WO2007099867A1 - 有機酸生産菌及び有機酸の製造法 - Google Patents
有機酸生産菌及び有機酸の製造法 Download PDFInfo
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- WO2007099867A1 WO2007099867A1 PCT/JP2007/053360 JP2007053360W WO2007099867A1 WO 2007099867 A1 WO2007099867 A1 WO 2007099867A1 JP 2007053360 W JP2007053360 W JP 2007053360W WO 2007099867 A1 WO2007099867 A1 WO 2007099867A1
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- C—CHEMISTRY; METALLURGY
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/44—Polycarboxylic acids
- C12P7/46—Dicarboxylic acids having four or less carbon atoms, e.g. fumaric acid, maleic acid
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- Organic acid producing bacteria and method for producing organic acid are organic acid producing bacteria and method for producing organic acid
- the present invention relates to organic acid-producing bacteria such as coryneform bacteria and production of organic acids such as succinic acid using the same.
- Non-Patent Document 1 When an organic acid such as succinic acid is produced by fermentation, anaerobic bacteria such as Anaerobiospirillum genus and Actinobacillus genus are usually used (Patent Documents 1 and 2, Non-Patent Document 1).
- anaerobic bacteria When anaerobic bacteria are used, the yield of the product is high, but on the other hand, a large amount of organics such as CSL (corn steep liquor) in the medium because it requires more nutrients to grow. It is necessary to add a nitrogen source. Adding a large amount of these organic nitrogen sources not only increases the cost of the medium, but also increases the purification cost when extracting the product, which is economical!
- Patent Document 2 With regard to 2-oxoglutarate dehydrogenase (also called at-ketoglutarate dehydrogenase), reports have shown that its activity has been confirmed in coryneform bacteria (Non-patent Document 2), and reports that genes have been cloned (non-patent) There is Patent Document 3). In addition, a method for producing an amino acid using a microorganism having a reduced 2-oxodaltalate dehydrogenase activity is disclosed (Patent Document 6).
- Patent Document 1 U.S. Pat.No. 5,143,834
- Patent Document 2 U.S. Pat.No. 5,504,004
- Patent Document 3 Japanese Patent Laid-Open No. 11-113588
- Patent Document 4 Japanese Patent Laid-Open No. 11 196888
- Patent Document 5 Japanese Patent Laid-Open No. 11-196887
- Patent Document 6 International Publication No. 95Z34672 Pamphlet
- Non-patent literature 1 International Journal of Systematic Bacteriology (1999), 49,207-216
- Non-patent literature 2 Shiio I, Ujigawa- Takeda K. 1980. Presence and regulation of -keto glutarate dehydrogenase complex in a glutamate— producing bacterium, Brevibacterium flavum. Agric. Biol. Chem. 44: 1897—1904.
- Non-Patent Document 3 Usuda Y. Tujimoto N, Abe CAsakura Y. Kimura E. Kawahara Y, O, Mat sui H. 1996. Molecular cloning of the Corynebacterium glutamicum ('Brevibacteriu m lactofermentum' AJ12036) odhA gene encoding a novel type of 2-oxoglutaratedeh ydrogenase. Microbiology. 142: 3347-54.
- An object of the present invention is to provide a method for producing an organic acid such as succinic acid with higher production efficiency.
- a bacterium having an ability to produce an organic acid and modified so that 2-oxodaltalate dehydrogenase activity is enhanced as compared to an unmodified strain of the enzyme.
- An organic acid is produced by allowing the bacterium of any one of (1) to (9) or a treated product thereof to act on an organic raw material in a reaction solution containing carbonate ion, bicarbonate ion or carbon dioxide gas. And producing the organic acid, and collecting the organic acid.
- a method for producing an organic acid-containing polymer comprising a step of producing an organic acid by the method of any one of (10) to (13), and a step of carrying out a polymerization reaction using the organic acid obtained in the step as a raw material .
- FIG. 1 shows the procedure for constructing plasmid pC3.14.
- FIG. 2 shows the procedure for constructing plasmid pODH3.2.
- the bacterium of the present invention is a bacterium having an organic acid-producing ability and modified so that 2-oxodaltalate dehydrogenase (hereinafter also referred to as ODH) activity is enhanced as compared with an unmodified strain.
- Organic acid-producing ability refers to the ability to accumulate organic acid in the medium when the bacterium of the present invention is cultured.
- Organic acids include organic acids that are metabolic intermediates of the TCA cycle, and include, for example, succinic acid, malic acid, fumaric acid, citrate, isocitrate, and cis-aconitic acid. Succinic acid, malic acid and fumaric acid are preferred, and succinic acid is more preferred.
- Such a bacterium may be a bacterium that originally has organic acid-producing ability or a bacterium that has been imparted with organic acid-producing ability by breeding and has been modified to enhance ODH activity. It may be modified so as to have an organic acid-producing ability by performing a modification that enhances. Examples of means for imparting an organic acid-producing ability by breeding include, for example, mutation treatment, gene recombination treatment, and the like. More specifically, modification or pyrubin that reduces ratate dehydrogenase activity as described later is used. Modifications that enhance the acid carboxylase activity are included.
- the bacterium used in the present invention may have the ability to produce two or more kinds of organic acids.
- Bacteria that can be used in the production method of the present invention is not particularly limited as long as it has an organic acid-producing ability, but coryneform bacteria, Bacillus bacteria, Escherichia bacteria, Lactobacillus bacteria, Among the succinobacteria and lysobium bacteria, coryneform bacteria are preferred.
- Escherichia bacteria include Escherichia coli
- Lactobacillus bacteria include Lactobacillus helveticus (J Appl Microbiol, 2001, 91, p846-852)
- Bacillus bacteria include Bacillus. Subtilis, Bacillus 'Amiloli cefaciens, Bacillus pumilus, Bacillus' steer mouth thermophilus, etc.
- the genus Rhizobium include Rhizobium etli.
- the coryneform bacterium is not particularly limited as long as it is classified as such, and examples thereof include bacteria belonging to the genus Corynebacterium, bacteria belonging to the genus Brevibaterum, and bacteria belonging to the genus Arthrobacter. Examples include those belonging to the genus Corynebataterum or Brevibaterium, more preferably ⁇ Bacteria classified as Brembacterium ammoniagenes or Brevibacterium ⁇ Breatbacterium lactofermentum.
- bacterial parent strain used in the present invention include Brevibaterium 'flavum MJ-233 (FERM BP-1497), MJ-233 AB-41 (FERM BP-1498), Brevibaterium 'Ammonia Genes ATCC6872, Corynebatarum' Glutamicum ATCC31831, Brevibaterumum 'Ratatomentumum ATCC13869, etc.
- Brevibaterium flavum is currently classified as Corynebacterium glutamicum (Lielbl, W., Ehrmann, M., Ludwig, W. and Sch leifer, KH, International Journal). of Systematic Bacteriology, 1991, vol.
- Brevibaterium 'flavum MJ-233 strain and its mutant MJ-233 AB-41 strain are each a corynebacterium.
- the above-mentioned bacteria used as parent strains are mutant strains obtained by ordinary mutation treatments such as UV irradiation and NTG treatment, and combinations induced by genetic techniques such as cell fusion or gene recombination. It may be a misplaced stock, such as a replacement stock.
- the bacterium of the present invention can be obtained by subjecting a bacterium having an organic acid-producing ability as described above to modification so that ODH activity is enhanced. However, the modification for enhancing the ODH activity may be performed after the modification for imparting the productive ability.
- ODH activity refers to the activity that catalyzes the reaction that oxidatively decarboxylates 2-oxodaltalic acid ( ⁇ -ketoglutaric acid) to produce succinylo CoA (succiny ⁇ CoA).
- the activity is enhanced means that the ODH activity is enhanced as compared with the non-modified ODH strain.
- the ODH activity is preferably enhanced 1.5 times or more per unit cell weight, more preferably 2 times or more, compared to the non-modified ODH strain.
- the ODH 7 tongue 'property can be measured according to the method of Shuo et al. (Isamu Shno and Kyoko Ujigawa-Takeda, Agnc. Biol. L ⁇ hem., 44 (8), 1897-1904, 1980).
- Modifications to enhance ODH activity using the ODH gene include, for example, transformation using a plasmid, integration of the ODH gene onto the chromosome by homologous recombination, modification of the ODH gene expression regulatory sequence, etc. Can be done by.
- the ODH gene that can be used when introducing an ODH gene into a host cell by transformation using a plasmid or homologous recombination is a gene that increases ODH activity when introduced into a host cell, that is,
- the gene is not particularly limited as long as it is a gene encoding a protein having ODH activity, and examples thereof include an ODH gene (odhA) derived from a coryneform bacterium having the base sequence shown in SEQ ID NO: 3.
- ODH gene encodes a protein having ODH activity, it is 90% or more, preferably 95% or more, more preferably more than the DNA having the above-mentioned base sequence and DNA that is stringent and hybridized under stringent conditions. May be a homologue gene such as DNA having a homology of 99% or more.
- stringent conditions are those for washing ordinary Southern hybridization60.
- ODH genes derived from bacteria other than coryneform bacteria, or from other microorganisms or plants and animals can also be used.
- ODH genes derived from microorganisms or plants and animals have already been sequenced, and have ODH activity based on genes, homology, etc.
- a gene coding for can be isolated from a chromosome of a microorganism, animal or plant, etc., and its nucleotide sequence determined.
- a gene synthesized according to the sequence can be used. These can be obtained by, for example, amplifying the region containing the promoter and ORF portion by a hybridization method or a PCR method.
- the ODH gene in the coryneform bacterium is introduced.
- a highly replaceable string-replaceable plasmid can be obtained.
- the promoter for expressing the ODH gene may be the promoter of the ODH gene itself, or may be replaced with another strong promoter that can function in the host bacteria.
- promoters derived from E. coli such as tac promoter and trc promoter can be mentioned.
- a plasmid vector that can be used is not particularly limited as long as it contains at least a gene that controls a replication function in the coryneform bacterium.
- Specific examples thereof include, for example, plasmid PCRY30 described in JP-A-3-210184; plasmids pCRY21, pCRY2KE, pCRY2KX, pCRY31 described in JP-A-2-72876 and US Pat. No. 5,185,262.
- PCRY3KE and PCRY3KX plasmids pCRY2 and pCRY3 described in JP-A-1 191686; JP-A-58-67679 [described in pAM330; JP-A-58-77895 described in PHM1519; PAJ655, pAJ611 and pAJ1844 described in JP-A-58-192900; pCGl described in JP-A-57-134500; PCG2 described in JP-A-58-35197; JP-A-57-183799 PCG4 and pCGll described in the publication No., pPK4 described in the pamphlet of International Publication No. 95Z34672, and the like.
- plasmid vectors used in the coryneform bacterium host vector system have a gene that controls the replication function of the plasmid in the coryneform bacterium and a gene that controls the stabilization function of the plasmid in the coryneform bacterium.
- plasmids pCRY30, pCRY21, pCRY2KE, pCRY2KX, pCRY31, pCRY3KE and pCRY3KX are preferably used.
- Enhancement of ODH activity can also be achieved by replacing a promoter on the host chromosome.
- the sequence information of the promoter region can be obtained from, for example, GenBank Database Accession No. AP005276.
- the type of promoter to be replaced is not particularly limited as long as it can function in the host bacterium, but a promoter that does not suppress transcriptional activity under anaerobic conditions is preferred.
- the tac promoter used in Escherichia coli the trc promoter Etc.
- an organic acid-producing bacterium modified so as to reduce acetic acid production may be modified to enhance ODH activity.
- 2-oxodaltalic acid may be a by-product.
- ODH catalyzes a reaction that acidally decarboxylates 2-oxoglutaric acid to produce succinyl-CoA, and by enhancing ODH activity, the by-product of 2-oxodaltalic acid is reduced. The production amount of the target organic acid is increased.
- modifications that reduce acetic acid production include, for example, acetate kinase (hereinafter also referred to as ACK), phosphotransacetylase (hereinafter also referred to as PTA), acetyl-CoA neuronase (hereinafter also referred to as ACH), Modifications that reduce the activity of pyruvate oxidase (hereinafter also referred to as POXB) can be mentioned.
- ACK acetate kinase
- PTA phosphotransacetylase
- ACH acetyl-CoA neuronase
- POXB pyruvate oxidase
- Acetic acid is an intermediate in the biosynthetic pathway of oxamouth acetic acid and oxamouth acetic acid derivatives. Since a Co-A force is also generated, it is preferable to reduce the activity of any one or all of the above enzymes in order to block the acetic acid synthesis pathway and reduce acetic acid by-products.
- PTA activity refers to an activity that catalyzes a reaction in which phosphoric acid is transferred to acetyl-CoA to form acetyl phosphonic acid. “Modified so that the PTA activity is decreased” means that the PTA activity has become lower than the specific activity of an unmodified strain, for example, a wild strain. It is more preferable that the PTA activity is decreased to 10% or less per microbial cell, which is preferably decreased to 30% or less per microbial cell as compared with the unmodified strain. Moreover, PTA activity may be completely lost. The decrease in PTA activity can be confirmed by measuring PTA activity by the method of Klotzsch et al. (Klotzsch, HR, Meth Enzymol. 12, 3 81-386 (1969)).
- ACK activity refers to the activity of catalyzing the reaction of producing acetic acid from acetylyl phosphate and ADP. “Modified so that the ACK activity decreases” means that the ACK activity has become lower than the specific activity of an unmodified strain, for example, a wild strain. It is more preferable that the ACK activity is reduced to 10% or less per microbial cell, which is preferably 30% or less per microbial cell, compared to the unmodified strain. Further, ACK activity may be completely lost. The decrease in ACK activity can be confirmed by measuring the ACK activity by the method of Ramponi et al. (Ramponi G., Meth. Enzymol. 42, 409-426 (1975)).
- the pta gene is disrupted according to a known method, for example, a method using homologous recombination or a method using the sacB gene (Schafer, A. et al. Gene 145 (1994) 69-73). be able to.
- Examples of the pta gene include DNA containing a base sequence consisting of base numbers 1-1383 of SEQ ID NO: 7.
- the homologous gene of the sequence should also be used. Can do.
- the homology that causes homologous recombination is preferably 70% or more, more preferably 80% or more. More preferably, it means homology of 90% or more, particularly preferably 95% or more.
- homologous recombination can occur if the DNAs can be hybridized with each other under stringent conditions.
- the activity when decreased by ACK alone, it may be modified using the ack gene.
- a gene having a nucleotide sequence of nucleotide numbers 1386 to 2579 of SEQ ID NO: 7 can be mentioned.
- a gene having homology to the sequence can be used to such an extent that homologous recombination with the ack gene on the chromosome occurs.
- the homology to the extent that homologous recombination occurs preferably means 70% or more, more preferably 80% or more, still more preferably 90% or more, particularly preferably 95% or more.
- homologous recombination can occur if the DNAs can be hybridized with each other under stringent conditions.
- the bacterium used in the present invention may be a bacterium obtained by combining two or more of the above modifications in addition to the modification that enhances the ODH activity.
- the number of one jet is not limited.
- ACH activity refers to an activity that catalyzes a reaction in which acetyl-CoA and hydropower also produce acetic acid.
- Modified to reduce ACH activity means that the ACH activity has become lower than the specific activity of an unmodified strain such as a wild strain. It is preferable that the ACH activity is reduced to 50% or less, preferably 30% or less, more desirably 10% or less per microbial cell as compared with the unmodified strain. The “decrease” includes the case where the activity is completely lost. ACH activity was decreased by measuring ACH activity with reference to Gergely J., et al. (Gergely J., Hele. P. & Ramkrishnan, CV (1952) J. Biol.
- Chem. 198 p323_334) As an example of a gene encoding ACH of coryneform bacteria, the sequence of Corynebaterum 'glutamicum registered in GenBank (NCgl2480 of GenBank Accession No.NC_003450 (complementary strand of 2729376..2730884 of NC_003450)) is (WO200 5/113744). The sequence of the ach gene of Corynebatatum glutamicum is shown in SEQ ID NO: 14 1037-2545. In addition, a gene having homology to the sequence to such an extent that homologous recombination with the ach gene on the chromosome can be used.
- the degree of homology that causes homologous recombination is preferably 70% or more, more preferably 80% or more, and even more preferably. Or 90% or more, particularly preferably 95% or more homology.
- homologous recombination can occur if the DNAs can be hybridized with each other under stringent conditions.
- POXB activity refers to the activity of catalyzing the reaction of generating acetic acid from pyruvic acid and water!
- “Modified so that the acupuncture activity is reduced” means that the specific activity of the acupuncture activity non-modified strain, for example, a wild strain is lowered.
- the sputum activity is preferably reduced to 50% or less per cell compared to the unmodified strain, more preferably 30% or less, and more preferably 10% or less. It is particularly preferred.
- “Decrease” includes the complete loss of activity. It is confirmed by measuring the POXB activity from Chang et al. (Chang Y. and Cronan JE JR, J. Bacteriol. 151, 1279-1289 (1982)) Can do.
- coryneform bacterium poxB gene is a sequence registered in GenBank (a complementary strand of 2776766-2778505 of GenBank Accession No. NC_003450) (WO2005 / 113745).
- GenBank a complementary strand of 2776766-2778505 of GenBank Accession No. NC_003450
- the sequence of the pox B gene of Corynebaterum 'glutamicum is shown in SEQ ID NO: 16 at 996-2735.
- a gene having homology to the sequence to such an extent that homologous recombination with the poxB gene on the chromosome can be used.
- the homology that causes homologous recombination preferably means 70% or more, more preferably 80% or more, still more preferably 90% or more, and particularly preferably 95% or more.
- homologous recombination can occur as long as the DNAs can hybridize with each other under stringent conditions.
- the ach gene and poxB gene can be disrupted by a publicly known method, similar to the above-described disruption of the pta gene.
- the bacterium of the present invention may be a bacterium modified so as to reduce the activity of latate dehydrogenase (hereinafter also referred to as LDH) in addition to the enhancement of ODH activity.
- LDH latate dehydrogenase
- Such bacteria are particularly effective when the organic acid is succinic acid.
- Such a bacterium can be obtained, for example, by preparing a bacterium in which the LDH gene is disrupted and further modifying the bacterium with the ODH gene. However, either modification for reducing LDH activity or modification for enhancing ODH activity may be performed first.
- LDH activity is reduced means that LDH activity is reduced compared to an LDH gene-unmodified strain. It means that The LDH activity is preferably reduced to 10% or less per cell as compared with the unmodified strain. LDH activity may be completely lost.
- the reduction in LDH activity can be confirmed by measuring LDH activity by a known method (L. Kanarek and R ⁇ Hill, J. Biol. Chem. 239, 4202 (1964)).
- a specific method for producing a strain having a reduced LDH activity of a coryneform bacterium a method by homologous recombination to a chromosome described in JP-A-11-206385 or a method using a sacB gene (Schafer , A. et al. Gene 145 (1994) 69-73).
- the bacterium used in the present invention may be a bacterium modified so that the activity of pyruvate carboxylase (hereinafter also referred to as PC) is enhanced in addition to the enhancement of ODH activity.
- PC pyruvate carboxylase
- the ⁇ C activity is enhanced means that the PC activity is preferably increased by 100% or more, more preferably by 300% or more, compared to a non-modified strain such as a wild strain or a parent strain.
- the PC activity can be measured, for example, by a method for measuring a decrease in NADH (WO2005Z021770).
- Such a bacterium can be obtained, for example, by introducing a PC gene into a coryneform bacterium with enhanced expression of the ODH gene.
- the modification operation for the introduction of the PC gene and the enhancement of the ODH activity may be performed in advance.
- PC gene can be performed, for example, by highly expressing the pyruvate carboxylase (PC) gene in coryneform bacteria in the same manner as described in JP-A-11-196888.
- PC pyruvate carboxylase
- a PC gene derived from Corynebacterium dartamicam (Peters- Wendisch, PG et al. Microbiology, vol. 14 4 (1998) p915-927 (SEQ ID NO: 5)) should be used. Can do.
- the DNA having a nucleotide sequence of SEQ ID NO: 5 is hybridized under stringent conditions, or the nucleotide sequence of SEQ ID NO: 5 is 90% or more, preferably 95% or more, more preferably 99% or more.
- a DNA encoding a protein having a PC activity can also be suitably used.
- Bacteria other than Corynebataterum dartamicum, or other bacteria or PC genes derived from animals and plants can also be used.
- the following PC genes derived from bacteria or animals and plants are known in sequence (documents are shown below), and the ORF part is amplified by hybridization genease or PCR method as described above. Can be obtained.
- Notillus' stearothermophilus (Bacillus stearothermophilus)
- the PC activity can be enhanced by the same method as the ODH activity enhancement described above.
- the bacterium or a processed product thereof is allowed to act on an organic raw material in a reaction solution containing carbonate ion, bicarbonate ion or carbon dioxide gas, thereby generating an organic acid. It is a method for producing an organic acid characterized by collecting.
- the organic acid to be produced is more preferably succinic acid, preferably succinic acid, fumaric acid, malic acid or pyruvic acid, which are the organic acids described above.
- a cultivated slant culture in a solid medium such as an agar medium for direct reaction, but the bacterium is cultured in advance in a liquid medium (seed culture). It is preferable to use the above.
- a medium used for seed culture a normal medium used for bacterial culture can be used.
- ammonium sulfate, potassium phosphate, sulfuric acid A general medium in which a natural nutrient source such as meat extract, yeast extract or peptone is added to the composition of inorganic salt such as magnesium can be used.
- the bacterial cells after seed culture are preferably collected by centrifugation, membrane separation, etc., and then used for organic acid production reaction.
- the organic acid may be produced by reacting the seed-cultured bacteria with the organic raw material while growing them in a medium containing the organic raw material, or the cells obtained by proliferating in advance contain the organic raw material.
- the organic acid may be produced by reacting with an organic raw material in the reaction solution.
- a processed product of bacterial cells can also be used.
- the treated product of cells include, for example, immobilized cells obtained by immobilizing cells with acrylamide, carrageenan, etc., crushed materials obtained by disrupting cells, centrifuged supernatants thereof, or supernatants thereof by ammonium sulfate treatment, etc. Examples include partially purified fractions.
- the organic raw material used in the production method of the present invention is not particularly limited as long as the bacterium can assimilate and produce an organic acid such as succinic acid, but usually galactose, ratatoose, gnolecose, Carbohydrates such as funolectose, glyceronole, sucrose, saccharose, starch, cellulose, etc .; fermentable saccharides such as glycerin, mannitol, xylitol, ribitol, and other polyalcohols are used, of which glucose or sucrose is preferred. In particular, glucose is preferable.
- a starch saccharified solution, molasses and the like containing the fermentable saccharide are also used. These fermentable carbohydrates can be used alone or in combination.
- concentration of the organic raw material used is not particularly limited, but it is advantageous to make it as high as possible within the range that does not hinder the production of organic acids such as succinic acid, and usually 5-30% ( 1 ⁇ ⁇ 7), The reaction is preferably carried out within the range of 10 to 20% ( 1 ⁇ 7).
- additional organic raw materials may be added as the organic raw materials decrease as the reaction progresses.
- the reaction solution containing the organic raw material is not particularly limited, and may be, for example, a medium for culturing bacteria or a buffer solution such as a phosphate buffer solution.
- the reaction solution is preferably an aqueous solution containing a nitrogen source or an inorganic salt.
- the nitrogen source is not particularly limited as long as the bacteria can be assimilated to produce organic acids such as succinic acid, but specifically, ammonium salt, nitrate, urea, soybean hydrolysis
- organic acids such as succinic acid, but specifically, ammonium salt, nitrate, urea, soybean hydrolysis
- organic products such as food, casein digest, peptone, yeast extract, meat extract, corn steep liquor, etc.
- An inorganic nitrogen compound is mentioned.
- inorganic salts include metal salts such as various phosphates, sulfates, magnesium, potassium, manganese, iron, and zinc.
- vitamins such as piotin, pantothenic acid, inositol, and nicotinic acid, nucleotides, amino acids and other factors that promote growth may be added as necessary.
- the reaction solution contains carbonate ion, bicarbonate ion, or diacid carbon gas.
- Carbonate or bicarbonate ions are also supplied with magnesium carbonate, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, etc., which can also be used as neutralizing agents. Acids or their salts or diacid carbon gas can also be supplied.
- Specific examples of the carbonate or bicarbonate salt include magnesium carbonate, ammonium carbonate, sodium carbonate, potassium carbonate, ammonium bicarbonate, sodium bicarbonate, potassium bicarbonate and the like.
- Carbonate ions and bicarbonate ions are added at a concentration of 1 to 500 mM, preferably 2 to 300 mM, more preferably 3 to 200 mM.
- carbon dioxide gas is contained, 50 mg to 25 g, preferably 100 mg to 15 g, more preferably 150 mg to 1 Og of diacid carbon dioxide gas per liter of the solution is contained.
- the pH of the reaction solution is adjusted by adding sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium hydroxide, calcium hydroxide, magnesium hydroxide, or the like. be able to.
- the pH in this reaction is usually pH 5 to 10, preferably pH 6 to 9.5, and the pH of the reaction solution is adjusted within the above range with an alkaline substance, carbonate, urea or the like as needed during the reaction.
- the optimal temperature for growth of bacteria used in this reaction is usually 25 ° C to 35 ° C.
- the temperature during the reaction is usually 25 ° C to 40 ° C, preferably 30 ° C to 37 ° C.
- the amount of cells used in the reaction is not particularly limited, but 1 to 700 gZL, preferably 10 to 500 gZL, more preferably 20 to 400 gZL is used.
- the reaction time is preferably 1 hour to 168 hours, more preferably 3 hours to 72 hours.
- the reaction for producing an organic acid such as succinic acid may be carried out by aeration and stirring. You may perform in the anaerobic atmosphere which does not supply.
- the anaerobic atmosphere here means to react with the dissolved oxygen concentration kept low. In this case, the dissolved oxygen concentration
- a container is sealed and reacted without aeration, a reaction is performed by supplying an inert gas such as a nitrogen gas, or a inert gas containing diacid-carbon gas is vented.
- an inert gas such as a nitrogen gas, or a inert gas containing diacid-carbon gas is vented. Can be used.
- organic acids such as succinic acid, fumaric acid, malic acid or pyruvic acid are generated and accumulated in the reaction solution.
- the organic acid accumulated in the reaction solution (culture solution) can be collected from the reaction solution according to a conventional method. Specifically, for example, solids such as bacterial cells are removed by centrifugation, filtration, etc., then desalted with ion exchange resin, etc., and purified from the solution by crystallization or column chromatography. Organic acids can be collected.
- an organic acid-containing polymer can be produced by producing an organic acid by the above-described method of the present invention and then performing a polymerization reaction using the obtained organic acid as a raw material.
- the succinic acid produced in the present invention is used after being processed into polymers such as polyester and polyamide.
- the succinic acid-containing polymer is obtained by polymerizing succinic acid with a succinic acid polyester obtained by polymerizing a diol such as butanediol or ethylene glycol with succinic acid, or a diamine such as hexamethylenediamine. Examples thereof include succinic acid polyamide.
- the succinic acid obtained by the production method of the present invention or a composition containing the succinic acid can be used for food additives, pharmaceuticals, cosmetics and the like.
- the obtained cells were suspended in 0.15 mL of a 10 mM NaCl / 20 mM Tris buffer (pH 8.0) / lmM EDTA ′ 2Na solution containing lysozyme at a concentration of lOmgZmL.
- proteinase K was added to the suspension so that the final concentration was 100 gZmL, and the mixture was incubated at 37 ° C for 1 hour.
- sodium dodecyl sulfate was added to a final concentration of 0.5%, and the mixture was incubated at 50 ° C for 6 hours for lysis.
- the E1 component gene (odhA) was obtained by using the DNA prepared in (A) above as a cage, and the entire genome sequence was reported.
- Synthetic DNA SEQ ID NO: 1 and SEQ ID NO: 2 designed on the basis of the sequence of the gene of Corynebaterium glutamicum ATCC13032 (complementary strand of 1172498... 1176271 of GenBank Database Accession No. BA000036) Performed by PCR used.
- Reaction solution composition vertical DNA 1 ⁇ PfxDNA polymerase (manufactured by Invitrogen) 0.5 ⁇ 1x concentrated buffer, 0.4 ⁇ each primer, ImM MgSO, 0.2 MdNTPs are mixed, and the total amount
- the recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 50 ⁇ g ZmL ampicillin and 50 ⁇ g ZmLX-Gal. Clones that formed white colonies on this medium were subjected to liquid culture by a conventional method, and then plasmid DNA was purified. By cutting the obtained plasmid DNA with restriction enzymes Ssel and BamHI, an inserted fragment of about 4.4 kb was observed, which was named pODHl.
- Coryneform bacterial vectors that can coexist with PTZ4 can be obtained by replacing the kanamycin metagene of the plasmid vector pC3 (Plasmid 36 62 (1996)), which has a replication region compatible with pTZ4, with a streptomycin ⁇ spectinomycin resistance gene. It was constructed. Note that ⁇ 4 is an original plasmid for a PC amplification plasmid introduced into the MJ233ZPCZ A LDH strain described later.
- reaction solution vertical DNA 10 ng, PfxDNA polymerase (manufactured by Invitrogen) 0.2 ⁇ L, 1 ⁇ concentration attached buffer, 0.3 M each primer (synthetic DNA shown in SEQ ID NO: 10 and SEQ ID NO: 11), ImM MgSO, mixed with 0.25 MdNTPs, total volume of 20
- DNA thermal cycler PTC-200 manufactured by MJ Research
- 20 cycles at 94 ° C for 20 seconds, 60 ° C for 20 seconds and 72 ° C for 60 seconds were repeated 20 times.
- the heat insulation at 94 ° C in the first cycle was 1 minute 20 seconds, and the heat insulation at 72 ° C in the final cycle was 2 minutes.
- Confirmation of the amplification product was carried out by separating by 0.8% agarose (SeaKem GTG agarose: manufactured by FMCBioProducts) gel electrophoresis, and then visualized by bromide zyme staining to detect a 937 bp fragment.
- the target DNA fragment is recovered from the gel using the QIAQuick Gel Extraction Kit (QIAGEN). After recovery, the DNA fragment is phosphorylated at the 5 'end with T4 Polynucleotide Kinase (Treasure Bio). did.
- plasmid DNA was prepared by a conventional method and analyzed by the above PCR using the synthetic DNAs of SEQ ID NO: 10 and SEQ ID NO: 11 as a result.
- streptomycin Z-spectinomycin resistance gene was It was confirmed that it was inserted, and this was named pC3.
- the DNA fragment prepared by cleaving pC3 with restriction enzymes BamHI and PvuII is blunt-ended with Klenow fragment, mixed with pBglll linker (Takara Bio: CAGATCTG), and ligation kit.
- E. coli DH5a strain
- E. coli DH5a strain
- plasmid DNA was prepared by a conventional method, and a plasmid that was cleaved with the restriction enzyme Bglll was selected and named pC3.1.
- the ⁇ -peptide gene containing the LacZ multicloning site was prepared by PCR using the E. coli plasmid pT7Blue (Novagen) as a saddle type and the synthetic DNAs shown in SEQ ID NO: 12 and SEQ ID NO: 13 as primers.
- Reaction solution composition vertical DNA 10 ng, PfxDNA polymerase (manufactured by Invitrogen) 0.2 ⁇ L, 1x concentration buffer, 0.3 ⁇ M each primer, ImM MgSO, 0. Md NTPs were mixed to make a total volume of 20 ⁇ L.
- Reaction temperature condition 94 using DNA thermal cycler PTC-200 (manufactured by MJ Research). C for 20 seconds, 60. C for 20 seconds, 72. The cycle of 30 seconds force in C was repeated 20 times. The heat insulation at 94 ° C in the first cycle was 1 minute 20 seconds, and the heat insulation at 72 ° C in the final cycle was 2 minutes.
- the amplification product was confirmed by separation by 1.0% agarose (SeaKem GTG agarose: manufactured by FMCBioProducts) gel electrophoresis, followed by visualization by bromide zyme staining to detect a 5777 bp fragment.
- the target DNA fragment is recovered from the gel using the QIAQuick Gel Extraction Kit (manufactured by QIAGEN). After recovery, the DNA fragment is phosphorylated at the 5 'end with T4 Polynucleotide Kinase (Treasure Bio). did.
- a DNA fragment of about 4.4 kb generated by cleaving the above pODHl.0 with restriction enzymes Ssel and BamHI was separated and recovered by 0.75% agarose gel electrophoresis, and the plasmid pC3.14 was separated from the restriction enzyme Pstl and The DNA was mixed with DNA prepared by cutting with BamHI, and ligated using Ligation Kit ver. 2 (Takara Bio).
- the Escherichia coli DH5 ⁇ strain was transformed with the plasmid DNA thus obtained, and smeared on an LB agar medium containing 50 gZmL spectinomycin and 50 ⁇ g ZmLX-Gal.
- ODH-enhancement plasmid pODH3.2 into Brevibaterium 'flavum MJ233 / PC / ⁇ LDH strain (a strain in which the PC gene is enhanced and the LDH gene is disrupted: JP 2005-951 69: WO2005Z21770) Transformants obtained by the electric pulse method (Res.
- the Brevibaterium flavum MJ233ZpODHZPCZ ⁇ LDH strain was cultured overnight in 100 ml of A medium containing 2% dulcose. 50 ml of the obtained culture solution was collected by centrifugation at 4,000 rpm ⁇ 10 minutes at 4 ° C. and then washed twice with 30 ml of 100 mM TES-NaOH buffer (pH 7.5). The washed cells were suspended in 10 ml of Buffer A (100 mM TES-NaOH buffer (pH 7.5), 30% glycerol).
- ODH activity was measured as follows with reference to the method described in Agric. Boil. Chem., 44 (8), 1897-1904, 1980, I Shiio and K Ujiga wa-Takeda. First, 100 mM TES (DOJINDO, # 344-02653)-NaOH buffer (pH 7.7), 0.2 mM coenzyme A (Wako, # 306-50481), 0.3 mM thiamin pyrophosphate (SIGMA, # C— 8754), 5 mM 2— oxoglutarate (S IGMA, # 305-72-6), 3 mM L-cysteine (Wako, # 033-05272), 5 mM MgCl, 1 mM 3-ac
- a reaction solution was prepared to be etylpyridine adenine dinucleotide (Wako, # 44047000). After transferring the reaction solution to the measurement cuvette, the reaction was started by adding the crude enzyme solution, and the increase in absorbance (A) at 365 nm was measured. 1U is the amount of enzyme when A force increases in 1 minute
- the ODH specific activity of Brevibaterium flavum MJ233ZpODHZPCZ ⁇ LDH strain was 0.028 U / mg-protein according to the above measurement method.
- the activity was 0.009 U / mg-protein, and in the ODH gene plasmid-enhanced strain approximately 3 times as much as ODH It was confirmed that the activity increased.
- Urea 4 g, ammonium sulfate: 14 g, 1 potassium phosphate: 0.5 g, 2 potassium phosphate. 5g, Magnesium sulfate heptahydrate: 0.5g, Ferrous sulfate ⁇ Heptahydrate: 20mg, Manganese sulfate ⁇ Hydrate: 20mg, D Piotin: 200g, Thiamine hydrochloride: 200g, yeast extract : 5 g, force Zamino acid: 5 g, and distilled water: lOOOOmL medium lOOmL was placed in a 500 mL Erlenmeyer flask and sterilized by heating at 120 ° C for 20 minutes.
- Ammonium sulfate 42 g, 1 potassium phosphate: 1.5 g, 1.5 potassium potassium phosphate, 1.5 g, magnesium sulfate heptahydrate: 1.5 g, ferrous sulfate, heptahydrate: 60 mg, Manganese sulfate ⁇ Hydrate: 60 mg, D-Piotin: 600 / ⁇ 8 , Thiamine hydrochloride: 600 g, Yeast extract 15 g, Casamino acid 15 g, Antifoaming agent (Ade force Nord LG294: manufactured by Asahi Denka): lmL and Distilled water: 2500 mL of medium was placed in a 5 L fermentor and sterilized by heating at 120 ° C for 20 minutes.
- antifoaming agent Ade force Nord L G294: manufactured by Asahi Denka
- the culture solution obtained by the above main culture is added to the cells collected by centrifugation at 8000 rpm for 5 minutes and resuspended so that the OD (660 ⁇ m) becomes 60. It became cloudy.
- 200 mL of this suspension and 200 mL of a 20% dalcose solution sterilized in advance were mixed in a 1 L jar mentor, and kept at 35 ° C.
- the pH was maintained at 7.6 using 2M ammonium carbonate, and the reaction was carried out without aeration and stirring at 400 rpm. The reaction was completed about 47 hours after the start of the reaction.
- the Brevibaterium flavum MJ233ZpODHZPCZ ALDH strain had a 3.4% increase in succinic acid yield and a 47% decrease in amino acid production relative to succinic acid production compared to the MJ 233ZPCZ ALDH strain.
- the enhancement of ODH showed a clear increase in succinic acid yield and a reduction in amino acid yield.
- Urea 4 g, ammonium sulfate: 14 g, 1 potassium phosphate: 0.5 g, 2 potassium phosphate. 5g, Magnesium sulfate heptahydrate: 0.5g, Ferrous sulfate ⁇ Heptahydrate: 20mg, Manganese sulfate ⁇ Hydrate: 20mg, D Piotin: 200g, Thiamine hydrochloride: 200g, yeast extract : 5 g, force Zamino acid: 5 g, and distilled water: lOOOOmL medium lOOmL was placed in a 500 mL Erlenmeyer flask and sterilized by heating at 120 ° C for 20 minutes.
- Ammonium sulfate 42 g, 1 potassium phosphate: 1.5 g, 1.5 potassium potassium phosphate, 1.5 g, magnesium sulfate heptahydrate: 1.5 g, ferrous sulfate, heptahydrate: 60 mg, Manganese sulfate Hydrate: 60 mg, D-Piotin: 600 / ⁇ 8 , thiamine hydrochloride: 600 / z g , yeast extract 15g, casamino acid 15g, antifoaming agent (Ade force Nord LG294: manufactured by Asahi Denka): lmL and distilled water: 2500mL Was placed in a 5 L fermenter and sterilized by heating at 120 ° C for 20 minutes.
- Ade force Nord LG294 manufactured by Asahi Denka
- Ade force Nord L G294 manufactured by Asahi Denka
- the culture solution obtained by the above main culture is added to the cells collected by centrifugation at 8000 rpm for 5 minutes and resuspended so that the OD (660 ⁇ m) becomes 60. It became cloudy.
- 200 mL of this suspension and 200 mL of a 20% dalcose solution sterilized in advance were mixed in a 1 L jar mentor, and kept at 35 ° C.
- the pH was kept at 7.6 using 2M sodium carbonate, and the reaction was conducted with no aeration and stirring at 400 rpm. The reaction was completed about 47 hours after the start of the reaction.
- strains with reduced acetic acid production include latipate dehydrogenase (Id h), acetylyl CoA nodose enzyme (ach), Phosphotransacetylase (pta), Tokinase (ack), a strain with reduced pyruvate oxidase (poxB) activity (2256 ⁇ (ldh, pta, ack, ach, poxB) strain) was used (International Publication WO2005 / 113744, WO2005 / 113745 Pamphlet) .
- Ratatofamentum 2256 A (ldh, pta, ack, ac h, poxB) strain was transformed by the electric pulse method and CM-Dex agar medium containing 25 ⁇ g / ml kanamycin (Gnorecose 5 g / L, Polypeptone 10 g / L) , East extra tart 10g / L, KH P
- the 2256 ⁇ (ldh, pta, ack, poxB, ach) / (pPKS-X) strain which is an ODH amplification strain, is the control strain 2256 ⁇ (ldh, pta, ack, poxB, ach) / (pPK4
- the yield was improved by about 3% on average, and the by-product a-KG decreased by about 1/2. From this, in cono and succinic acid-producing bacteria modified to reduce the production of acetic acid, by enhancing the ODH activity, by-product a-KG may be reduced, and the amount of succinic acid produced may be increased. Indicated.
- an organic acid such as succinic acid can be produced quickly and efficiently.
- the obtained organic acid such as succinic acid can be used for food additives, pharmaceuticals, cosmetics and the like.
- An organic acid-containing polymer can also be produced by conducting a polymerization reaction using the obtained organic acid as a raw material.
Abstract
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BRPI0707674-6A BRPI0707674A2 (pt) | 2006-02-24 | 2007-02-23 | bactÉria capaz de produzir Ácido orgÂnico e processo para produÇço do mesmo |
EP07714831.0A EP1995308B1 (en) | 2006-02-24 | 2007-02-23 | Bacterium capable of producing organic acid, and method for production of organic acid |
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Publication number | Publication date |
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CN101389752B (zh) | 2015-08-05 |
CN101389752A (zh) | 2009-03-18 |
EP1995308A4 (en) | 2010-03-24 |
EP1995308A1 (en) | 2008-11-26 |
US7993888B2 (en) | 2011-08-09 |
JP5180060B2 (ja) | 2013-04-10 |
US20090156779A1 (en) | 2009-06-18 |
EP1995308B1 (en) | 2014-07-30 |
JPWO2007099867A1 (ja) | 2009-07-16 |
BRPI0707674A2 (pt) | 2011-05-10 |
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