KR20170024324A - Gene coding lysine decarboxylase derived from H. alvei, recombinant vector, host cell and method for producing cadaverine using the same - Google Patents

Abstract

Provided are a cadA2 gene encoding lysine decarboxylase derived from Hafnia alvei, a recombinant vector including the same, a host cell, and a method for producing cadaverine using the host cell.

Description

A gene encoding lysine decarboxylase derived from Hapnia alba, a recombinant vector containing the same, a host cell and a method for producing the same, method for producing cadaverine using the same}

The present invention relates to a gene coding for lysine dicarboxylase derived from Hapnia alba , a recombinant vector comprising the same, a host cell and a method for producing cadaverine using the same, and more particularly, alvei) relates to cadA2 gene, recombinant vector, host cells, and method for producing cadaverine by the host cells containing the same coding for lysine decarboxylase derived.

Plastics, textiles, automobiles and construction The polyamide industry, with its diverse industrial applications, is a noteworthy industry with a steadily increasing market share of $ 22 billion in 2013, (Kind et al., 2014: 25., Metal Eng .; Thielen et al., 2010: 5) have been making efforts to produce biochemical processes through carbon dioxide production and global warming. , Bioplastic Mag .; Kuciel et al., 2012: 57., Polimery).

In particular, adaverine, a 5-carbon diamine that is a raw material for polyamides and plays an important role in bioplastics and new nylon production, is also known as 1,5-diaminopentane or pentamethylenediamine.

Cadaverine can be formed directly by L-lysine decarboxylase, which catalyzes the decarboxylation of L-lysine in some microorganisms, and lysine decarboxylase can be formed directly from plant and E. coli , (Soda et al., 1969: 34., Biochem. Biophys. Res. Com .; Sabo et al., 1974 (13), Biochemistry; Brink et al., 1990: Int. J. Food Microbiol.).

In addition, polyamines and significant amounts of cadaverine have also been detected in cheese, dairy products, most of the published foods, kimchi and salted fish (Summer et al., 1991: 54, al., 1992: 17, Int. J. Food Microbiol.).

There have been many reports on the industrial use of coryneform bacteria, which are widely used industrially for industrial production of cadaverine. Especially, the mutant Corynebacterium glutamicum, which is an important precursor for the production of cadaverine, (Japanese Patent Application Laid-Open No. 2002-223770, International Patent Publication No. WO2008 / 092720, International Patent Publication No. WO2012 / 077744).

Since the presence of the lysine dicarboxylase gene has not been found in the coryneform bacteria glutamicum, the ldcC gene encoding the heterologous lysine dicarboxylase of the heterologous origin has been replaced with the hom gene to produce lysine decarboxylase (WO 2007/113127, WO < RTI ID = 0.0 > 2011/073278), < / RTI > having homoserine requirement, promoting cadaverine production through a coryneform strain with AEC resistance, (Mimitsuka et al., 2007: 71 (9), Biosci. Biotechnol. Biochem), which has been used for the production of cadaverine (International Patent Publication WO2012 / 077744).

In addition, deletion of genes involved in the degradation of cadaverine and / or pathway ( speE , speG , ygjG, puuP, puuA ) and the like were amplified and the inducible lysine decarboxylase (cadA) gene derived from Escherichia coli was amplified, (Korean Patent Laid-Open No. 10-2010-0075066, Japanese Laid-open Patent No. 2009-531042, International Patent Publication No. WO2012 / 018226). Simultaneous amplification of lysine dicarboxylase gene and lysine cadaverine reverse transporter produced cadaverine (Japanese Patent Laid-Open No. 2002-223770), the acetyltransferase and lysine transporter were deleted , ldcC was inserted to increase the productivity of cadaverine, and there was also a cadaverine producing strain produced by replacing the promoter of the ldc gene with the sod gene promoter (International Publication No. WO2007-113127).

It has also been reported that cadabelin production was induced by simultaneous expression of E. coli lysine decarboxylase (cadA) and α-amylase (AmyA) of Streptococcus bovis using a carbon source as a starch (Toshihiro et al., 2007: 74. , Appl. Microbiol. Biotechnol.).

In addition, wild-type Corynebacterium has no known cadavirin biosynthetic pathway, and there are reports of tolerance for the production of cadarylin that can grow at a concentration of 0.2-0.3M cadavirin (Mimitsuka et al., 2007: 71., Biosci. Biotechnol., Biochem), it is also necessary to prepare resistant strains resistant to high concentrations of cadaverine in order to be industrially available.

In addition, it has been confirmed that the cultivation of a microorganism secreting lysine dicarboxylase enzyme to cells to produce cadaverine increases the concentration of cadaverine accumulation three times or more as compared with the case where lysine decarboxylase is added directly from the outside (International Patent Publication No. WO2011 / 1053440).

And, Hafnia alvei (Ming et al., 2013: 5 (8)., Int. J. Adv. Comput. Technol.), In which the ldc gene derived is produced using coryneform bacteria or Escherichia coli

Hafnia Alvei is a Gram-negative strain, rod-shaped, known as a tuber -anaerobic strain and intestinal bacteria, and is also known as a strain having strong lysine and ornithine decarboxylase activity (Greipsson et al. 1983: 33 (3), Int. J. Syst. Evol., Microbiol.).

However, since there is a problem that the production efficiency of the cadaverine according to the conventional techniques is not high, development of a strain capable of producing cadaverine at a high yield and development of a production method of the cadaverine using the same is urgently required It is true.

The present inventors have discovered that Hafnia alvei The novel gene cadA2 encoding the lysine dicarboxylase derived from lysine can increase the activity of lysine decarboxylase to produce a high yield of cadaverine, thus completing the present invention.

Accordingly, an object of the present invention is to provide a novel gene encoding lysine decarboxylase.

Another object of the present invention is to provide a recombinant vector containing the gene.

It is still another object of the present invention to provide a transformed host cell transformed with the recombinant vector, wherein the activity of the gene is increased over the intrinsic enzyme activity.

It is another object of the present invention to provide a method for producing cadaverine using the transformed host cells.

In order to achieve the above object, the present invention provides a gene derived from Hafnia alvei , which encodes lysine dicarboxylase and comprises cadA2 having the nucleotide sequence of SEQ ID NO: 1 Gene.

The present invention also relates to the above cadA2 A recombinant vector containing the gene is provided.

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

In addition, the present invention provides a method for producing cadaverine comprising culturing using transformed host cells.

The recombinant vector and the transformed host cell of the present invention have an increased activity of the lysine decarboxylase enzyme as compared to the wild type, so that the production efficiency of the cataractine can be increased.

Figure 1 shows the results of PK- cadA2 Fig.

As used herein, the term " vector and plasmid "refers to any nucleic acid that is inserted into a host cell and recombined with the host cell genome, inserted into it, or comprising a competent nucleotide sequence that replicates spontaneously as an episome. Such vectors include linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, and viral vectors.

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

The term "operably linked" in the present invention means that the nucleic acid sequence having the promoter activity is linked to the transcription initiation and promoter sequence of the target gene encoding the enzyme such as ldcC , cadA2 etc. related to the cadaverine biosynthesis, Means that the gene requiring expression and its regulatory sequence are operatively linked to each other in such a manner as to enable gene expression.

In the present invention, "host cell" may be a receptor of any recombinant vector (s) or isolated polynucleotide of the present invention, or includes individual cells or cell cultures that are receptors. The host cell may be a progeny of a single host cell, and the progeny may not be completely identical to the original parent cell (either in form or in a total DNA image) due to natural, accidental or artificial mutations and / or alterations. Host cells include cells transfected, transformed or infected with a recombinant vector or polynucleotide of the invention in vivo or in vitro. The host cell comprising the recombinant vector of the present invention is a recombinant host cell, a recombinant cell, a recombinant microorganism or a mutant microorganism.

The term "transformed" in the present invention means that DNA is introduced into a host and the DNA becomes replicable as a factor other than a chromosome or by insertion into a chromosome.

"Primer" in the present invention refers to a nucleic acid that hybridizes to a complementary RNA or DNA target polynucleotide and functions as a starting point for the stepwise synthesis of a polynucleotide from a mononucleotide by, for example, the action of a nucleotidyltransferase that occurs in a polymerase chain reaction Oligonucleotide < / RTI >

In the present invention, the term " ldc promoter " means a promoter of a lysine decarboxylase ( ldc ) derived from a hapnia- type bacterium, preferably Hapnia alba , Nucleotide sequence, which corresponds to the NCBI accession No. < RTI ID = 0.0 > Same as AIU71453.

In the present invention, the novel " cadA2 " of SEQ ID NO: 1 is a gene coding for a lysine decarboxylase derived from Hapnia albicans and a lysine decarboxylase (NCBI accession No. AIU73989) of H. alvei FB1, The sequence has about 99% similarity.

"Function" and "functional" in the present invention mean biological or enzymatic functions. "Increased" or "increase" refers to a greater amount of a given product or molecule (e.g., a generic chemical, biofuel, or intermediate product thereof) relative to a control microorganism, such as a non- Lt; RTI ID = 0.0 > of the < / RTI > recombinant microorganism.

The present invention can be implemented by culturing a recombinant microorganism transformed by inserting a gene encoding a specific enzyme into a basic vector, using standard cloning techniques and conventional methods known to those of ordinary skill in the art. Therefore, the present invention includes all of the related gene cloning methods, recombinant microorganisms, and microbial systems.

The present invention relates to a method for the preparation of < RTI ID = 0.0 & aldehyde- encoding gene encoding lysine dicarboxylase and encodes cadA2 having the nucleotide sequence of SEQ ID NO: 1 Gene.

The cadA2 of the present invention In the gene, the Hapnia alba may be Hafnia alvei ATCC 13337 .

In addition, cadA2 Lt; RTI ID = 0.0 > gene. ≪ / RTI >

In the recombinant vector of the present invention, the cadA2 The gene may be operably linked to a promoter having the nucleotide sequence of SEQ ID NO: 2 as a promoter of the ldc gene encoding a decarboxylase .

The recombinant vector of the present invention can be cloned into the genome itself, as well as the Half-type bacteria, after being transformed into a suitable host cell, regardless of the genome of the host cell. At this time, the suitable host cell may contain a replication origin which is a specific nucleic acid sequence at which replication of the vector is possible.

In addition, the recombinant vector according to the present invention may include a selection marker, which is used for screening a transformant (host cell) transformed with a vector, wherein the selection marker is a medium Since only the cells expressing the selectable marker can survive, selection of transformed cells is possible.

Typical examples of the selection marker include kanamycin, streptomycin, chloramphenicol, etc. In the present invention, kanamycin can be used.

The present invention also relates to a host cell transformed with said recombinant vector.

The transformed host cells of the present invention can be used for the production of < RTI ID = 0.0 > alvei ) PKC0303-0001 (LCTC 12882BP).

The transformed host cell of the present invention may have enhanced gene activity encoding intracellular lysine decarboxylase than intrinsic activity.

The present invention also relates to a method for producing cadaverine comprising culturing the transformed host cells.

In the method for producing cadaverine of the present invention, cultivation of the transformed host cell (transformant) can be carried out according to a conventional method known in the art. These known culture methods are described in Biotechnol Bioeng., Qian et al., 2011: 108 (1) 93; J.Micobiol.Biotechnol., Kim et al., 2015: 25 (7)

In the method for producing cadaverine of the present invention, the medium used for the culture must meet the requirements of the specific strain in an appropriate manner. Culture media for Hapnia alba strains are known, for example, Fecker et al., 1986: 203, 177, Mol Gen Genent; Greipsson et al., 1983: 33 (3) 470., Int'l Syst. Evol. microbiol. And the like.

In the production method of the cadaverine of the present invention, the saccharide that can be used for the culture includes sugar and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch and cellulose, soybean oil, sunflower oil, castor oil, Fatty acids such as 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.

In the method for producing cadaverine of the present invention, examples of nitrogen sources that can be used for culturing include peptone, yeast extract, gravy, malt extract, corn steep liquor, soybean meal and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, Ammonium, ammonium carbonate and ammonium nitrate. The nitrogen sources may also be used individually or as a mixture.

In the production method of the cadaverine of the present invention, the number of usable for culturing includes potassium dihydrogenphosphate or dipotassium hydrogenphosphate or the corresponding sodium-containing salt.

In the method for producing cadaverine of the present invention, the culture medium may contain a metal salt such as magnesium sulfate or iron sulfate necessary for growth. In addition, essential growth materials such as amino acids and vitamins can be used in addition to the above materials.

In addition, suitable precursors may be used in the culture medium. The above-mentioned raw materials can be added to the culture in a batch manner or in a continuous manner by an appropriate method.

In the method for producing cadaverine of the present invention, 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. An oxygen or oxygen-containing gas (e.g., air) can be injected into the culture to maintain aerobic conditions.

In the method for producing cadaverine of the present invention, 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.

In addition, the method for producing cadaverine of the present invention may further include a method for recovering the cadaverine produced in the culturing step.

In the method for producing cadaverine of the present invention, the method for recovering the cadaverine can separate L-cadaverine from a cell or a culture medium by a method well known in the art.

In the production method of the cadaverine of the present invention, examples of the cadaverine recovery method include filtration, ion exchange chromatography, crystallization and HPLC, but the present invention is not limited thereto.

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  1> Hafnia alvei  Origin lysine decarboxylase  ( cadA2 ) Of the gene Cloning  And a recombinant vector (PK- cadA ) Production

Hafnia alvei A genome sequencing analysis of ATCC 13337 was performed (Genotech, Daejeon, Korea), and the thus analyzed Hafnia alvei The nucleotide sequence of the gene cadA2 encoding the novel lysine decarboxylase in the entire nucleotide sequence of ATCC13337 was confirmed.

The primers shown in Table 1 were designed with the nucleotide sequences of the genes identified above. At this time, the design of the primer was performed using the CLC Main Workbench program .

The cadA2 gene and the previously reported Hafnia lt; RTI ID = 0.0 &gt; ldc &lt; / RTI &gt; gene encoding lysine decarboxylase of alvei FB1 cadA2 A vector containing the gene was constructed as follows.

In order to prepare a recombinant vector, pB plasmids obtained from SEQ ID NOS: 3 and 4, and PCR fragments cadA2 , SEQ ID NOS: 7 and 8, and SEQ ID NOS: 9 and 10 obtained from SEQ ID NOS: 5 and 6, WO2001 / 066573) and PCR fragments of about 5000 Kb in size including the promoter, respectively.

At this time, PCR was performed under the following conditions.

Primer GXL DNA Polymerase (Takara Bio Inc., Shiga, Japan) was used as the polymerase. The PCR conditions were denaturation at 98 ° C for 10 seconds, annealing at 60 ° C for 15 seconds, and polymerization at 68 ° C for 1 minute Was repeated 30 times and the resulting fragments were purified with a PCR purification kit (Qiagen, Hilden, Germany).

In order to clone the fragment fragments obtained here, PCR fragments already reported in several papers were conveniently cloned without restriction enzyme treatment (Matsumoto et al., 2011: 51 (1), BioTechniques ;, Jakobi et al., 2012: 7 (6)., PLOS one, etc.).

Here's how to do it. The concentration of each of the obtained PCR fragments was adjusted to about 200 ng, respectively, and the final volume was adjusted to 10 mu l in a tube to which DpnI was added, followed by assembly using Bio-Rad C1000, 2 hours, 5 minutes at 98 占 폚, 3 minutes at 65 占 폚, 5 minutes at 22 占 폚 for 5 minutes, and 5 minutes at 30 占 폚, where the obtained lyaches were resolved in E. coli DH5a (RBC Bioscience, Taiwan Cat. No. RH 618. Hanahan, D. 1983 J. Mol. Biol. 166: 557-580).

Selection of plasmid-containing cells was carried out on LB (Luria-Bertani) agar plates containing kanamycin (50 占 퐂 / ml) and allowed to stand at 37 占 폚 for 16 hours.

Plasmid extraction of the generated colonies was carried out using a plasmid miniprep kit (Qiagen, Hilden, Germany). The purification of the purified plasmid was confirmed by sequencing analysis method, and this construct was named "PK- cadA2 " .

< Example  2 > Recombinant plasmid PK- cadA2 in  Production of transformed cadaverine producing strain

In order to transform the clone identified as PK- cadA2 plasmid prepared in Example 1 into Hafnia alvei , H. alvei ( Hafnia alvei Moller ATCC13337) and SCS110 (Stratagene, Heidelberg, Germany) were prepared as competent cells by the method described below.

One colony was inoculated in 5 ml of LB [10 g of Tryptone, 5 g of Yeast extract, 10 g of Nacl (1 liter of distilled water, pH 7.0)] and incubated at 37 ° C overnight at 200 rpm.

The pre-cultured cells were inoculated so that the OD _{(600 nm)} was 0.1, and the cells were cultured in 100 ml LB-broth medium at 18 rpm at 200 rpm until the OD (600 nm) reached 0.6-0.7.

The culture was placed in a prechilled tube and allowed to stand for 10 minutes in ice and then repeatedly washed with Inoue buffer (BioWorld, Seongnam, Korea) at 2500 g for 2 minutes at 4 ° C to recover the cells. , And then suspended in 10 ml of Inoue buffer. Then, 1.5 ml of DMSO solution was added thereto, and 100 μl of each solution was dispensed into a micro-e tube and stored at -70 ° C.

Using the above water-soluble cell SCS110, the construct PK- cadA2 prepared in Example 1 was allowed to stand in ice for 30 minutes and then left to stand in a pre-warmed thermoblock at 42 ° C for 40 seconds, After incubation for 2 minutes, 1 ml of LB-broth medium was added, followed by shaking culture at 200 rpm and 37 ° C for 1 hour, and then plated on an LB-agar plate containing kanamycin (50 μg / ml) Lt; 0 &gt; C for 20 hours.

The resultant colonies were confirmed by colony PCR using the primers of SEQ ID NO: 11 and SEQ ID NO: 12 in Table 1, and the confirmed colonies were confirmed by plasmid miniprep kit (Qiagen, Hilden, Germany) Respectively.

The recombinant plasmid extracted using the final water-soluble cell, H. alvei, was left to stand in ice for 30 minutes and then left to stand for 40 seconds in a pre-warmed thermoblock at 42 ° C, After adding 1 ml of LB-broth medium, the mixture was shake-cultured at 200 rpm and 37 ° C for 1 hour, and then plated on an LB-agar plate containing kanamycin (50 μg / ml) Respectively.

The resultant colonies were identified as " Hafnia alvei PKC0303-0001" by colony PCR using the primers of SEQ ID NO: 11 and SEQ ID NO: 12 and designated as " Hafnia alvei PKC0303-0001" It was deposited with the Korea Research Institute of Bioscience and Biotechnology as deposit number KCTC 12882BP.

< Example  3> PKC0303 - 0001 strain of Lysine  Analysis of lysine decarboxylase activity and method of producing cadaverine

Hafnia alvei for measuring the parent strain (ATCC 13337) and carrying out the activity of the enzyme cadA2 PKC0303-0001 strain prepared in Example 2 were cultured as follows.

The strains were inoculated into 100 ml LB-broth medium containing kanamycin (50 / / ml), cultured at 30 캜 until OD _{(600 nm)} reached about 20, The cells were separated by centrifugation for 10 minutes, washed twice with 10% glycerol and then resuspended in 10% glycerol containing 0.1 mM DTT to an OD (600 nm) of 100.

Then, 1 g of glass beads and 1 ml of suspension were added to a 2 ml microtube, and the cells were disrupted by repeating 12 times for 1 minute with a cell crusher (Life real, Shandong, China) at intervals of 3 minutes , Bradford protein assay (Bradford, MM et al., 1976: 72). After the supernatant was collected by centrifugation at 12,000 rpm for 30 minutes at 4 ° C, Anal. Biochem .; Zor, T et al., 1996: 236, Anal. BSA (0, 2, 4, 6, 8, 10 mg / ml) was used as the standard.

The lysine decarboxylase activity was measured by adding 0.5 ml of 0.5 M potassium phosphate (pH 6.5), 0.2 mM of pyridoxal phosphate, 0.5 M of Lysine-HCl and about 1 mg of crude extract. After reacting at 37 ° C for 1 hour, the enzyme was inactivated at 95 ° C for 5 minutes, and then the residual amount of lysine and the amount of cadaverine produced were simultaneously analyzed.

At this time, the analysis was analyzed by the following method.

The reaction solution was centrifuged at 13000 rpm for 5 minutes using HPLC (Shimadzu, Kyoto, Japan), and a supernatant was obtained. A sample was prepared through a HPLC filter, and the consumed lysine and the produced catarbine For the simultaneous analysis, mobile phase methanesulfonic acid was prepared at a concentration of 1: 200 with distilled water. The pH of the mobile phase was adjusted to 1.32 in consideration of the pKa of the analytes, and the refractive index detector (RI: Shimadzu, Kyoto , Japan) were used.

In addition, the temperature of the column for analysis was pre-warmed to 50 ° C and analyzed using a cation exchange column, IonPacCS12A (Thermo Fisher, MA, US) column at 0.8 ml / min. We confirmed that the analysis was done well.

In addition, as shown in the lysine decarboxylase Table 2 below the active sample, Hafnia than the parental strain alvei PKC0303-0001 strain showed a 55% increase in the conversion of lysine to cadaverine.

Since the recombinant vector comprising the cadA2 gene encoding the lysine decarboxylase derived from the genus Hapnia according to the present invention has the property that the cadA2 gene is increased over the parent strain, It is useful for production and is effective and economically available by simultaneous analysis of lysine and cadaverine.

Korea Biotechnology Research Institute KCTC12882BP 20150813

<110> Paik Kwang Industrial Co. Ltd. <120> Gene coding lysine decarboxylase derived from H. alvei,          recombinant vector, host cell and method for producing cadaverine          using the same <130> 10194 <160> 12 <170> Kopatentin 2.0 <210> 1 <211> 2142 <212> DNA <213> cadA2 gene of Hafnia alvei <400> 1 atgaatatta ttgccatctt gaatcacatg ggcgtctact tcaaagaaga gcctatccgt 60 gaactgcaca aggcactgga agcactcgat tttcagattg tttatccaaa cgaccgtgaa 120 gacctgctga aactcatcga caacaacgca cgtctgtgcg gcgttatctt cgactgggat 180 acttacaatc tcgacctgtg cgaagaaatc agcgcgatga acgaacatct gcctgtctat 240 gcgttcgcca acacccactc tactctggat gttagcctga acgatctgcg tctgaacgtt 300 gagttcttcg aatacgcact gggcgccgct caggatatcg cacagaaaat ccgtcagagc 360 accgacgcat acatcgacga aatcctgcct ccgctgacca aagcactgtt caactacgtt 420 aaagaaggta aatacacctt ctgtactccg ggtcacatgg gcggtactgc gttccagaaa 480 tccccagtgg gcagcatctt ctatgatttc ttcggcgcta acgcgatgaa atctgatatc 540 tccatctctg tgggtgaact gggttctctg cttgaccact caggtccaca caaagaagct 600 gaagaataca ttgcgcgtac tttcaacgca gaacgcagct acatggtgac taacggtact 660 tctaccgcga acaaaatcgt tggtatgtac tcagcacccg ctggcagcac cgttctgatt 720 gaccgtaact gccataagtc tctgactcac ctgatgatga tgagcgacat cactcctatt 780 tacttccgtc caacccgtaa cgcttacggt atcttgggtg gtattcctaa gagtgaattc 840 cagcacgaca ccatcgctga acgcgttgca cagactccaa atgcaacctg gccagttcac 900 gccgtagtga ccaactctac ctacgacggt ctgctgtaca acactgatta catcaaagaa 960 gcgctggacg ttaaatccat ccactttgac tctgcatggg ttccttacac caacttcagc 1020 cctatctaca aaggtctgtg tggtatgagc ggtggccgtg tagaaggcaa agttatttat 1080 gaaactcagt ctactcacaa actgctggca gcattctctc aggcttcaat gattcacgtt 1140 aaaggtgaca tcaacgaaga aaccttcaac gaagcctaca tgatgcacac ctctacttct 1200 cctcactacg gcatcgtggc ttccaccgaa accgctgctg caatgatgaa aggtaatgcc 1260 ggtaaacgtc tgatcaacgg ttctatcgaa cgtgcgattc gtttccgtaa agaaatcaaa 1320 cgtctgaact ccgagtctga aggctggttc ttcgacgtat ggcagccaga aggtatcgac 1380 gaagcgaaat gctggccttt ggattccaaa gacagctggc atggctttaa agatatcgat 1440 aacgaccaca tgtatctgga cccaatcaaa gtcactctgt tgactccagg gatgcagaaa 1500 gatggttcaa tggctgatac cggtatccca gcgtctatcg tttctaaata cttagacgaa 1560 cacggcatca tcgttgagaa aactggtcca tacaacttgc tgttcctgtt cagcatcggt 1620 atcgacaaaa ctaaagcact gagcctgctg cgtgcgctga ccgaattcaa acgttcatac 1680 gacttgaacc tgcgcgttaa gaatatgctg ccttcactgt atcgtgaaga tccagagttc 1740 tatgaaaaca tgcgtattca ggacttggca cagggcatcc atgcgctgat ccaacaccac 1800 aacctgccgg acctgatgta ccgtgcattt gaagtgttgc caaccatggt aatgaaccca 1860 catgcagcgt tccaaaaaga actgcgtggc cagactgaag aagtttatct ggaagagatg 1920 atcggcaaag ttaatgccaa catgatcctg ccatatcctc caggagttcc tttggtaatg 1980 ccaggtgaaa tgctgaccga agaaagccgc ccagttctgg agttcttgca gatgctgtgc 2040 gaaatcggtg cacattaccc aggctttgaa actgatatcc acggtgcata tcgtcaggct 2100 gacggtcgct acactgttaa agttatcaaa gaccagaagt aa 2142 <210> 2 <211> 162 <212> DNA <213> Idc gene promoter of Hafnia alvei <400> 2 cgtaagatgg gtagaaacca agagaagtta accacgccga cggatcgtca gtcttaatcc 60 actcaaccat taacacgcaa gttggtaaca gcacacagca cagaggatga atttccgcct 120 ctgtgctaat aacccagccc acagaagtct ggagagtttt gc 162 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 3 atgaatatta ttgccatctt gaatc 25 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 4 ttacttctgg tctttgat 18 <210> 5 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 5 agagttttgc atgaatatta ttgccatctt gaatc 35 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 6 tttttgataa ctttaacagt gtag 24 <210> 7 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 7 gctgttttgg cggatgag 18 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 8 gcaaaactct ccagacttct 20 <210> 9 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 9 ccagaagtaa gctgttttgg cggatgag 28 <210> 10 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 10 ccagacttct gtgggctg 18 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 11 agaagtctgg agagttttgc 20 <210> 12 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 12 ctcatccgcc aaaacagc 18

Claims (10)

Hafnia aldehyde- encoding gene encoding lysine dicarboxylase and encodes cadA2 having the nucleotide sequence of SEQ ID NO: 1 gene. The method of claim 1, wherein the Hafnia Alba is a Hafnia &lt; RTI ID = 0.0 &gt; cadA2 gene wherein alvei) ATCC13337. CadA2 of claim 1 A recombinant vector containing the gene. 4. The method of claim 3, wherein the cadA2 Wherein the gene is a promoter of an ldc gene encoding a decarboxylase , operatively linked to a promoter having the nucleotide sequence of SEQ ID NO: 2. A host cell transformed with the recombinant vector of claim 3 or 4 6. The method of claim 5, wherein the host cell is selected from the group consisting of Hafnia & alvei ) PKC0303-0001 (KCTC 12882BP). 6. The transformed host cell according to claim 5, wherein the host cell is enhanced in gene activity encoding lysine decarboxylase in the cell than intrinsic activity. A method for producing cadaverine, comprising culturing using the transformed host cell of claim 5. A method for producing cadaverine, comprising culturing the transformed host cell of claim 6. A method for producing cadaverine, comprising culturing the transformed host cell of claim 7.

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