Art
[1]
Herein is putrescine in microorganisms to produce shoes and use them to a method of producing putrescine.
[2]
BACKGROUND
[3]
Putrescine worn is known as one of the raw materials of the polyamide. Putrescine worn and producing oil compound so far as the chemical method by which the raw material, the fermentation putrescine production technology have been studied using a genetic engineering technique and fermentation techniques.
[4]
For example, a Corynebacterium strain capable of putrescine produced by manipulating the metabolic pathway of Solarium sp known group (Republic of Korea Patent Publication No. 2014-0115244 call, International Publication No. WO2014-148743).
[5]
On the other hand, formate dehydrogenase to catalyze the oxidation of formic acid, the second substrate, NAD + reduction and the NADH and to CO resulting 2 is an enzyme that generates. NADH is known as an important matter throughout the metabolism of the strains. It is possible to increase the reducing power microbial NADH through the increase because it can be advantageous to produce the target substance.
[6]
Strengthen NADH using a formate dehydrogenase, a method of producing succinic acid in anaerobic conditions, and bio-alcohol is known. Succinic acid can be produced by using a reducing TCA (TCA reverse) path under anaerobic fermentation conditions. NADH has a production amount of succinic acid and direct relevance in the reductive TCA path, oxaloacetic NADH two moles of succinic acid to acetic acid from the path (oxaloacetate) is consumed. When actually enhance the production of succinic acid in anaerobic conditions FDH from glucose it has been reported to increase the yield of 20% or more bars (Appl Environ Microbiol, 2012, 78 (9):. 3325-3337). However, unlike the acid in putrescine biosynthesis NADH is not used as a direct substrate, the bar does not report about the association between a formate dehydrogenase and putrescine production.
[7]
Detailed Description of the Invention
SUMMARY
[8]
The inventors have herein by confirming that the increase in the cases efforts result in NADH and ATP levels of strain by over-expressing the formate dehydrogenase to increase putrescine production in putrescine production strain and, putrescine is increased renal production through which It was completed.
[9]
Problem solving means
[10]
One object of the present application is to provide a microorganism of the genus Corynebacterium, which increases the production of putrescine activity of formate dehydrogenase (formate dehydrogenase, Fdh).
[11]
It is another object of the present application is to provide a method for producing putrescine using the microorganism.
[12]
Effects of the Invention
[13]
Putrescine improved Corynebacterium spp ability production of the present application is to putrescine mass production of God's can be modified to increase the activity of formate dehydrogenase and increase in the NADH and ATP generation, increase putrescine produced by this It can be effectively utilized.
[14]
Brief Description of the Drawings
[15]
1 is an SDS-PAGE gel photograph showing the result of overexpression of the CbFdh using an E. coli host. Column 1 is the result of cell protein milling liquid was 24 hours expressed in 18 ℃ condition using Escherichia coli BL21 DE3. Second column is a result of soluble protein expression 24 hours at 18 ℃ condition using Escherichia coli BL21 DE3. Column 3 is the result of cellular protein expression grinding liquid was 8 hours at 30 ℃ condition using Escherichia coli BL21 DE3. Column 4 is the result of soluble protein expression 8 hours at 30 ℃ condition using Escherichia coli BL21 DE3. Column 5 is the result of cell protein milling liquid was 24 hours expressed in 18 ℃ condition using Escherichia coli Rosetta DE3. 6 column is a result of soluble protein expression 24 hours at 18 ℃ condition using Escherichia coli Rosetta DE3. Column 7 is a protein results in crushed cells in which the liquid phase expressed in 8 hours 30 ℃ conditions using E. coli Rosetta DE3. 8 is a heat-soluble protein results were expressed in 8 hours 30 ℃ conditions using E. coli Rosetta DE3.
[16]
Figure 2 is a graph showing the amount of NADH over time. Buffer was used to 100mM phosphate buffer (pH 7.2), the control sample is a reaction other than the soluble protein. CbFdh is a soluble protein having a 10% reaction sample overexpressing formate dehydrogenase at 30 ℃ using the E. coli BL21 DE3. For CbFdh Over time, it was confirmed that the reaction product of CbFdh NADH is continuously increasing.
[17]
Figure 3 is a graph showing the formic acid concentration over time. The control group is a strain pSCEC_CJ7 vector is inserted into Corynebacterium glutamicum strains. CbFdh is C. boidinii is derived from formic acid, a strain capable of expressing the insert of the plasmid pSCEC_CJ7_CbFdh dehydrogenase gene. Into the culture medium in the formic acid of 0, 2, and 10 g / ℓ was observed a change in the control and the formic acid concentration CbFdh.
[18]
Best Mode for Carrying Out the Invention
[19]
In some embodiments for achieving the above object, the present provides a genus Corynebacterium microorganism producing put the activity of the formate dehydrogenase (formate dehydrogenase, Fdh) increases, putrescine.
[20]
By the term, the formic acid "formate dehydrogenase (formate dehydrogenase, hereinafter" Fdh "written in)" herein as a substrate, to catalyze the oxidation NAD + reduction and the NADH and CO 2 is known as an enzyme that generates.
[21]
The formate dehydrogenase is not limited to those derived sequences or because there is the case that the difference in the amino acid sequence of a protein showing the activity depending on the species or strain of microorganism present.
[22]
Specifically, the Fdh is serie Poly option cis sub suberic miss Fora ( Ceriporiopsis subvermispora), methyl tumefaciens X Torr quenched switch ( Methylobacterium extorquens ), Sinners tricot sports Solarium (methyl Methylosinus trichosporium ), the queue-free father Douce oxalato tea kusu ( Cupriavidus oxalaticus ), Candida methyl Rica ( Candida methylica ), methyl tropic tumefaciens ( the Methylotrophic bacterium ), not really bakteo aqua tee Syracuse ( Ancylobacter aquaticus ), Komagata Ella Paz Laboratories ( Komagataella pastoris ), Mycobacterium bassi kids ( Mycobacterium vaccae ), and Arabidopsis Italia ( Arabidopsis thaliana ) May be derived from such, it may be the last known Corynebacterium glutamicum (Microbiology (2012), 158, 2428-2439) origin. Specifically Candida seen dini ( Candida boidinii may be a) derived from, but not limited thereto.
[23]
Further, in the present Fdh a protein comprising the amino acid sequence of SEQ ID NO: 10, or the sequence that is at least 70%, specifically at least 80%, more specifically at least 90%, still more specifically at least 95%, most specifically, as the amino acid sequence shown by at least 99% homologous, if substantially protein which is active as a formate dehydrogenase can be included without limitation.
[24]
If the amino acid sequence having a biological activity substantially the same as or correspond to the protein of SEQ ID NO: 10 as a sequence having a homology with the sequence, also the scope of the present application, if some sequence having a deletion, modified, substituted or added in the amino acid sequence included in it is self-evident.
[25]
Polynucleotides encoding Fdh of the present application is one having a similar activity as the Fdh protein, SEQ ID NO: 10 for the amino acid sequence or a sequence with at least 70%, specifically at least 80%, more specifically at least 90%, still more specifically, a 95% or more, and most specifically may comprise a polynucleotide encoding a protein showing a homology of 99% or more. It may comprise, for example, the nucleotide sequence of SEQ ID NO: 9.
[26]
Further, be of mutations encoding a Fdh to polynucleotides encoding Fdh of the present application may be hybridized at a probe (probe) under stringent conditions (stringent conditions) derived from the base sequence or the base sequence of SEQ ID NO: 9, normally function can.
[27]
The term "homology" in the above refers to the degree of matching the given amino acid sequence or nucleotide sequence and can be expressed as a percentage. As used herein, a homologous sequence thereof having the same or similar activity with a given amino acid sequence or nucleotide sequence is represented by the "% homology". For example, the sequence by Southern hybridization experiment under the score (score), identities (identity) and similarity (similarity) standard software, stringent conditions specifically used, define the BLAST 2.0 calculating a parameter (parameter), such as appropriate hybridization conditions is to check, by comparing the definition is within the skill of a method well known to those skilled in the art (e.g., J. Sambrook et al., Molecular Cloning, a Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989;. FM Ausubel et al, may be determined in Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York). And in said it means a condition that allows for specific hybridization between the terms "stringent conditions" is a polynucleotide. For example, such conditions are described in detail in the literature (e.g., J. Sambrook et al., Above).
[28]
[29]
The term herein, the term "increased activity" means that the microorganism is active compared to the endogenous activity or modified before activity with improved. The increased activity may comprise both to that for introducing a foreign Fdh, enhance the activity of the intrinsic Fdh. Specifically, it is possible to increase capability means that the putrescine production is increased, the activity of the formate dehydrogenase.
[30]
Specifically, the increased activity herein,
[31]
1) increasing the copy number of the polynucleotide encoding the enzyme,
[32]
2) transformation of the expression control sequences to increase the expression of the polynucleotide,
[33]
3) modification of the polynucleotide sequence on the chromosome so that the activity is enhanced in the enzyme, or
[34]
4) but it can be carried out by such method that modified to enhanced by the combination thereof, but is not limited thereto.
[35]
[36]
Wherein 1) increasing the copy number of the polynucleotide is not particularly limited, be performed as operably linked to a vector form, it can be carried out by being inserted into the chromosome in the host cell. Specifically, the vector can be a polynucleotide encoding the enzyme of the present operably linked, and independently of the host replication functions may be performed by being introduced into the host cell. Or can be done in a way to increase the copy number of within the polynucleotide chromosome of the host cell by being incorporated in the polynucleotide is operably linked, a host vector the host cell capable of inserting the polynucleotide into the chromosome in the cell have.
[37]
Also may be performed by in one aspect of the increased copy number, the introduction of a foreign polynucleotide or mutant polynucleotide with codon optimization of the polynucleotide indicates the activity of the enzyme. The introduction of a foreign polynucleotide sequence may be performed by introducing an exogenous polynucleotide encoding an enzyme exhibiting the enzyme and the same / similar activity into a host cell. The exogenous polynucleotide may be used without limitation to the origin and a sequence showing the same enzymatic and / similar activity. May also be introduced into the foreign poly nucleotide is optimized in a host cell transcription, translation is to occur to optimize its codon host cell. The introduction can be carried out by any person skilled in the art to adequately select a known transfection method, whereby the introduction of the polynucleotide is expressed in a host cell enzyme is generated may be the activity increase.
[38]
Next, 2) modification of the polynucleotide expression control sequences so as to express the increase in the, particularly for but not limited to, the expression deletion of nucleic acid sequence so as to further enhance the activity of the regulatory sequence, insertion, Vivo wholly or conservative substitutions thereof performed in combination to induce a mutation on the sequence, or may be performed by replacing as a nucleic acid sequence having a stronger activity. The expression control sequence may comprise a particularly useful for but not limited to promoters, operator sequences, such as sequences that control the termination of the sequence, the transcription and translation coding for a ribosome binding site.
[39]
Specifically, there polynucleotide upper portion of the expression unit, may have a strong heterologous promoters connected instead of the original promoter, and examples of the strong promoter may include CJ7 promoter, lysCP1 promoter, EF-Tu promoter, groEL promoters, aceA or aceB promoter. More specifically, the genus Corynebacterium-derived promoter lysCP1 promoter (WO2009 / 096689) or the CJ7 promoter (Republic of Korea Patent No. 0,620,092 No. and WO2006 / 065095) and operation expression of the polynucleotide is possible to connect encoding the enzyme but it can be improved, and the like.
[40]
In addition, 3) modification of the polynucleotide sequence on the chromosome is especially useful for but not limited to, deletion of a nucleic acid sequence so as to further enhance the activity of the polynucleotide sequence, expression control by insertion, Vivo wholly or conservative substitution or a combination of these sequences performed by inducing a mutation on, or can be performed by replacing as in the polynucleotide sequence improved so as to have a stronger activity.
[41]
Finally, 4) the above 1) to 3) how modified to enhanced by the combination of the increased copy number of the polynucleotide encoding the enzyme, modification of an expression regulatory sequence such that its expression is increased, the polynucleotides on the chromosome variants of the sequence and it can be carried out by applying with one or more methods of transformation of the foreign polynucleotide or a polynucleotide variant thereof optimized codon representing the activity of the enzyme.
[42]
The term "vector" as used herein refers to a DNA preparation containing the nucleotide sequence of the polynucleotide encoding the desired protein operably linked to suitable control sequences so as to express the desired protein in a suitable host. The control sequences may include any operator sequence, sequences that control the termination of the sequence, and a transcription and translation encoding a suitable mRNA ribosome-binding site for regulating the promoter, such that transcription can initiate transcription. Vector may then be transformed into a suitable host cell, replicate independently of the host genome, or functions, may be integrated into the genome itself.
[43]
Vector as used herein, as long as it can replicate in a host cell is not particularly limited, it is possible to use any vector known in the art. Examples of the normal vector to be used may be a naturally occurring or recombinant plasmid of the state, cosmid, virus and bacteriophage. For example, the phage vector or course as mid vector may be used. PWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, etc. Charon4A, and Charon21A, pBR series, pUC system, pBluescriptII system as plasmid vector It may be used based pGEM, pTZ-based, such as pCL and pET-based system. Specifically, it may be used pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vector or the like.
[44]
With a polynucleotide via a vector for cell insertion mutations within the chromosome of a polynucleotide encoding a protein of interest in the chromosome in one example it can be replaced. Insertion into the chromosome of the above polynucleotides is any method known in the art, for example, but may be made by homologous recombination, but is not limited to this.
[45]
The term "transgenic" herein is meant to allow the protein to the polynucleotide encoding the expression in a host cell by introducing a vector comprising a polynucleotide encoding the target protein in the host cell. If the transformed polynucleotide can be as long as expression in a host cell, is inserted in the chromosome of the host cell may be located, or include both of these positions in addition to the chromosome, or no matter what. In addition, the polynucleotides include DNA and RNA encoding the target protein. The polynucleotide so long as it can be expressed is introduced into a host cell, it does not matter whether it is to be introduced in any form. For example, the polynucleotide may be introduced into a host cell in the form of there is expressed by itself in an expression cassette (cassette expression) gene construct containing all the elements required. The expression cassette may include a promoter that is normally operably linked to the polynucleotide (promoter), a transcription termination signal, ribosome binding site and translation termination signal. The expression cassette may be an expression vector form a self-replicable. In addition, the polynucleotide is introduced into a host cell in the form of itself, and may be, which is possibly connected with the operation sequence necessary for the expression in a host cell, and the like.
[46]
In addition, it means that the promoter sequence and the gene sequence to initiate and mediate the transcription of a polynucleotide encoding a protein of interest in terms rea herein "operably linked" in the above is operatively connected to.
[47]
[48]
The term "putrescine producing shoes microorganisms" or "putrescine microorganisms with new production capacity" is, naturally putrescine parent strain no ability Shin-producing microorganism or putrescine produced with the capability to be used in the present putrescine It refers to the microbial production capability granted.
[49]
Microorganism producing put the putrescine is not particularly limited, for example, a glutamate to enhance the biosynthetic route to the ornithine from glutamate-acetyl glutamate-acetyl glutamate synthase, or acetyl ornithine to switch to the (N-acetylglutamate) to the ornithine acetyl transferase dehydratase (ArgJ), acetyl glutamate to ornithine converted to tin acetyl-glutamyl-phosphate (N-acetylglutamyl phosphate) acetyl glutamate kinase (ArgB), acetyl glutamyl phosphate acetyl glutamate semi-aldehyde (N to switch to implicitly a -acetylglutamate semialdehyde) acetyl gamma glutamyl phosphate reductase kinase (ArgC), acetyl glutamate semi-aldehyde acetyl ornithine amino transferase activity of the kinase (ArgD) to switch to acetyl ornithine (N-acetylornithine) to switch to Gender is deformed so as to increase than this may be increased productivity of ornithine is used as a raw material gods putrescine biosynthesis.
[50]
Further, the microorganism ornithine ornithine acid that in tin involved in arginine synthesis carbamoyl transferase dehydratase (ornithine carbamoyltransfrase, ArgF), glutamate X Porter (glutamate exporter) protein showing the activity, and / or putrescine put acetyl crystallized acetyltransferase la endogenous activity of the kinase activity is mutated to inactivate / or can be modified to the introduction of the ornithine decarboxylase (ornithine decarboxylase, ODC) activity.
[51]
In this case, the ornithine carbamoyl transferase dehydratase (ArgF), glutamate X Porter (glutamate exporter) protein indicates the active, ornithine decarboxylase (ODC), ornithine acetyl transferase dehydratase (ArgJ), acetyl glutamate kinase (ArgB), acetyl-gamma-glutamyl-phosphate reductase kinase (ArgC), and acetyl ornithine aminotransferase (ArgD) is especially useful for but not limited to, specifically, are shown in SEQ ID nO: 11, 12, 13, 14 , 15, 16, and amino acids that are described as 17 sequence, or the 70% or more, specifically 80% or more, more specifically 90% or more, still more specifically at least 95%, most specifically at least 99% homologous having a can comprise the amino acid sequence.
[52]
In addition, putrescine put acetyl crystallized acetyl transferase kinase is especially useful for but not limited, specifically SEQ ID NO: 18 or the amino acid sequence, or the 70% or more is described as 19, specifically at least 80%, more specifically more than 90%, the still more specifically less than 95%, and most specifically may comprise an amino acid sequence having at least 99% homology.
[53]
In addition, the activity of the protein represents the putrescine discharge activity, but may be increased compared to the intrinsic activity, but is not limited thereto. Putrescine, but not protein representing the new discharge activity is limited specifically to, specifically, SEQ ID NO: 20 or the amino acid sequence, or the 70% or more is described as 21, specifically at least 80%, more specifically at least 90% , it can be made still more specifically at least 95%, and most specifically includes the amino acid sequence having at least 99% homology.
[54]
On the other hand, the microorganism of the present application is a microorganism having the putrescine-producing ability, may include a prokaryotic microorganism that the Fdh protein is expressed, and examples thereof include genus Escherichia ( Escherichia sp.), Shigella genus ( Shigella sp. ), in bakteo a sheet ( Citrobacter sp.), Salmonella in ( Salmonella sp.), Enterococcus bakteo in ( Enterobacter sp.) yeosi California in ( Yersinia sp.), keurep when Ella in ( Klebsiella genus sp.), air Winiah ( Erwinia sp.), the genus Corynebacterium ( of Corynebacterium sp.), Brevibacterium genus ( Brevibacterium sp.), Lactobacillus genus ( Lactobacillus sp.), in Selena Nomo Nas ( Selenomanas in sp.), Vibrio ( Vibriosp.), Pseudomonas species ( Pseudomonas sp.), Streptomyces in cis ( Streptomyces sp.), O Kano in bacteria ( Arcanobacterium sp.), alkali Zen in ( Alcaligenes may be a microorganism belonging to the like sp.). More specifically, may be a microorganism of the present application is a microorganism belonging to the genus Corynebacterium or the genus Escherichia, more specifically, Corynebacterium glutamicum ( Corynebacterium glutamicum may be a), and the like.
[55]
As another aspect of the present application is to provide for producing putrescine, the use of a Corynebacterium microorganism. Wherein the microorganism is formate dehydrogenase and the activity is increased compared to before modification of the active microbe (formate dehydrogenase, Fdh), the purpose can be designed to produce putrescine.
[56]
[57]
As another aspect of the present application is, (a) culturing a microorganism of the genus Corynebacterium to produce putrescine put an increased activity of formate dehydrogenase (formate dehydrogenase, Fdh) in a medium; And (b) from microorganisms or culture medium obtained in the above step provides a putrescine production process comprising the step of collecting putrescine.
[58]
For the enhanced ability microorganism formate dehydrogenase and putrescine production as described above.
[59]
In the method, the method comprising culturing the microorganism, and may be particularly, but not limited thereto, carried out by the known batch culture method, the continuous culture method, a fed-batch culture method. At this time, the culture conditions, particularly for but not limited to, a basic compound to an appropriate pH using: (phosphoric acid or sulfuric acid for example) (for example, pH 5 to 9, in particular (for example, sodium hydroxide, potassium hydroxide or ammonia) or an acidic compound may be adjusted to pH 6 to 8, most specifically at pH 6.8), oxygen or oxygen-containing gas mixture is introduced to the culture to maintain the aerobic conditions. The culture temperature is 20 to 45 ℃, specifically, can be maintained for 25 to 40 ℃, but can be cultured for about 10 to 160 hours, without being limited thereto. The worn by the culture production of putrescine can be secreted into the culture medium or remains in the cell.
[60]
In addition, the medium for the culture to be used per a carbon source and a carbohydrate (such as glucose, sucrose trehalose, lactose, fructose, maltose, know three, starch and cellulose), maintenance, and fat (such as soybean oil, sunflower seed oil, peanut oil and coconut oil), fatty acids (e.g. palmitic acid, stearic acid and linoleic acid), alcohols (for example, glycerol and ethanol) and organic acids (e.g. acetic acid) can be used by using individually or mixed, such as, but , but it is not limited thereto. The nitrogen source may include nitrogen-containing organic compounds (e.g., peptone, yeast extract, gravy, malt extract, corn steep liquor, soybean bakbun and urea), or inorganic compounds (e.g., ammonium ammonium ammonium sulfate, chloride, phosphate, ammonium carbonate and ammonium nitrate), but it can be used by using individually or mixed, such as, but not limited thereto. Although the source of the phosphate can be individually used, or a mixture of monobasic potassium phosphate, potassium susoyi, such as the corresponding sodium-containing salts to, but is not limited thereto. In addition, the culture medium and other metal salts (such as magnesium sulfate or iron sulfate), essential amino acids, and growth, such as vitamins can include a promoting material.
[61]
How to put the recovery of putrescine produced by the culture step of the present application can be by using a suitable method known in the art according to the culture method to collect the desired amino acid from the culture broth. For example, centrifugation, filtration, anion exchange chromatography, crystallization and HPLC, and the like can be used, may be by using a suitable method known in the art to recover the desired putrescine from the culture medium or the microorganism.
[62]
Mode for the Invention
[63]
And more specifically described by the herein below in the Examples. However, these examples are for explaining the present application by way of example, not intended to limit the scope of the present application to these examples.
[64]
[65]
Example 1: E. coli expression and reactivity of the evaluation CbFdh
[66]
[67]
1) E. coli expression of genes CbFdh
[68]
CbFdh ( Candida boidinii the formate dehydrogenase) Candida seen dini (in order to over-expression in E. coli, Candida boidinii the genome DNA was obtained by culturing a) KCTC17776 strain. Formate dehydrogenase genes in pET28a vector by using primers of SEQ ID NOS: 1 and 2 (CbFdh) was inserted (SEQ ID NO: 9).
[69]
Specifically, PCR condition were performed denaturation (denaturation) 30 cho 95 ℃, annealing (annealing) 55 ℃ 30 seconds and chain extension (extension) 30 TIMES 72 ℃ 1 minute. After 1.0% agarose gel electrophoresis in a loss from the PCR product was purified by elution of the 1.1 kb sized band. After cutting the purified PCR product and the pET28a vector solution sample NcoI and XhoI the insert 37 ℃, 4 a restriction enzyme treatment to the time the reaction and after electrophoresis using a 1.5% agarose gel the nucleic acid fragments of CbFdh vector size on Gel prep to give a nucleic acid fragment purified by using a kit (GeneAll, Seoul, Korea). After the resulting fragment CbFdh pET28a vector fragment and 1 mg of each was ligated using the T4 ligase (ligase) E. coliWere electroporation (electrophoration) to 2500 V in the DH5α strain. After the electroporation, the subject to a number of strains spread on LB plate medium containing 50 ㎍ / ℓ spectinomycin (spectinomycin) incubated at 37 ℃ 1 days were selected for the strain showing resistance to spectinomycin. A smear by the number of strain on LB plate medium containing 50 ㎍ / ℓ kanamycin (kanamycin) cultures in 37 ℃ 1 days were selected for the strain showing resistance. The selected strain is CbFdh inserted by verifying that the size of the gene observed in the 1.3 kb on the T7 promoter and terminator (terminator) after PCR with the above conditions by using the sequence number of primers 3 and 4 of the sequence, the 1.0% agarose gel It was OK.
[70]
Put 50 mg / ㎖ concentration of ampicillin for the strains confirmed the insertion of the CbFdh in LB medium solution of 3 ㎖ were incubated for 12 hours at 37 ℃. If the insert and the cultured strain in LB containing 50 ㎖ antibiotics optical intensity of 0.8 (600 nm wavelength) and incubated at 37 ℃, to induce expression in a variety of temperature / time conditions by the addition of 0.2 mM IPTG. Washing the cultured strain, which was pulverized cells using Sony locator (sonicator). After grinding it was confirmed that the over-expression CbFdh (41kDa, SEQ ID NO: 10) through a SDS-PAGE gel results (Fig. 1).
[71]
[72]
2) Activity of the expressed gene CbFdh
[73]
In order to evaluate the activity of CbFdh were used 100 mM phosphate buffer (pH 7.2) in reaction buffer. NAD 10 mM of the buffer solution + the standard solution into a 0.1% sodium formate (sodium formate) was used as a control. On the other hand, to give CbFdh overexpressing cells pulverized mixture identified in Example 1-1) to make 10% control rates were evaluated in CbFdh activity. The reaction solution was confirmed that change in value at 339 nm wavelength using a 96-well plate reader. 340 nm wavelength of light is known to selectively absorb to NADH.
[74]
As a result, it was confirmed that the NADH is continuously generated for a few minutes (Figure 2). CbFdh and can be overexpressed in E. coli via the present embodiment, the expressed protein was estimated to have a specific activity.
[75]
[76]
Example 2: CbFdh expressing Corynebacterium spp production
[77]
[78]
Next, to enhance the capabilities of CbFdh the genus Corynebacterium strains producing putrescine was to determine whether the putrescine produced performance improvements. Expressing CbFdh from Corynebacterium sp and was introduced into the CJ7 promoter (KCCM10617, Republic of Korea Patent No. 10-0620092 call) before the initiation codon of the gene CbFdh to confirm its activity.
[79]
First, in order to obtain a gene comprising a CJ7 promoter sequence PCR was performed using the primer pair of Corynebacterium glutamicum ATCC13032 SEQ ID NO: genomic DNA as a template, 5 and 6. PCR reaction was performed by repeating 95 ℃ denaturation 30 sec, annealing 55 ℃ 30 seconds, and elongation for 30 sec 72 ℃ process 30 times. Using a 1.5% agarose gel to confirm the PCR results nucleic acid having a size of 400 bp (base pair) after electrophoresis. Secured to the CJ7 promoter nucleic acid fragment purified using PCR prep kit (GeneAll, Seoul) from the resulting PCR product. Into the BamHI and XbaI to CJ7 purified nucleic acid fragments and vectors pSCEC sample solution was treated with restriction enzymes 37 ℃, 4 hours of reaction. Of then 1.5% agarose gel by using by using a cut and a CJ7 promoter and pSCEC vector size nucleic acid fragments of having a size of 400 bp after electrophoresis, Gel prep kit (GeneAll, Seoul) CJ7 promoter fragment and pSCEC vector to give a nucleic acid fragment. Wherein after each 1 mg of the CJ7 promoter fragment and vector pSCEC connection using T4 ligase E. coliWere electroporation (electrophoration) to 2500 V in the DH5α strain. The electroporation and then by the number of strains spread on LB plate medium containing 50 ㎍ / ℓ spectinomycin (spectinomycin) incubated at 37 ℃ 1 days of the visible resistant to spectinomycin were selected 18 strains thereof. After colony PCR using primers for the selected 18 strains SEQ ID NO: 5 and 6, it was confirmed the PCR product having a size of 400 bp. From the colony PCR results confirmed the production of pSCEC_CJ7 with the CJ7 promoter.
[80]
Using the above-described conditions CbFdh PCR results obtained with the same primer in SEQ ID NO: 7 and 8 obtained in Example 1 to obtain an CbFdh PCR results that can be inserted into the pSCEC_CJ7. By connecting the pSCEC_CJ7 and CbFdh PCR results treated with restriction enzymes XbaI and SalI E. coli was inserted into DH5α strain. Obtaining a pSCEC_CJ7_CbFdh from the selected strains and putrescine-producing strain of the genus Corynebacterium microorganism KCCM11240P (Republic of Korea Patent Publication No. 2013-0082478 call) with electroporation to 2500 V in KCCM11401P (Republic of Korea Patent Publication No. 2014-0017243 No.) was (electrophoration).
[81]
Colonies by culturing smears a strain obtained by the electroporation in 50 ㎍ / ℓ spectinomycin BHIS plate medium (Braine heart infusion 37 g / ℓ, sorbitol 91 g / ℓ, agar 2%), including (spectinomycin) to form. The selected strain is 50 ㎍ / ℓ spectinomycin CM medium containing hygromycin (spectinomycin) (glucose 10 g / ℓ, polypeptone 10 g / ℓ, yeast extract 5 g / ℓ, beef extract 5 g / ℓ, NaCl 2.5 g / end was selected and cultured with shaking at ℓ, Urea 2 g / ℓ, pH 6.8). KCCM11240P named the strain is inserted into the pSCEC_CJ7_CbFdh KCCM11240P / pSCEC_CJ7_CbFdh (CC04-0081) and was named the strain KCCM11240P the pSCEC_CJ7 is inserted into KCCM11240P / pSCEC_CJ7. Similarly KCCM11401P was named the strain is inserted into the pSCEC_CJ7_CbFdh KCCM11401P / pSCEC_CJ7_CbFdh KCCM11401P, named the strain is inserted into the pSCEC_CJ7 KCCM11401P / pSCEC_CJ7.
[82]
This one was, in a strain deposited with the accession number CC04-0081 KCCM11798P the international deposit under the Budapest Treaty on microorganisms organization Conservation Center Korea (Korea Culture center of Microorganisms, KCCM) dated 8 January 2016.
[83]
[84]
Example 3: Activity of four Corey CbFdh at tumefaciens spp
[85]
[86]
CbFdh is analyzed formic acid concentration in the medium was added to formic acid (formic acid) to confirm that the inserted Corynebacterium formic acid decarboxylase enzyme activity of Solarium sp (Fig. 3). CbFdh is a 0 g / ℓ, 2 g / ℓ, formic acid 10 g / ℓ concentration in the culture solution of the strain with the empty vector inserted enhanced strain was added to the culture medium. Enhanced strain and empty vector inserted strain was used KCCM11240P / pSCEC_CJ7_CbFdh and KCCM11240P / pSCEC_CJ7.
[87]
In the case of the strain containing the empty vector culture results, it was confirmed that formic acid will remain in the culture solution when a 2 g / ℓ and 10 g / ℓ of formic acid was added. While strains CbFdh the reinforcement was confirmed that all of the decomposition of 2 g / ℓ of formic acid in 24 hours. [0114] In the case of formic acid of 10 g / ℓ added, but not all decomposed in 32 hours, it was confirmed that the continuously formic acid is reduced. The reaction in second 32 hours it was confirmed that the degree of conversion of 80% formic acid compared with the control strain.
[88]
CbFdh enhanced strain by formic acid variation analysis with CbFdh enhanced strain and the control strain culture was found to decompose formic acid. As a result the via was confirmed that the maintain the ability to express the CbFdh is normally introduced into the Corynebacterium sp.
[89]
[90]
Example 4: Enhanced CbFdh putrescine-producing ability evaluation of novel strain
[91]
[92]
CbFdh enhanced putrescine four of the four kinds of production in order to evaluate the production performance of the novel strain Corey tumefaciens glutamicum mutant (KCCM11240P / pSCEC_CJ7_CbFdh, KCCM11240P / pSCEC_CJ7 , KCCM11401P / pSCEC_CJ7_CbFdh, KCCM11401P / pSCEC_CJ7) containing each 1 mM arginine CM plate medium (glucose 1%, polypeptone 1% , yeast extract 0.5%, beef extract 0.5%, NaCl 0.25%, urea 0.2%, 50% NaOH 100 ㎕, 50 μg spectinomycin, agar 2%, pH 6.8, 1 ℓ and plated on standard) it was incubated at 30 ℃ for 24 hours. Each strain cultured therefrom titer medium (Glucose 8% of 25 ㎖, Soy Protein 0.25%, corn solids 0.50% (NH 4 ) 2 SO 4 4% KH 2 PO 4 0.1%, MgSO 4 · 7H 2 O 0.05%, urea 0.15%, biotin 100 ㎍, thiamine hydrochloride 3 mg, calcium pantothenate 3 mg, nicotinamide 3 mg, CaCO 3 5%, 50 ㎍ spectinomycin, 1 ℓ basis) a platinum as after 200 rpm them at 30 ℃ inoculated so on, in the case of KCCM11240P / pSCEC_CJ7_CbFdh and KCCM11240P / pSCEC_CJ7 strains 98 hours, KCCM11401P / pSCEC_CJ7_CbFdh and KCCM11401P / pSCEC_CJ7 strain for cultured with shaking for 104 hours.
[93]
Measuring the concentration of putrescine produced from each of the cultures, and the results are shown in Table 1 below.
[94]
[95]
TABLE 1
Strain name The amount of formic acid (g / ℓ) Putrescine (g / ℓ)
KCCM11240P/pSCEC_CJ7 0 12.2
KCCM11240P/pSCEC_CJ7 5 12.3
KCCM11240P/pSCEC_CJ7_CbFdh 0 13.4
KCCM11240P/pSCEC_CJ7_CbFdh 5 13.1
KCCM11401P/pSCEC_CJ7 0 11.4
KCCM11401P/pSCEC_CJ7 5 10.7
KCCM11401P/pSCEC_CJ7_CbFdh 0 12.0
KCCM11401P/pSCEC_CJ7_CbFdh 5 12.0

[96]
[97]
Putrescine concentration in the culture broth was analyzed by HPLC. As shown in Table 1, KCCM11240P / For pSCEC_CJ7 strain did not putrescine production amount greatly changes depending on the presence or absence of 5 g / ℓ of formic acid. On the other hand KCCM11240P / pSCEC_CJ7_CbFdh the introduced strain was confirmed that the increase in the KCCM11240P / pSCEC_CJ7 strain compared to production, regardless of 5g / ℓ or without formic acid over 7%. Regardless of the presence of formic acid it was confirmed that the Pew increase production of gods Trail Fdh enhanced strains.
[98]
In the case of the KCCM11401P / pSCEC_CJ7 strain evaluated in the same medium without formic acid addition 11.4 g / ℓ of putrescine was god production, the strains yields reduced by 6% cultured on of 5 g / ℓ of formic acid was added culture medium (10.7 g / ℓ) was found to be. On the other hand, it was confirmed that the putrescine god produced (12.0 g / ℓ) in the case of KCCM11401P / pSCEC_CJ7_CbFdh CbFdh the same strain is enhanced regardless of the existence of the amount of formic acid. KCCM11401P / pSCEC_CJ7_CbFdh strain and KCCM11401P / pSCEC_CJ7 when analyzed put the production of putrescine from the strain, the strain is enhanced CbFdh was confirmed to be more than 5% of putrescine production increase than KCCM11401P / pSCEC_CJ7. Irrespective of the presence of formic acid was confirmed that the strain CbFdh enhance the production capability increased putrescine.
[99]
[100]
Taken together, the above results, putrescine formate dehydrogenase (CbFdh) the transduction switch isolates the producing strain was confirmed to further increase the putrescine production, which is the effect appears regardless of the addition of Status of formic acid. Thus, it is expected to mass-produce putrescine efficiently through the present application.
[101]
[102]
From the above description, those skilled in the present application will appreciate that the present application without changing the technical spirit or essential features may be embodied in other specific forms. In this regard, the embodiments described above are only to be understood as exemplary rather than limiting in all aspects. Scope of the present application is to be the meaning and scope, and all such modifications as derived from the equivalent concepts of the claims to be described later, rather than the foregoing description be construed as included within the scope of the present application.

Claims
[Claim 1]
Formate dehydrogenase (formate dehydrogenase, Fdh) The genus Corynebacterium microorganism producing the shoes, putrescine increased compared with the activity before modification activity.
[Claim 2]
2. The method of claim 1, wherein the Candida Fdh show dini ( Candida boidinii ) of origin, microorganisms.
[Claim 3]
The method of claim 1, wherein the microorganism is Fdh the configuration of the amino acid sequence of SEQ ID NO: 10.
[Claim 4]
According to claim 1, further ornithine decarboxylase, the microbial activity is the introduction of (ornithine decarboxylase, ODC).
[Claim 5]
The method of claim 1 wherein the add-acetyl transferase activity of the kinase as compared with the endogenous activity of weakening, the microorganism.
[Claim 6]
The method of claim 1, wherein the protein is active indicating the additional putrescine discharge activity to increase compared to the endogenous activity, microorganisms.
[Claim 7]
The method of claim 1, wherein the microorganism is Corynebacterium glutamicum ( Corynebacterium glutamicum ) of, microorganisms.
[Claim 8]
(A) the method comprising the microorganism of any one claim 1 to claim 7, wherein the culture in a medium; And (b), putrescine production method comprising the step of collecting putrescine from the microorganism or the medium obtained in the above step.
[Claim 9]
9. The method of claim 8, wherein the culture is a method to culture in a medium that does not contain the microorganism formate.
[Claim 10]
10. The method of claim 8, wherein the step of culturing the microorganism is a method to culture under aerobic conditions.