JP4428999B2 - Method for producing non-amino organic acid - Google Patents

Description

  The present invention relates to a method for producing a non-amino organic acid using a bacterium such as a coryneform bacterium.

  When non-amino 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). 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, and non-amino organic acids are used as stationary cells without aeration of oxygen. A production 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 an aerobic bacterium is used, amino acids are by-produced in addition to the target non-amino organic acid, which should be improved in order to further improve the yield of the non-amino organic acid. In addition, for microorganisms used for the production of non-amino organic acids, genetically modified microorganisms (see Patent Document 4 or 5) have also been reported. However, in order to further improve the yield of non-amino organic acids, Creation was required.

  In general, microorganisms are known to have at least the following two biosynthetic pathways in order to assimilate ammonia to produce glutamic acid. The former is a pathway that produces glutamate from ammonia and α-ketoglutarate (GDH pathway) by glutamate dehydrogenase (GDH), and the latter first produces glutamine from ammonia and glutamate by glutamine synthetase (GS). This is a pathway (GS-GOGAT pathway) in which two molecules of glutamate are generated from α-ketoglutarate by glutamate synthase (GOGAT) (see, for example, Non-Patent Document 2).

  In the coryneform bacterium, a GDH gene-only defective strain (for example, see Non-patent Document 3) or a GOGAT gene-only defective strain (for example, see Non-Patent Document 4) has been reported. No glutamic acid requirement was exhibited, and there was no significant change in glutamic acid production compared to non-deficient strains. The reason for this is that glutamic acid may be generated by the other biosynthetic pathway of the above two types of biosynthetic pathways, or glutamic acid may be generated by other ammonia anabolic enzymes and aminotransferases such as alanine dehydrogenase. Although the possibility etc. are considered, it is not clarified actually (for example, refer nonpatent literature 5). Therefore, from these findings alone, it was difficult to predict whether glutamate production would actually be reduced when both of the two ammonia assimilation pathways were destroyed. Therefore, a method for efficiently destroying the glutamate biosynthesis pathway has not been known.

The glutamic acid biosynthetic system synthesizes glutamic acid using α-ketoglutaric acid, which is one of the precursors of succinic acid, as a substrate, but the succinic acid biosynthetic system is complex. Since the acid and isocitric acid are synthesized as precursors, the relationship between the succinic acid biosynthetic system and the glutamic acid biosynthetic system has not been sufficiently studied. Therefore, a disrupted strain of the glutamate biosynthesis gene as described above has not been used for the production of non-amino organic acids such as succinic acid.
US Pat. No. 5,142,834 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 By Gottschalk G., Spriner-Verlag 2nd ed., New York, NY, 1986 Bormann-El Kholy ER, ER, Eikmanns BJ, Gutmann M., Sahm H., Appl.Environ.Microbiol., Vol. 59, p2329-2331, 1993 Beckers G., Nolden L., Burkovski A., Microbiology, vol. 147, p2961-2970, 2001 By M. Tesch, AA de Graaf, and H. Sahm, Applied and Environmental Microbiology vol. 65, p1099-1109, 1999

  An object of the present invention is to provide a method for producing a non-amino organic acid having a higher production efficiency by reducing amino acid by-products.

  As a result of intensive studies to solve the above-mentioned problems, the present inventor has obtained a bacterium or a processed product thereof modified so that glutamate synthase (GOGAT) activity and glutamate dehydrogenase (GDH) activity are reduced. It was found that by reacting with organic raw materials in a reaction solution containing ions, bicarbonate ions, or carbon dioxide gas, by-product amino acids are reduced and the amount of non-amino organic acid produced is increased, and the present invention is completed. It came to do.

That is, according to the present invention, the following inventions are provided.
(1) A bacterium modified so that the activity of one or both of glutamate synthase and glutamine synthetase, and glutamate dehydrogenase activity is reduced to 30% or less compared to the unmodified strain, or a treated product thereof, Collecting and collecting non-amino organic acids from the reaction liquid by generating and accumulating non-amino organic acids in the reaction liquid by acting on organic raw materials in the reaction liquid containing ions, bicarbonate ions or carbon dioxide gas A process for producing a non-amino organic acid, characterized in that
(2) The production method of (1), wherein the bacterium is modified so that glutamate synthase activity and glutamate dehydrogenase activity are each reduced to 30% or less as compared to the unmodified strain.
(3) The bacterium is any bacterium selected from the group consisting of coryneform bacteria, Bacillus bacteria, Rhizobium bacteria, Escherichia bacteria, Lactobacillus bacteria, and Actinobacillus bacteria (1) or ( 2) Production method.
(4) The bacterium according to any one of (1) to (3), wherein the bacterium is further modified so that lactate dehydrogenase activity is reduced to 10% or less compared to an unmodified strain of the enzyme. Production method.
(5) The method according to any one of (1) to (4), wherein the bacterium is further modified so that pyruvate carboxylase activity is enhanced.
(6) The method according to any one of (1) to (5), wherein the organic raw material is allowed to act in an anaerobic atmosphere.
(7) The manufacturing method in any one of (1)-(6) whose said organic raw material is glucose or sucrose.
(8) The production method of any one of (1) to (7), wherein the non-amino organic acid is succinic acid.
(9) A non-amino organic comprising a step of producing a non-amino organic acid by any one of the methods (1) to (8), and a step of performing a polymerization reaction using the non-amino organic acid obtained in the step as a raw material A method for producing an acid-containing polymer.

  By producing a non-amino organic acid using a bacterium modified to reduce glutamate synthase and / or glutamine synthetase activity, and glutamate dehydrogenase (GDH) activity or a treated product thereof, Raw amino acids can be reduced and the production of non-amino organic acids can be increased.

  Hereinafter, embodiments of the present invention will be described in detail.

1. Bacteria used in the present invention Bacteria that can be used in the production method of the present invention have reduced activity of one or both of glutamate synthase (GOGAT) and glutamine synthetase (GS), and glutamate dehydrogenase (GDH) activity. And a bacterium having a non-amino organic acid-producing ability. Such a bacterium may be a bacterium that originally has a non-amino organic acid-producing ability or a bacterium that has been imparted with a non-amino organic acid-producing ability by breeding, and may be modified as described above. It is also possible to have a non-amino organic acid-producing ability by performing various modifications. Here, “having the ability to produce a non-amino organic acid” means that a non-amino organic acid can be produced and accumulated in the medium when the bacterium is cultured in the medium. A non-amino organic acid means an organic acid other than an amino acid, but succinic acid, malic acid, fumaric acid or pyruvic acid is preferable, and succinic acid is more preferable.

  This bacterium can be obtained by using a bacterium as shown below as a parent strain and modifying the parent strain. The kind of the parent strain is not particularly limited, but Coryneform Bacterium, Bacillus, Rhizobium, Escherichia, Lactobacillus, or Actinobacillus ) Genus bacteria are preferred, and coryneform bacteria are more preferred. Examples of coryneform bacteria include microorganisms belonging to the genus Corynebacterium, microorganisms belonging to the genus Brevibacterium, or microorganisms belonging to the genus Arthrobacter, and among these, those belonging to the genus Corynebacterium or Brevibacterium More preferably, Corynebacterium glutamicum, Brevibacterium flavum, Brevibacterium ammoniagenes or Brevibacterium lactofermentum And microorganisms belonging to

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.

  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 modifies the parent strain as described above so that the activity of one or both of glutamate synthase (GOGAT) and glutamine synthetase (GS), and glutamate dehydrogenase (GDH) activity is reduced. Can be obtained. That is, the bacteria used in the present invention include strains modified to reduce the activities of GOGAT and GDH, strains modified to reduce the activities of GS and GDH, and GOGAT, GS and GDH. Mention may be made of strains modified to reduce activity.

  "Glutamate synthase activity" refers to the activity of catalyzing the reaction of converting glutamine into α-ketoglutaric acid and glutamic acid, "Glutamine synthetase activity" refers to the activity of catalyzing the reaction of generating glutamine from glutamic acid and ammonia, “Glutamate dehydrogenase activity” refers to the activity of catalyzing the reaction of producing glutamic acid from α-ketoglutaric acid and ammonia, respectively.

  “Glutamate synthase activity is reduced”, “Glutamine synthetase activity is reduced” and “Glutamate dehydrogenase activity is reduced” means that the activity of these enzymes is lower than that of an unmodified strain of these enzymes. It means being reduced. The activities of these enzymes are each preferably reduced to 30% or less of the unmodified strain, and more preferably 10% or less. These enzyme activities may be completely lost. The activities of glutamate synthase and glutamate dehydrogenase can be measured by a method based on the decrease in NADH as shown in Example 3 or 4 described later, respectively. Moreover, glutamine synthetase activity can be measured by the method described in Journal of Fermentation and Bioengineering vol 70, p182-184 (1990). In the present specification, the “non-modified strain” does not have to be the original strain itself, but may be a wild strain of the same kind or a parent strain as described above.

  Preparation of strains with reduced glutamate synthase (GOGAT) activity and / or glutamine synthetase (GS) activity and glutamate dehydrogenase (GDH) activity can be carried out by known homologous recombination methods, for example, in JP-A-11-206385. It can be carried out by disrupting the gene by the described method by homologous recombination to the chromosome or the method using the SacB gene (Schafer, A. et al. Gene 145 (1994) 69-73). Specific production methods include those described in Example 3 of this specification for GOGAT and Example 4 for GDH, respectively. On the other hand, since the GS gene sequence is known (base numbers 11680 to 13113 or 25666 to 27006 of GenBank Accession No. AP005281), the GS gene is also disrupted in the same manner as GOGAT and GDH. Can do.

  The enzyme activity can also be reduced by a known mutagenesis method such as a PCR method or a mutagen treatment. Specifically, it can be obtained by introducing a mutation into the region encoding the active site of the enzyme by PCR and introducing the obtained mutant gene into the parent strain. In addition, the parent strain is subjected to mutation treatment with ultraviolet irradiation, X-ray irradiation, radiation irradiation, N-methyl-N′-nitrosoguanidine (NTG) or EMS (ethyl methanesulfonate), etc. It can also be obtained by selecting bacteria with reduced activity. Note that any of the GOGAT, GS, and GDH genes may be disrupted or mutated first.

  Furthermore, the bacterium used in the present invention may have lactate dehydrogenase activity in addition to reduced glutamate synthase (GOGAT) activity and / or glutamine synthetase (GS) activity and glutamate dehydrogenase (GDH) activity. It may be a modified bacterium. Such bacteria are particularly effective when the non-amino organic acid is succinic acid. Such a bacterium, for example, produces a bacterium in which the LDH gene is disrupted as in Example 2 described later, and further disrupts the GOGAT gene and / or GS gene and GDH gene of the bacterium by the above-described method. Can be obtained. However, the modification operation for reducing LDH activity and the modification operation for reducing glutamate synthase (GOGAT) activity and / or glutamine synthetase (GS) activity and glutamate dehydrogenase (GDH) activity should be performed first. May be.

“Lactate dehydrogenase activity is reduced” means that lactate dehydrogenase activity is reduced as compared to a non-modified strain of lactate dehydrogenase. It is preferable that the lactate dehydrogenase activity is reduced to 10% or less per microbial cell as compared with the lactate dehydrogenase non-modified strain. Moreover, the lactate dehydrogenase activity may be completely lost. Reduction of lactate dehydrogenase activity is confirmed by measuring lactate dehydrogenase activity by a known method (L. Kanarek and RL Hill, J. Biol. Chem. 239, 4202 (1964)). Can do. As a specific method for producing a mutant having reduced lactate dehydrogenase activity of coryneform bacteria, a method by homologous recombination to a chromosome described in JP-A-11-206385, or Examples of the present specification 2. A method using the SacB gene described in 2 (Schafer, A. et
al. Gene 145 (1994) 69-73).

  In addition, the bacterium used in the present invention is such that glutamate synthase (GOGAT) activity and / or glutamine synthetase (GS) activity and glutamate dehydrogenase (GDH) activity are reduced, and pyruvate carboxylase activity is enhanced. It may be a modified bacterium. “The activity of pyruvate carboxylase is enhanced” means that the activity of pyruvate carboxylase is preferably increased by 100% or more, more preferably by 300% or more with respect to an unmodified strain such as a wild strain or a parent strain. Say. The activity of pyruvate carboxylase can be measured, for example, by a method for measuring the decrease in NADH as in Example 6 described later. Such a bacterium can be obtained, for example, by introducing a PC gene into a coryneform bacterium having a disrupted GOGAT gene and / or GS gene and GDH gene. Any of the introduction of the PC gene and the modification operation for destroying these genes by homologous recombination may be performed first.

The introduction of the PC gene can be carried out, for example, by highly expressing the pyruvate carboxylase (PC) gene in coryneform bacteria in the same manner as described in JP-A-11-196888. Specific examples of the PC gene gene include, for example, a PC gene derived from Corynebacterium glutamicum ATCC13032 (Peters-Wendisch, PG et al. Microbiology, vol. 144 (1998) p915-927, GenBank Database Accession No. AP005276 ( The DNA fragment containing the PC gene is not limited to those isolated from Corynebacterium glutamicum chromosomal DNA, but can also be used with commonly used DNA synthesizers such as Applied Biosystems ( It may be synthesized using a 394 DNA / RNA synthesizer manufactured by Applied Biosystems, Inc. The PC gene obtained from coryneform bacterial chromosomal DNA as described above is the function of the encoded PC, that is, carbon dioxide. Unless the property involved in fixation is substantially impaired, the nucleotide sequence of SEQ ID NO: 15 In which a part of the base may be substituted with another base or may be deleted, or a new base may be inserted, and a part of the base sequence is rearranged. Any of these derivatives may be used in the present invention, and the DNA hybridizes under stringent conditions with the DNA having the base sequence of SEQ ID NO: 15 or the base sequence of SEQ ID NO: 15. A DNA having a homology of 90% or more, preferably 95% or more, more preferably 99% or more, which encodes a protein having PC activity, can be suitably used. The conditions are 60 ° C., 1 × SSC, 0.1% SDS, preferably 0.1 × SSC, 0.1% SDS, which is the usual washing condition for Southern hybridization. Hybridizing conditions are mentioned those salt concentrations.

Moreover, bacteria other than Corynebacterium glutamicum, or PC genes derived from other microorganisms or animals and plants can also be used. In particular, the following PC genes derived from microorganisms or animals and plants are known in sequence (documents are shown below), and the ORF portion is amplified by hybridization as described above or by PCR. Can be obtained.
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)]

  High expression of PC in coryneform bacteria 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 responsible for the replication and replication function of the plasmid in coryneform bacteria. Possible recombinant plasmids can be obtained. Here, in the above recombinant plasmid, the promoter for expressing the PC gene can be a promoter possessed by coryneform bacteria, but is not limited thereto, and has a base sequence for initiating transcription of the PC gene. Any promoter can be used. For example, a TZ4 promoter as described in Example 3 described later can be mentioned.

  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 growth function in coryneform bacteria. Specific examples thereof include, for example, a plasmid pCRY30 described in JP-A-3-210184; plasmids pCRY21, pCRY2KE, pCRY2KX described in JP-A-2-72876 and US Pat. No. 5,185,262. pCRY31, pCRY3KE and pCRY3KX; 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; pCG2 described in JP-A-58-35197; described in JP-A-57-183799 PCG And pCG11, etc. can be mentioned. 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 expression of the PC gene can be obtained. The PC activity can also be enhanced by introducing a PC gene on the chromosome by a known homologous recombination method, making it highly expressed by substitution, amplification or the like. Transformation can be performed by, for example, the electric pulse method (Res. Microbiol., Vol. 144, p.181-185, 1993).

  The bacterium used in the present invention is modified so that glutamate synthase (GOGAT) activity and / or glutamine synthetase (GS) activity and glutamate dehydrogenase (GDH) activity are reduced, pyruvate carboxylase activity is enhanced, and lactate Bacteria modified to reduce dehydrogenase activity are particularly preferred. Such a bacterium can be obtained, for example, by transforming a coryneform bacterium having a disrupted LDH gene, GOGAT gene and / or GS gene, and GDH gene with a recombinant vector containing a PC gene. Note that any of these modification operations may be performed first.

2. Production method of the present invention In the production method of the present invention, the bacterium or a treated product thereof is allowed to act on an organic raw material in a reaction solution containing carbonate ion, bicarbonate ion, or carbon dioxide gas to produce a non-amino organic acid. This is a method for producing a non-amino organic acid. The non-amino organic acid to be produced is preferably succinic acid, fumaric acid, malic acid or pyruvic acid, more preferably succinic acid.

  When using the above-mentioned bacteria for the production of non-amino organic acids, slant cultures in solid media such as an agar medium may be used for the direct reaction, but the bacteria previously cultured in a liquid medium (seed culture) It is preferable to use it. As a medium used for seed culture, a normal medium used for culturing microorganisms 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 a reaction for producing a non-amino organic acid. In addition, a non-amino 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 the cells obtained by proliferating in advance contain the organic raw material. Non-amino organic acids may also be produced by reacting with organic raw materials in the reaction solution.

  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 it is a carbon source that can be assimilated by the microorganism to produce succinic acid, but is usually galactose, lactose, glucose, fructose, glycerol, sucrose, sucrose. 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 saccharides 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 the microorganism can be assimilated to produce succinic acid, and 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, and inorganic salt. 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 supply from these salts or a 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 0.001 to 5M, preferably 0.1 to 3M, and more preferably 1 to 2M. 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 reaction for producing succinic acid may be carried out with aeration and stirring, but may be carried out in an anaerobic atmosphere without aeration and without supplying oxygen. The anaerobic atmosphere referred to here means that the reaction is performed while the dissolved oxygen concentration in the solution is kept low. In this case, the dissolved oxygen concentration is desirably 0 to 2 ppm, preferably 0 to 1 ppm, more preferably 0 to 0.5 ppm. As a method therefor, for example, a method in which the container is sealed and reacted without aeration, an inert gas such as nitrogen gas is supplied and reacted, a carbon dioxide-containing inert gas is aerated, and the like can be used. .

By the microbial reaction as described above, non-amino organic acids such as succinic acid, fumaric acid, malic acid or pyruvic acid are generated and accumulated in the reaction solution. The non-amino 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, crystallization from the solution or purification by column chromatography, etc. Amino organic acids can be collected.

  Furthermore, in this invention, after manufacturing a non-amino organic acid by the method of this invention mentioned above, a non-amino organic acid containing polymer can be manufactured by performing a polymerization reaction using the obtained non-amino organic acid as a raw material. . In recent years, as the number of environmentally friendly industrial products has increased, attention has been paid to polymers using plant-derived raw materials. Non-amino organic acids produced in the present invention, particularly succinic acid, include polyesters and polyamides. It can be used after being processed into a polymer. The non-amino organic acid obtained by the production method of the present invention or the composition containing the non-amino 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.

<Construction of vector for gene disruption>
(A) Extraction of Bacillus subtilis genomic DNA In 10 mL of LB medium [composition: tryptone 10 g, yeast extract 5 g, NaCl 5 g dissolved in distilled water 1 L], Bacillus subtilis ISW1214 was cultured until the late logarithmic growth phase, The cells were collected. The obtained bacterial cells were suspended in 0.15 mL of a 10 mM NaCl-20 mM Tris buffer (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) Amplification and cloning of SacB gene by PCR Acquisition of the Bacillus subtilis SacB gene (Schafer, A. et al. Gene 145 (1994) 69-73) was already reported using the DNA prepared in (A) above as a template. PCR was performed using synthetic DNA (SEQ ID NO: 1 and SEQ ID NO: 2) designed based on the base sequence of the gene (GenBank Database Accession No. X02730).

  Composition of reaction solution: 1 μL of template DNA, 0.2 μL of Pfx DNA polymerase (manufactured by Invitrogen), 1 × concentration attached buffer, 0.3 μM each primer, 1 mM MGSO4, 0.25 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 94 ° C. for 20 seconds and 68 ° C. for 2 minutes was repeated 35 times. However, the heat retention at 94 ° C. in the first cycle was 1 minute 20 seconds, and the heat retention at 68 ° C. in the final cycle was 5 minutes.

  Confirmation of the amplified product was carried out by separating by 0.75% agarose (SeaKem GTG agarose: manufactured by FMC BioProducts) gel electrophoresis and then visualized by ethidium bromide staining, and a fragment of about 2 kb was detected. The DNA fragment of interest was recovered from the gel using a QIAQuick Gel Extraction Kit (manufactured by QIAGEN).

  The recovered DNA fragment was phosphorylated at the 5 ′ end with T4 polynucleotide kinase (T4 Polynucleotide Kinase: manufactured by Takara Bio Inc.), and then ligated kit ver. 2 (manufactured by Takara Bio Inc.) was used to bind to the EcoRV site of the Escherichia coli vector (pBluescript II: manufactured by STRATEGENE), and Escherichia coli (DH5α strain) was transformed with the resulting plasmid DNA. The recombinant Escherichia coli thus obtained was smeared on an LB agar medium [10 g tryptone, 5 g yeast extract, 5 g NaCl, and 15 g agar dissolved in 1 L distilled water] containing 50 μg / mL ampicillin and 50 μg / mL X-Gal. did.

  Clones that formed white colonies on this medium were then transferred to LB agar medium containing 50 μg / mL ampicillin and 10% sucrose and cultured at 37 ° C. for 24 hours. Among these clones, those that could not grow on a medium containing sucrose were subjected to liquid culture by a conventional method, and then plasmid DNA was purified. Strains in which the SacB gene is functionally expressed in E. coli should be unable to grow on sucrose-containing media. The obtained plasmid DNA was cleaved with restriction enzymes SalI and PstI, whereby an inserted fragment of about 2 kb was observed, and the plasmid was named pBS / SacB.

(C) Construction of chloramphenicol resistant SacB vector 500 ng of E. coli plasmid vector pHSG396 (Takara Bio Inc .: chloramphenicol resistant marker) was reacted with the restriction enzyme PshBI10units at 37 ° C. for 1 hour, followed by phenol / chloroform extraction and ethanol Collected by precipitation. After blunting both ends with Klenow fragment (manufactured by Takara Bio Inc.), ligation kit ver. 2 (manufactured by Takara Bio Inc.) was used to connect and circularize the MluI linker (Takara Bio Inc.) to transform Escherichia coli (DH5α strain). The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 34 μg / mL chloramphenicol. Plasmid DNA was prepared from the obtained clones by a conventional method, and a clone having a restriction enzyme MluI cleavage site was selected and named pHSG396Mlu.

  On the other hand, pBS / SacB constructed in (B) above was cleaved with restriction enzymes SalI and PstI, and then the ends were blunted with Klenow fragment. Ligation kit ver. After linking the MluI linker using 2 (manufactured by Takara Bio Inc.), a DNA fragment of about 2.0 kb containing the SacB gene was separated and recovered by 0.75% agarose gel electrophoresis. This SacB gene fragment was cleaved with the restriction enzyme MluI, then the pHSG396Mlu fragment dehydrated with alkaline phosphatase (Alkaline Phosphatase Calf intestine) and ligation kit ver. 2 (manufactured by Takara Bio Inc.) was used to transform E. coli (DH5α strain). The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 34 μg / mL chloramphenicol. The colonies thus obtained were then transferred to an LB agar medium containing 34 μg / mL chloramphenicol and 10% sucrose and cultured at 37 ° C. for 24 hours. Among these clones, plasmid DNA was purified by a conventional method for those that could not grow on a medium containing sucrose. As a result of analyzing the plasmid DNA thus obtained by MluI digestion, it was confirmed that it had an inserted fragment of about 2.0 kb, and this was named pCMB1.

(D) Acquisition of kanamycin resistance gene The kanamycin resistance gene was acquired using the DNA of E. coli plasmid vector pHSG299 (Takara Bio Inc .: Kanamycin resistance marker) as a template and the synthetic DNAs shown in SEQ ID NO: 3 and SEQ ID NO: 4 as primers. The PCR method was used.

Composition of reaction solution: 1 ng of template DNA, Pyrobest DNA polymerase (Takara Bio Inc.)
0.1 μL, 1 × concentration buffer, 0.5 μM each primer, and 0.25 μM dNTPs were mixed to make a total volume of 20 μL.

  Reaction temperature condition: DNA thermal cycler PTC-200 (manufactured by MJ Research) was used, and a cycle consisting of 94 ° C. for 20 seconds, 62 ° C. for 15 seconds, and 72 ° C. for 1 minute 20 seconds was repeated 20 times. However, the heat retention at 94 ° C. in the first cycle was 1 minute 20 seconds, and the heat retention at 72 ° C. in the final cycle was 5 minutes.

  Confirmation of the amplification product was carried out by separating by 0.75% agarose (SeaKem GTG agarose: manufactured by FMCBioProducts) gel electrophoresis and then visualized by ethidium bromide staining to detect a fragment of about 1.1 kb. The DNA fragment of interest was recovered from the gel using a QIAQuick Gel Extraction Kit (manufactured by QIAGEN). The recovered DNA fragment was phosphorylated at the 5 ′ end with T4 polynucleotide kinase (T4 Polynucleotide Kinase: manufactured by Takara Bio Inc.).

(E) Construction of kanamycin-resistant SacB vector The approximately 3.5 kb DNA fragment obtained by cleaving pCMB1 constructed in (C) above with restriction enzymes Van91I and ScaI was separated and recovered by 0.75% agarose gel electrophoresis. did. This was mixed with the kanamycin resistance gene prepared in (D) above, and ligation kit ver. 2 (manufactured by Takara Bio Inc.) and Escherichia coli (DH5α strain) was transformed with the resulting plasmid DNA. The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 50 μg / mL kanamycin.

  It was confirmed that the strain grown on this kanamycin-containing medium was unable to grow on the sucrose-containing medium. Further, the plasmid DNA prepared from the same strain produced fragments of 354, 473, 1807, and 1997 bp by digestion with the restriction enzyme HindIII. Therefore, it was determined that the structure shown in FIG. 1 was correct, and the plasmid was designated as pKMB1. Named.

<Preparation of LDH gene disruption strain>
(A) Extraction of Brevibacterium flavum MJ233-ES 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 0.5g, 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] in 10mL Brevibacterium flavum strain MJ-233 was cultured until the late logarithmic growth phase, and genomic DNA was prepared from the cells obtained by the method shown in (A) of Example 1 above.

(B) Cloning of lactate dehydrogenase gene The MJ233 strain lactate dehydrogenase gene was obtained by using the DNA prepared in (A) above as a template and a synthetic DNA designed on the basis of the base sequence of the gene described in JP-A-11-206385 ( It was performed by PCR using SEQ ID NO: 5 and SEQ ID NO: 6).
Composition of reaction solution: Template DNA 1 μL, Taq DNA polymerase (Takara Bio Inc.) 0.2 μL, 1 × concentration attached buffer, 0.2 μM each primer, 0.25 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 94 ° C. for 20 seconds, 55 ° C. for 20 seconds and 72 ° C. for 1 minute was repeated 30 times. However, the heat retention at 94 ° C. in the first cycle was 1 minute 20 seconds, and the heat retention at 72 ° C. in the final cycle was 5 minutes.

  Confirmation of the amplification product was carried out by separating by 0.75% agarose (SeaKem GTG agarose: manufactured by FMCBioProducts) gel electrophoresis and then visualized by ethidium bromide staining to detect a fragment of about 0.95 kb. The DNA fragment of interest was recovered from the gel using a QIAQuick Gel Extraction Kit (manufactured by QIAGEN).

  The recovered DNA fragment was mixed with a PCR product cloning vector pGEM-TEasy (Promega) and ligation kit ver. After ligation using 2 (manufactured by Takara Bio Inc.), Escherichia coli (DH5α strain) was transformed with the obtained plasmid DNA. The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 50 μg / mL ampicillin 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 the plasmid DNA was purified. By cutting the obtained plasmid DNA with restriction enzymes SacI and SphI, an insert fragment of about 1.0 kb was recognized, and this was named pGEMT / CgLDH.

(C) Construction of plasmid for disrupting lactate dehydrogenase gene The coding region of lactate dehydrogenase consisting of about 0.25 kb was excised by cutting the pGEMT / CgLDH prepared in (B) above with restriction enzymes EcoRV and XbaI. The ends of the remaining approximately 3.7 kb DNA fragment were blunted with Klenow fragment, and ligated kit ver. 2 (manufactured by Takara Bio Inc.) was used to transform into E. coli (DH5α strain). The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 50 μg / mL ampicillin. The strain grown on this medium was subjected to liquid culture by a conventional method, and then the plasmid DNA was purified. By cutting the obtained plasmid DNA with restriction enzymes SacI and SphI, a clone in which an insert fragment of about 0.75 kb was recognized was selected and named pGEMT / ΔLDH.

  Next, a DNA fragment of about 0.75 kb generated by cleaving the above pGEMT / ΔLDH with restriction enzymes SacI and SphI is separated and recovered by 0.75% agarose gel electrophoresis, and a lactate dehydrogenase gene fragment containing a defective region Was prepared. This DNA fragment was mixed with pKMB1 constructed in Example 1 cleaved with restriction enzymes SacI and SphI, and ligation kit ver. After ligation using 2 (manufactured by Takara Bio Inc.), Escherichia coli (DH5α strain) was transformed with the obtained plasmid DNA. The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 50 μg / mL kanamycin 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 the plasmid DNA was purified. The obtained plasmid DNA was cleaved with restriction enzymes SacI and SphI, and a plasmid in which an insert fragment of about 0.75 kb was recognized was selected and named pKMB1 / ΔLDH (FIG. 2).

(D) Preparation of lactate dehydrogenase gene disruption strain derived from Brevibacterium flavum MJ-233 strain Plasmid DNA used for transformation of Brevibacterium flavum MJ-233 strain was obtained by the calcium chloride method (Journal of using pKMB1 / ΔLDH). Molec
(Early Biology, 53, 159, 1970).

Brevibacterium flavum strain MJ-233 (FERM BP-1497) was prepared by a conventional method (Wolf H et al., J. Bacteriol. 1983, Vol. 156 (3), p1165-1170, Kurusu Y et al., Agric Biol.
Chem. 1990, vol. 54 (2), p443-447), the endogenous plasmid was removed (curing), and the resulting plasmid curing strain, ie, Brevibacterium flavum MJ233-ES Used for conversion. The Brevibacterium flavum MJ233-ES strain was transformed by the electric pulse method (Res. Microbiol., Vol. 144, p.181-185, 1993), and the obtained transformant was treated with kanamycin 50 μg / mL. LBG agar medium containing [tryptone 10 g, yeast extract 5 g, NaCl 5 g, glucose 20 g, and agar 15 g dissolved in 1 L of distilled water] was smeared.

  Since the strain grown on this medium is a plasmid in which pKMB1 / ΔLDH cannot be replicated in Brevibacterium flavum MJ233-ES, the lactate dehydrogenase gene of the plasmid and Brevibacterium flavum MJ-233 strain As a result of homologous recombination with the same gene on the genome, 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 kanamycin 50 μg / mL. A portion equivalent to about 1 million cells of this culture was smeared on a 10% sucrose-containing LBG medium. As a result, about 10 strains that were considered to be sucrose-insensitive due to elimination of the SacB gene by the second homologous recombination were obtained.

  Among the strains thus obtained, those in which the lactate dehydrogenase gene is replaced with a mutant derived from pKMB1 / ΔLDH and those in which the wild type is restored are included. Confirmation of whether the lactate dehydrogenase gene is a mutant type or a wild type is easily confirmed by subjecting the cells obtained by liquid culture in LBG medium to direct PCR reaction and detecting the lactate dehydrogenase gene. it can. When the lactate dehydrogenase gene is analyzed using primers (SEQ ID NO: 7 and SEQ ID NO: 8) for PCR amplification, a wild-type DNA fragment of 720 bp and a mutant type having a deletion region should have a DNA fragment of 471 bp.

  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 / ΔLDH.

(E) Confirmation of lactate dehydrogenase activity The Brevibacterium flavum MJ233 / ΔLDH strain prepared in (D) above was inoculated in medium A and cultured with shaking aerobically at 30 ° C. for 15 hours. The obtained culture is centrifuged (3,000 × g, 4 ° C., 20 minutes) to recover the bacterial cells, and then sodium-phosphate buffer [composition: 50 mM sodium phosphate buffer (pH 7.3)]. Washed with.

  Next, 0.5 g (wet weight) of washed cells was suspended in 2 mL of the sodium-phosphate buffer, and the cells were crushed by applying an ultrasonic crusher (manufactured by Branson) under ice cooling. The crushed material was centrifuged (10,000 × g, 4 ° C., 30 minutes), and the supernatant was obtained as a crude enzyme solution. As a control, a crude enzyme solution of Brevibacterium flavum MJ233-ES strain was similarly prepared and subjected to the following activity measurement.

For the confirmation of the lactate dehydrogenase enzyme activity, the coenzyme NADH was oxidized to NAD ⁺ with the production of lactic acid using pyruvic acid as a substrate for both crude enzyme solutions as a change in absorbance at 340 nm [L. Kanarek and R.L.Hill, J. Biol. Chem. 239, 4202 (1964
)]. The reaction was performed at 37 ° C. in the presence of 50 mM potassium-phosphate buffer (pH 7.2), 10 mM pyruvic acid, 0.4 mM NADH. As a result, the lactate dehydrogenase activity in the crude enzyme solution prepared from Brevibacterium flavum MJ233 / ΔLDH was 10 minutes compared to the lactate dehydrogenase activity in the crude enzyme solution prepared from Brevibacterium flavum MJ233-ES strain. 1 or less.

<Preparation of GOGAT gene disruption strain>
(A) Cloning of GOGAT gene MGO233 strain GOGAT gene was obtained by using the DNA prepared in Example 2 (A) as a template and the sequence of the gene of Corynebacterium glutamicum ATCC13032 strain for which the entire genome sequence has been reported ( This was performed by PCR using synthetic DNA (SEQ ID NO: 9 and SEQ ID NO: 10) designed based on GenBank Database Accession No. AP005276).

  Composition of reaction solution: 1 μL of template DNA, 0.2 μL of Pfx DNA polymerase (manufactured by Invitrogen), 1 × concentration attached buffer, 0.3 μM each primer, 1 mM MGSO4, 0.25 μM dNTPs were mixed to make a total volume of 20 μL.

  Reaction temperature condition: DNA thermal cycler PTC-200 (manufactured by MJ Research) was used, and a cycle consisting of 94 ° C. for 20 seconds, 60 ° C. for 20 seconds and 72 ° C. for 2 minutes was repeated 35 times. However, the heat retention at 94 ° C. in the first cycle was 1 minute 20 seconds, and the heat retention at 72 ° C. in the final cycle was 5 minutes.

  Confirmation of the amplification product was carried out by separating by 0.75% agarose (SeaKem GTG agarose: manufactured by FMCBioProducts) gel electrophoresis and then visualized by ethidium bromide staining, and a fragment of about 1.7 kb was detected. The DNA fragment of interest was recovered from the gel using a QIAQuick Gel Extraction Kit (manufactured by QIAGEN).

  The recovered DNA fragment was phosphorylated at the 5 ′ end with T4 polynucleotide kinase (T4 Polynucleotide Kinase: manufactured by Takara Bio Inc.), and then ligated kit ver. 2 (manufactured by Takara Bio Inc.) was used to bind to the HincII site of E. coli vector pHSG396 (manufactured by Takara Bio Inc.), and Escherichia coli (DH5α strain) was transformed with the resulting plasmid DNA. The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 34 μg / mL chloramphenicol 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 the plasmid DNA was purified. By cutting the obtained plasmid DNA with restriction enzymes XhoI and BamHI, an insert fragment of about 1.7 kb was observed, which was named pGOGAT / 396.

(B) Construction of GOGAT gene disruption plasmid The pGOGAT / 396 prepared in (A) above was cleaved with the restriction enzyme PstI to excise the coding region of the 0.43 kb GOGAT gene. The remaining DNA fragment of about 3.5 kb was blunted with Klenow fragment, and ligated kit ver. 2 (manufactured by Takara Bio Inc.) was used to transform into E. coli (DH5α strain). The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 50 μg / mL ampicillin. The strain grown on this medium was subjected to liquid culture by a conventional method, and then the plasmid DNA was purified. By cutting the obtained plasmid DNA with restriction enzymes XhoI and BamHI, a clone in which an insert fragment of about 1.2 kb was recognized was selected and named pGOGAT / 396-pst.

  Next, a DNA fragment of about 1.2 kb generated by cleaving the above pGOGAT / 396-pst with restriction enzymes XhoI and BamHI was separated and recovered by 0.75% agarose gel electrophoresis, and the GOGAT gene containing the defective region Fragments were prepared. This DNA fragment was mixed with pKMB1 constructed in Example 1 cleaved with restriction enzymes XhoI and BamHI, and ligation kit ver. After ligation using 2 (manufactured by Takara Bio Inc.), Escherichia coli (DH5α strain) was transformed with the obtained plasmid DNA. The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 50 μg / mL kanamycin 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 the plasmid DNA was purified. The obtained plasmid DNA was cleaved with restriction enzymes XhoI and BamHI, so that an insert fragment of about 1.2 kb was selected and named pKMB1 / ΔGOGAT (FIG. 3).

(C) Preparation of a GOGAT gene disruption strain derived from Brevibacterium flavum MJ233-ES strain Plasmid DNA used for transformation of Brevibacterium flavum MJ-233 strain was obtained by the calcium chloride method (Journal of Molecular using pKMB1 / ΔGOGAT). Biology, 53, 159, 1970) and prepared from E. coli strain JM110.

  The test strain for GOGAT gene disruption was the Brevibacterium flavum MJ233 / ΔLDH strain prepared in Example 2. The selection method using transformation and the SacB gene was performed according to the method described in Example 2.

Among the second homologous recombination strains thus obtained, those in which the GOGAT gene has been replaced with a mutant type derived from pKMB1 / ΔGOGAT and those in which the wild type has been restored are included. Whether the GOGAT gene is a mutant type or a wild type can be confirmed by analyzing the GOGAT gene using primers (SEQ ID NO: 9 and SEQ ID NO: 10) for PCR amplification. In the mutant type possessed, a DNA fragment of 1229 bp should be recognized.
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 / ΔGOGATΔLDH strain.

(D) Measurement of GOGAT activity The GOGAT gene-disrupted strain Brevibacterium flavum MJ233 / ΔGOGATΔLDH obtained in (C) above was cultured overnight in 100 mL of A medium containing 2% glucose. The obtained cells were collected, washed with 50 mL of 50 mM potassium phosphate buffer (pH 7.5), and resuspended in 20 mL of the same composition. The suspension was crushed with SONIFIER 350 (manufactured by BRANSON), and the centrifuged supernatant was used as a cell-free extract. GOGAT activity was measured using the obtained cell-free extract. The enzyme activity was measured in a reaction solution containing 100 mM Tris / HCl buffer (pH 7.5), 1 mM dithiothreitol, 5 mM sodium 2-oxoglutarate, 5 mM L-glutamine, 0.32 mM NADH, and a cell-free extract. The reaction was carried out at 0 ° C. 1 U was defined as the amount of enzyme that catalyzes the decrease of 1 μmol of NADH per minute. As a result, the GOGAT activity in the GOGAT gene-disrupted strain was below the detection limit of 0.001 U / mg protein. The specific activity of GOGAT activity in a cell body obtained by similarly culturing the marker-free strain Brevibacterium flavum MJ233 / ΔLDH strain in which the GOGAT gene was not disrupted using the A medium was 0.017 U / mg protein.

<Production of glutamate dehydrogenase gene disruption strain>
(A) Cloning of glutamate dehydrogenase gene The MJ233 strain glutamate dehydrogenase gene was obtained by using the DNA prepared in Example 2 (A) as a template and the gene of Corynebacterium glutamicum ATCC13032 strain for which the entire genome sequence was reported. PCR was performed using synthetic DNA (SEQ ID NO: 11 and SEQ ID NO: 12) designed based on the sequence (GenBank Database Accession No. AP005276).

  Composition of reaction solution: 1 μL of template DNA, 0.2 μL of Pfx DNA polymerase (manufactured by Invitrogen), 1 × concentration attached buffer, 0.3 μM each primer, 1 mM MGSO4, 0.25 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 94 ° C. for 20 seconds, 60 ° C. for 20 seconds, and 72 ° C. for 1 minute and 20 seconds was repeated 35 times. However, the heat retention at 94 ° C. in the first cycle was 1 minute 20 seconds, and the heat retention at 72 ° C. in the final cycle was 5 minutes.

  The amplification product was confirmed by separation by 0.75% agarose (SeaKem GTG agarose: manufactured by FMC BioProducts) gel electrophoresis and visualization by ethidium bromide staining, and a fragment of about 1.2 kb was detected. The DNA fragment of interest was recovered from the gel using a QIAQuick Gel Extraction Kit (manufactured by QIAGEN).

  The recovered DNA fragment was phosphorylated at the 5 ′ end with T4 polynucleotide kinase (T4 Polynucleotide Kinase: manufactured by Takara Bio Inc.), and then ligated kit ver. 2 (manufactured by Takara Bio Inc.) was used to bind to the HincII site of E. coli vector pHSG396 (manufactured by Takara Bio Inc.), and Escherichia coli (DH5α strain) was transformed with the resulting plasmid DNA. The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 34 μg / mL chloramphenicol 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 the plasmid DNA was purified. The obtained plasmid DNA was cleaved with restriction enzymes XhoI and EcoRI, and an inserted fragment of about 1.2 kb was recognized, which was named pGDH / 396.

(B) Acquisition of chloramphenicol resistance gene The acquisition of the chloramphenicol resistance gene was performed using the DNA of E. coli plasmid vector pHSG396 (manufactured by Takara Bio Inc.) as a template and the synthetic DNAs shown in SEQ ID NO: 13 and SEQ ID NO: 14. The PCR method was used as a primer.

Composition of reaction solution: 1 ng of template DNA, Pyrobest DNA polymerase (Takara Bio Inc.)
0.1 μL, 1 × concentration buffer, 0.5 μM each primer, and 0.25 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 94 ° C. for 20 seconds, 62 ° C. for 15 seconds and 72 ° C. for 1 minute was repeated 20 times. However, the heat retention at 94 ° C. in the first cycle was 1 minute 20 seconds, and the heat retention at 72 ° C. in the final cycle was 5 minutes.

Confirmation of the amplified product was carried out by separating by 0.75% agarose (SeaKem GTG agarose: manufactured by FMC BioProducts) gel electrophoresis and then visualized by ethidium bromide staining to detect a fragment of about 0.9 kb. Recovery of the target DNA fragment from the gel is performed using the QIAQuick Gel Extraction Kit (manufactured by QIAGEN)
It was performed using. The recovered chloramphenicol resistance gene fragment was phosphorylated at the 5 ′ end with T4 polynucleotide kinase (T4 Polynucleotide Kinase: manufactured by Takara Bio Inc.).

(C) Construction of a plasmid for disrupting glutamate dehydrogenase gene The pGDH / 396 constructed in (A) above was cleaved with restriction enzymes EcoRI and PstI, and an about 0.9 kb glutamate dehydrogenase gene fragment was extracted with pHSG299 (Takara Bio Inc.). Manufactured by the ligation kit ver. 2 (manufactured by Takara Bio Inc.), and Escherichia coli (DH5α strain) was transformed with the obtained plasmid DNA. The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 34 μg / mL chloramphenicol 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 the plasmid DNA was purified. By cutting the obtained plasmid DNA with restriction enzymes PstI and EcoRI, an inserted fragment of about 0.9 kb was observed, and this was named pGDH / 299.

  Next, the DNA fragment linearized by cleaving pGDH / 299 with the restriction enzyme EcoRV and the chloramphenicol resistance gene fragment prepared in (B) above were ligated with the ligation kit ver. 2 (manufactured by Takara Bio Inc.) was used to transform E. coli (DH5α strain). The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 34 μg / mL chloramphenicol.

  The strain grown on this medium was subjected to liquid culture by a conventional method, and then the plasmid DNA was purified. By cutting the obtained plasmid DNA with restriction enzymes PstI and EcoRI, a clone in which an insert fragment of about 1.8 kb was recognized was selected and named pGDH81 (FIG. 4).

(D) Production of Glutamate Dehydrogenase Gene Disrupted Strain from Brevibacterium flavum MJ233-ES Strain Plasmid DNA used for transformation of Brevibacterium flavum MJ233 strain was obtained by using the calcium chloride method (Journal of Molecular Biology, 53 , 159, 1970).

  The test strain for disrupting the glutamate dehydrogenase gene was the Brevibacterium flavum MJ233 / ΔGOGATΔLDH strain prepared in Example 4. The transformation method was performed according to the method described in Example 2 to obtain a strain that grew on an LBG agar medium containing 50 μg / mL kanamycin. The strain thus obtained was considered to have once caused homologous recombination between the glutamate dehydrogenase gene on the genome of Brevibacterium flavum MJ233 / ΔGOGATΔLDH and the gene of pGDH81.

  Next, the above strain was cultured overnight in an LBG liquid medium containing only 5 μg / mL chloramphenicol, and then smeared on an LBG agar medium containing only 5 μg / mL chloramphenicol. About 2000 colonies obtained were replicated on an LBG agar medium containing 25 μg / mL kanamycin according to a conventional method, and cultured at 30 ° C. overnight. As a result, strains sensitive to kanamycin were selected from chloramphenicol resistance.

As a result of the second homologous recombination, the strain obtained in this way left a mutant gene in which the chloramphenicol resistance gene was inserted into the glutamate dehydrogenase gene, and the kanamycin resistance gene and the sequence derived from pHSG299. It is thought to have been deleted.

  Direct confirmation of whether the glutamate dehydrogenase gene is mutant or wild type can be performed by analyzing the glutamate dehydrogenase gene using primers (SEQ ID NO: 12 and SEQ ID NO: 15) for PCR amplification. From the wild type, a PCR amplification product of 402 bp should be obtained, and from the mutant type having a chloramphenicol resistance gene insertion region, a 1292 bp PCR amplification product should be obtained.

  As a result of analyzing the strain that showed resistance to chloramphenicol and became insensitive to kanamycin by the above method, a strain having only the mutant gene was selected, and the strain was named Brevibacterium flavum MJ233 / ΔGDHΔGOGATΔLDH. .

(E) Measurement of glutamate dehydrogenase (GDH) activity GDH gene-disrupted strain Brevibacterium flavum MJ233 / ΔGDHΔGOGATΔLDH obtained in (D) above was overnight in 100 mL of A medium containing 2% glucose and 10 μg / mL chloramphenicol. Culture was performed. The obtained cells were collected, washed with 50 mL of 50 mM potassium phosphate buffer (pH 7.5), and resuspended in 20 mL of the same composition. The suspension was crushed with SONIFIER 350 (manufactured by BRANSON), and the centrifuged supernatant was used as a cell-free extract. GOGAT activity was measured using the obtained cell-free extract. The enzyme activity was measured by reacting at 25 ° C. in a reaction solution containing 100 mM Tris / HCl buffer (pH 8.0), 20 mM ammonium chloride, 5 mM sodium 2-oxoglutarate, 0.32 mM NADH, and cell-free extract. went. 1 U was defined as the amount of enzyme that catalyzes the decrease of 1 μmol of NADH per minute. As a result, the specific activity of GDH in the GDH gene-disrupted strain Brevibacterium flavum MJ233 / ΔGDHΔGOGATΔLDH was 0.012 U / mg protein. The specific activity of GDH was 0.795 U / mg protein in cells cultured in the same manner using the parent strain Brevibacterium flavum MJ233 / ΔGOGATΔLDH, which was not gene-disrupted, using A medium.

<Construction of Coryneform Bacterial Expression Vector>
(A) Preparation of Promoter Fragment for Coryneform Bacteria Utilizing the DNA fragment (hereinafter referred to as TZ4 promoter) described in SEQ ID NO: 4 of JP-A-7-95891 reported to have a strong promoter activity in coryneform bacteria It was decided. The promoter fragment was obtained by using a synthetic DNA designed based on the sequence described in SEQ ID NO: 4 of JP-A-7-95891 using Brevibacterium flavum MJ233 genomic DNA prepared in Example 2 (A) as a template. It was carried out by PCR using SEQ ID NO: 16 and SEQ ID NO: 17).

  Composition of reaction solution: 1 μL of template DNA, 0.2 μL of Pfx DNA polymerase (manufactured by Invitrogen), 1 × concentration attached buffer, 0.3 μM each primer, 1 mM MGSO4, 0.25 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 94 ° C. for 20 seconds, 60 ° C. for 20 seconds, and 72 ° C. for 30 seconds was repeated 35 times. However, the heat retention at 94 ° C. in the first cycle was 1 minute and 20 seconds, and the heat retention at 72 ° C. in the final cycle was 2 minutes.

The amplification product was confirmed by separation by 2.0% agarose (SeaKem GTG agarose: manufactured by FMC BioProducts) gel electrophoresis and visualization by ethidium bromide staining, and a fragment of about 0.25 kb was detected. The DNA fragment of interest was recovered from the gel using a QIAQuick Gel Extraction Kit (manufactured by QIAGEN).

  The recovered DNA fragment was phosphorylated at the 5 ′ end with T4 polynucleotide kinase (T4 Polynucleotide Kinase: manufactured by Takara Bio Inc.), and then ligated kit ver. 2 (manufactured by Takara Bio Inc.) was used to bind to the SmaI site of E. coli vector pUC19 (Takara Bio Inc.), and the resulting plasmid DNA was used to transform E. coli (DH5α strain). The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 50 μg / mL ampicillin and 50 μg / mL X-Gal.

  Six clones that formed white colonies on this medium were subjected to liquid culture by a conventional method, and then the plasmid DNA was purified and the nucleotide sequence was determined. Among them, a clone in which the TZ4 promoter was inserted so as to have a transcriptional activity in the reverse direction to the lac promoter of pUC19 was selected and named pUC / TZ4.

  Next, a DNA fragment prepared by cleaving pUC / TZ4 with restriction enzymes BamHI and PstI comprises synthetic DNA (SEQ ID NO: 18 and SEQ ID NO: 19) phosphorylated at the 5 ′ end, and BamHI and A DNA linker having a sticky end against PstI was mixed, and ligation kit ver. After ligation using 2 (manufactured by Takara Bio Inc.), Escherichia coli (DH5α strain) was transformed with the obtained plasmid DNA. This DNA linker contains a ribosome binding sequence (AGGAGG) and a cloning site (PacI, NotI, ApaI in this order from the upstream).

  Clones that formed white colonies on this medium were subjected to liquid culture by a conventional method, and then the plasmid DNA was purified. From the obtained plasmid DNA, one that was cleaved by the restriction enzyme NotI was selected and named pUC / TZ4-SD. The thus constructed pUC / TZ4-SD was digested with the restriction enzyme PstI, blunted with Klenow fragment, and then cleaved with the restriction enzyme KpnI to obtain a promoter fragment of about 0.3 kb. Separated and collected by 0% agarose gel electrophoresis.

(B) Assembly of Coryneform Bacterial Expression Vector pHSG298par-rep described in JP-A-12-93183 is used as a plasmid that can stably replicate independently in coryneform bacteria. This plasmid comprises a replication region of the natural plasmid pBY503 possessed by Brevibacterium stachynis IFO12144 strain and a region having a stabilizing function, a kanamycin resistance gene derived from the Escherichia coli vector pHSG298 (Takara Bio Inc.), and a replication region of Escherichia coli. .

  The DNA prepared by cleaving pHSG298par-rep with the restriction enzyme SseI, blunting the ends with Klenow fragment, and then cleaving with the restriction enzyme KpnI is mixed with the TZ4 promoter fragment prepared in (A) above and ligated. Kit ver. After ligation using 2 (manufactured by Takara Bio Inc.), Escherichia coli (DH5α strain) was transformed with the obtained plasmid DNA. The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 50 μg / mL kanamycin.

  The strain grown on this medium was subjected to liquid culture by a conventional method, and then the plasmid DNA was purified. From the obtained plasmid DNA, one that was cleaved by the restriction enzyme NotI was selected, and the plasmid was named pTZ4 (construction procedure is shown in FIG. 5).

<Preparation of a strain with enhanced pyruvate carboxylase activity>
(A) Acquisition of pyruvate carboxylase gene The acquisition of pyruvate carboxylase gene derived from Brevibacterium flavum MJ233 strain has been reported for the whole genome sequence using the DNA prepared in (A) of <Example 2> as a template. It was carried out by PCR using synthetic DNAs (SEQ ID NO: 20 and SEQ ID NO: 21) designed based on the gene sequence (GenBank Database Accession No. AP005276) of Corynebacterium glutamicum ATCC13032 strain.
Composition of reaction solution: 1 μL of template DNA, 0.2 μL of Pfx DNA polymerase (manufactured by Invitrogen), 1 × concentration attached buffer, 0.3 μM each primer, 1 mM MGSO4, 0.25 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 94 ° C. for 20 seconds and 68 ° C. for 4 minutes was repeated 35 times. However, the heat retention at 94 ° C. in the first cycle was 1 minute and 20 seconds, and the heat retention at 68 ° C. in the final cycle was 10 minutes. After completion of the PCR reaction, 0.1 μL of Takara Ex Taq (Takara Bio Inc.) was added, and the mixture was further incubated at 72 ° C. for 30 minutes.

  Confirmation of the amplification product was carried out by separating by 0.75% agarose (SeaKem GTG agarose: manufactured by FMC BioProducts) gel electrophoresis and then visualized by ethidium bromide staining, and a fragment of about 3.7 kb was detected. The target DNA fragment was recovered from the gel using QIAQuick Gel Extraction Kit (manufactured by QIAGEN).

  The recovered DNA fragment was mixed with a PCR product cloning vector pGEM-TEasy (Promega) and ligation kit ver. After ligation using 2 (manufactured by Takara Bio Inc.), Escherichia coli (DH5α strain) was transformed with the obtained plasmid DNA. The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 50 μg / mL ampicillin 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 the plasmid DNA was purified. The obtained plasmid DNA was cleaved with restriction enzymes PacI and ApaI, whereby an inserted fragment of about 3.7 kb was observed, which was designated as pGEM / MJPC.

  The base sequence of the insert fragment of pGEM / MJPC was determined using a base sequencing device (Model 377XL) manufactured by Applied Biosystems and a Big Dye Terminator cycle sequence kit ver3. The resulting DNA base sequence and deduced amino acid sequence are set forth in SEQ ID NO: 22. Since this amino acid sequence shows extremely high homology (99.4%) with that derived from Corynebacterium glutamicum ATCC13032, the insert fragment of pGEM / MJPC is a pyruvate carboxylase gene derived from Brevibacterium flavum MJ233. I determined that there was.

(B) Construction of Pyruvate Carboxylase Activity Enhancement Plasmid A pyruvate carboxylase gene fragment consisting of about 3.7 kb generated by cleaving the pGEM / MJPC prepared in (A) above with restriction enzymes PacI and ApaI It was separated and collected by% agarose gel electrophoresis. This DNA fragment was mixed with pTZ4 constructed in <Example 3> cleaved with restriction enzymes PacI and ApaI, and ligation kit ver. After ligation using 2 (manufactured by Takara Bio Inc.), Escherichia coli (DH5α strain) was transformed with the obtained plasmid DNA. The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 50 μg / mL kanamycin.

The strain grown on this medium was subjected to liquid culture by a conventional method, and then the plasmid DNA was purified. The obtained plasmid DNA was cleaved with restriction enzymes PacI and ApaI, so that an insert fragment of about 3.7 kb was selected and named pMJPC1 (FIG. 6).

(C) Transformation into Brevibacterium flavum MJ233 / ΔGDHΔGOGATΔLDH and Brevibacterium flavum MJ233 / ΔLDH strains Plasmid DNA for transformation with pMJPC1 capable of replicating in Brevibacterium flavum MJ233 strain is (B ) And transformed from E. coli (DH5α strain).
Transformation into Brevibacterium flavum MJ233 / ΔGDHΔGOGATΔLDH strain and Brevibacterium flavum MJ233 / ΔLDH strain was performed by the electric pulse method (Res. Microbiol., Vol. 144, p.181-185, 1993). The transformant thus obtained was smeared on an LBG agar medium containing 50 μg / mL kanamycin [10 g tryptone, 5 g yeast extract, 5 g NaCl, 20 g glucose, and 15 g agar dissolved in 1 L distilled water].

  From the strain grown on this medium, liquid culture was performed by a conventional method, and then plasmid DNA was extracted and analyzed by restriction enzyme digestion. As a result, it was confirmed that the strain retained pMJPC1. The strains were designated as bacterial flavum MJ233 / PC / ΔGDHΔGOGATΔLDH strain and Brevibacterium flavum MJ233 / PC / ΔLDH strain.

(D) Pyruvate carboxylase enzyme activity The transformant Brevibacterium flavum MJ233 / PC / ΔGDHΔGOGATΔLDH strain obtained in (C) above was cultured overnight in 100 mL of A medium containing 2% glucose and 25 mg / L kanamycin. . The obtained cells were collected, washed with 50 mL of 50 mM potassium phosphate buffer (pH 7.5), and resuspended in 20 mL of the same composition. The suspension was crushed with SONIFIER 350 (manufactured by BRANSON), and the centrifuged supernatant was used as a cell-free extract. The pyruvate carboxylase activity was measured using the obtained cell-free extract. Enzyme activity was measured at 100 mM Tris / HCl buffer (pH 7.5), 0.1 mg / 10 mL biotin, 5 mM magnesium chloride, 50 mM sodium bicarbonate, 5 mM sodium pyruvate, 5 mM adenosine triphosphate, 0.32 mM NADH. The reaction was performed at 25 ° C. in a reaction solution containing 20 units / 1.5 mL malate dehydrogenase (manufactured by WAKO, derived from yeast) and the enzyme. 1 U was defined as the amount of enzyme that catalyzes the decrease of 1 μmol of NADH per minute. The specific activity of the cell-free extract of Brevibacterium flavum MJ233 / PC / ΔGDHΔGOGATΔLDH strain expressing pyruvate carboxylase was 0.2 U / mg protein. In addition, in the cells cultured in the same manner using the parent strain Brevibacterium flavum MJ233 / ΔGDHΔGOGATΔLDH using A medium, the pyruvate carboxylase activity was below the detection limit of 0.001 U / mg protein. .

<Bacteria reaction (neutralization of ammonium carbonate, anaerobic reaction)>
Brevibacterium flavum MJ233 / PC / ΔGDHΔGOGATΔLDH strain is cultured in the same manner as in Example 7 above, a reaction suspension is prepared, the pH is maintained at 7.6 using 2M ammonium carbonate, and aeration is performed. The reaction was carried out with stirring at 200 rpm. The sugar consumption rate at this time was 2.0 g / L / h, the succinic acid production rate was 1.2 g / L / h, and the yield was 79%. The amino acid production was glutamic acid 0 g / L, alanine 1.3 g / L, valine 0.73 g / L, and leucine 0 g / L.

On the other hand, when the Brevibacterium flavum MJ233 / PC / ΔLDH strain prepared in Example 6 was reacted in the same manner as described above, the sugar consumption rate was 1.8 g / L / h, and the succinic acid production rate was 0. .93 g / L / h, yield was 73%. The amino acid production was 0 g / L glutamic acid, 2.0 g / L alanine, 1.2 g / L valine, and 0.14 g / L leucine.

  From these results, by using coryneform bacteria modified to reduce glutamate synthase (GOGAT) activity and glutamate dehydrogenase (GDH) activity, by-products of amino acids such as alanine, valine, and leucine are reduced. It was confirmed that the production rate and yield of succinic acid can be produced efficiently.

  According to the production method of the present invention, a non-amino organic acid can be produced quickly and with high efficiency. The obtained non-amino organic acid can be used for food additives, pharmaceuticals, cosmetics and the like. Moreover, a non-amino organic acid containing polymer can also be manufactured by performing a polymerization reaction using the obtained non-amino organic acid as a raw material.

The figure which shows the construction procedure and restriction enzyme map of plasmid pKMB1. The figure which shows the construction | assembly procedure of plasmid pKMB1 / (DELTA) LDH. The figure which shows the construction | assembly procedure of plasmid pKMB1 / (DELTA) GOGAT. The figure which shows the construction procedure of plasmid pGDH81. The figure which shows the construction procedure of plasmid pTZ4. The figure which shows the construction procedure of plasmid pMJPC1.

Claims (4)

Glutamate synthase gene, glutamate dehydrogenase gene, and E. coli lactate dehydrogenase gene is disrupted Corynebacterium glutamicum (Corynebacterium glutamicum) or Brevibacterium flavum (Brevibacterium flavum) or processed product thereof, a carbonate ion, bicarbonate ion or carbon dioxide gas by acting under anaerobic atmosphere on an organic raw material in a reaction solution containing, to produce and accumulate succinic acid in the reaction solution, the production of succinic acid and collecting succinic acid from the reaction solution Method. The Corynebacterium glutamicum or Brevibacterium flavum further, pyruvate carboxylase activity was modified to enhance, the production method according to claim 1. The organic raw material is glucose or sucrose, a manufacturing method according to claim 1 or 2. The manufacturing method of a succinic-acid containing polymer including the process of manufacturing a succinic acid by the method as described in any one of Claims 1-3 , and the process of performing a polymerization reaction using the succinic acid obtained at the said process as a raw material.

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