JP5034395B2 - Organic acid producing bacteria and method for producing organic acid - Google Patents

Description

  The present invention relates to a novel organic acid-producing bacterium and a method for producing an organic acid using the same.

  When organic acids such as succinic acid are produced by fermentation, anaerobic bacteria such as Anaerobiospirillum genus and Actinobacillus genus are usually used (for example, Patent Document 1 or 2, Non-patent document 1) Patent Document 1). When anaerobic bacteria are used, the yield of the product is high, but on the other hand, a large amount of organic nitrogen sources such as CSL (corn steep liquor) in the medium because it requires many nutrients to grow. Need to be added. Adding a large amount of these organic nitrogen sources not only leads to an increase in culture medium cost, but also increases the purification cost when taking out the product, which is not economical.

  In addition, aerobic bacteria such as coryneform bacteria are once cultured under aerobic conditions, and after growing the cells, they are collected and washed to produce organic acids as a stationary cell without aeration of oxygen. A method is also known (see, for example, Patent Document 3 or 4). In this case, the amount of organic nitrogen added may be small in order to grow the cells, and it is economical because it can be sufficiently grown on a simple medium. There was room for improvement, such as improvement of the production speed per hit and simplification of the manufacturing process. In addition, when aerobic bacteria are used, the fact that amino acids are by-produced in addition to the target organic acid is another point that should be improved in order to further increase the yield of organic acid. In addition, genetically modified microorganisms (see Patent Document 4 or 5) have been reported for microorganisms used in the production of organic acids. However, the creation of new microorganisms is required to further improve the yield of organic acids. It was.

The cg1630 gene is a gene registered under the number of cg1630 (base number is 1521068-1521499) in the sequence of GenBank accession number NC_006958 showing the genome sequence of Corynebacterium glutamicum ATCC13032. Although little research has been done on this gene and its function is unknown, it has recently been reported that phosphorylation of the gene product OdhI is associated with the regulation of 2-oxoglutarate dehydrogenase activity (non- Patent Document 2). However, the relationship between this gene and organic acid production was completely unknown.
US Pat. No. 5,143,834 US Pat. No. 5,504,004 JP-A-11-113588 JP 11-196888 A JP-A-11-206385 International Journal of Systematic Bacteriology, vol. 49, p207-216, 1999 Journal of Biological Chemistry, vol. 281, no. 18, p12300-12307, 2006

  An object of the present invention is to provide a novel method for efficiently producing an organic acid such as succinic acid.

As a result of intensive studies to solve the above problems, the present inventor contains a bacterium modified so as to reduce the expression of the cg1630 gene or a treated product thereof, containing carbonate ion, bicarbonate ion or carbon dioxide gas. It was found that the amount of organic acid produced was increased by acting on the organic raw material in the reaction solution, and the present invention was completed.

That is, according to the present invention, the following inventions are provided.
(1) A bacterium that has an organic acid-producing ability and has been modified so that the expression of the cg1630 gene is reduced compared to an unmodified strain.
(2) The bacterium according to (1), wherein the cg1630 gene on the chromosome is disrupted.
(3) The bacterium according to (1) or (2), wherein the cg1630 gene is a gene having 80% or more homology with the nucleotide sequence of SEQ ID NO: 17.
(4) The bacterium according to any one of (1) to (3), further modified so that lactate dehydrogenase activity is reduced as compared with an unmodified strain.
(5) The bacterium according to any one of (1) to (4), further modified so that pyruvate carboxylase activity is enhanced as compared with an unmodified strain.
(6) Further, the activity of one or more enzymes selected from acetate kinase, phosphotransacetylase, pyruvate oxidase and acetyl-CoA hydrolase is modified so as to be reduced as compared with the unmodified strain. The bacterium according to any one of 1) to (5).
(7) The bacterium according to any one of (1) to (6), which is a coryneform bacterium.
(8) An organic acid is produced by allowing the bacterium of any one of (1) to (7) 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 collecting the organic acid.
(9) The method for producing an organic acid according to (8), wherein the organic raw material is allowed to act in an anaerobic atmosphere.
(10) The method for producing an organic acid according to (8) or (9), wherein the organic raw material is glucose or sucrose.
(11) The method for producing an organic acid according to any one of (8) to (10), wherein the organic acid is succinic acid.
(12) A method for producing an organic acid-containing polymer, comprising a step of producing an organic acid by any one of the methods (8) to (11), and a step of carrying out a polymerization reaction using the organic acid obtained in the step as a raw material. .

By producing an organic acid using a bacterium modified so as to reduce the expression of the cg1630 gene or a processed product thereof, by-products can be reduced, and the amount of organic acid produced can be increased.

Hereinafter, embodiments of the present invention will be described in detail.
The bacterium of the present invention is a bacterium modified so that the expression of the cg1630 gene is reduced, and is a bacterium having an organic acid-producing ability.
Here, “having the ability to produce an organic acid” means that an organic acid can be produced and accumulated in the medium when the bacterium is cultured in the medium.
Examples of the organic acid include lactic acid, succinic acid, malic acid, fumaric acid, oxaloacetic acid, citric acid, isocitric acid, 2-oxoglutaric acid, cis-aconitic acid, pyruvic acid, acetic acid, amino acid, and the like. Among them, succinic acid, malic acid, fumaric acid, citric acid, isocitric acid, 2-oxoglutaric acid, cis-aconitic acid and pyruvic acid are preferable, and succinic acid is particularly preferable.

The bacterium of the present invention may be one that has been modified to reduce the expression of the cg1630 gene in a bacterium that originally has an organic acid-producing ability or a bacterium that has been given organic acid-producing ability by breeding,
It may be an organic acid-producing ability that has been modified by reducing the expression of the cg1630 gene. Examples of means for imparting organic acid-producing ability by breeding include mutation treatment, gene recombination treatment, and the like, and publicly known methods such as enhanced expression of biosynthetic enzyme genes can be adopted for each organic acid. In the case of imparting succinic acid-producing ability, examples include a modification that reduces lactate dehydrogenase activity as described below, and a means that enhances pyruvate carboxylase activity.

The bacterium used in the present invention can be obtained by using a bacterium as shown below as a parent strain and modifying the parent strain. The type of parent strain is not particularly limited as long as it is a bacterium that retains the cg1630 gene and is capable of producing organic acids, but is not limited to Coryneform Bacterium, Mycobacterium, Rhodococcus, Examples include bacteria of the genus Cardia or bacteria of the genus Streptomyces, with coryneform bacteria being more preferred.
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 Brevibacterium, and bacteria belonging to the genus Arthrobacter. Are those belonging to the genus Corynebacterium or Brevibacterium, more preferably, Corynebacterium glutamicum, Brevibacterium flavum, Brevibacterium ammoniagenes (Brevibacterium) ammoniagenes) or bacteria classified as Brevibacterium lactofermentum.

Particularly preferable specific examples of the bacterial parent strain used in the present invention include Brevibacterium flavum MJ-233 (FERM BP-1497), MJ-233 AB-41 (FERM BP-1498), Brevibacterium ammoniagenes. ATCC6872, Corynebacterium glutamicum ATCC31831, Brevibacterium lactofermentum ATCC13869, etc. are mentioned. Brevibacterium flavum is currently sometimes classified as Corynebacterium glutamicum (Lielbl, W., Ehrmann, M., Ludwig, W. and Schleifer, KH, International Journal of
Systematic Bacteriology, 1991, vol. 41, p255-260), in the present invention, Brevibacterium flavum MJ-233 strain and its mutant MJ-233 AB-41 strain are each Corynebacterium glutamicum MJ- It shall be the same strain as the 233 strain and the MJ-233 AB-41 strain.
Brevibacterium flavum MJ-233 was established on April 28, 1975, by the Ministry of International Trade and Industry, Institute of Industrial Science, Microbial Industrial Technology Research Institute (currently the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center) (305-8566 Japan) Deposited as FERM P-3068 at Tsukuba City, Ibaraki Prefecture, 1-chome, 1-chome, 1st 6th), transferred to an international deposit based on the Budapest Treaty on May 1, 1981, and deposited with a deposit number of FERM BP-1497 Has been.
In addition, the above-mentioned bacterium used as a parent strain is induced not only by wild strains but also by mutants obtained by ordinary mutation treatment such as UV irradiation and NTG treatment, genetic methods such as cell fusion or gene recombination methods. Any strain such as a recombinant strain may be used.

The bacterium used in the present invention can be obtained by modifying the strain as described above so that the expression of the cg1630 gene is reduced.
The “cg1630 gene” is a gene registered under the number of cg1630 in the sequence of GenBank accession number NC_006958 or a homologous gene thereof, and by reducing its expression, the ability of the host bacterium to produce organic acid is reduced. A gene that can be improved. In the present specification, “cg1630 gene” is also referred to as “OdhI gene”.
“The expression of the cg1630 gene is reduced” means that the expression of this gene is reduced as compared to an unmodified strain such as a wild strain or a parent strain. The expression of the cg1630 gene is preferably reduced to 30% or less of the unmodified strain, more preferably 10% or less. The expression of the cg1630 gene may be reduced below the detection limit.
The reduction in the expression of the cg1630 gene can be confirmed by measuring the amount of mRNA by Northern hybridization or RT-PCR.

For the strain with reduced expression of cg1630 gene, the parent strain is treated with a mutagen such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG) or nitrous acid and usually used for mutagenesis. Can be obtained by selecting strains with reduced.
Moreover, you may modify | change using cg1630 gene. Specifically, this can be achieved by disrupting the cg1630 gene on the chromosome or modifying expression control sequences such as a promoter or Shine-Dalgarno (SD) sequence.

  Hereinafter, a method for disrupting the cg1630 gene in coryneform bacteria will be described. Examples of the cg1630 gene on the chromosome include DNA containing the base sequence shown in SEQ ID NO: 17. The cg1630 gene can be obtained, for example, by cloning a synthetic oligonucleotide based on the above sequence and performing a PCR reaction using the chromosomal DNA of Corynebacterium glutamicum as a template. Chromosomal DNA is obtained from bacteria that are DNA donors, for example, the method of Saito and Miura (H. Saito and K. Miura, Biochem. Biophys. Acta, 72, 619 (1963), Biotechnology Experiments, Nippon Biotechnology, Ed., Pp. 97-98, Bafukan, 1992).

  The cg1630 gene prepared as described above or a part thereof can be used for gene disruption. However, since the gene used for gene disruption only needs to have a degree of homology that causes homologous recombination with the cg1630 gene on the chromosomal DNA of the bacterium to be disrupted, a homologous gene having homology with SEQ ID NO: 17 is also used. can do. Here, the homology that causes homologous recombination is preferably 80% or more, more preferably 90% or more, and particularly preferably 95% or more. In addition, homologous recombination can occur as long as it is a DNA that can hybridize with the above gene under stringent conditions. Here, as stringent conditions, it corresponds to 60 ° C., 1 × SSC, 0.1% SDS, preferably 0.1 × SSC, 0.1% SDS, which is a normal washing condition for Southern hybridization. The conditions for hybridizing at a salt concentration are included.

  Using a gene as described above, for example, deleting a partial sequence of the cg1630 gene, preparing a deleted cg1630 gene modified so as not to produce a normal Cg1630 protein, and using a DNA containing the gene to coryneform bacteria The cg1630 gene on the chromosome can be destroyed by transforming and causing recombination between the deleted gene and the gene on the chromosome. Such gene disruption by gene replacement using homologous recombination has already been established, and includes a method using linear DNA and a method using a plasmid containing a temperature-sensitive replication origin (US Pat. No. 6,303,383). Or Japanese Patent Laid-Open No. 05-007491). In addition, gene disruption by gene replacement using homologous recombination as described above can also be performed using a plasmid that does not have replication ability on the host. As a plasmid that does not have replication ability in coryneform bacteria, A plasmid having replication ability in Escherichia coli is preferable, and examples thereof include pHSG299 (manufactured by Takara Bio Inc.), pHSG399 (manufactured by Takara Bio Inc.), and the like.

The bacterium of the present invention may be a bacterium modified so that lactate dehydrogenase (also referred to as LDH) activity is reduced in addition to the decrease in the expression of the cg1630 gene. Here, “LDH activity is reduced” means that LDH activity is reduced as compared to an unmodified strain. The LDH activity is preferably reduced to 10% or less per unit cell weight as compared to the unmodified strain. Also, LDH activity may be completely lost. The decrease in LDH activity can be confirmed by measuring LDH activity by a known method (L. Kanarek and RL Hill, J. Biol. Chem. 239, 4202 (1964)). As 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). A coryneform bacterium with reduced LDH activity and reduced cg1630 gene expression can be obtained, for example, by preparing a bacterium with a disrupted LDH gene and reducing the expression of the cg1630 gene in the bacterium. However, either the modification operation for reducing LDH activity or the modification operation for reducing cg1630 gene expression may be performed first.

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 decrease in the expression of the cg1630 gene. “The PC activity is enhanced” means that the PC activity is preferably increased by 1.5 times or more, more preferably by 3 times or more per unit cell weight relative to an unmodified strain such as a wild strain or a parent strain. Say. The enhanced PC activity can be confirmed by measuring the PC activity by a known method (Magasanik's method [J. Bacteriol., 158, 55-62, (1984)]).
Such a bacterium can be obtained, for example, by introducing the pc gene into a coryneform bacterium in which the expression of the cg1630 gene is reduced. Any modification operation for enhancing PC activity and reducing cg1630 gene expression may be performed first.

  Introduction of the pc gene can be carried out, for example, by highly expressing the pc gene in a host bacterium in the same manner as described in JP-A-11-196888. As a specific pc gene, for example, a pc gene derived from Corynebacterium glutamicum (Peters-Wendisch, PG et al. Microbiology, vol. 144 (1998) p915-927) (SEQ ID NO: 19) can be used. it can. In addition, the pc gene is DNA that hybridizes under stringent conditions with DNA having the nucleotide sequence of SEQ ID NO: 19, or 80% or more, preferably 90% or more, more preferably 95% or more with the nucleotide sequence of SEQ ID NO: 19. Particularly preferably, DNA having a homology of 99% or more and encoding a protein having PC activity can also be suitably used.

Furthermore, pc genes derived from coryneform bacteria other than Corynebacterium glutamicum, or from other microorganisms or animals and plants can also be used. In particular, the microorganism or animal or plant-derived pc gene shown below has a known sequence (shown below), and by amplifying the ORF portion by hybridization or PCR as described above, Can be acquired.
Human [Biochem.Biophys.Res.Comm., 202, 1009-1014, (1994)]
Mouse [Proc.Natl.Acad.Sci.USA., 90, 1766-1779, (1993)]
Rat [GENE, 165, 331-332, (1995)]
Yeast; Saccharomyces cerevisiae
[Mol.Gen.Genet., 229, 307-315, (1991)]
Schizosaccharomyces pombe
[DDBJ Accession No .; D78170]
Bacillus stearothermophilus
[GENE, 191, 47-50, (1997)]
Rhizobium etli
[J. Bacteriol., 178, 5960-5970, (1996)]

By introducing a DNA fragment containing the pc gene as described above into an appropriate plasmid, for example, a plasmid vector containing at least a gene that controls replication and replication of the plasmid in the host bacterium, high expression of the pc gene in the host bacterium can be achieved. Possible recombinant plasmids can be obtained. Here, in the above recombinant plasmid, the promoter for expressing the pc gene may be any promoter as long as it is a base sequence for initiating transcription of the pc gene. For example, tac promoter, trc promoter, etc. Can be mentioned.

  Specific examples of plasmids that can be used for introducing genes into coryneform bacteria include, for example, plasmid pCRY30 described in JP-A-3-210184; JP-A-2-72876 and US Pat. No. 5,185,262. Plasmids pCRY21, pCRY2KE, pCRY2KX, pCRY31, pCRY3KE and pCRY3KX described in the specification gazette; plasmids pCRY2 and pCRY3 described in JP-A-1-191686; pAM330 described in JP-A-58-67679; PHM1519 described in JP-A-58-77895; pAJ655, pAJ611 and pAJ1844 described in JP-A-58-192900; pCG1 described in JP-A-57-134500; described in JP-A-58-35197 of CG2; may be mentioned pCG4 and pCG11 like described in JP-57-183799 publication. Among them, plasmid vectors used in the host-vector system of coryneform bacteria have a gene responsible for the replication and replication function of the plasmid in coryneform bacteria and a gene responsible for the plasmid stabilization function in coryneform bacteria. For example, plasmids pCRY30, pCRY21, pCRY2KE, pCRY2KX, pCRY31, pCRY3KE and pCRY3KX are preferably used.

A recombinant vector obtained by inserting the pc gene into an appropriate site of such a plasmid vector is used to transform a coryneform bacterium such as Brevibacterium flavum MJ-233 (FERM BP-1497). Thus, a coryneform bacterium with enhanced pc gene expression is obtained. The PC activity can also be enhanced by making multiple copies of the pc gene on the chromosome by a known homologous recombination method. Transformation can be performed by, for example, the electric pulse method (Res. Microbiol., Vol. 144, p.181-185, 1993).
In addition, enhancement of PC activity can also be achieved by replacing the promoter of pc gene with a strong promoter on the chromosome.

  Furthermore, the bacterium of the present invention, in addition to decreased expression of cg1630 gene, acetate kinase (hereinafter also referred to as ACK), phosphotransacetylase (hereinafter also referred to as PTA), pyruvate oxidase (hereinafter also referred to as POXB). And a bacterium modified so as to reduce the activity of one or more enzymes selected from the group consisting of acetyl-CoA hydrolase (hereinafter also referred to as ACH).

Either PTA or ACK may decrease the activity, but it is more preferable to decrease both activities in order to efficiently reduce the byproduct of acetic acid.
“PTA activity” refers to the activity of catalyzing the reaction of transferring phosphoric acid to acetyl-CoA to produce acetyl phosphate. “Modified to reduce PTA activity” means that the PTA activity is lower than that of an unmodified strain such as a wild strain. The PTA activity is preferably reduced to 30% or less per unit cell weight, more preferably 10% or less, compared to 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, 381-386 (1969)).

“ACK activity” refers to the activity of catalyzing the reaction of producing acetic acid from acetyl phosphate and ADP. “Modified to reduce ACK activity” means that ACK activity is lower than that of an unmodified strain, for example, a wild strain. The ACK activity is preferably reduced to 30% or less per unit cell weight, more preferably 10% or less, compared to the unmodified strain. Further, the 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)).

  In Corynebacterium glutamicum (including those classified as Brevibacterium flavum), both enzymes are pta as described in Microbiology. 1999 Feb; 145 (Pt 2): 503-13. Since it is encoded by the ack operon (GenBank Accession No. X89084), when the pta gene is disrupted, the activities of both PTA and ACK enzymes can be reduced.

  The decrease in PTA and ACK activity can be achieved by disrupting these genes according to known methods, for example, using homologous recombination or using the sacB gene (Schafer, A. et al. Gene 145 (1994) 69-73). Can be done. Specifically, it can be performed according to the method disclosed in JP-A-2006-000091 and the examples described later. As the pta gene and the ack gene, in addition to the gene having the base sequence of the above GenBank Accession No. X89084, a gene having a degree of homology that causes homologous recombination with the pta gene and the ack gene on the host chromosome can also be used. . Here, the homology that causes homologous recombination is preferably 80% or more, more preferably 90% or more, and particularly preferably 95% or more. In addition, homologous recombination can occur if the DNAs can hybridize with each other under stringent conditions.

  “POXB activity” refers to the activity of catalyzing the reaction of producing acetic acid from pyruvic acid and water. “Modified to reduce POXB activity” means that POXB activity is lower than that of an unmodified strain such as a wild strain. The POXB activity is preferably reduced to 30% or less per unit cell weight, more preferably 10% or less, compared to the unmodified strain. “Decrease” includes the case where the activity is completely lost. POXB activity can be confirmed by measuring the activity according to the method of Chang et al. (Chang Y. and Cronan J. E. JR, J. Bacteriol. 151, 1279-1289 (1982)).

  The decrease in POXB activity can be achieved by disrupting the poxB gene according to a known method such as a method using homologous recombination or a method using the sacB gene (Schafer, A. et al. Gene 145 (1994) 69-73). It can be carried out. Specifically, it can be performed according to the methods disclosed in WO2005 / 113745 and the examples described later. Examples of the poxB gene include GenBank Accession No. A gene having the base sequence of Cgl2610 (the complementary strand of 2776766-2778505 of GenBank Accession No. BA000036) can be mentioned. Therefore, a homologous gene of the sequence can also be used. Here, the homology that causes homologous recombination is preferably 80% or more, more preferably 90% or more, and particularly preferably 95% or more. In addition, homologous recombination can occur if the DNAs can hybridize with each other under stringent conditions.

  “ACH activity” refers to the activity of catalyzing the reaction of producing acetic acid from acetyl-CoA and water. “Modified to reduce ACH activity” means that ACH activity is lower than that of a non-modified strain such as a wild-type strain. The ACH activity is preferably reduced to 30% or less per unit cell weight, more preferably 10% or less, compared to the unmodified strain. The “decrease” includes the case where the activity is completely lost. ACH activity can be measured by the method of Gergely, J., et al. (Gergely, J., Hele, P. & Ramkrishnan, C.V. (1952) J. Biol. Chem. 198 p323-334).

The decrease in ACH activity can be achieved by disrupting the ach gene 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). It can be carried out. Specifically, it can be carried out according to the methods disclosed in WO2005 / 113744 and examples described later. Examples of the ach gene include GenBank Accession No.
. A gene having the base sequence of Cgl2569 (the 2729376..2730917 complementary strand of GenBank Accession No. BA000036) is mentioned, but it has homology to the extent that homologous recombination occurs with the ach gene on the chromosomal DNA of the host bacteria Therefore, a homologous gene of the sequence can also be used. Here, the homology that causes homologous recombination is preferably 80% or more, more preferably 90% or more, and particularly preferably 95% or more. In addition, homologous recombination can occur if the DNAs can hybridize with each other under stringent conditions.

  Note that the bacterium used in the present invention may be a bacterium obtained by combining two or more kinds of the above modifications in addition to the modification that reduces the cg1630 gene. When making a plurality of modifications, the order does not matter.

2. Organic acid production method The organic acid production method of the present invention is to produce an organic acid by allowing the bacterium or its treated product to act on an organic raw material in a reaction solution containing carbonate ion, bicarbonate ion or carbon dioxide gas. And collecting this, a method for producing an organic acid. Examples of organic acids that can be produced and examples of preferred organic acids are as described above.

  When using the above-mentioned bacterium for the production of organic acid, a slant culture in a solid medium such as an agar medium may be used for the direct reaction. However, the above bacterium previously cultured in a liquid medium (seed culture) is used. Is preferred. As a medium used for seed culture, a normal medium used for bacterial culture can be used. For example, a general medium in which a natural nutrient source such as meat extract, yeast extract or peptone is added to a composition composed of inorganic salts such as ammonium sulfate, potassium phosphate and magnesium sulfate can be used. The bacterial cells after seed culture are preferably collected by centrifugation, membrane separation, etc., and then used for the reaction for producing an organic acid. In addition, an organic acid may be produced by reacting a seed-cultured bacterium with an organic raw material while growing it in a medium containing the organic raw material, or a bacterial cell obtained by proliferating in advance is a reaction solution containing the organic raw material. The organic acid may also be produced by reacting with an organic raw material.

  In the present invention, a treated product of bacterial cells can also be used. Examples of treated cells include, for example, immobilized cells obtained by immobilizing cells with acrylamide, carrageenan, etc., crushed materials obtained by crushing cells, centrifugation supernatants thereof, or partial supernatants thereof by ammonium sulfate treatment, etc. Examples include 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 succinic acid. Usually, galactose, lactose, glucose, fructose, glycerol, sucrose, sucrose are used. Carbohydrates such as starch and cellulose; fermentable carbohydrates such as polyalcohols such as glycerin, mannitol, xylitol and ribitol are used, among which glucose or sucrose is preferable, and glucose is particularly preferable.

  Moreover, the starch saccharified liquid, molasses, etc. containing the said fermentable saccharide | sugar are also used. These fermentable carbohydrates can be used alone or in combination. The use concentration of the organic raw material is not particularly limited, but it is advantageous to make it as high as possible within a range not inhibiting the production of succinic acid, and is usually 5 to 30% (W / V), preferably 10 to 20%. The reaction is carried out within the range of (W / V). Further, additional addition of organic raw materials may be performed in accordance with the decrease of the organic raw materials as the reaction proceeds.

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, an inorganic salt, and the like. Here, the nitrogen source is not particularly limited as long as it is a nitrogen source that can be assimilated by the bacterium to produce succinic acid. Specifically, ammonium salt, nitrate, urea, soybean hydrolysate, casein degradation product And various organic and inorganic nitrogen compounds such as peptone, yeast extract, meat extract and corn steep liquor. As the inorganic salt, various phosphates, sulfates, magnesium, potassium, manganese, iron, zinc, and other metal salts are used. In addition, factors that promote growth such as vitamins such as biotin, pantothenic acid, inositol, and nicotinic acid, nucleotides, and amino acids are added as necessary. In order to suppress foaming during the reaction, it is desirable to add an appropriate amount of a commercially available antifoaming agent to the culture solution.

  The reaction liquid contains, for example, carbonate ions, bicarbonate ions, or carbon dioxide gas (carbon dioxide gas) in addition to the organic raw material, nitrogen source, inorganic salt, and the like. Carbonate ions or bicarbonate ions are supplied from magnesium carbonate, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, etc., which can also be used as a neutralizing agent. It can also be supplied from these salts or carbon dioxide gas. Specific examples of the carbonate or bicarbonate salt include magnesium carbonate, ammonium carbonate, sodium carbonate, potassium carbonate, ammonium bicarbonate, sodium bicarbonate, and potassium bicarbonate. 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. When carbon dioxide gas is contained, 50 mg to 25 g, preferably 100 mg to 15 g, more preferably 150 mg to 10 g of carbon dioxide gas is contained per liter of the solution.

  The pH of the reaction solution can be adjusted by adding sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium hydroxide, calcium hydroxide, magnesium hydroxide or the like. Since the pH in this reaction is usually pH 5 to 10, preferably 6 to 9.5, the pH of the reaction solution is adjusted within the above range by an alkaline substance, carbonate, urea or the like as needed during the reaction. To do.

  The optimal growth temperature for 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. Although the quantity of the microbial cell used for reaction is not prescribed | regulated, 1-700 g / L, Preferably it is 10-500 g / L, More preferably, 20-400 g / L is used. The reaction time is preferably 1 hour to 168 hours, more preferably 3 hours to 72 hours.

  During bacterial seed culture, oxygen must be supplied by aeration and agitation. On the other hand, the production reaction of an organic acid such as succinic acid may be carried out by aeration and stirring, but may be carried out in an anaerobic atmosphere without aeration and without supplying oxygen. Under the anaerobic atmosphere here, for example, the container is sealed and reacted without aeration, supplied with an inert gas such as nitrogen gas and reacted, or the inert gas containing carbon dioxide gas is vented. Can be obtained.

  By the bacterial reaction as described above, 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, after removing solids such as bacterial cells by centrifugation, filtration, etc., desalting with an ion exchange resin or the like, and crystallization from the solution or purification by column chromatography, etc. Acid can be collected.

Furthermore, in this invention, after manufacturing organic acids, such as a succinic acid, by the above-mentioned method of this invention, an organic acid containing polymer can be manufactured by performing a polymerization reaction using the obtained organic acid as a raw material. In recent years, with the increase in the number of environmentally friendly industrial products, attention has been focused on polymers using plant-derived raw materials. In particular, succinic acid produced in the present invention is processed into polymers such as polyester and polyamide. Can be used. Specific examples of succinic acid-containing polymers include succinic acid polyesters obtained by polymerizing diols such as butanediol and ethylene glycol and succinic acid, succinic acid polyamides obtained by polymerizing diamines such as hexamethylenediamine and succinic acid, and the like. Is mentioned.
The organic acid obtained by the production method of the present invention or the composition containing the organic acid can be used for food additives, pharmaceuticals, cosmetics and the like.

[Example]
Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples.

<Preparation of PoxB deficiency, ACH deficiency, PTA deficiency, ACK deficiency>
(A) Extraction of MJ233 strain genomic DNA A medium [urea 2 g, (NH ₄ ) ₂ SO ₄ 7 g, KH ₂ PO ₄ 0.5 g, K ₂ HPO ₄ 0.5 g, MgSO ₄ .7H ₂ O 0.5 g, _{FeSO 4 · 7H 2 O 6mg,} MnSO 4 · 4-5H 2 O6mg, biotin 200 [mu] g, thiamine 100 [mu] g, yeast extract 1g, casamino acid 1g, glucose 20g, dissolved in distilled water 1L] 10 mL, Brevibacterium flavum MJ The -233 strain was cultured until the late logarithmic growth phase, and the cells were collected by centrifugation (10000 g, 5 minutes). The obtained cells were suspended in 0.15 mL of a 10 mM NaCl / 20 mM Tris buffer solution (pH 8.0) / 1 mM EDTA · 2Na solution containing lysozyme at a concentration of 10 mg / mL. Next, proteinase K was added to the suspension so that the final concentration was 100 μg / mL, and the mixture was incubated at 37 ° C. for 1 hour. Further, 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. To this lysate, add an equal amount of phenol / chloroform solution, shake gently at room temperature for 10 minutes, and then centrifuge (5,000 × g, 20 minutes, 10-12 ° C.) to obtain a supernatant. The fraction was collected and sodium acetate was added to a concentration of 0.3 M, and then twice as much ethanol was added and mixed. The precipitate collected by centrifugation (15,000 × g, 2 minutes) was washed with 70% ethanol and then air-dried. To the obtained DNA, 5 mL of a 10 mM Tris buffer (pH 7.5) -1 mM EDTA · 2Na solution was added and left overnight at 4 ° C., and used as template DNA for subsequent PCR.

(B) Construction of a plasmid for destruction of poxB Obtaining a DNA fragment lacking the internal sequence of the poxB gene derived from Brevibacterium flavum MJ233 strain was reported using the DNA prepared in (A) above as a template and the entire genome sequence. Synthetic DNA (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4) designed based on the sequence around the gene (GenBank Database Accession No. BA00000036) of Corynebacterium glutamicum ATCC13032 Performed by crossover PCR. PCR was performed using the DNA fragments in the 5 ′ end region of the poxB gene as SEQ ID NO: 1 and SEQ ID NO: 2, and the DNA fragments in the 3 ′ end region as synthetic primers of SEQ ID NO: 3 and SEQ ID NO: 4, respectively. Composition of reaction solution: Template DNA 1 μL, PfxDNA polymerase (manufactured by Invitrogen) 0.5 μL, 1 × concentration attached buffer, 0.4 μM each primer, 1 mM MgSO ₄ , 0.2 μM dNTPs were mixed to make a total volume of 50 μL. Reaction temperature condition: DNA thermal cycler PTC-200 (manufactured by MJ Research) was used, and a cycle consisting of 94 ° C. for 15 seconds, 55 ° C. for 30 seconds, and 68 ° C. for 45 seconds was repeated 30 times. However, the heat retention at 94 ° C. in the first cycle was 2 minutes, and the heat retention at 68 ° C. in the final cycle was 3 minutes. Next, PCR was performed using the obtained two amplification products as templates and the synthetic DNAs of SEQ ID NO: 1 and SEQ ID NO: 4 as primers. Composition of reaction solution: Template DNA 1 μL, PfxDNA polymerase (manufactured by Invitrogen) 0.5 μL, 1 × concentration attached buffer, 0.4 μM each primer, 1 mM MgSO ₄ , 0.2 μM dNTPs were mixed to make a total volume of 50 μL. Reaction temperature condition: DNA thermal cycler P
Using TC-200 (manufactured by MJ Research), a cycle consisting of 94 ° C. for 15 seconds, 55 ° C. for 30 seconds, and 68 ° C. for 1 minute 20 seconds was repeated 30 times. However, the heat retention at 94 ° C. in the first cycle was 2 minutes, and the heat retention at 68 ° C. in the final cycle was 3 minutes. The obtained DNA fragment from which the internal sequence of the poxB gene was deleted was QIAquick PCR Purification.
After purification using Kit (manufactured by QIAGEN), restriction enzymes XhoI and Sa
Cut with cI. The resulting DNA fragment of about 1.0 kb was separated by 0.8% agarose (SeaKem GTG agarose: manufactured by FMCBioProducts) gel electrophoresis and then visualized by staining with ethidium bromide, and QIAquick Gel Extraction Kit (QIAGEN From the gel. This DNA fragment was mixed with DNA prepared by cleaving pKMB1 (plasmid containing sacB gene: JP-A-2005-95169) with restriction enzymes XhoI and SacI, and ligation kit ver. 2 (made by Takara Bio Inc.). Escherichia coli (DH5α strain) was transformed with the obtained plasmid DNA and smeared on an LB agar medium containing 50 μg / mL kana machine and 50 μg / mL X-Gal. Clones that formed white colonies on this medium were subjected to liquid culture by a conventional method, and then plasmid DNA was purified. The obtained plasmid DNA was cleaved with restriction enzymes XhoI and SacI, and an inserted fragment of about 1.0 kb was recognized, and this was named pOXB11 (FIG. 1).

(C) Construction of plasmid for ach disruption Obtaining a DNA fragment lacking the internal sequence of the ach gene from Brevibacterium flavum MJ233 strain was reported using the DNA prepared in (A) above as a template and the entire genome sequence. Synthetic DNA (SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8) designed based on the sequence around the gene (GenBank Database Accession No. BA00000036) of Corynebacterium glutamicum ATCC13032 Performed by crossover PCR. The DNA fragments in the 5 ′ terminal region of the ach gene were subjected to PCR using SEQ ID NO: 5 and SEQ ID NO: 6, and the DNA fragments in the 3 ′ terminal region were subjected to PCR using the synthetic DNAs of SEQ ID NO: 7 and SEQ ID NO: 8, respectively. Composition of reaction solution: Template DNA 1 μL, PfxDNA polymerase (manufactured by Invitrogen) 0.5 μL, 1 × concentration attached buffer, 0.4 μM each primer, 1 mM MgSO ₄ , 0.2 μM dNTPs were mixed to make a total volume of 50 μL. Reaction temperature condition: DNA thermal cycler PTC-200 (manufactured by MJ Research) was used, and a cycle consisting of 94 ° C. for 15 seconds, 55 ° C. for 30 seconds, and 68 ° C. for 45 seconds was repeated 30 times. However, the heat retention at 94 ° C. in the first cycle was 2 minutes, and the heat retention at 68 ° C. in the final cycle was 3 minutes. Next, PCR was performed using the obtained two amplification products as templates and the synthetic DNAs of SEQ ID NO: 5 and SEQ ID NO: 8 as primers. Composition of reaction solution: Template DNA 1 μL, PfxDNA polymerase (manufactured by Invitrogen) 0.5 μL, 1 × concentration attached buffer, 0.4 μM each primer, 1 mM MgSO ₄ , 0.2 μM dNTPs were mixed to make a total volume of 50 μL. Reaction temperature condition: DNA thermal cycler PTC-200 (manufactured by MJ Research) was used, and a cycle consisting of 94 ° C. for 15 seconds, 55 ° C. for 30 seconds, and 68 ° C. for 1 minute 20 seconds was repeated 30 times. However, the heat retention at 94 ° C. in the first cycle was 2 minutes, and the heat retention at 68 ° C. in the final cycle was 3 minutes. The obtained DNA fragment lacking the internal sequence of the ach gene was purified using QIAquick PCR Purification Kit (manufactured by QIAGEN) and then cleaved with restriction enzymes SalI and SacI. The resulting DNA fragment of about 1.0 kb was separated by 0.8% agarose (SeaKem GTG agarose: manufactured by FMCBioProducts) gel electrophoresis and then visualized by staining with ethidium bromide, and QIAquick Gel Extraction Kit (QIAGEN From the gel. From this DNA fragment, pKMB1 (Japanese Patent Laid-Open No. 2005-95169) was converted into restriction enzymes XhoI and Sa
mixed with DNA prepared by digestion with cI, and ligation kit ver. 2 (made by Takara Bio Inc.). Escherichia coli (DH5α strain) was transformed with the obtained plasmid DNA and smeared on an LB agar medium containing 50 μg / mL kana machine and 50 μg / mL X-Gal. Clones that formed white colonies on this medium were subjected to liquid culture by a conventional method, and then plasmid DNA was purified. The obtained plasmid DNA was cleaved with restriction enzymes MluI and SacI, and an insertion fragment of about 1.1 kb was observed, which was designated as pACH22 (FIG. 2).

(D) Construction of plasmid for destroying pta-ack The DNA fragment lacking the internal sequence of pta-ack gene derived from Brevibacterium flavum MJ233 strain was obtained using the DNA prepared in (A) above as a template and the entire genome. Synthetic DNA (SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12) designed based on the sequence around the gene of Corynebacterium glutamicum ATCC13032 strain (GenBank Database Accession No. BA00000036) whose sequence has been reported ) Was used for crossover PCR. PCR was carried out using the DNA fragments of the 5 ′ terminal region of the pta-ack gene as SEQ ID NO: 9 and SEQ ID NO: 10, and the DNA fragments of the 3 ′ terminal region of the synthetic DNAs of SEQ ID NO: 11 and SEQ ID NO: 12, respectively. Composition of reaction solution: Template DNA 1 μL, PfxDNA polymerase (manufactured by Invitrogen) 0.5 μL, 1 × concentration attached buffer, 0.4 μM each primer, 1 mM MgSO ₄ , 0.2 μM dNTPs were mixed to make a total volume of 50 μL. Reaction temperature condition: DNA thermal cycler PTC-200 (manufactured by MJ Research) was used, and a cycle consisting of 94 ° C. for 15 seconds, 55 ° C. for 30 seconds, and 68 ° C. for 45 seconds was repeated 30 times. However, the heat retention at 94 ° C. in the first cycle was 2 minutes, and the heat retention at 68 ° C. in the final cycle was 3 minutes. Next, PCR was performed using the obtained two amplification products as templates and the synthetic DNAs of SEQ ID NO: 9 and SEQ ID NO: 12 as primers. Composition of reaction solution: Template DNA 1 μL, PfxDNA polymerase (manufactured by Invitrogen) 0.5 μL, 1 × concentration attached buffer, 0.4 μM each primer, 1 mM MgSO ₄ , 0.2 μM dNTPs were mixed to make a total volume of 50 μL. Reaction temperature condition: DNA thermal cycler PTC-200 (manufactured by MJ Research) was used, and a cycle consisting of 94 ° C. for 15 seconds, 55 ° C. for 30 seconds, and 68 ° C. for 1 minute 20 seconds was repeated 30 times. However, the heat retention at 94 ° C. in the first cycle was 2 minutes, and the heat retention at 68 ° C. in the final cycle was 3 minutes. The obtained DNA fragment lacking the internal sequence of the pta-ack gene was purified using QIAquick PCR Purification Kit (manufactured by QIAGEN), and then cleaved with restriction enzymes SacI and SphI. The resulting DNA fragment of about 1.1 kb was separated by gel electrophoresis with 0.8% agarose (SeaKem GTG agarose: manufactured by FMCBioProducts) and then visualized by staining with ethidium bromide, and QIAquick Gel Extraction Kit (QIAGEN From the gel. This DNA fragment was mixed with DNA prepared by cleaving pKMB1 (Japanese Patent Laid-Open No. 2005-95169) with restriction enzymes SacI and SphI, and ligation kit ver. 2 (made by Takara Bio Inc.). Escherichia coli (DH5α strain) was transformed with the obtained plasmid DNA and smeared on an LB agar medium containing 50 μg / mL kana machine and 50 μg / mL X-Gal. Clones that formed white colonies on this medium were subjected to liquid culture by a conventional method, and then plasmid DNA was purified. The obtained plasmid DNA was cleaved with restriction enzymes SacI and SphI, and an insertion fragment of about 1.1 kb was observed, which was named pTACK1 (FIG. 3).

<Preparation of PoxB / ACH / PTA / ACK deficient strain>
(A) Preparation of PoxB-deficient strain The test strain for preparation of the poxB gene-deficient strain was Brevibacterium flavum MJ233 / ΔLDH (LDH disrupted strain: JP-A-2005-95169), and the plasmid DNA for transformation was Using the pOXB11 constructed in (B) of Example 1 above, the calcium chloride method (Journal of Molecular Biology, 53, 159, 1)
970) and prepared from E. coli strain JM110. Brevibacterium was transformed by the electric pulse method (Res. Microbiol. Vol. 144, p.181-185, 1993), and the obtained transformant was smeared on an LBG agar medium containing 50 μg / mL of kanamycin. . Since the strain grown on this medium is a plasmid in which pOXB11 cannot replicate in the Brevibacterium flavum MJ233 strain, the plasmid poxB gene and the same gene on the genome of Brevibacterium flavum MJ233 strain As a result of homologous recombination, the kanamycin resistance gene and sacB gene derived from the plasmid should be inserted on the same genome. Next, the homologous recombination strain was subjected to liquid culture in an LBG medium containing 25 μg / mL kanamycin. As a result of smearing about 1 million cells of this culture on LBG medium containing 10% sucrose, sacB gene was eliminated by the second homologous recombination, and several tens of strains considered to be insensitive to sucrose were obtained. I got it. Among the homologous recombination strains obtained in the second round as described above, those in which the poxB gene is replaced with a mutant type derived from pOXB11 and those in which the wild type is restored. Whether the poxB gene is a mutant type or a wild type can be determined by analyzing the poxB gene using primers (SEQ ID NO: 1 and SEQ ID NO: 4) for PCR amplification. In the case of a mutant having a deletion region of 1518 bp, a DNA fragment of 981 bp is generated. As a result of analyzing the strain that became insensitive to sucrose by the above method, a strain having only the mutant gene was selected, and the strain was named Brevibacterium flavum MJ233 / ΔPoxB / ΔLDH.

(B) Preparation of ACH-deficient strain The test strain for preparation of the ach gene-deficient strain was Brevibacterium flavum MJ233 / ΔPoxB / ΔLDH prepared in (A) above, and the plasmid DNA for transformation was that of Example 1 above. It was prepared from E. coli strain JM110 transformed by the calcium chloride method (Journal of Molecular Biology, 53, 159, 1970) using pACH22 constructed in (C). Brevibacterium transformation and selection of homologous recombinants twice were performed by the method shown in (A) above, and several sucrose-insensitive colonies were obtained. Of these, the determination of whether the ach gene is a mutant type or a wild type can be performed by analyzing using primers (SEQ ID NO: 5 and SEQ ID NO: 8) for PCR amplification of the ach gene, In the wild type, a DNA fragment of 1228 bp is generated, and in the mutant type having a deletion region, a DNA fragment of 1003 bp is generated. As a result of analyzing the strain that became insensitive to sucrose by the above method, a strain having only the mutant gene was selected, and the strain was named Brevibacterium flavum MJ233 / ΔACH / ΔPoxB / ΔLDH.

(C) Preparation of PTA-ACK deficient strain The test strain for preparation of the pta-ack gene deficient strain was Brevibacterium flavum MJ233 / ΔACH / ΔPoxB / ΔLDH prepared in (B) above, and the plasmid DNA for transformation was It was prepared from E. coli strain JM110 transformed by the calcium chloride method (Journal of Molecular Biology, 53, 159, 1970) using pTACK1 constructed in (D) of Example 1 above. Brevibacterium transformation and selection of homologous recombinants twice were performed by the method shown in (A) above, and several sucrose-insensitive colonies were obtained. Of these, the determination of whether the pta-ack gene is a mutant type or a wild type is performed by analyzing the pta-ack gene using primers (SEQ ID NO: 9 and SEQ ID NO: 12) for PCR amplification. In the wild type, a DNA fragment of 1645 bp is generated, and in a mutant type having a deletion region, a DNA fragment of 1086 bp is generated. As a result of analyzing the strain that became insensitive to sucrose by the above method, a strain having only the mutant gene was selected, and the strain was named Brevibacterium flavum MJ233 / ΔPTA / ΔACK / ΔACH / ΔPoxB / ΔLDH. .

<Preparation of OdhI disruption strain>
(A) Construction of plasmid for OdhI disruption Obtaining the OdhI gene fragment derived from Brevibacterium flavum MJ233 strain was performed using the DNA prepared in (A) above as a template and Corynebacterium glutamicum ATCC13032 for which the entire genome sequence has been reported. It was performed by PCR using a synthetic DNA (SEQ ID NO: 13 and SEQ ID NO: 14) designed based on the sequence of the gene of the strain (GenBank Database Accession No. BA000036 (base number 1519601..1520032)). Composition of reaction solution: Template DNA 1 μL, PfxDNA polymerase (manufactured by Invitrogen) 0.5 μL, 1-fold concentration buffer, 0.4 μM each primer, 1 mM MgSO ₄ , 0.2 μM dNTPs were mixed to make a total volume of 50 μL. Reaction temperature condition: DNA thermal cycler PTC-200 (manufactured by MJ Research) was used, and a cycle consisting of 94 ° C. for 15 seconds, 55 ° C. for 30 seconds, and 68 ° C. for 45 seconds was repeated 30 times. However, the heat retention at 94 ° C. in the first cycle was 2 minutes, and the heat retention at 68 ° C. in the final cycle was 3 minutes. The obtained DNA fragment of the internal sequence of the OdhI gene was purified using ChargeSwitch PCR Clean-Up Kit (manufactured by Invitrogen), and then cleaved with restriction enzymes KpnI and SphI. The resulting DNA fragment of about 0.4 kb was separated by 0.9% agarose (SeaKem GTG agarose: manufactured by FMCBioProducts) gel electrophoresis and then visualized by ethidium bromide staining, and DNA Fragment Purification Kit MagExtractor ( And collected from the gel using Toyobo Co., Ltd. This DNA fragment was mixed with DNA prepared by cleaving E. coli vector pHSG298 (manufactured by Takara Bio Inc.) with restriction enzymes KpnI and SphI, and ligation kit ver. 2 (manufactured by Takara Bio Inc.). Escherichia coli (DH5α strain) was transformed with the obtained plasmid DNA and smeared on an LB agar medium containing 50 μg / mL kana machine and 50 μg / mL X-Gal. Clones that formed white colonies on this medium were subjected to liquid culture by a conventional method, and then plasmid DNA was purified. The obtained plasmid DNA was cleaved with restriction enzymes KpnI and SphI, and a plasmid in which an insertion fragment of about 0.4 kb was recognized was selected and named pOdhI1 (FIG. 4).

(B) Preparation of OdhI-disrupted strain Test strains for preparing the odhI gene-disrupted strain were Brevibacterium flavum MJ233 / ΔLDH (strain in which the LDH gene was disrupted: JP-A-2005-95169) and (C) of Example 2 above. Brevibacterium flavum MJ233 / ΔPTA / ΔACK / ΔACH / ΔPoxB / ΔLDH prepared in (1) was prepared, and the plasmid DNA used for transformation was prepared from E. coli MJ110 strain transformed with pOdhI1 constructed in (A) above. Brevibacterium flavum MJ233 / ΔLDH strain, Brevibacterium flavum MJ233 / ΔPTA / ΔACK / ΔACH / ΔPoxB / ΔLDH is transformed by the electric pulse method (Res. Microbiol. Vol. 144, p. 181-185, 1993). The obtained transformant was smeared on an LBG agar medium containing 10 μg / mL kanamycin [10 g of tryptone, 5 g of yeast extract, 5 g of NaCl, 20 g of glucose, and 15 g of agar dissolved in 1 L of distilled water]. Since the strain grown on this medium is a plasmid in which pOdhI1 cannot replicate in the Brevibacterium flavum MJ233 strain, the same gene on the genome of the OdhI gene of the plasmid and the Brevibacterium flavum MJ233 strain is used. As a result of the homologous recombination with the kanamycin, a kanamycin resistance gene derived from the plasmid should be inserted on the same genome. Whether or not the kanamycin resistant strain thus obtained has undergone homologous recombination between the odhI gene present on its genome and the gene present on the plasmid pOdhI1 is confirmed by SEQ ID NO: 15 and This was performed by colony PCR using SEQ ID NO: 16. The template DNA was a supernatant obtained by suspending colonies in 50 μL of sterilized water and boiling for 5 minutes. Reaction solution composition: Template DNA 1 μL, Ex-Taq DNA polymerase (manufactured by Takara Bio Inc.) 0.2 μL, 1-fold concentration buffer, 0.2 μM each primer, 0.2 μM dNTPs were mixed to make a total volume of 20 μL. Reaction temperature conditions: DNA thermal cycler PTC-200 (manufactured by MJ Research) was used, and a cycle consisting of 98 ° C. for 10 seconds, 60 ° C. for 20 seconds and 72 ° C. for 1 minute was repeated 30 times. However, the heat retention at 95 ° C. in the first cycle was 2 minutes, and the heat retention at 72 ° C. in the final cycle was 3 minutes. As a result of analyzing kanamycin-resistant strains by the above method, strains that obtain PCR amplification products of 966 bp were selected, which were selected from Brevibacterium flavum MJ233 / ΔOdhI / ΔLDH and Brevibacterium flavum MJ233 / ΔOdhI / ΔPTA / ΔACK, respectively. / ΔACH / ΔPoxB / ΔLDH.

<Evaluation of OdhI disruption strain>
100 mL seed culture medium (urea: 4 g, ammonium sulfate: 14 g, monopotassium phosphate: 0.5 g, dipotassium phosphate 0.5 g, magnesium sulfate heptahydrate: 0.5 g, ferrous sulfate-7 water (Japanese product: 20 mg, manganese sulfate / hydrate: 20 mg, D-biotin: 200 μg, thiamine hydrochloride: 200 μg, yeast extract: 1 g, casamino acid: 1 g, and distilled water: 100 mL of 1000 mL medium) in a 500 mL Erlenmeyer flask The mixture was sterilized by heating at 120 ° C. for 20 minutes. This was cooled to room temperature, 4 mL of 50% glucose aqueous solution sterilized in advance and 50 μL of 5% kanamycin aqueous solution filtered aseptically were added, and the Brevibacterium flavum MJ233 / ΔOdhI / ΔLDH strain, Brevibacterium prepared in Example 3 (B) Bacterium flavum MJ233 / ΔOdhI / ΔPTA / ΔACK / ΔACH / ΔPoxB / ΔLDH strains were inoculated and cultured at 30 ° C. for 24 hours. However, when culturing the Brevibacterium flavum MJ233 / ΔLDH strain as a control strain and the Brevibacterium flavum MJ233 / ΔPTA / ΔACK / ΔACH / ΔPoxB / ΔLDH strain prepared in (C) of Example 2 above, The kanamycin addition was excluded.
The obtained whole culture solution was collected by centrifugation at 10,000 × g for 5 minutes, and a cell suspension medium (magnesium sulfate 7 hydrate: 1 g, ferrous sulfate 7 hydrate: 40 mg, sulfuric acid Manganese hydrate: 40 mg, D-biotin: 400 μg, thiamine hydrochloride: 400 μg, monoammonium phosphate: 0.8 g, diammonium phosphate: 0.8 g, potassium chloride: 0.3 g, ammonium sulfate 66 g, and distilled water : 1000 mL) was suspended so that the absorbance of OD ₆₆₀ was 80. Add 0.5 mL of the substrate solution (glucose: 100 g, magnesium carbonate: 194 g, and distilled water: 1000 mL) to 0.5 mL of the above cell suspension in a 4 mL reactor, and in a 5 to 6% carbon dioxide atmosphere, The reaction was carried out at 35 ° C. for 22 hours.
After the reaction, the mixture was centrifuged under the conditions described above, and the organic acid concentration of the supernatant was analyzed. As a result, the Brevibacterium flavum MJ233 / ΔOdhI / ΔLDH strain was compared with the control strain Brevibacterium flavum MJ233 / ΔLDH. The succinic acid yield per glucose consumed is increased by 7.5%, and the amount of dicarboxylic acid by-product per succinic acid is 84% (however, dicarboxylic acid is the total value of malic acid and fumaric acid), The by-product amount of α-ketoglutaric acid was reduced by 88%, and the by-product amount of amino acid was reduced by 78% (however, amino acids are the total value of alanine, valine and glutamic acid). The Brevibacterium flavum MJ233 / ΔOdhI / ΔPTA / ΔACK / ΔACH / ΔPoxB / ΔLDH strain is compared to the control strain Brevibacterium flavum MJ233 / ΔPTA / ΔACK / ΔACH / ΔPoxB / ΔLDH. Per succinic acid yield increased by 15%, by-product amount of dicarboxylic acid per succinic acid was 39% (however, dicarboxylic acid is the total value of malic acid and fumaric acid), α-ketoglutaric acid by-product The amount was 89% and the amount of amino acid by-products was reduced by 62% (however, amino acids are the total value of alanine, valine and glutamic acid). A clear improvement in the yield of succinic acid and a reduction in the amount of by-products of malic acid, fumaric acid, α-ketoglutaric acid, and amino acids were observed by disruption of the odhI gene.

The figure which shows the construction procedure of plasmid pOXB11. The figure which shows the construction procedure of plasmid pACH22. The figure which shows the construction procedure of plasmid pTACK1. The figure which shows the construction procedure of plasmid pOdhI1.

Claims (10)

Bacteria that have an organic acid-producing ability and have been modified so that the expression of the cg1630 gene is reduced as a result of the disruption of the cg1630 gene on the chromosome compared to the unmodified strain , A method for producing an organic acid, comprising producing an organic acid by acting on an organic raw material in a reaction solution containing carbonate ion or carbon dioxide gas, and collecting the organic acid . The method for producing an organic acid according to claim 1 , wherein the cg1630 gene on the bacterial chromosome is a gene having 80% or more homology with the nucleotide sequence of SEQ ID NO: 17. The method for producing an organic acid according to claim 1 or 2 , wherein the bacterium is further modified so that lactate dehydrogenase activity is reduced as compared to an unmodified strain by disrupting a lactate dehydrogenase gene on a chromosome. . The bacterium further enhances pyruvate carboxylase activity compared to an unmodified strain by introducing a pyruvate carboxylase gene or replacing the pyruvate carboxylase gene promoter with a strong promoter on the chromosome. The method for producing an organic acid according to any one of claims 1 to 3 , wherein the organic acid is modified. The bacterium further has an activity of one or more enzymes selected from acetate kinase, phosphotransacetylase, pyruvate oxidase and acetyl CoA hydrolase, and the gene encoding the enzyme on the chromosome has been disrupted. The method for producing an organic acid according to any one of claims 1 to 4 , wherein the organic acid is modified so as to be reduced as compared with an unmodified strain. The method for producing an organic acid according to any one of claims 1 to 5 , wherein the bacterium is a coryneform bacterium. The method for producing an organic acid according to any one of claims 1 to 6, wherein the organic raw material is allowed to act in an anaerobic atmosphere. The method for producing an organic acid according to any one of claims 1 to 7 , wherein the organic raw material is glucose or sucrose. The manufacturing method of the organic acid as described in any one of Claims 1-8 whose organic acid is a succinic acid. The manufacturing method of an organic acid containing polymer including the process of manufacturing an organic acid by the method as described in any one of Claims 1-9 , and the process of performing a polymerization reaction using the organic acid obtained at the said process as a raw material.

Images