcaccaagt acttttgcga agcgccatct 240 gacggatttt caaaagatgt atatgctcgg tgcggaaacc tacgaaagga ttttttaccc 300                                                                          300 <210> 2 <211> 300 <212> DNA <213> Artificial Sequence <220> <223> SOD promoter variant (SDm1) <400> 2 aagcgcctca tcagcggtaa ccatcacggg ttcgggtgcg aaaaaccatg ccataacagg 60 aatgttcctt tcgaaaattg aggaagcctt atgcccttca accctactta gctgccaatt 120 attccgggct tgtgacccgc tacccgataa ataggtcggc tgaaaaatcc cgttgcaata 180 tcaacaaaaa ggcctatcat tgggaggtgt cgcaccaagt acttttgcga agcgccatct 240 gacggatttt caaaagatgt atatgctcgg tgcggaaacc tacgaaagga ttttttaccc 300                                                                          300 <210> 3 <211> 300 <212> DNA <213> Artificial Sequence <220> <223> SOD promoter variant (SDm2) <400> 3 aagcgcctca tcagcggtaa ccatcacggg ttcgggtgcg aaaaaccatg ccataacagg 60 aatgttcctt tcgaaaattg aggaagcctt atgcccttca accctactta gctgccaatt 120 attccgggct tgtgacccgc tacccgataa ataggtcggc tgaaaaattt cgttgcaata 180 tcaacaaaaa ggtgtataat ggggaggtgt cgcaccaagt acttttgcga agcgccatct 240 gacggatttt caaaagatgt atatgctcgg tgcggaaacc tacgaaagga ttttttaccc 300                                                                          300 <210> 4 <211> 300 <212> DNA <213> Artificial Sequence <220> <223> SOD promoter (SDm3) <400> 4 aagcgcctca tcagcggtaa ccatcacggg ttcgggtgcg aaaaaccatg ccataacagg 60 aatgttcctt tcgaaaattg aggaagcctt atgcccttca accctactta gctgccaatt 120 attccgggct tgtgacccgc tacccgataa ataggtcggc tgaaaaatcc cgttgcaata 180 tcaacaaaaa ggtgtacaat tgggaggtgt cgcaccaagt acttttgcga agcgccatct 240 gacggatttt caaaagatgt atatgctcgg tgcggaaacc tacgaaagga ttttttaccc 300                                                                          300 <210> 5 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 5 gggatcctcg aaggatcgta aacaacctcc ac 32 <210> 6 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 6 ggatcctcct ttcgtaggtt tccgcaccga g 31 <210> 7 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 7 cgtctagact gcagctttaa gcgcctcatc agcgg 35 <210> 8 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 8 cgcgaattct tgcttcgtcg atgaagttga c 31 <210> 9 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 9 taggtcggct gaaaaatccc gttgcaatat caacaa 36 <210> 10 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 10 ttgttgatat tgcaacggga tttttcagcc gaccta 36 <210> 11 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 11 atcaacaaaa aggtgtataa tggggaggtg tcgca 35 <210> 12 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 12 tgcgacacct ccccattata cacctttttg ttgat 35 <210> 13 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 13 atatcaacaa aaaggtgtac aattgggagg tgtcgc 36 <210> 14 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 14 gcgacacctc ccaattgtac acctttttgt tgatat 36

Claims (10)

Promoter variants of genes encoding superoxide dismutase (SOD) derived from coryneform bacteria, comprising any one or more nucleotide sequences selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4 . The promoter variant according to claim 1, which is composed of any one nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4. A recombinant vector comprising the promoter variant of claim 1. 4. The recombinant vector of claim 3, wherein the promoter variant is operably linked to a coding sequence of the gene of interest. 5. The method according to claim 4, wherein the target gene is a ddh gene encoding diamino pimelate dehydrogenase, an ask gene encoding aspartokinase, a tkt gene encoding transketolase, a dihydropicolate synthase (DapA gene), a gene coding for dihydropicolylateductase (dapB gene), a gene coding for diaminopimelate decarboxylase (lysA gene), a gene coding for aspartic semialdehyde (asd gene), a gene encoding aspartate transaminase (aspB gene), a gene encoding glucose 6-phosphate dehydrogenase (zwf gene), a gene encoding phosphate gluconate dehydrogenase (gnd gene) , A gene encoding fructose bisphosphatase (fbp gene), pyruvate carboxylase La gene encoding a dehydratase recombinant vector comprising the one or more kinds of genes selected from the group consisting of (pycA gene). A host cell transformed with the recombinant vector of claim 3. 7. The host cell of claim 6, wherein the promoter variant of claim 1 is inserted into the chromosome by homologous recombination. The host cell according to claim 6, wherein the host cell is a bacterium of Corynebacterium. Method for producing an amino acid comprising culturing using the host cell of claim 6. 10. The method according to claim 9, wherein the amino acid is L-lysine.

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