WO2012077995A2 - 퓨트레신을 생산하는 미생물 및 이를 이용하여 퓨트레신을 생산하는 방법 - Google Patents
퓨트레신을 생산하는 미생물 및 이를 이용하여 퓨트레신을 생산하는 방법 Download PDFInfo
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- WO2012077995A2 WO2012077995A2 PCT/KR2011/009478 KR2011009478W WO2012077995A2 WO 2012077995 A2 WO2012077995 A2 WO 2012077995A2 KR 2011009478 W KR2011009478 W KR 2011009478W WO 2012077995 A2 WO2012077995 A2 WO 2012077995A2
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- WIPO (PCT)
- Prior art keywords
- putrescine
- ornithine
- microorganism
- activity
- gene
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- C12Y203/00—Acyltransferases (2.3)
- C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
- C12Y203/01035—Glutamate N-acetyltransferase (2.3.1.35)
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- C12Y—ENZYMES
- C12Y206/00—Transferases transferring nitrogenous groups (2.6)
- C12Y206/01—Transaminases (2.6.1)
- C12Y206/01013—Ornithine aminotransferase (2.6.1.13)
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- C12Y—ENZYMES
- C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
- C12Y207/02—Phosphotransferases with a carboxy group as acceptor (2.7.2)
- C12Y207/02008—Acetylglutamate kinase (2.7.2.8)
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- C12Y—ENZYMES
- C12Y401/00—Carbon-carbon lyases (4.1)
- C12Y401/01—Carboxy-lyases (4.1.1)
- C12Y401/01017—Ornithine decarboxylase (4.1.1.17)
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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/15—Corynebacterium
Definitions
- the present invention relates to a microorganism producing putrescine and a method for producing putrescine using the same.
- Polyamines are substances present in most living cells.
- Spermidine or spermine belonging to polyamines, is found in various species, including bacteria, fungi, and animals.
- Putrescine putrescine or 1,4-butanediamine
- a precursor to spermidine and spermine metabolism is found in Gram-negative bacteria or fungi, and is thought to play an important role in metabolic pathways because of its high concentration in various species. .
- Putrescine is an important raw material for synthesizing polyamine nylon-4,6 by reacting with adipic acid. It is mainly used in the production of propylene from acrylonitrile and succinate. Produced by chemical synthesis via succinonitrile. The chemical synthesis consists of a three-step process that includes energy-consuming catalytic oxidation reactions, toxic compounds such as cyanide, and hydrogenation using high pressure hydrogen. Putrescine chemical production is not environmentally friendly and has a problem of depletion of petroleum resources. Therefore, there is a need for a method using biomass that is more environmentally friendly and can reduce energy consumption for the production of putrescine.
- the putrescine biosynthetic pathway in microorganisms is the same as the arginine biosynthetic pathway from glutamate to ornithine synthesis.
- Two pathways are known which are synthesized into putrescine via decarboxylation of the ornithine produced as an intermediate, or throughacceptine from arginine through agmatine. These two pathways produce the energy needed for metabolism or confer stress resistance to acidity.
- a method of producing putrescine using microorganisms a method of producing putrescine at high concentration by transforming Escherichia coli and Coritebacterium has been disclosed. The production of putrescine in E.
- coli can be achieved by increasing the expression levels of ornithine decarboxylase and glutamate acetyltransferase, and the putrescine degradation or use pathways of spermidine and acetyl It is possible to produce high concentrations of putrescine by deleting the acetylspermidine synthesis pathway (Qian. ZD. Et al., Biotechnol. Bioeng. 104: 4, 651-662, 2009, International Patent Publication No. WO06 / 005603. Patent Publication No. WO09 / 125924).
- Escherichia coli can grow in the presence of 44 g / L of putrescine, and Corynebacterium glutamicum can be grown at a concentration of 66 g / L. Therefore, it is expected to develop a strain for producing putrescine using a microorganism strain of Corynebacterium that can grow in the presence of a higher concentration of putrescine than Escherichia coli.
- Strains of the genus Corynebacterium are industrial microorganisms widely used in the production of amino acids, nucleic acids, enzymes, and antibiotic analogs.
- L-arginine is synthesized by an enzyme expressed from a gene of an arginine operon structure consisting of argCJBDFRGH form from glutamate.
- Arginine operon genes that play the most important role in arginine biosynthesis synthesize arginine using L-glutamate synthesized in cells. 2 shows the synthetic pathway of arginine from glutamate in a strain of genus Corynebacterium.
- argJ converts glutamate to N-acetylglutamate
- argB converts N-acetylglutamate to N-acetylglutamyl phosphate
- argC converts to N-acetyl Convert glutamylphosphate to N-acetylglutamate semialdehyde
- argD converts N-acetylglutamate semialdehyde to N-acetylornithine
- argJ raises N-acetyl ornithine
- ArgF converts ornithine to citrulline
- argG converts citrulline to argininosuccinate
- argH encodes an enzyme that converts arginosuccinate to arginine.
- arginine producing strains have been developed by introducing mutations into arginine operons or by increasing the expression level of enzymes involved in arginine biosynthesis through mutations such as promoters.
- argR which regulates and inhibits the expression of arginine operon gene
- argB inhibited by arginine concentration
- ornithine is produced by ornithine decarboxylase (ODC) from the synthesized ornithine. Therefore, ornithine, a putrescine precursor, must be sufficiently made to make a strain having a high productivity of putrescine. In the argF- and argR-deleting strains of wild-type E.
- glutamate addition increased ornithine production by 20%, and in addition to the route from glutamate to ornithine, gamma glutamyl kinase was involved in the first step in the production of proline from glutamate.
- Ornithine is also known to increase when the gene proB, which encodes ⁇ -glutamylkinase), is deleted. This shows that increasing the amount of glutamate has a positive effect on ornithine production.
- NCgl1221 protein from Corynebacterium glutamicum wild type strain (Cgl 13032) promotes betain release and is known to have an amino acid sequence similar to that of yggB, a mechanosensitive channel protein of Escherichia coli. Patent Publication No. 2010-0017581).
- One object of the present invention is to provide a microorganism endowed with putrescine production capacity.
- Another object of the present invention is to provide a method for producing putrescine using the microorganism.
- Microorganisms endowed with putrescine production capacity of the present invention may be widely used for the production of putrescine which is more effective.
- Figure 3 shows the vector pDZ vector for intrachromosomal insertion of the microorganism of the genus Corynebacterium.
- the present invention is the activity of ornithine carbamoyl transferase and protein involved in glutamate release (NCgl1221) is weakened compared to the intrinsic activity, ornithine dicarboxyl Provided is a microorganism that produces putrescine incorporating the activity of a lagase.
- ornithine carbamoyltransferase refers to an enzyme that exhibits a catalytic activity for producing citrulline and phosphoric acid through the reaction of carbamoyl phosphate with ornithine. It is present in plants and microorganisms as well as in the liver of excretory animals, and is involved in the synthesis of arginine in microorganisms.
- the enzyme includes a catalytic function region and a regulatory function site, ornithine binding to the regulatory function site is inhibited the activity of the enzyme.
- E. coli K12 strains have two types of OCTs (argF, argI), and enteric microorganisms, including E. coli B and W strains, have one type of OCT similar to argI.
- OCTs encoded by argF and argI are different isoenzymes with different amino acid sequences but have the same function (EMBO J. (1982) 1: 853-857).
- Microorganisms of the genus Corynebacterium contain only OCT encoded by the argF gene. Since OCT acts on the pathway for synthesizing arginine from ornithine, attenuating the activity of OCT can increase the production of ornithine in cells.
- the present invention provides a microorganism of the genus Corynebacterium in which the path of conversion of ornithine to arginine is blocked in order to block the conversion of ornithine, which is a putrescine precursor, to arginine, and for this purpose, the ornithine carbamoyl transferra
- a transgenic strain was prepared in which the gene encoding the aze was deleted.
- the ornithine carbamoyl transferase is not particularly limited, but the amino acid sequence of SEQ ID NO: 28 or more than 70%, more preferably 80% or more, more preferably 90% or more homology It may be a protein having a sequence.
- homology refers to a similar degree of a nucleotide sequence or an amino acid sequence of a gene encoding a protein. When homology is sufficiently high, expression products of the gene may have the same or similar activity.
- glycosyl transferer refers to a kind of mechanosensitive channels as a protein that plays a role in releasing glutamate produced in a cell to the outside of the cell.
- the present invention provides a microorganism of the genus Corynebacterium endowed with the ability to produce putrescine, and for this purpose, a gene encoding a protein that releases glutamate, which is a raw material for the synthesis of ornithine, a precursor of putrescine to the outside of cells, is deleted. To prepare a transformant strain capable of maintaining high levels of intracellular glutamate.
- glutamate release may be reduced or eliminated by weakening the activity of NCgl1221 protein.
- the protein involved in the deletion glutamate discharge is not particularly limited, but the amino acid sequence of SEQ ID NO: 30 or more than 70%, more preferably at least 80%, more preferably at least 90% homology I can.
- Deactivation of the protein may include 1) deletion of part or all of the gene encoding the protein, 2) modification of expression control sequences to reduce expression of the gene, 3) the sequence of the gene on the chromosome so that the activity of the protein is attenuated. 4) or a combination thereof, but is not particularly limited thereto.
- the method for deleting part or all of the polynucleotide encoding the protein is performed by replacing a polynucleotide encoding an intrinsic target protein in a chromosome with a polynucleotide or a marker gene which has deleted some nucleic acid sequences through a bacterial chromosomal insertion vector.
- "part" differs according to the kind of polynucleotide, it is specifically 1-300 pieces, Preferably it is 1-100 pieces, More preferably, it is 1-50 pieces.
- the method of modifying the expression control sequence to reduce the expression of the polynucleotide is to delete, insert, non-conservative or conservative substitution or combination of nucleic acid sequences to further weaken the activity of the expression control sequence on the expression control sequence
- the mutation can be carried out by inducing or by replacing with a nucleic acid sequence having weaker activity.
- the expression control sequences include promoters, operator sequences, sequences encoding ribosomal binding sites, and sequences that control the termination of transcription and translation.
- a method of modifying a polynucleotide sequence on a chromosome, which encodes an enzyme of the present invention involves altering the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, to further weaken the activity of the enzyme. Or by replacing with a polynucleotide sequence modified to have weaker activity.
- argR which is a transcriptional inhibitor of arginine biosynthetic pathway.
- intrinsic activity refers to the active state of an enzyme that a microorganism has in its natural state, and in the present invention, the ornithine carbamoyl transferase and glutamate excreted in the microorganism have a natural state.
- Mean activity of the involved protein means that the protein involved in ornithine carbamoyl transferase and glutamate release by the deletion or mutation of the gene (NCgl1221) refers to a state in which the activity of the protein involved in ornithine carbamoyl transferase and glutamate excretion (NCgl1221), which microorganisms have in their natural state, is weakened.
- ornithine decarboxylase (ODC) refers to an enzyme that produces putrescine using ornithine, wherein the ODC is a coenzyme pyridoxal phosphate (Pyridoxal 5'-phosphate). , PLP) and are present in most Gram-negative bacteria and may be present in some of the intestinal bacteria such as Lactobacillus among Gram-positive bacteria.
- ODC pyridoxal phosphate
- ODCs Some species have two types of ODCs, such as E. coli, and others have only one type. Two species are Escherichia sp., Shigella sp., Citroacter sp., Salmonella sp. And Enterobacter sp. Strains of ODC (speC, speF), Yersinia sp., Klebsiella sp., Erwinia sp., Etc. have one type of ODC (speC). . In the case of lactic acid bacteria, ODC is expressed in one type of gene (speF), and it is known that expression is induced at low pH or rich conditions of ornithine and histidine.
- the ODC is not particularly limited, but may be a protein encoded by the amino acid sequence of SEQ ID NO: 41 or an amino acid sequence having at least 70%, more preferably 80%, more preferably 90% or more homology thereof.
- introducing the activity of ornithine decarboxylase (ODC) into the microorganism can be carried out by various methods well known in the art, for example, a polynucleotide containing a base sequence encoding the ODC A method of inserting a nucleotide into a chromosome, A method of introducing the polynucleotide into a vector system, and a microorganism, A method of introducing a promoter showing improved activity upstream of an nucleotide sequence encoding an ODC, or introducing a modified ODC into a promoter Method, a method of introducing a variant of the nucleotide sequence encoding the ODC can be used, and more preferably, when introducing the nucleotide sequence encoding the ODC, the CJ7 promoter of SEQ ID NO: 42 as a promoter for controlling its expression Can be used.
- ODC ornithine decarboxylase
- the present invention provides a microorganism of the genus Corynebacterium endowed with the ability to produce putrescine, and for this purpose the base sequence encoding ornithine decarboxylase that can synthesize putrescine from ornithine A transgenic strain capable of introducing into the chromosome to produce putrescine was produced.
- Putrescine produced by microorganisms may be decomposed into spermidine, acetyl putrescine, and gamma aminobutyric acid (GABA) by E. coli in the intracellular degradation pathway. do.
- ODCs are present in most Gram-negative bacteria, but not in Corynebacterium microorganisms. Therefore, when developing putrescine-producing strains using microorganisms of the genus Corynebacterium, it is necessary to introduce ODC of foreign species.
- microorganisms endowed with the ability to produce putrescine or "microorganisms producing putrescine” refers to a microorganism to which putrescine has been produced, but is not given the ability to produce putrescine.
- Microorganisms to which production capacities are endowed or produce putrescine are not particularly limited, but acetylglutamate synthase or acetyl ornithine, which converts glutamate to acetyl glutamate to enhance biosynthetic pathways from glutamate to ornithine synthesis Ornithine acetyl transferase (ArgJ) which converts to ornithine, acetyl glutamate kinase (ArgB) which converts acetyl glutamate to acetyl glutamyl phosphate, acetyl glutamate semialdehyde (Nacetylglutamate) semialdehyde) Acetyl gamma glutamyl phosphate reductase (ArgC), which converts to acetyl glutamate semialdehyde, to acetyl ornithine (Nacetylornithine) to increase the activity of
- ornithine which is transformed to be used as a raw material of putrescine, is improved, and a gene encoding ornithine decarboxylase (speC) is transformed to be introduced to produce putrescine using the ornithine. It may be a microorganism.
- One or more methods selected from may be carried out by.
- various methods can be generally used to increase the activity of a protein in a microorganism. Modulations that increase the number of copies of a polynucleotide, modify the expression control sequence of a polynucleotide, or increase the expression of a polynucleotide by, for example, transformation with plasmid introduction, homologous recombination, conjugation, translocation, and the like.
- the expression level of a polynucleotide can be increased by amplifying a gene encoding a factor or destroying or attenuating a polynucleotide encoding a regulator that reduces expression of the polynucleotide.
- gene fragments comprising polynucleotides are operably linked to multiple copy vectors capable of replicating within Corynebacterium microorganisms, introducing one or multiple polynucleotide copies into a chromosome, or Expression control sequences, including promoters of nucleotides, can be replaced with more advanced activities.
- the vector pHC139T is used to transform microorganisms into the argCJBD family of genes to produce microorganisms with significantly improved ornithine production compared to wild type, or to improve promoter sites that regulate the expression of the argCJBD gene present in the chromosome of the microorganism. Can be enhanced or replaced with a promoter that exhibits more improved activity, resulting in a microorganism that has enhanced the biosynthetic pathway to ornithine.
- the method of improving the promoter site is not particularly limited, but the promoter replacement in the chromosome may be performed by constructing both terminal sequences of the site to be replaced and the promoter to be introduced in the same form as the original position in the chromosome, and then known pDZ.
- the method of performing the same method as the gene deletion method using a vector can be used.
- the improved promoter is not particularly limited, but it is preferable to use a pcj7 (or P (CJ7)) promoter (Korean Patent Registration No. 0620092) having a nucleotide sequence of SEQ ID NO: 42, and the pDZ vector is particularly
- a vector represented by the cleavage map of FIG. 3 may be used.
- the term "vector” refers to a DNA preparation containing the nucleotide sequence of a gene operably linked to a suitable regulatory sequence to allow expression of the gene of interest in a suitable host.
- the regulatory sequence includes a promoter capable of initiating transcription, any operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosomal binding site, and a sequence regulating termination of transcription and translation. Examples of commonly used vectors include natural or recombinant plasmids, cosmids, viruses and bacteriophages.
- pWE15, M13, [lambda] EMBL3, [lambda] EMBL4, [lambda] FIXII, [lambda] DASHII, [lambda] ZAPII, [lambda] gt10, [lambda] 11, Charon4A, and Charon21A can be used as phage vectors or cosmid vectors.
- System pBluescriptII system, pGEM system, pTZ system, pCL system, pET system and the like can be used.
- the vector which can be used is not specifically limited, A well-known expression vector can be used.
- a pDZ vector or the like can be used.
- the microorganism is not particularly limited thereto, but the microorganism of the present invention does not have a putrescine metabolic pathway and exhibits the activity of ornithine carbamoyl transferase and protein (NCgl1221) involved in glutamate release.
- Genus Esscherichia sp.
- Shigella sp. Citrobacter sp.
- Salmonella sp. Enterobacter sp. Yersinia sp.
- the genus Corynebacterium sp. The genus Brevibacterium sp., The genus Lactobacillus sp.
- It may be a microorganism which transformed a microorganism belonging to the genus Selenomanas sp., Or Vibrio sp.
- ornithine accumulates by reducing or inactivating the activity of ornithine carbamoyltransferase, and the amount of glutamate in the cell is increased by decreasing or inactivating the activity of glutamate exporter.
- Increasing the expression level of the argCJBD gene family involved in arginine biosynthesis produces ornithine in excess, and the activity of ornithine decarboxylase (ODC), which converts ornithine to putrescine, Microorganisms introduced and transformed to have putrescine production capacity.
- ODC ornithine decarboxylase
- the KCCM-10785P strain is a glutamate over-producing strain that has deleted cg2624 (NCBI LOCUS ID YP_226636) and cg2115 (NCBI LOCUS ID YP_226173) based on the glutamate producing strain (KFCC-11074) selected through a mutagen such as NTG. .
- cg2624 is pcaR, which is named IclR family regulatory protein
- cg2115 is sugR, which regulates the expression factor of glucose metabolism. It is termed a protein (transcriptional regulators of sugar metabolism).
- ornithine As can be seen in the synthesis route of ornithine shown in Figure 2, in order to increase the amount of ornithine to increase the amount of the starting material glutamate, block the route to convert the synthesized ornithine to arginine, orni It is required to increase the amount or activity of enzymes involved in the biosynthesis of chitin. As such, the productivity of ornithine is improved, and a gene encoding ornithine decarboxylase (ODC), an enzyme capable of biosynthesizing putrescine from ornithine, is introduced into a microorganism that does not have a putrescine metabolic pathway. Subsequently, an excessively produced ornithine can be used to prepare a transformed microorganism to which putrescine productivity is given.
- ODC ornithine decarboxylase
- Corynebacterium glutamicum strains (ATCC 13032 ⁇ argF and KCCM-10785P ⁇ argF) (Example 1), in which the argF gene is deleted, the Corynebacterium lacking the argF gene and the NCgl1221 gene Glutamicum strains (ATCC 13032 ⁇ argF ⁇ NCgl1221 and KCCM-10785P ⁇ argF ⁇ NCgl1221) (Example 2), Corynebacterium glutamicum strains in which the argF and NCgl1221 genes were deleted and the argCJBD gene was introduced (ATCC 13032 ⁇ argF ⁇ NCgl1221 pHC139T-argCJBD (Cgl) and KCCM-10785P ⁇ argF ⁇ NCgl1221 / pHC139T-argCJBD (Cgl)) (Example 3-1), the argF gene and NCgl1221 gene were deleted and the Coryne
- Corynebacterium glutamicum strain (ATCC 13032) in which the argF gene and the NCgl1221 gene were deleted, the promoter of the argCJBD gene group in the chromosome was substituted, and the speC gene was introduced into the chromosome.
- Productivity was confirmed (Table 7 and Table 8).
- the present inventors refer to the putrescine producing strain which is confirmed to have excellent putrescine production ability as "CC01-0064 (ATCC 13032 ⁇ argF ⁇ NCgl1221 P (CJ7) -argCJBD bioAD :: P (CJ7) -speC (Ec))". It was deposited under the Treaty of Budapest under the accession number KCCM11138P on November 24, 2010 to the Korean Culture Center of Microorganisms (KCCM) in Hongje 1-dong, Seodaemun-gu, Seoul.
- the present invention comprises the steps of (i) culturing the microorganisms given the putrescine production capacity to obtain a culture; And (ii) provides a method for producing putrescine comprising the step of recovering putrescine from the cultured microorganism or culture.
- the step of culturing the microorganism is not particularly limited thereto, but is preferably performed by a known batch culture method, a continuous culture method, a fed-batch culture method, and the like, and the culture conditions are not particularly limited thereto.
- Adjusting the appropriate pH pH 5-9, preferably pH 6-8, most preferably pH 6.8 using a compound (e.g. sodium hydroxide, potassium hydroxide or ammonia) or an acidic compound (e.g. phosphoric acid or sulfuric acid)
- the incubation temperature can be maintained at 20 to 45 °C, preferably 25 to 40 °C, culture for about 10 to 160 hours It is preferable to.
- Putrescine produced by the culture may be secreted into the medium or remain intracellular.
- the culture medium used may include sugars and carbohydrates (e.g. glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose), fats and fats (e.g. soybean oil, sunflower seeds) as carbon sources.
- sugars and carbohydrates e.g. glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose
- fats and fats e.g. soybean oil, sunflower seeds
- Oils, peanut oils and coconut oils fatty acids (e.g. palmitic acid, stearic acid and linoleic acid), alcohols (e.g. glycerol and ethanol) and organic acids (e.g. acetic acid), etc.
- Nitrogen sources include nitrogen-containing organic compounds such as peptone, yeast extract, gravy, malt extract, corn steep liquor, soybean meal and urea, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and Ammonium nitrate) and the like can be used individually or in combination;
- As a source of phosphorus, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, a corresponding sodium-containing salt, and the like can be used individually or in combination;
- Other metal salts such as magnesium sulfate or iron sulfate, and essential growth-promoting substances such as amino acids and vitamins.
- ArgF deletion strains were constructed in order to block the route of arginine synthesis from ornithine in publication 2008-0034334).
- the arginine biosynthetic genes of Corynebacterium glutamicum ATCC 13032 consist of the argCJBDFRGH type of operon structure, and the deletion-target argF gene (SEQ ID NO: 27) is parallel to the gene encoding the enzyme involved in the ornithine synthesis pathway on the chromosome. Because it exists, we tried to construct a plasmid to delete the argF part based on the nucleotide sequence of argD and argR located outside.
- homologous recombination fragments of the N-terminal outer portion of argF and homologous recombinant fragments of the C-terminal outer portion of argF were obtained based on the argD and argR base sequences of the ATCC 13032 strain.
- the fragment of the N-terminal outer portion of argF is a genomic DNA obtained from the ATCC 13032 strain as a template and by PCR using primers (SEQ ID NOS: 1 and 2) (94 °C 30 seconds denaturation, 55 °C 30 seconds annealing And 72 ° C. 30 sec.
- the homologous recombinant fragment of the obtained N-terminal outer portion of argF was digested with BamHI and SalI, and the homologous recombinant fragment of the C-terminal outer portion of argF was digested with SalI and XbaI to obtain respective fragments.
- Each of these truncated fragments was treated with BamHI and XbaI and introduced into the truncated pDZ vector to obtain plasmid pDZ-argF (K / O).
- the obtained plasmid pDZ-argF (K / O) was introduced into ATCC 13032 strain and KCCM-10785P strain, and the introduced strains were kanamycin (25 ⁇ g / ml) and X-gal (5-bromo-4-chloro-3). Colonies were formed by smearing and incubating in BHIS plate medium (Braine heart infusion 37g / l, sorbitol 91g / l, agar 2%) containing -indolin- ⁇ -D-galactoside, and the blue color among the formed colonies. By selecting the colonies shown, strains into which the plasmid pDZ-argF (K / O) was introduced were selected.
- the selected strain was shaken culture (30 °C) in CM medium (glucose 10g / l, polypeptone 10g / l, yeast extract 5g / l, beef extract 5g / l, NaCl 2.5g / l, urea 2g / l, pH 6.8) , 8 hours), and diluted sequentially from 10 -4 to 10 -10 , and plated and cultured in a solid medium containing X-gal to form colonies. Of the colonies formed, white colonies appearing at relatively low rates were selected to select strains lacking argF.
- Example 2 Construction of Corynebacterium glutamicum strains deleted with argF gene and NCgl1221 gene
- the NCgl1221 gene which is a glutamate exporter gene, was further deleted, thereby increasing the intracellular content of glutamate, an ornithine precursor.
- NCgl1221 SEQ ID NO: 29
- homologous recombinant fragments of the N-terminal outer portion of NCgl1221 and homologous recombinant fragments of the C-terminal outer portion of NCgl1221 were obtained.
- the fragment of the N-terminal outer portion of NCgl1221 was obtained by performing genomic DNA obtained from the ATCC 13032 strain as a template and performing PCR using primers (SEQ ID NOs: 5 and 6), and the fragment of the C-terminal outer portion of NCgl1221.
- SEQ ID NOS: 7 and 8 Table 2
- the homologous recombinant fragment of the obtained N-terminal outer portion of NCgl1221 was digested with BamHI and SalI, and the homologous recombinant fragment of the C-terminal outer portion of NCgl1221 was digested with SalI and XbaI to obtain respective fragments.
- Each of these truncated fragments was treated with BamHI and XbaI and introduced into the truncated pDZ vector to obtain plasmid pDZ-NCgl1221 (K / O).
- the obtained plasmid pDZ-NCgl1221 (K / O) was introduced into ATCC 13032 ⁇ argF strain and KCCM-10785P ⁇ argF strain, and the introduced strain was smeared on a BHIS plate medium containing kanamycin (25 ⁇ g / ml) and X-gal. Colonies were formed by culturing, and a strain in which the plasmid pDZ-NCgl1221 (K / O) was introduced was selected by selecting a colony showing blue color among the formed colonies.
- the selected strains were shaken in a CM medium (30 ° C., 8 hours), diluted sequentially from 10 ⁇ 4 to 10 ⁇ 10 , respectively, and plated and cultured on a solid medium containing X-gal to form colonies. I was. Among the colonies formed, white colonies appearing at a relatively low rate were selected to select strains that lack NCgl1221.
- ArgC, argJ, argB, argD to enhance the ornithine production pathway by increasing the copy number of the argCJBD operon (including the promoter region, SEQ ID NO: 31) encoding an enzyme involved in the pathway for synthesizing ornithine from glutamate.
- Vectors into which the genes (SEQ ID NOs: 32, 34, 36 and 38, respectively) were introduced were prepared, and transformants into which the genes were introduced were prepared.
- a chromosome of the ATCC 13032 strain was used as a template, and PCR was performed using primers (SEQ ID NOs: 9 and 10) (95 ° C. 40 sec denaturation, 55 ° C. 40 sec annealing, and 72 ° C. 150 sec extension). , 30 cycle), 4,900bp gene fragment was obtained.
- the obtained gene fragment was electrophoresed on a 0.8% agarose gel, followed by eluting a band of a desired size, and treating the eluted band with restriction enzymes KpnI and XbaI to obtain a fragment, and the fragment was obtained at pHC139T-gfp.
- Cloning into a vector Karl Patent Publication No. 2008-0074286), the expression vector pHC139T-argCJBD (Cgl) was prepared.
- the expression vector pHC139T-argCJBD (Cgl) prepared above was introduced into the ATCC 13032 ⁇ argF ⁇ NCgl1221 strain and KCCM-10785P ⁇ argF ⁇ NCgl1221 strain using an electroporation method to increase ornithine production in the strain, and 25 ⁇ g / ml
- the transformants were selected by smearing a BHIS plate medium containing kanamycin, and each of the selected transformants was ATCC 13032 ⁇ argF ⁇ NCgl1221 / pHC139T-argCJBD (Cgl) and KCCM-10785P ⁇ argF ⁇ NCgl1221 / pHC139T-argCJBD (Cgl). Named it.
- Example 3-2 Promoter substitution of the argCJBD gene group in the chromosome
- the chromosome argCJBD itself promoter was intended to be replaced by the newly developed CJ7 promoter of the applicant.
- a homologous recombination fragment comprising a CJ7 promoter, the terminal ends of which had the original sequence on the chromosome.
- the 5'-terminal portion of the CJ7 promoter is a genomic DNA of the ATCC 13032 strain as a template and PCR using primers (SEQ ID NOs: 11 and 12) (94 °C 30 seconds denaturation, 55 °C 30 seconds annealing and 72 30 seconds extension, 28 cycles) was obtained, the site of the CJ7 promoter was obtained by performing PCR under the same conditions using the primers (SEQ ID NO: 13 and 14), and the 3'-terminal part of the CJ7 promoter was the primer (SEQ ID NO: 15 and It was obtained by performing PCR under the same conditions using 16).
- primers SEQ ID NOs: 11 and 12
- the 5'-terminal site (argC-L) of the obtained promoter was treated with restriction enzymes BamHI and EcoRI, the CJ7 promoter site was treated with restriction enzymes EcoRI and XbaI, and the 3'-terminal site of the promoter (argC-R).
- the prepared expression vector pDZ-CJ7 (arg) was transformed into ATCC 13032 ⁇ argF ⁇ NCgl1221 and KCCM-10785P ⁇ argF ⁇ NCgl1221 strains by electroporation.
- the prepared transformant was shaken in a culture medium (30 ° C., 8 hours), and the culture was sequentially diluted from 10 ⁇ 4 to 10 ⁇ 10 , containing 25 ⁇ g / ml kanamycin and X-gal. Colonies were formed by plating and incubating in BHIS plate media.
- a strain was finally selected in which the arg promoter was replaced by CJ7 by the second crossing, and the genomic DNA obtained from the strain was used as a template.
- PCR was performed using primers (SEQ ID NOs. 13 and 16) (94 ° C. 30 sec denaturation, 55 ° C. 30 sec annealing and 72 ° C. 60 sec extension, 28 cycles) by the introduced expression vector pDZ-CJ7 (arg).
- ODC E. coli ornithine decarboxylase
- E. coli-derived speC gene (SEQ ID NO: 40) was cloned to be expressed using the CJ7 promoter of SEQ ID NO: 42 to introduce the Corynebacterium glutamicum strain.
- the CJ7 promoter site was templated with p117-CJ7-gfp (Korean Patent Registration No. 10620092) and subjected to PCR using primers (SEQ ID NOs: 17 and 18) (94 ° C. 40 sec denaturation, 55 ° C. 40 sec annealing and 72 ° C. 60 sec extension, 30 cycles), and the coding region of the speC gene was obtained by performing chromosomes of the wild type Escherichia coli W3110 strain as a template and performing PCR using primers (SEQ ID NOs: 19 and 20) under the same conditions.
- CJ7 promoter region and the coding region of the speC gene were treated with restriction enzymes KpnI and XbaI, cloned into KpnI and XbaI-treated pHC139T-gfp vectors, and the expression comprising the gene linked to the ODC coding region after the CJ7 promoter.
- Vector pHC139T-P (CJ7) -speC (Ec) was prepared.
- E. coli-derived speC gene was introduced between bioA and bioD.
- both ends of the P (CJ7) -speC (Ec) gene fragment included in the expression vector pHC139T-P (CJ7) -speC (Ec) prepared in Example 4-1 were microorganisms of the genus Corynebacterium. Both ends were cloned to have bioA and bioD sequences, respectively, so that they could be used as homologous recombination sites of chromosomes.
- the genome of the ATCC 13032 strain as a template and PCR using primers (SEQ ID NOs: 21 and 22) (94 ° C. 40 sec denaturation, 55 ° C. 30 sec annealing and 72 ° C.
- bioA gene fragment PCR was performed using the same template and primers (SEQ ID NOs: 25 and 26) under the same conditions to obtain a bioD gene fragment, and the expression vector pHC139T-P (CJ7) -speC (Ec) was used as a template. PCR using (SEQ ID NOs: 23 and 24) was carried out under the same conditions to obtain P (CJ7) -speC (Ec) gene fragment.
- the obtained bioA gene fragment was treated with restriction enzymes BamHI and ScaI
- the P (CJ7) -speC (Ec) gene fragment was treated with restriction enzymes ScaI and EcoRI
- the bioD gene fragment was treated with restriction enzymes EcoRI and XbaI
- Each PCR product treated with these restriction enzymes was cloned into a pDZ vector treated with BamHI and XbaI to prepare an expression vector pDZ-bioAD-P (CJ7) -speC (Ec) for introducing the speC gene into the chromosome.
- the prepared expression vector pDZ-bioAD-P (CJ7) -speC (Ec) was used as the ATCC 13032 ⁇ argF ⁇ NCgl1221 strain, ATCC 13032 ⁇ argF ⁇ NCgl1221 P (CJ7) -argCJBD, KCCM-10785P ⁇ argF ⁇ NCgl1221 and KCCM-10785C ⁇ argF
- Each transformant was prepared by introducing each of the) -argCJBD strains using electroporation.
- Each of the transformants prepared above was shake-cultured in a CM medium (30 ° C., 8 hours), and the cultures were sequentially diluted from 10 ⁇ 4 to 10 ⁇ 10 to give 25 ⁇ g / ml kanamycin and X-gal. Colonies were formed by smearing and incubating in the BHIS plate medium contained.
- strains in which P (CJ7) -speC was introduced into the chromosome by the second crossing were selected.
- Genomic DNA obtained from each of the above selected strains was used as a template, and PCR was performed using primers (SEQ ID NOs: 21 and 26) (94 ° C. 30 sec denaturation, 55 ° C. 30 sec annealing and 72 ° C. 120 sec extension, 28cycle).
- the introduced expression vector pDZ-bioAD-P (CJ7) -speC (Ec) was confirmed that the insertion of the P (CJ7) -speC gene fragment between the bioA and bioD in the chromosome, each of the identified strains ATCC 13032 ⁇ argF ⁇ NCgl1221 bioAD :: P (CJ7) -speC (Ec), ATCC 13032 ⁇ argF ⁇ arggl ⁇ NCgl1221 P (CJ7) -argCJBD bioAD :: P (CJ7) -speC (Ec), KCCM-10785P ⁇ argF ⁇ NCgl1221 bioAD :: P (C -speC (Ec) and KCCM-10785P ⁇ argF ⁇ NCgl1221 P (CJ7) -argCJBD bioAD :: P (CJ7) -speC (Ec).
- the pHC139T-argCJBD (Cgl) vector prepared in Example 3-1 was prepared using the ATCC 13032 ⁇ argF ⁇ NCgl1221 bioAD :: P (CJ7) -speC (Ec) strain and KCCM-10785P ⁇ argF ⁇ NCgl1221 bioAD :: P (CJ7) -speC (Ec) Transformants prepared by introducing into strain were ATCC 13032 ⁇ argF ⁇ NCgl1221 bioAD :: P (CJ7) -speC (Ec) / pHC139T-argCJBD (Cgl) and KCCM-10785P ⁇ argF ⁇ NCgl1221 bioAD :: P (CJ7) -speC (Ec) / pHC139T-argCJBD (Cgl).
- each strain prepared in Examples 2 to 4 (ATCC 13032 ⁇ argF ⁇ NCgl1221 (Experimental Group 1), ATCC 13032 ⁇ argF ⁇ NCgl1221 / pHC139T-argCJBD (Cgl) (Experimental Group 2), ATCC 13032 ⁇ argF ⁇ NCgl1221 P (CJ7) -argCJBD (Experimental Group 3), ATCC 13032 ⁇ argF ⁇ NCgl1221 bioAD :: P (CJ7) -speC (Ec) (Experimental Group 4), ATCC 13032 ⁇ argF ⁇ NCgl1221 bioAD :: P (CJ7) -speC (Ec) / HC139T-argCJBD (Cgl) ( Experimental Group 5) and ATCC 13032 ⁇ argF ⁇ NCgl1221 P (CJ7) -argCJBD bioAD :: P (CJ7) -speC (Ec) (Experiment 6)) were
- ornithine was produced when the argF and NCgl1221 genes were deleted or the argF and NCgl1221 genes were deleted and the expression level of the argCJBD gene was increased, but putrescine was not produced. This was attributed to the absence of the speC gene encoding ornithine decarboxylase (ODC), an enzyme that synthesizes putrescine from ornithine in the Corynebacterium glutamicum strain.
- ODC ornithine decarboxylase
- Example 4-2 the three strains prepared in Example 4-2, in which the speC gene derived from E. coli was introduced, showed little ornithine and putrescine was produced, which was introduced into the speC gene derived from E. coli.
- the ODC expressed therefrom was analyzed to be due to the synthesis of putrescine from ornithine.
- the production amount of putrescine when comparing the production amount of ornithine produced in the strains of the experimental groups 1 to 3 and the production amount of putrescine produced in the strains of the experimental groups 4 to 6 into which the speC gene was introduced into the strains of the experimental groups 1 to 3, was found to be proportional to the production of ornithine.
- the production of ornithine and putrescine was improved when the expression level of the endogenous argCJBD gene was increased, rather than when the foreign argCJBD gene was additionally introduced.
- Putrescine production capacity was compared for each strain prepared in.
- each strain prepared in Examples 2 to 4 (KCCM-10785P ⁇ argF ⁇ NCgl1221 (Experimental Group 1), KCCM-10785P ⁇ argF ⁇ NCgl1221 / pHC139T-argCJBD (Cgl) (Experimental Group 2), KCCM-10785P ⁇ argF ⁇ NCgl1221 P (CJ7) ) -argCJBD (Experimental Group 3), KCCM-10785P ⁇ argF ⁇ NCgl1221 bioAD :: P (CJ7) -speC (Ec) (Experimental Group 4), KCCM-10785P ⁇ argF ⁇ NCgl1221 bioAD :: P (CJ7) -speC (Ec) / HC139T- argCJBD (Cgl) (Experimental Group 5) and KCCM-10785P ⁇ argF ⁇ NCgl1221 P (CJ7) -argCJBD bioAD :::
- ornithine was produced when the argF and NCgl1221 genes were deleted or the argF and NCgl1221 genes were deleted and the expression level of the argCJBD gene was increased even in the glutamate overproducing strain base, but putrescine was not produced. Confirmed.
- putrescine is produced only in three strains prepared in Example 4-2, in which the speC gene derived from E. coli was introduced, which indicates that the speC gene derived from E. coli is introduced so that the ODC expressed from ornithine is It was analyzed because of the synthesis of putrescine.
- Comparing ornithine with glutamate produced by the strains of Experimental Groups 1 to 3 showed an increase in the amount of ornithine produced in proportion to the amount of glutamate produced in the strain.
- the production amount of putrescine when comparing the production amount of ornithine produced in the strains of the experimental groups 1 to 3 and the production amount of putrescine produced in the strains of the experimental groups 4 to 6 into which the speC gene was introduced into the strains of the experimental groups 1 to 3 was found to be proportional to the production of ornithine.
- the production of ornithine and putrescine was improved when the expression level of the endogenous argCJBD gene was increased, rather than when the foreign argCJBD gene was additionally introduced. In conclusion, as the amount of glutamate produced in the cell increases, the amount of ornithine increases, and finally, the amount of putrescine produced increases.
- Example 4-2 the strain produced in Example 4-2 with excellent putrescine ability "CC01-0064 (ATCC 13032 ⁇ argF ⁇ NCgl1221 P (CJ7) -argCJBD bioAD :: P (CJ7) -speC (Ec It was deposited under the Treaty of Budapest under the Treaty No. KCCM11138P on November 24, 2010 to the Korean Culture Center of Microorganisms (KCCM) in Hongje 1-dong, Seodaemun-gu, Seoul.
- KCCM11138P Korean Culture Center of Microorganisms
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Abstract
Description
명칭 | 서열번호 | 서열(5'-3') |
argF-del-F1_BamHIargF-del-R1_SalIargF-del-F2_SalIargF-del-R2_XbaI | 1234 | CGGGATCCTGGCCGTACCGGCGATTTCTCGCGTCGACAAGTTTGAGTCCTTTATGCGCGCGTCGACGACATGTCCCTTGGCTCAACTGCTCTAGAAGTAATTCACCTAGTTCTTTACC |
명칭 | 서열번호 | 서열(5'-3') |
NCgl1221-del-F1_BamHINCgl1221-del-R1_SalINCgl1221-del-F2_SalINCgl1221-del-R2_XbaI | 5678 | CGGGATCCGTCCAAGCCAAGCCGATTTCAACACGCGTCGACCCACTCGGCGCTTGATAATACACGCGTCGACCTGGAACAAGAACTCTCCAGCCTAGTCTAGA GGTTGGTGCTTCCACTGCTG |
명칭 | 서열번호 | 서열(5'-3') |
P_argC-5-KpnIargD-3_XbaI | 910 | CGGGGTACCCTCCTCCAGCAGCTCTAGCTCTGCTCTAGAAAGTTTGAGTCCTTTATGCG |
명칭 | 서열번호 | 서열(5'-3') |
argC-L-5-BamHIargC-L-3-EcoRICJ7-5-EcoRICJ7-3-XbaIargC-R-5-XbaIargC-R-3-SalI | 111213141516 | CGGGATCCGCAACGCTTGCGGTGAGAGACCGGAATTCCTGGAAGTGGTCGAAGAAGACCGGAATTCGCCGGCATAGCCTACCGATGTGCTCTAGAGATATCAGTGTTTCCTTTCGTGCTCTAGAATGATAATGCATAACGTGTAACGCGTCGACGCTTTCCGGAGGTGTTGTAC |
명칭 | 서열번호 | 서열(5'-3') |
CJ7-5-KpnICJ7-3speC(Ec)-5speC(Ec)-3_XbaI | 17181920 | CGGGGTACCGCCGGCATAGCCTACCGATGp-GATATCAGTGTTTCCTTTCGp-ATCATGAAATCAATGAATATTGCCGTGCTCTAGATTACTTCAACACATAACCGTACAAC |
명칭 | 서열번호 | 서열(5'-3') |
bioA-5-BamHIbioA-3-ScaIP(CJ7)-5-ScaIspeC(Ec)-3-EcoRIbioD-5-EcoRIbioD-3-XbaI | 212223242526 | CGGGATCCTGCGCGAGCTTGATCACCGAAAAAGTACTGCCTTGCCCACACACATGATAAAAGTACTGCCGGCATAGCCTACCGATGCCGGAATTCTTACTTCAACACATAACCGTACAACCCGGAATTCGCTGTTTTGGCGGATGAGAGTGCTCTAGACGCAAAAAGGCCATCCGTCA |
실험군 | 오르니틴(g/L) | 퓨트레신(g/L) |
대조군123456 | 0.06.06.47.70.10.10.2 | 0.00.00.00.05.26.28.1 |
실험군 | 글루타메이트(g/L) | 오르니틴(g/L) | 퓨트레신(g/L) |
대조군123456 | 15.55.24.82.01.40.10.0 | 0.07.67.99.01.71.30.1 | 0.00.00.00.05.96.89.5 |
Claims (13)
- 오르니틴 카르바모일 트랜스퍼라아제 및 글루타메이트 배출에 관여하는 단백질(NCgl1221)의 활성이 내재적 활성에 비하여 약화되도록 변형되고, 오르니틴 디카르복실라아제(ODC)의 활성이 도입된, 퓨트레신을 생산하는 미생물.
- 제1항에 있어서,상기 오르니틴 카르바모일 트랜스퍼라아제는 서열번호 28의 아미노산 서열 또는 이와 70% 이상의 상동성을 가지는 아미노산 서열을 갖는 것인 퓨트레신을 생산하는 미생물.
- 제1항에 있어서,상기 글루타메이트 배출에 관여하는 단백질은 서열번호 30의 아미노산 서열 또는 이와 70% 이상의 상동성을 가지는 아미노산 서열을 갖는 것인 퓨트레신을 생산하는 미생물.
- 제1항에 있어서,단백질의 활성 약화는 1) 상기 단백질을 암호화하는 유전자의 일부 또는 전체의 결실, 2) 상기 유전자의 발현이 감소하도록 발현조절 서열의 변형, 3) 상기 단백질의 활성이 약화되도록 염색체 상의 상기 유전자 서열의 변형 및 4) 이의 조합으로 이루어진 군으로부터 선택되는 방법에 의하여 수행되는 것인, 퓨트레신을 생산하는 미생물.
- 제1항에 있어서,상기 오르니틴 디카르복실라아제는 서열번호 40의 아미노산 서열 또는 이와 70% 이상의 상동성을 가지는 아미노산 서열을 갖는 것인 퓨트레신을 생산하는 미생물.
- 제1항에 있어서,ODC의 활성 도입은 ODC를 코딩하는 염기서열을 포함하는 폴리뉴클레오티드를 염색체에 삽입하는 방법, 상기 폴리뉴클레오티드를 벡터 시스템에 도입하여 미생물에 도입하는 방법, ODC를 코딩하는 염기서열의 상류에 개량된 활성을 나타내는 프로모터를 도입하거나 프로모터에 변이를 준 ODC를 도입하는 방법 및 ODC를 코딩하는 염기서열의 변이체를 도입하는 방법으로 이루어진 군으로부터 선택되는 것인, 퓨트레신을 생산하는 미생물.
- 제1항에 있어서,추가적으로 오르니틴의 생합성에 관여하는 아세틸 감마 글루타밀 포스페이트 리덕타아제(ArgC), 아세틸글루타메이트 신타아제 또는 오르니틴 아세틸트랜스퍼라아제(ArgJ), 아세틸글루타메이트 키나아제(ArgB), 및 아세틸오르니틴 아미노트랜스퍼라아제(ArgD)의 활성이 내재적 활성에 비하여 강화된 것인 퓨트레신을 생산하는 미생물.
- 제7항에 있어서,상기 ArgC, ArgJ, ArgB 및 ArgD는 각각 서열번호 33, 35, 37, 및 39의 아미노산 서열 또는 이와 70% 이상의 상동성을 가지는 아미노산 서열을 가지는 것인 퓨트레신을 생산하는 미생물.
- 제7항에 있어서,단백질의 활성 증가는 1) 상기 단백질을 암호화하는 폴리뉴클레오티드의 카피수 증가, 2) 상기 폴리뉴클레오티드의 발현이 증가하도록 발현조절 서열의 변형, 3) 상기 효소의 활성이 강화되도록 염색체 상의 상기 폴리뉴클레오티드 서열의 변형 및 4) 이의 조합으로 이루어진 군으로부터 선택되는 방법에 의하여 수행되는 것인, 퓨트레신을 생산하는 미생물.
- 제1항에 있어서,상기 미생물은 코리네박테리움 속 미생물인 것인 퓨트레신을 생산하는 미생물.
- 제10항에 있어서,상기 코리네박테리움 속 미생물은 코리네박테리움 글루타미쿰인 것인 퓨트레신을 생산하는 미생물.
- 제10항에 있어서,상기 코리네박테리움 속 미생물은 코리네박테리움 글루타미쿰(KCCM11138P)인 것인 퓨트레신을 생산하는 미생물.
- (i) 오르니틴 카르바모일 트랜스퍼라아제 및 글루타메이트 배출에 관여하는 단백질(NCgl1221)의 활성이 약화되도록 변형되고, 오르니틴 디카르복실라아제(ODC)의 활성이 도입된, 퓨트레신 생산능이 부여된 미생물을 배양하여 배양물을 수득하는 단계; 및(ii) 상기 배양된 미생물 또는 배양물로부터 퓨트레신을 회수하는 단계를 포함하는, 퓨트레신의 생산방법.
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EP11846494.0A EP2650357B1 (en) | 2010-12-08 | 2011-12-08 | Microorganisms for producing putrescine and method for producing putrescine using same |
RU2013131033/10A RU2573923C2 (ru) | 2010-12-08 | 2011-12-08 | Микроорганизмы для получения путресцина и способ получения путресцина с их использованием |
AU2011339096A AU2011339096B2 (en) | 2010-12-08 | 2011-12-08 | Microorganisms for producing putrescine and method for producing putrescine using same |
US13/992,242 US9890404B2 (en) | 2010-12-08 | 2011-12-08 | Microorganisms for producing putrescine and method for producing putrescine using same |
BR112013014442A BR112013014442A2 (pt) | 2010-12-08 | 2011-12-08 | micro-organismos para a produção de putrescina e método para a produção de putrescina utilizando o mesmo |
JP2013543103A JP6219168B2 (ja) | 2010-12-08 | 2011-12-08 | プトレシンを生産する微生物、及びそれを用いてプトレシンを生産する方法 |
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US10415068B2 (en) | 2013-07-17 | 2019-09-17 | Korea Advanced Institute Of Science And Technology | Microorganism for production of putrescine and methods for production of putrescine using the same |
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RU2653453C1 (ru) * | 2014-04-25 | 2018-05-08 | СиДжей ЧеилДжеданг Корпорейшн | Микроорганизм для продуцирования путресцина и способ получения путресцина с его использованием |
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Also Published As
Publication number | Publication date |
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JP6219168B2 (ja) | 2017-10-25 |
RU2573923C2 (ru) | 2016-01-27 |
US20140004577A1 (en) | 2014-01-02 |
KR101348461B1 (ko) | 2014-01-08 |
JP2016119901A (ja) | 2016-07-07 |
JP2014500728A (ja) | 2014-01-16 |
MY165886A (en) | 2018-05-18 |
WO2012077995A3 (ko) | 2012-09-07 |
US9890404B2 (en) | 2018-02-13 |
AU2011339096A1 (en) | 2013-07-18 |
BR112013014442A2 (pt) | 2016-09-13 |
EP2650357B1 (en) | 2019-02-20 |
AU2011339096B2 (en) | 2016-02-25 |
EP2650357A2 (en) | 2013-10-16 |
CN103403147B (zh) | 2016-12-07 |
CN103403147A (zh) | 2013-11-20 |
RU2013131033A (ru) | 2015-01-20 |
EP2650357A4 (en) | 2014-05-14 |
KR20120064046A (ko) | 2012-06-18 |
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