WO2005113744A1 - コハク酸生産菌及びコハク酸の製造方法 - Google Patents
コハク酸生産菌及びコハク酸の製造方法 Download PDFInfo
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- WO2005113744A1 WO2005113744A1 PCT/JP2005/009232 JP2005009232W WO2005113744A1 WO 2005113744 A1 WO2005113744 A1 WO 2005113744A1 JP 2005009232 W JP2005009232 W JP 2005009232W WO 2005113744 A1 WO2005113744 A1 WO 2005113744A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/77—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Corynebacterium; for Brevibacterium
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/02—Thioester hydrolases (3.1.2)
- C12Y301/02001—Acetyl-CoA hydrolase (3.1.2.1)
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G63/00—Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule
- C08G63/02—Polyesters derived from hydroxycarboxylic acids or from polycarboxylic acids and polyhydroxy compounds
- C08G63/12—Polyesters derived from hydroxycarboxylic acids or from polycarboxylic acids and polyhydroxy compounds derived from polycarboxylic acids and polyhydroxy compounds
- C08G63/16—Dicarboxylic acids and dihydroxy compounds
Definitions
- the present invention relates to the fermentation industry, and relates to a method for efficiently producing succinic acid by a fermentation method using coryneform bacteria.
- Anaerobic bacteria such as the genus Anaerobiospirillum (Anaerobiospirillum) and the genus Actinobacillus (Actinobacillus) have been used (see Patent Documents 1 and 2 or Non-Patent Document 1).
- anaerobic bacteria When anaerobic bacteria are used, the product yield is high, but on the other hand, large amounts of organic compounds such as CSL (corn steep liquor) are required in the medium due to the need for many nutrients to grow. It is necessary to add a nitrogen source. Adding a large amount of these organic nitrogen sources not only raises the cost of culture but also raises the cost of refining when extracting products, which is not economical.
- Aerobic bacteria such as coryneform bacteria
- the cells are grown, then collected, washed, and produced as quiescent cells without aeration with oxygen to produce succinic acid.
- Patent Documents 3 and 4 There is also a known method (see Patent Documents 3 and 4).
- the amount of organic succinic acid to be produced, the production concentration, and the cell concentration are economical because the amount of organic nitrogen added is small and the cells can be sufficiently grown in a simple medium.
- improvement such as improvement of the production speed per unit and simplification of the manufacturing process.
- Patent Document 8 a method for producing succinic acid using Escherichia coli deficient in phosphoacetyltransferase (pta) and ratate dehydrogenase (ldh) ( Patent Document 8), an amino acid production method using an intestinal bacterial group deficient in pyruvate oxidase (poxB), and D-pantothenic acid using an enteric bacterial group deficient in pyruvate oxidase (poxB) A method for producing is known (see Patent Document 9).
- Acetate kinase (ack) and phosphotransacetylase (pta) have been reported as enzymes involved in acetic acid utilization of coryneform bacteria.
- a plurality of enzymes such as pyruvate oxidase (poxB) (see Patent Document 10), acyl phosphatase (acp), aldehyde dehydrogenase, acetyl CoA-noidase and the like are involved. It is clear that which enzymes are contributing to acetic acid synthesis. Therefore, until now, there has been no known method for producing succinic acid by using a coryneform bacterium with a reduced acetic acid biosynthetic enzyme.
- Acetyl-CoA nodose is an enzyme that produces acetic acid from acetyl-CoA and water
- Patent Document 1 U.S. Pat.No. 5,143,833
- Patent Document 2 U.S. Pat.No. 5,504,004
- Patent Document 3 JP-A-11-113588
- Patent Document 4 JP-A-11 196888
- Patent Document 5 JP-A-11-206385
- Patent Document 6 JP-A-6-14781
- Patent Document 7 JP-A-7-67683
- Patent Document 8 International Publication No. 99Z06532 pamphlet
- Patent Document 9 International Publication No. 02Z36797 pamphlet
- Patent Document 10 European Patent Application Publication No. 1096013
- Patent Document 11 European Patent Application Publication No. 1108790
- Non-Patent Document 2 Microbiology.1999 Feb; 145 (Pt 2): 503-13
- An object of the present invention is to provide a coryneform bacterium having an improved ability to produce succinic acid.
- the present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, have found that reducing acetyl-CoA hydrase activity in coryneform bacteria improves succinic acid-producing ability. I found it. In addition, they have found that by reducing phosphotransacetylase and acetate kinase activities in addition to acetyl-CoA nodulase activity, by-products of acetic acid are reduced, thereby completing the present invention.
- the present invention is as follows.
- a coryneform bacterium having a succinic acid-producing ability the coryneform bacterium being modified so that the activity of acetyl CoA nodose is reduced.
- Coryneform bacterium according to (3) which is the DNA according to the following (a) or (b), (A) DNA containing a nucleotide sequence consisting of nucleotide numbers 1037 to 2542 of SEQ ID NO: 44, or
- coryneform bacterium according to any one of (1) to (4), further modified so that the activity of one or both of phosphotransacetylase and acetate kinase is reduced.
- the coryneform bacterium according to any one of (1) to (7) or a treated product thereof is allowed to act on an organic raw material in a reaction solution containing carbonate ions, bicarbonate ions or carbon dioxide, whereby the reaction is carried out.
- a method for producing succinic acid comprising producing and accumulating succinic acid in a solution, and collecting succinic acid from the reaction solution.
- a method for producing a succinic acid-containing polymer comprising a step of producing succinic acid by the method of (8) or (9) and a step of polymerizing the obtained succinic acid.
- FIG. 1 shows a procedure for constructing plasmid pBS3.
- FIG. 2 is a view showing a procedure for constructing a plasmid pBS4S.
- FIG. 3 is a view showing a procedure for constructing a plasmid pBS5T.
- FIG. 4 is a view showing a procedure for constructing a plasmid p ⁇ ldh-l.
- FIG. 5 is a diagram showing a procedure for constructing plasmid pBS5T :: ⁇ ack.
- FIG. 6 is a diagram showing a procedure for constructing plasmid pBSCT :: ⁇ ptaack.
- FIG. 7 is a view showing a procedure for constructing plasmid pBS5T :: ⁇ poxB.
- FIG. 8 is a view showing a procedure for constructing plasmid pBS4S :: ⁇ ach.
- coryneform bacterium includes bacteria which are conventionally classified into the genus Brevipacterium and are currently classified into the genus Corynebacterium (Int. J. Syst. BacterioL, 41, 255). (1981)), and also includes bacteria of the genus Brevibataterirum, which is closely related to the genus Corynebacterium. Examples of such coryneform bacteria include the following.
- succinic acid-producing ability refers to the ability to accumulate succinic acid in a medium when the coryneform bacterium of the present invention is cultured. This succinic acid-producing ability may be a property of a wild-type coryneform bacterium or a property imparted by breeding. Good.
- coryneform bacterium having the ability to produce succinic acid include Brevibataterim. Flavam MJ233 ⁇ ldh strain having reduced ratatate dehydrogenase activity (JP-A-11-206385) and pyruvate carboxylase. Alternatively, Brevibataterum flavum MJ233 / pPCPYC strain (WO 01/27258 pamphlet, JP-A-11-196887) in which phosphoenol pyruvate carboxylase activity is enhanced, or Brevibatate terium flavum MJ-233 (FERM) BP—1497), MJ—233 AB—41 (FERM
- Brevibataterim 'Ammoniagenes ATCC6872 Corynebacterium' Glutamicum ATCC31831 '
- Brevibataterim' ratatofarmentum ATC C 13869 Brevibataterum 'Flavum' may now be classified as Corynebataterum 'glutamicum (Lielbl, W., Ehrmann, M., Ludwig, W. and Schleifer, KH, International Journal of Systematic Bacteriology, 1991, vol.
- the coryneform bacterium of the present invention is a coryneform bacterium having the succinic acid-producing ability as described above, and a coryneform bacterium modified so as to reduce acetyl-CoA-noid-mouth enzyme activity.
- ACH activity refers to an activity that catalyzes a reaction for producing acetic acid from acetyl-CoA and water.
- Modified to reduce acetyl-CoA hydratase activity means that the activity of acetyl-CoA hydridase is lower than the specific activity of an unmodified strain, for example, a wild-type coryneform bacterium.
- the ACH activity is preferably reduced to 50% or less per cell, preferably 30% or less, and more preferably 10% or less per cell, as compared to the unmodified strain.
- a control wild type coryneform bacterium for example, as a wild type strain, Brevibataterium 'ratatofarmentum ATCC13869, and as a non-modified strain, as a wild type strain, Brevibataterium' ratatofarmentum Alddh Strains.
- Acetyl CoA hydrase activity can be measured with reference to the method of Gergely J., et al. (Gergely J., Hele. P. & Ramkrishnan, CV (1952) J. Biol. Chem.
- the coryneform bacterium of the present invention preferably has a lower acetyl-CoA-noidase activity than a wild-type strain or a non-modified strain, but further has an improved accumulation of succinic acid as compared to these strains. It is more desirable! / ,.
- Examples of the acetyl CoA hydrase having the above activity include a protein having the amino acid sequence shown in SEQ ID NO: 45.
- a protein having the amino acid sequence shown in SEQ ID NO: 45 As long as it has acetyl CoA nodulase activity, it has an amino acid sequence containing substitution, deletion, insertion or addition of one or several amino acids in the amino acid sequence shown in SEQ ID NO: 45. There may be. Here, several means, for example, 2 to 20, preferably 2 to 10, and more preferably 2 to 5.
- Modified to reduce acetyl-CoA hydrase activity means, for example, that the number of acetyl-CoA nodose enzymes per cell is reduced, or that acetyl-CoA hydrase per molecule is reduced. This corresponds to the case where the oral enzyme activity is reduced. Specifically, it can be achieved by deleting a gene encoding acetyl-CoA or idolase on a chromosome, or modifying an expression control sequence such as a promoter Shine-Dalgarno (SD) sequence.
- SD Shine-Dalgarno
- Examples of the acetyl-CoA noidal mouth gene on the chromosome include a DNA containing a base sequence consisting of base numbers 1037 to 2542 of SEQ ID NO: 44. Further, as long as it encodes a protein having acetyl CoA hydrolase activity, a base sequence consisting of base numbers 1037 to 2542 of SEQ ID NO: 44 or a base sequence having the same base sequence strength can be prepared. It may be DNA that hybridizes under lob and stringent conditions! “Stringent conditions” refer to conditions under which a so-called specific hybrid is formed and a non-specific hybrid is not formed. It is difficult to numerically express this condition, but one example is 60. C, 1 ⁇ SSC, 0.1% SDS, preferably, 0.1 ⁇ SSC, 0.1% SDS, and a salt concentration corresponding to 0.1%, more preferably 2 to 3 times.
- the acquisition of the acetyl CoA noidal mouth gene (hereinafter referred to as the ach gene) is carried out by the sequence of Corynebatadium 'glutamicum registered in GenBank (NCgl2480 of GenBank Accession No. NC_003450 (2729376..2730884th of NC_003450). Based on the complementary strand))), synthetic oligonucleotides can be synthesized and cloned by performing a PCR reaction using the chromosome of Corynebacterium 'daltamicum' as type I.
- sequences of coryneform bacteria such as Brevibatatellium 'ratatofamentum, whose base sequence has been determined by the genome project in recent years, can also be used.
- Chromosomal DNA can be obtained from bacteria that are DNA donors, for example, by the method of Saito and Miura. Saito and K. Miura, Biochem. Biophys. Acta, 72, 619 (1963), Bioengineering Experiments, Japan Society for Biotechnology. Ed., Pp. 97-98, Baifukan, 1992).
- the ach gene prepared as described above or a part thereof can be used for gene disruption.
- the gene to be used for gene disruption is such that it undergoes homologous recombination with the ach gene on the chromosome DNA of the coryneform bacterium to be disrupted (for example, a gene having the nucleotide sequence of nucleotides 1037 to 2542 of SEQ ID NO: 44).
- Such a homologous gene can also be used, as long as it has the same homology.
- the homology at which homologous recombination occurs is preferably 70% or more, more preferably 80% or more, further preferably 90% or more, and particularly preferably 95% or more.
- homologous recombination can occur between DNAs capable of hybridizing with the above genes under stringent conditions.
- Stringent conditions refer to conditions under which a so-called specific hybrid is formed and a non-specific hybrid is not formed. It is difficult to clearly numerically I spoon this condition causes shows an example ⁇ or ,, 60 ° C, 1 X SSC , 0. 1 0/0 SDS, preferably ⁇ or, 0. 1 X SSC, 0.1 0/0 Ne th at those salt concentrations SDS, preferably include conditions of washing 2-3 times than once.
- a modified ach gene modified so as not to produce a functional acetyl CoA hydrolase is produced, and a coryneform bacterium is transformed with the DNA containing the gene.
- a coryneform bacterium is transformed with the DNA containing the gene.
- the ach gene on the chromosome can be disrupted.
- Such gene disruption by gene replacement using homologous recombination has already been established, and there are a method using linear DNA and a method using a plasmid containing a temperature-sensitive replication origin (US Pat. No. 6,303,383). Specification or JP-A-05-007491).
- Gene disruption by gene replacement using homologous recombination as described above can also be performed using a plasmid that does not have a replication ability on a host.
- a plasmid for recombination is prepared by inserting a temperature-sensitive replication origin, a deletion type ach gene, a sacB gene encoding levan sucrose, and a marker gene showing drug resistance such as chloramuecole.
- the SacB gene encoding levan sucrose is a gene used to efficiently select a strain from which the vector portion has been eliminated from the chromosome (Schafer. A. et al. Gene 145). (1994) 69-73). That is, in the case of coryneform bacterium, when levan eucase is expressed, levan produced by assimilating sucrose works lethally and cannot grow. Therefore, if the strain carrying the levan cyclase-loaded vector remaining on the chromosome is cultured on a sucrose-containing plate, it cannot grow, and only the strain from which the vector has been removed can be selected on the sucrose-containing plate.
- sacB gene or its homologous gene a gene having the following sequence can be used.
- Bacillus' subtilis sacB GenBank Accession Number X02730 (SEQ ID NO: 35)
- Bacillus ami liquorifaciens sacB GenBank Accession Number X52988
- Zymomonas mobilis sacB GenBank Accession Number L33402
- Notyl's stearothermophilus surB GenBank Accession Number U34874 Lactobacillus ⁇ San Francisensis: frfA GenBank Accession Number AJ508391 acetopacta ⁇ ⁇ ⁇ Xylinas: lsxA GenBank Accession Number AB034152
- a coryneform bacterium is transformed with the above-mentioned recombinant plasmid. Transformation may be performed according to the transformation method reported so far. For example, as described in Escherichia coli K-12, a method for increasing the permeability of DNA by treating recipient cells with salt calcium (Mandel, M. and Higa. , J. Mol. Biol., 53, 159 (1970)), and a method for preparing DNA and introducing DNA by preparing a cell in a growth stage, such as that reported for Bacillus' subtilis (Dancan, CH, Wilson. GA and Young.
- coryneform bacterium can also be transformed by the electric pulse method (Sugimoto et al., JP-A-2-207791).
- the temperature-sensitive plasmids of coryneform bacteria include p48K and pSFKT2 (the
- JP-A-2000-262288 pHSC4 (see French Patent Publication 1992 2667875, JP-A-5-7491), and the like.
- These plasmids are capable of autonomous replication at least at 25 ° C in coryneform bacteria and cannot replicate autonomously at the power of 37 ° C.
- Escherichia coli _112571 which retains pHSC4, was established on October 11, 1990 by the National Institute of Advanced Industrial Science and Technology, National Institute of Advanced Industrial Science and Technology (currently the National Institute of Advanced Industrial Science and Technology, Patent Microorganisms Depositary) (Dai 305-5466 Japan).
- the transformant obtained as described above is cultured at a temperature (25 ° C) in which the temperature-sensitive replication origin does not function, and a strain into which the plasmid has been introduced is obtained. Incubate the plasmid-introduced strain at high temperature to remove the temperature-sensitive plasmid, and spread the strain on a plate containing antibiotics. Since temperature-sensitive plasmids cannot replicate at high temperatures, strains from which plasmids have been eliminated cannot grow on plates containing antibiotics, but, with very low frequency, acetic acid biosynthesis genes on plasmids and chromosomes. A strain that has undergone recombination with the ach gene Appear.
- the strain in which the recombinant DNA has been integrated into the chromosome thus undergoes recombination with the ach gene sequence originally present on the chromosome, and two fusion genes of the chromosomal ach gene and the deletion type ach gene are obtained. It is inserted into the chromosome with the other part of the recombinant DNA (vector part, temperature-sensitive replication origin and drug resistance marker) interposed therebetween.
- the normal ach gene is left on the chromosomal DNA and the deleted ach gene is cut out, and conversely, the deleted ach gene is left on the chromosome DNA and the normal ach gene is cut out.
- the cut DNA is retained in the cells in the form of a plasmid.
- the ach gene on the plasmid loses its cellular power along with the plasmid. Then, a strain in which the ach gene has been disrupted can be obtained by selecting a strain in which the deletion type ach gene remains on the chromosome by PCR, Southern hybridization, or the like.
- the same gene disruption can be performed by using a plasmid having no replication ability in coryneform bacteria instead of the above-mentioned temperature-sensitive plasmid.
- a plasmid having no replication ability in coryneform bacteria a plasmid capable of replication in Escherichia coli is preferred, for example, pHSG299 (manufactured by Takara Bio Inc.) and pHSG399 (manufactured by Takara Bio Inc.).
- acetyl-CoA noidal mouth enzyme in addition to the above-described gene manipulation method, for example, coryneform bacteria are irradiated with ultraviolet light or N-methyl-N'-two-trough N-- A method may be used in which the strain is treated with a mutagen used in the usual mutagenesis treatment such as torsogazine (NTG) or nitrite, and the strain having reduced acetyl-CoA noidose activity is selected.
- a mutagen used in the usual mutagenesis treatment such as torsogazine (NTG) or nitrite
- phosphotransacetylase hereinafter referred to as PTA
- ACK acetate kinase
- phosphotransacetylase (PTA) activity refers to the conversion of phosphate to acetyl CoA.
- Acetate kinase (ACK) refers to the activity that catalyzes the reaction that produces acetic acid from acetyl phosphate and ADP (EC: 2.3.1.8). 2.7.2.1).
- the gene disruption method can be performed in the same manner as the above-described ach gene disruption method.
- each enzyme is, for example, Corynebacterium registered in GenBank.
- PTA activity has been reduced means that PTA activity has been reduced as compared to a non-modified PTA strain.
- the PTA activity may be reduced as compared with the non-modified PTA strain or the wild strain, but is preferably reduced to 50% or less per cell, more preferably 10% or less. Further, the PTA activity may be completely deleted.
- the decrease in PTA activity was determined by the method of Klotzsch et al. (Klotzsch, HR, Meth Enzymol. 12,
- a coryneform bacterium with reduced ACH and PTA activities can be obtained by preparing a coryneform bacterium with reduced ACH activity and further modifying it to reduce PTA activity. However, the modification for decreasing PTA activity and the modification for decreasing ACH activity should be performed first.
- ACK activity is decreased means that ACK activity is decreased as compared with a wild-type strain or an ACK-unmodified strain.
- the ACK activity may be reduced as compared to the ACK non-modified strain or the wild-type strain, but is 50% lower per cell than the ACK non-modified strain.
- concentration be reduced to 10% or less per cell.
- the ACK activity may be completely lost.
- the decrease in ACK activity can be confirmed by measuring ACK activity according to the method of Ramponi et al. (Ramponi G., Meth. Enzymol. 42, 409-426 (1975)).
- a coryneform bacterium with reduced ACH and ACK activities can be obtained by constructing a coryneform bacterium with reduced ACH activity and further modifying it to reduce ACK activity.
- any of the modification for decreasing the ACK activity and the modification for decreasing the ACH activity may be performed first.
- Ratate dehydrogenase activity refers to the activity of catalyzing the reaction of reducing pyruvic acid to produce lactic acid using NADH as a coenzyme.
- the ratate dehydrogenase activity is reduced means that the LDH activity is reduced as compared to the LDH non-modified strain.
- the LDH activity may be reduced as compared to the non-modified LDH strain or the wild strain, but is preferably reduced to 50% per cell, desirably 10% or less per cell.
- LDH activity is completely deficient!
- the decrease in LDH activity can be confirmed by measuring LDH activity by the method of Kanarek et al. (L. Kanarek and R ⁇ . Hill, J. Biol. Chem. 239, 4202 (1964)). it can.
- the coryneform bacterium of the present invention can be obtained by producing a coryneform bacterium having a reduced LDH activity and further modifying the coryneform bacterium so that the ACH activity is reduced.
- the modification for reducing the LDH activity and the modification for reducing the ACH activity may be performed first.
- the ldh gene for example, a gene having the sequence shown in SEQ ID NO: 37 can be used, and gene disruption and the like can be performed in the same manner as in the ach gene.
- a bacterium modified so as to increase the activity of pyruvate carboxylase (hereinafter, referred to as PC) in addition to decreasing the activity of ACH may be used.
- PC pyruvate carboxylase
- the activity of pyruvate carboxylase is enhanced means that the activity of PC is increased with respect to a wild-type strain or an unmodified strain such as a parent strain.
- the activity of PC can be measured by the method of Peters-Wendisch PG et al. (Peters-Wendisch PG et al. Microbiology 143, 1095-1103 (1997)).
- the pc gene encoding the PC protein used in the method of the present invention encodes a gene whose base sequence has already been determined or a protein having PC activity by the method described below.
- a DNA fragment isolated from a chromosome of a microorganism, animal, plant, or the like and having a determined base sequence can be used.
- a gene synthesized according to the sequence can also be used.
- the pyruvate carboxylase gene of Corynebacterium glutamicum ATCC13032 GenBank Accession No. NCgl0659 gene SEQ ID NO: 46
- PC genes derived from the following organisms can also be used.
- the DNA fragment containing the pc gene is inserted into an appropriate expression plasmid, for example, pUC118 (manufactured by Takara Bio), and introduced into an appropriate host microorganism, for example, Escherichia coli JM109 (manufactured by Takara Bio). Can be expressed.
- the expression of pyruvate carboxylase, which is the expressed pc gene product, is confirmed by measuring the PC activity of the transformant by the above-mentioned known method and comparing the non-transforming activity with the PC activity of the extracted crude enzyme solution. , Can be confirmed.
- a DNA fragment containing the pc gene into an appropriate plasmid, for example, a plasmid vector containing at least a gene responsible for the replication and replication function of the plasmid in a coryneform bacterium, recombination capable of highly expressing pc in a coryneform bacterium
- a promoter for expressing the pc gene in the recombinant plasmid was used.
- the promoter is not limited to the power that can be the promoter possessed by the coryneform bacterium, but may be any promoter as long as it is a nucleotide sequence that initiates transcription of the pc gene.
- the plasmid vector into which the pc gene can be introduced is not particularly limited as long as it contains at least a gene that controls the replication and growth function in coryneform bacteria. Specific examples thereof include plasmid pCRY30 described in JP-A-3-210184; plasmids pCRY21, pCRY2KE and pCRY2KX described in JP-A-2-72876 and US Pat. No. 5,185,262.
- the plasmid vector used in the coryneform bacterium host vector system includes a gene that controls the replication replication function of the plasmid in the coryneform bacterium and a gene that controls the plasmid stabilization function in the coryneform bacterium.
- plasmids having plasmids pCRY30, pCRY21, pCRY2KE, pCRY2KX, pCRY31, pCRY3KE, and pCRY3KX are preferably used.
- the recombinant vector is a coryneform bacterium, for example, brevinocacterium.ratatofarmentum ( Brevibacterium lactofermentum) 2256 strains (
- a coryneform bacterium with enhanced pc gene expression can be obtained. Transformation can be performed, for example, by the electric pulse method (Res. Microbiol, Vol. 144, p. 181-185, 1993).
- the PC activity can also be enhanced by introducing, replacing, or amplifying a pc gene on the chromosome by a known homologous recombination method to achieve high expression. By disrupting the ach gene in the thus obtained PC gene highly expressing strain, the PC activity is enhanced and the ACH activity is reduced. Bacterial strains can be obtained. Either reduction of ACH activity or enhancement of PC activity may be performed first.
- ACH activity or ACH, PTA activity, or ACK activity is modified so as to be reduced, LDH activity is further modified so as to be further reduced, or PC activity is further enhanced.
- the use of a bacterium that has been modified to be particularly effective is particularly effective in producing substances, particularly in producing succinic acid.
- succinic acid By culturing the coryneform bacterium obtained as described above in a medium, producing and accumulating succinic acid in the medium, and collecting succinic acid from the medium, succinic acid can be efficiently produced.
- the above bacteria are used in the production reaction of succinic acid, those obtained by slant culture on a solid medium such as an agar medium may be directly used for the reaction, but the above bacteria are preliminarily cultured in a liquid medium (seed culture). It is preferable to use one that has been used. Bacteria that have been seed-cultured in this way can be produced by reacting the bacteria with an organic material while growing the same in a medium containing the organic material. Alternatively, the cells can be produced by collecting the cells obtained by growing the cells and reacting the cells with an organic material in a reaction solution containing an organic material.
- the bacterium be cultured under ordinary aerobic conditions and then used in the reaction.
- a medium used for the culture a medium usually used for culturing a microorganism can be used.
- a general medium obtained by adding a natural nutrient source such as a meat extract, a yeast extract, or peptone to a composition comprising inorganic salts such as ammonium sulfate, potassium phosphate, and magnesium sulfate. it can.
- a natural nutrient source such as a meat extract, a yeast extract, or peptone
- a composition comprising inorganic salts such as ammonium sulfate, potassium phosphate, and magnesium sulfate.
- a processed product of bacterial cells can also be used.
- the treated cells include immobilized cells in which the cells are immobilized with acrylamide, carrageenan, etc., crushed cells, crushed cells, centrifuged supernatant, or supernatant treated with ammonium sulfate. A partially purified fraction and the like can be mentioned.
- the organic raw material used in the production method of the present invention is not particularly limited as long as it is a carbon source capable of assimilating succinic acid by assimilation of the present microorganism, and usually, galactose, ratatose, and dal Carbohydrates such as kose, funolectose, glyceronole, sucrose, saccharose, starch, cenorexose; fermentable carbohydrates such as polyalcohols such as glycerin, mannitol, xylitol, ribitol, and glucose and fructose are used. Glycerol is preferred, and glucose is particularly preferred.
- a starch saccharified solution, molasses, or the like containing the above fermentable carbohydrate is also used.
- These fermentable carbohydrates can be used alone or in combination.
- the concentration of the organic raw material used is not particularly limited, but it is advantageous to make it as high as possible without impairing the production of succinic acid, usually 5 to 30% ( 1 ⁇ ⁇ 7), preferably 10 to 10%.
- the reaction is performed within the range of 20% ( 1 ⁇ 7). Further, the organic raw material may be additionally added in accordance with the decrease of the organic raw material with the progress of the reaction.
- the reaction solution containing the organic raw material is not particularly limited, and a force medium using water, a buffer, a medium, or the like is most preferable.
- the reaction solution is preferably a reaction solution containing a nitrogen source, an inorganic salt, and the like.
- the nitrogen source is not particularly limited as long as the microorganism can assimilate and produce succinic acid, and specific examples thereof include ammonium salts, nitrates, urea, soybean hydrolyzate, and casein hydrolyzate. , Peptone, yeast extract, meat extract, corn steep liquor, and other various organic and inorganic nitrogen compounds.
- the inorganic salt various phosphates, sulfates, and metal salts such as magnesium, potassium, manganese, iron, and zinc are used.
- growth promoting factors such as vitamins such as biotin, pantothenic acid, inositol and nicotinic acid, nucleotides and amino acids are added as necessary.
- 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. Since the pH in this reaction is usually pH 5 to 10, preferably pH 6 to 9.5, the pH of the reaction solution may be adjusted within the above range depending on the need for alkaline substances, carbonates, urea, etc., even during the reaction. Adjust within.
- the medium preferably contains carbonate ions, bicarbonate ions or carbon dioxide (carbon dioxide).
- Carbonate or bicarbonate ions also provide magnesium carbonate, sodium carbonate, sodium bicarbonate, potassium carbonate and potassium bicarbonate which can also be used as neutralizing agents. However, if necessary, it can be supplied from carbonic acid or bicarbonate or a salt or carbon dioxide gas.
- Specific examples of the carbonate or bicarbonate salts include magnesium carbonate, ammonium carbonate, sodium carbonate, potassium carbonate, ammonium bicarbonate, sodium bicarbonate, potassium bicarbonate and the like.
- the carbonate ions and bicarbonate ions are added at a concentration of 0.001 to 5M, preferably 0.1 to 3M, and more preferably 1 to 2M. When carbon dioxide is contained, 50 mg to 25 g, preferably 100 mg to 15 g, and more preferably 150 mg to 10 g per liter of the solution is contained.
- the optimal growth temperature of the 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 the cells used for 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 succinic acid may be carried out with aeration and stirring, or may be carried out under anaerobic conditions without aeration and without supply of oxygen.
- the anaerobic condition means that the reaction is performed with the dissolved oxygen concentration in the solution kept low.
- a method for this purpose for example, a method in which the reaction is carried out with the container hermetically sealed and air-free, a method in which an inert gas such as nitrogen gas is supplied and the reaction is performed, or a method in which an inert gas containing carbon dioxide gas is passed is used. it can.
- Succinic acid accumulated in the reaction solution can be separated and purified from the reaction solution according to a conventional method. Specifically, after removing solids such as bacterial cells by centrifugation, filtration, etc., desalting with ion-exchange resin, etc., the succinic acid is separated and purified by crystallization or column chromatography. Can be.
- a succinic acid-containing polymer can be produced by producing succinic acid by the above-mentioned method of the present invention and then conducting a polymerization reaction using the obtained succinic acid as a raw material.
- the succinic acid-containing polymer may be a homopolymer or a copolymer with another polymer raw material.
- succinic acid-containing polymer examples include a succinic polyester obtained by polymerizing a diol such as butanediol and ethylene glycol and succinic acid, and a succinic acid obtained by polymerizing a diamine such as hexamethylene diamine and succinic acid. Acid polyamide and the like.
- succinic acid or a composition containing the succinic acid obtained by the production method of the present invention can be used for food additives, pharmaceuticals, cosmetics, and the like.
- the sacB gene (SEQ ID NO: 35) was obtained by PCR using Bacillus subtilis chromosomal DNA as type I and SEQ ID NOs: 1 and 2 as primers.
- LA taq (TaKaRa) was used for one cycle of incubation at 94 ° C for 5 minutes, followed by denaturation at 94 ° C for 30 seconds, association at 49 ° C for 30 seconds, and extension at 72 ° C for 2 minutes. Repeated 25 times.
- the resulting PCR product was purified by a conventional method, digested with Bglll and BamHI, and blunt-ended. This fragment was digested with Avail of pHSG299 and inserted into the blunted site.
- a plasmid in which the Smal site in the kanamycin resistance gene sequence present on PBS3 was disrupted by base substitution without amino substitution was obtained by crossover PCR.
- the synthetic DNA of SEQ ID NOS: 3 and 4 was And perform PCR to obtain an amplification product on the N-terminal side of the kanamycin resistance gene.
- PCR was performed using pBS3 as type III and the synthesized DNAs of SEQ ID NOS: 5 and 6 as type III.
- the PCR reaction was performed using Pyrobest DNA Polymerase (Takara Bio Inc.) for 1 cycle at 98 ° C for 5 minutes, followed by denaturation at 98 ° C for 10 seconds, association at 57 ° C for 30 seconds, and extension at 72 ° C for 1 minute.
- the desired PCR product can be obtained by repeating the following cycle 25 times.
- SEQ ID NOS: 4 and 5 are partially complementary, and the Smal site present in this sequence is destroyed by base substitution without amino acid substitution.
- the above kanamycin resistance gene N-terminal and C-terminal gene products are mixed so as to be approximately equimolar, and this is mixed.
- PCR was performed using the synthetic DNAs of SEQ ID NOS: 3 and 6 as primers to obtain a mutated Km-resistant gene amplification product.
- the PCR reaction uses Pyrobest DNA Polymerase (Takara Bio Inc.), after one cycle of incubation at 98 ° C for 5 minutes, denaturation 98 ° C 10 seconds, association 57 ° C 30 seconds, extension 72 ° C 1.5 minutes.
- the desired PCR product can be obtained by repeating the cycle 25 times.
- the PCR product was purified by a conventional method, digested with Banll, and inserted into the Banll site of pBS3 described above.
- the temperature-sensitive replication origin of coryneform bacteria obtained by BamHI and Smal treatment of 1 "[3-4 (see Patent Publication 5-7491) was obtained by smoothing the Ndel site of PBS4S.
- transformation was performed using a competent cell of Escherichia coli JM109 (manufactured by Takara Bio Inc.), applied to an LB medium containing 25 g / ml of Km, and cultured overnight. Thereafter, the emerged colonies were picked up, a single colony was separated to obtain a transformant, a plasmid was extracted from the obtained transformant, and the target PCR product was inserted.
- PBS5T The temperature-sensitive replication origin of coryneform bacteria obtained by BamHI and Smal treatment of 1 "[3-4 (see Patent Publication 5-7491) was obtained by smoothing the
- Figure 3 shows the construction of pBS5T.
- ldh gene The gene fragment from the ORF of ratate dehydrogenase (hereinafter abbreviated as ldh gene) derived from Brevibatadium ratatofamentum 2256 strain is a previously released corynebate teratium 'glutamicum ATCC13032 (GenBank Database Accession
- NC_003450 was obtained by crossover PCR using a synthetic DNA designed with reference to the nucleotide sequence of the gene (SEQ ID NO: 37) (SEQ ID NO: 37). Specifically, PCR was performed by a conventional method using the chromosomal DNA of Brevibatarum 'ratatophamentum 2256 strain' as ⁇ type and the synthetic DNAs of SEQ ID NOS: 7 and 8 as primers to obtain an amplification product on the N-terminal side of the ldh gene.
- PCR was performed by a conventional method using the genomic DNA of Brevibata terium 'ratatofamentum 2256 strain genomic DNA and the synthetic DNAs of SEQ ID NOS: 9 and 10 as primers. .
- SEQ ID NOS: 8 and 9 are complementary to each other and have a structure in which the entire sequence of the ORF of ldh is deleted.
- the 2256 strains of Brevi Bata Terimu 'Ratoto Armamentum' can be obtained from the American 'Type-Carcia I' collection (ATCC) (Address ATCC, PO Box 1549, Manassas, VA 20108, United States of America) .
- ATCC American 'Type-Carcia I' collection
- PCR was performed by a conventional method using the synthetic DNAs of Nos. 11 and 12 as primers to obtain a mutated ldh gene amplification product.
- the resulting PCR product was purified by a conventional method, digested with Sail, and inserted into the above-mentioned Sail site of PBS4S.
- FIG. 4 shows a construction diagram of the plasmid.
- p ⁇ ldh56-l obtained in (A) above does not contain a region that enables autonomous replication in coryneform bacterium cells, the frequency is extremely low when transforming coryneform bacterium with this plasmid.
- a strain in which this plasmid has been integrated into the chromosome by homologous recombination appears as a transformant.
- Brevibata terium 'ratatofarmentum 2256 strain was transformed by the electric pulse method using a high-concentration plasmid P ⁇ ldh56-l, and CM-Dex medium containing 25 ⁇ g / ml of kanamycin (glucose 5 g / L, polypeptone) was used.
- these first fibrils were cultured overnight at 31.5 ° C in a CM-Dex liquid medium containing no kanamycin and appropriately diluted, and then kanamycin-free 10% Dex-S10 medium containing sucrose (sucrose 10 g / L, polypeptone 10 g / L, yeast extratato 10 g / L, KH
- the strains thus obtained include those in which the ldh gene has been replaced by a mutant derived from pAldh56-l and those which have returned to the wild type. Whether the ldh gene is mutant or wild-type can be easily confirmed by directly subjecting cells obtained by culturing on Dex-S10 agar medium to PCR and detecting the ldh gene. it can. When the ldh gene was analyzed using primers (SEQ ID NO: 7 and SEQ ID NO: 10) for PCR amplification, the ldh-deficient PCR product was found to have a smaller PCR product size than the chromosomal DNA of the 2256 strain. The strain was used in subsequent experiments.
- ack a gene fragment in which the ORF of the acetate kinase gene (hereinafter referred to as ack) of Brevibatatermium ratatophamentum 2256 strain has been obtained is based on the previously published Corynebacterium glutamicum ATCC13032 (GenBank Database Accession
- NC_003450 was obtained by crossover PCR using a synthetic DNA designed as a primer with reference to the nucleotide sequence of the gene (1945 to 3135 of SEQ ID NO: 39). Specifically, PCR was performed using Brevibata terium 'ratatophamentum 2256 strain genomic DNA as a type I and the synthetic DNAs of SEQ ID NOS: 13 and 14 as primers to obtain an amplification product on the N-terminal side of the ack gene.
- PCR was performed using genomic DNA of Brevibata terium 'ratatofamentumum 2256 strain type II and synthetic DNAs of SEQ ID NOS: 15 and 16 as primers.
- SEQ ID NOs: 14 and 15 are partially complementary.
- the PCR reaction was carried out using KOD-plus- (TOYOBO) for one cycle of incubation at 94 ° C for 2 minutes, and then N-terminal side was denatured at 94 ° C for 10 seconds, association at 55 ° C for 30 seconds, and extended 68
- the cycle at 30 ° C for 30 seconds and the cycle for denaturation at 94 ° C for 10 seconds, association at 55 ° C for 30 seconds, and extension at 68 ° C for 2 minutes were repeated 30 times on the C-terminal side.
- PCR was performed using 18 synthetic DNAs as primers to obtain mutated ack gene amplification products.
- the PCR reaction was performed using KOD-plus- (manufactured by Toyobo Co., Ltd.) after one cycle of incubation at 94 ° C for 2 minutes, followed by denaturation at 94 ° C for 10 seconds, association at 55 ° C for 30 seconds, and extension at 68 ° C for 2.5 minutes. This cycle was repeated 30 times to obtain the target mutated ack gene amplification product.
- the generated PCR product was purified by a conventional method, digested with Xbal, and inserted into the Xbal site of PBS5T constructed in Example 1 (C) above.
- transformation was performed using a combinatorial cell of Escherichia coli JM109 (manufactured by Takara Bio Inc.), and an LB medium containing IPTG 100 ⁇ M, X-Gal 40 ⁇ g / ml and kanamycin 25 g / ml. And cultured. afterwards, Appeared white colonies were picked and separated into single colonies to obtain transformants. Plasmids were extracted from the obtained transformants, and those into which the desired PCR product had been inserted were named pBS5T :: Aack.
- the construction process of pBS5T :: A ack is shown in FIG.
- the origin of replication of coryneform bacteria in pBS5T :: ⁇ ack obtained in (A) above is temperature-sensitive.
- the plasmid is capable of autonomous replication in coryneform bacteria at 25 ° C, but not at 31.5 ° C (or 34 ° C).
- Brevibataterium 'ratatophamentum 2256 ⁇ (Wh) strain was transformed by an electric pulse method, applied to a CM-Dex medium containing 25 ⁇ g / ml of kanamycin, and incubated at 25 ° C. ⁇ After culturing, the colonies that appeared were isolated and used as transformants. This transformant carries the plasmid.
- CM-Dex medium containing 25 ⁇ g / ml of kanamycin, and incubate at 34 ° C. Culture was performed for about 30 hours. The strain grown on this medium underwent homologous recombination between the ack gene fragment of the plasmid and the same gene on the genome of Brevibataterium 'ratatophamentum 2256 ⁇ (Wh) strain, and as a result, The kanamycin resistance gene and SacB gene derived from the plasmid have been inserted.
- the strains thus obtained include those in which the ack gene has been replaced with a mutant type derived from pBS5T :: Aack and those in which the ack gene has returned to the wild type. Whether the ack gene is mutant or wild type can be easily confirmed by directly subjecting the cells obtained by culturing on Dex-S10 agar medium to PCR and detecting the ack gene. it can. ack gene Analysis using primers for PCR amplification (SEQ ID NO: 13 and SEQ ID NO: 16) should reveal a 3.7 kb DNA fragment for the wild type and a 2.5 kb DNA fragment for the mutant with the deletion region. As a result of analyzing the sucrose-insensitive strain by the above method, a strain having only the mutant gene was selected, and the strain was named 2256A (Wh, ack).
- the ORF of acetate kinase (ack) and phosphotransacetylase gene (hereinafter referred to as pta) of Brevibata terium 'ratatophamentum 2256 strain has an operon structure, and these ORFs are deleted simultaneously. It is possible to do. Acquisition of these gene fragments was performed by referring to the nucleotide sequence (pta-ack gene; SEQ ID NO: 39) of the gene of Corynebacterium glutamicum ATCC13032 (GenBank Database Accession No. NC_003450) which has already been published. The DNA was obtained by crossover PCR using DNA as a primer.
- PCR was performed using Brevibata terium 'ratatophamentum 2256 strain genomic DNA as type I and synthetic DNAs of SEQ ID NOS: 19 and 20 as primers to obtain an amplification product at the N-terminal end of the pta gene.
- PCR was performed using Brevi Batterellium 'ratatophamentum 2256 genomic DNA as a type I and the synthetic DNAs of SEQ ID NOS: 21 and 16 as primers.
- SEQ ID NOs: 20 and 21 are partially complementary.
- the PCR reaction was carried out using KOD-plus- (TOYOBO) for one cycle of incubation at 94 ° C for 2 minutes, and then the N-terminal side was denatured at 94 ° C for 10 seconds, association at 55 ° C for 30 seconds, and extended 68 A cycle consisting of 30 ° C. for 30 seconds and a cycle consisting of denaturation at 94 ° C. for 10 seconds, association at 55 ° C. for 30 seconds, and extension at 68 ° C. for 2 minutes were repeated 30 times on the C-terminal side.
- KOD-plus- TOYOBO
- PCR was performed using the synthetic DNAs of SEQ ID NOS: 22 and 18 as primers to obtain the amplified pta-ack gene amplification product.
- the PCR reaction was performed using KOD-plus- (TOYOBO), one cycle of incubation at 94 ° C for 2 minutes, followed by denaturation at 94 ° C for 10 seconds, association at 55 ° C for 30 seconds, and extension at 68 ° C.
- the cycle with 2.5 minutes of force was repeated 30 times to obtain the target mutagenized pta-ack gene amplified product.
- the resulting PCR product was purified by a conventional method, digested with Xbal, and inserted into the Xbal site of pBS5T.
- transformation was carried out using a competent cell of Escherichia coli JM109 (manufactured by Takara Bio Inc.), and an LB medium containing IPTG 100 M, X-Gal 40 g / ml and kanamycin 25 g / ml was used. And cultured overnight. Thereafter, the emerged white mouth was caught and single colonies were separated to obtain a transformant.
- a plasmid was extracted from the obtained transformant, and the one into which the desired PCR product had been inserted was named pBS5T :: A pta-ack.
- the construction process of pBS5T :: ⁇ pta-ack is shown in FIG.
- the origin of replication of coryneform bacteria in pBS5T is temperature-sensitive.
- the plasmid is capable of autonomous replication in coryneform bacteria at 25 ° C, but not at 31.5 ° C (or 34 ° C).
- the plasmid was used to transform Brevibataterium 'ratatophamentum 2256 A (ldh) strain by the electric pulse method, and the transformant was applied to a CM-Dex medium containing 25 ⁇ g / ml of kanamycin and incubated at 25 ° C. ⁇ After culturing, the colonies that appeared were isolated and used as transformants. This transformant carries the plasmid.
- transformants were cultured at ⁇ 34 ° C. in a CM-Dex liquid medium without kanamycin, diluted appropriately, and spread on a CM-Dex medium containing 25 ⁇ g / ml of kanamycin.
- the cells were cultured at ° C for about 30 hours.
- the strain grown on this medium undergoes homologous recombination between the pta-ack gene fragment of the plasmid and the same gene on the genome of Brevibataterium 'ratatophamentum 2256 ⁇ (Wh) strain, resulting in the same genome.
- the kanamycin resistance gene and SacB gene derived from the plasmid have been inserted.
- these first fibrils were cultured in a kanamycin-free! / ⁇ ⁇ CM-Dex liquid medium at 31.5 ° C, diluted appropriately, and then kanamycin-free 10%
- the cells were applied to sucrose-containing Dex-S10 medium, and cultured at 31.5 ° C for about 30 hours.
- about 50 strains that were considered to be sucrose-insensitive by dropping the SacB gene by the second homologous recombination were obtained.
- the strains thus obtained include those in which the pta and ack genes have been replaced with a mutant derived from pBS5T :: A pta-ack and those in which the pta and ack have reverted to the wild type.
- ack gene is mutated Whether it is a type or a wild type can be easily confirmed by directly subjecting cells obtained by culturing on Dex-S10 agar medium to a PCR reaction and detecting pta and ack genes.
- sucrose-insensitive strain As a result of analysis of the sucrose-insensitive strain by the above method, a strain having only the mutant gene was selected, and the strain was named 2256A (ldh, pta, ack).
- PCR was performed using Brevibacterium 'ratatophamentum 2256 strain genomic DNA as a type II and synthetic DNAs of SEQ ID NOS: 23 and 24 as primers to obtain an amplification product on the N-terminal side of poxB gene.
- PCR is performed using genomic DNA of Brevibata terium 'ratatofamentum 2256 strain type II and synthetic DNAs of SEQ ID NOS: 25 and 26 as primers.
- SEQ ID NOS: 24 and 25 are complementary to each other.
- KOD-plus- TOYOBO
- the cycle of elongation at 68 ° C for 40 seconds was repeated 30 times.
- the above-mentioned poxB N-terminal and C-terminal gene products were mixed so as to be approximately equimolar, respectively, and the resulting mixture was designated as ⁇ type and SEQ ID NO: 27.
- PCR was performed using the synthetic DNAs of the above and primers as primers to obtain a mutated poxB gene amplification product.
- the PCR reaction was carried out using KOD-plus- (TOYOBO) after one cycle of incubation at 94 ° C for 2 minutes, followed by denaturation at 94 ° C for 10 seconds, association at 55 ° C for 30 seconds, and extension at 68 ° C for 70 seconds.
- the generated PCR product was purified by a conventional method, digested with Xbal, and inserted into the Xbal site of PBS5T constructed in Example 1 (C) above. Using this DNA, transformation was carried out using a combinatorial cell of Escherichia coli JM109 (manufactured by Takara Bio Inc.), and LB medium containing IPTG 100 ⁇ ⁇ , X-Gal 40 ⁇ g / ml and kanamycin 25 g / ml was used. And cultured overnight. After that, the emerged white colonies were picked and separated into single colonies to obtain transformants. Plasmids were extracted from the resulting transformants, and those into which the desired PCR product had been inserted were named pBS5T :: ⁇ poxB. The construction process of pBS5T :: ⁇ is shown in FIG.
- the origin of replication of coryneform bacteria in pBS5T :: ⁇ poxB obtained in (B) above is temperature-sensitive.
- the plasmid is capable of autonomous replication in coryneform bacterium cells at 25 ° C and becomes incapable of autonomous replication at a power of 31.5 ° C (or 34 ° C).
- the plasmid was used to transform Brevibataterium 'ratatophamentum 2256 A (ldh, pta, ack) strain by the electric pulse method, and the transformant was applied to a CM-Dex medium containing 25 ⁇ g / ml of kanamycin.
- the cells were cultured at 25 ° C for 2 ⁇ , and the resulting colonies were isolated and used as transformants. This transformant should carry the plasmid.
- CM-Dex liquid medium containing no kanamycin at 34 ° C and diluted appropriately, and then CM-Dex medium containing 25 ⁇ g / ml of kanamycin was appropriately diluted. And cultured at 34 ° C for about 30 hours.
- the strain grown on this medium was the result of homologous recombination between the poxB gene fragment of the plasmid and the same gene on the genome of Brevibataterium 'ratatophamentum 2256A (ldh, pta, ack) strain.
- a kanamycin resistance gene and a SacB gene derived from the plasmid were inserted.
- these first recombinants were cultured in CM-Dex liquid medium without kanamycin at 31.5 ° C, diluted appropriately, and kanamycin-free! / ⁇ Dex containing 10% sucrose.
- the cells were applied to -S10 medium and cultured at 31.5 ° C for about 30 hours. As a result, about 50 strains that were considered to be sucrose-insensitive due to the loss of the SacB gene due to the second homologous recombination were obtained.
- the strains thus obtained include those in which the poxB gene has been replaced with a mutant derived from pBS5T :: A poxB and those in which the poxB has returned to the wild-type. Whether the acetate kinase gene is mutant or wild type can be confirmed by culturing on Dex-S10 agar medium. The obtained cells can be easily confirmed by directly subjecting them to a PCR reaction and detecting the poxB gene. When the poxB gene is analyzed using primers for PCR amplification (SEQ ID NO: 23 and SEQ ID NO: 26), a DNA fragment of 2.4 kb can be detected in the wild type and a 1.2 kb DNA fragment can be detected in the mutant having the deletion region. As a result of analyzing a sucrose-insensitive strain by the above method, a strain having only the mutant gene was selected, and the strain was named 2256A (Wh, pta, ack, poxB).
- Brevibata terium 'ratatophamentum 2256 strain's acetyl-CoA-noid-mouth-lase gene (hereinafter referred to as “ach”) was obtained by deleting the gene fragment of ORF from the previously published corynebacterium.
- ach acetyl-CoA-noid-mouth-lase gene
- NC_003450 was obtained by crossover PCR using a synthetic DNA designed with reference to the nucleotide sequence (SEQ ID NO: 44) of the gene. Specifically, PCR is performed using Brevibata terium 'ratatophamentum 2256 strain genomic DNA as a type I and synthetic DNAs of SEQ ID NOS: 29 and 30 as primers to obtain an amplification product on the C-terminal side of the ach gene. On the other hand, in order to obtain an amplification product on the N-terminal side of the ach gene, PCR is performed using genomic DNA of Brevibata terium 'ratatofamentum 2256 strain ⁇ as a type III and synthetic DNAs of SEQ ID NOS: 31 and 32 as primers.
- SEQ ID NOS: 30 and 31 are complementary to each other.
- KOD-plus- manufactured by Toyobo Co., Ltd.
- both N-terminal side and C-terminal side were incubated at 94 ° C for 2 minutes for 1 cycle, denaturation 94 ° C for 10 seconds, association 55 ° C A cycle of 30 seconds and an extension of 68 ° C for 50 seconds was repeated 30 times.
- the above ach N-terminal and C-terminal gene products were mixed so as to be almost equimolar, respectively, and the resulting mixture was designated as type I and SEQ ID NO: 33.
- PCR was performed using 34 synthetic DNAs as primers to obtain mutated ach gene amplification products.
- the PCR reaction was carried out using KOD-plus- (TOYOBO) for 2 cycles of incubation at 94 ° C for 2 minutes, followed by denaturation at 94 ° C for 10 seconds, association at 55 ° C for 30 seconds, and extension at 68 ° C for 90 seconds. Another cycle was repeated 30 times to obtain the desired mutated ach gene amplification product.
- the resulting PCR product was purified by a conventional method, digested with Xbal, and the pBS4S constructed in Example 1 (B) above was purified. Inserted at Xbal site.
- the strains grown on this medium were the ach gene fragment of the plasmid and the Brevibataterium 'lactofermentum 2256 ⁇ (ldh), 2256 ⁇ (ldh, pta, ack), 2256 ⁇ (ldh, pta, ack, poxB) strain and 2256 ⁇ (ldh, pta, ack, poxB, acp) strain.Homologous recombination with the same gene on the genome resulted in a kanamycin resistance gene derived from the plasmid in the same genome. And the SacB gene has been inserted!
- the strains thus obtained include those in which the ach gene has been replaced with a mutant derived from pBS4S :: A ach and those in which the ach gene has returned to the wild type. Whether the ach gene is mutant or wild type can be easily confirmed by directly subjecting cells obtained by culturing on Dex-S10 agar medium to PCR and detecting the ach gene. it can. ach gene Analysis using primers for PCR amplification (SEQ ID NO: 29 and SEQ ID NO: 32) should reveal a DNA fragment of 2.9 kb in the wild type and 1.4 kb in the mutant with the deletion region.
- Culture for succinic acid production was carried out as follows using Brevibata terium 'ratatophamentum 2256 ⁇ (ldh) strain and 2256 ⁇ (ldh, ach) strain.
- Cells of 2256 ⁇ (ldh) strain and 2256 ⁇ (ldh, ack) strain obtained by culturing in CM-Dex plate medium were seeded with 3 ml of seed medium (Dalcos 10 g / L ⁇ (NH) SO 2.5 g / L, KH PO 0.5g / L, MgSO-7H O 0.25g / L, urea 2g / L,
- shaking culture was performed in a test tube at 31.5 ° C under aerobic conditions for about 15 hours.
- succinic acid production culture was carried out with a silicone stopper tightly closed. The culture was shaken at 31.5 ° C for about 24 hours to terminate the culture before the sugar in the medium was exhausted.
- Culture for succinic acid production was performed using Brevibata terium 'ratatophamentum 2256A (ldh) strain and 2256A (ldh, ach, pta, ack) strain as follows.
- the cells of the 2256 A (ldh) strain and the 2256 A (ldh, ack, pta, ack) strain obtained by culturing in the CM-Dex plate medium were inoculated into 3 ml of the above-described seed medium, and subjected to aerobic conditions. At 31.5 ° C for about 15 hours in a test tube.
- test tube was inoculated with 3 ml of the above-mentioned main medium, and sealed with a silicon stopper to prevent aeration, and succinic acid production culture was performed.
- the culture was shaken at 31.5 ° C for about 24 hours to terminate the culture before the sugar in the medium was exhausted.
- Brevibataterium 'ratatofu amentum 2256 ⁇ (Idh) strain, 2256 A (Wh, pta-ack, ach) strain, 2256 ⁇ (ldh, pta-ack, poxB) strain, and 2256 ⁇ (ldh, pta-ack , poxB, ach) strain was cultured for succinic acid production as follows.
- the test tube was inoculated with 3 ml of the above-mentioned main medium, and sealed with a silicon stopper to prevent aeration, and succinic acid production culture was performed.
- the culture was shaken at 31.5 ° C for about 24 hours to terminate the culture before the sugar in the medium was exhausted.
- the accumulated amount of succinic acid and by-product acetic acid in the culture solution was analyzed by liquid chromatography after appropriately diluting the culture solution.
- the column used was two Shim-pack SCR-102H (Simazu) connected in series, and the sample was eluted at 40 ° C using 5 mM p-toluenesulfonic acid.
- the eluate was neutralized with a 20 mM Bis-Tris aqueous solution containing 5 mM p-toluenesulfonic acid and 100 ⁇ M EDTA.
- Succinic acid is useful as a raw material for biodegradable polymers and the like.
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Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
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EP05741285A EP1760143B1 (en) | 2004-05-20 | 2005-05-20 | Succinic acid-producing bacterium and process for producing succinic acid |
BRPI0510919-1A BRPI0510919A (pt) | 2004-05-20 | 2005-05-20 | bactéria produtora de ácido succìnico e processo para produzir ácido succìnico |
CN2005800246195A CN1989238B (zh) | 2004-05-20 | 2005-05-20 | 生产琥珀酸的细菌和生产琥珀酸的方法 |
JP2006513736A JP5023701B2 (ja) | 2004-05-20 | 2005-05-20 | コハク酸生産菌及びコハク酸の製造方法 |
US11/561,011 US7972823B2 (en) | 2004-05-20 | 2006-11-17 | Succinic acid-producing bacterium and process for producing succinic acid |
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JP5023701B2 (ja) | 2012-09-12 |
CN1989238B (zh) | 2010-05-26 |
BRPI0510919A (pt) | 2008-05-20 |
US20080293112A1 (en) | 2008-11-27 |
US7972823B2 (en) | 2011-07-05 |
JPWO2005113744A1 (ja) | 2008-03-27 |
CN1989238A (zh) | 2007-06-27 |
EP1760143B1 (en) | 2012-05-16 |
EP1760143A4 (en) | 2007-06-27 |
EP1760143A1 (en) | 2007-03-07 |
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