JP2008067623A - Method for producing non-amino organic acids - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing non-amino organic acids, with which non-amino organic acids, such as succinic acid, can be produced efficiently. <P>SOLUTION: This method for producing the non-amino organic acids comprises treating an organic raw material in a reaction solution containing carbonate ions, bicarbonate ions or carbon dioxide gas with a bacterium whose glutamate dehydrogenase activity is reduced so as to be 30% or lower, as compared to that of a strain whose enzyme has not been modified and whose glutamate synthase activity and glutamine synthase activity have not been reduced to 30% or lower, in comparison with that of the non-modified stain of the enzyme, or its treated product. <P>COPYRIGHT: (C)2008,JPO&INPIT

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.

The glutamic acid biosynthesis system synthesizes glutamic acid using α-ketoglutaric acid, which is one of the precursors of succinic acid, as a substrate.
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).
A non-amino organic acid is produced using a bacterium or a treated product thereof modified so that the activity of either or both of GOGAT and GS and GDH activity are reduced to 30% or less compared to the unmodified strain, respectively. A method has been reported (Patent Document 6). In this document, research has been conducted on the recognition that the decrease in the activity of GOGAT and / or GS and GDH is important for improving the production of non-amino organic acids, and the decrease in the activity of GDH alone is the production of non-amino organic acids. It was not expected to be effective for improvement.
On the other hand, a strain lacking the GDH gene alone of coryneform bacteria (for example, see Non-patent Document 3) has been reported, but this strain does not show glutamic acid requirement, and is much larger in glutamate production than non-deficient strains. There was no change. Furthermore, the succinic acid biosynthetic system is complex, and succinic acid is synthesized by using fumaric acid and isocitric acid as precursors in addition to α-ketoglutaric acid. The production of non-amino organic acids could not be expected to improve, and a GDH gene-deficient strain was never used for the production of non-amino organic acids.
US Pat. No. 5,143,834 US Pat. No. 5,504,004 JP-A-11-113588 JP 11-196888 A JP-A-11-206385 JP-A-2005-168401 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

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

  As a result of intensive studies to solve the above problems, the inventor has maintained a certain level of glutamate synthase activity and glutamine synthetase activity, and has been modified to reduce glutamate dehydrogenase (GDH) activity or It was found that the amount of non-amino organic acid produced was increased by allowing the treated product to act on the organic raw material in a reaction solution containing carbonate ion, bicarbonate ion or carbon dioxide gas, and the present invention was completed. It was.

That is, according to the present invention, the following inventions are provided.
(1) A bacterium modified so that glutamate dehydrogenase activity is reduced to 30% or less compared to an unmodified strain of the enzyme, wherein the glutamate synthase activity and the glutamine synthetase activity are compared with the unmodified strain of the enzyme. The non-amino organic acid is produced by allowing a bacterium that has not been reduced to 30% or less or a processed product thereof to act on an organic raw material in a reaction solution containing carbonate ion, bicarbonate ion, or carbon dioxide gas. A method for producing a non-amino organic acid, which comprises collecting an organic acid.
(2) The method for producing a non-amino organic acid according to (1), wherein the bacterium is further modified so that lactate dehydrogenase activity is reduced.
(3) The method for producing a non-amino organic acid according to (1) or (2), wherein the bacterium is further modified so that pyruvate carboxylase activity is enhanced.
(4) The bacterium is any bacterium selected from the group consisting of coryneform bacteria, Bacillus bacteria, Rhizobium bacteria, Escherichia bacteria, Lactobacillus bacteria, and Succinobacillus bacteria, (1) The manufacturing method of the non-amino organic acid in any one of-(3).
(5) The method for producing a non-amino organic acid according to any one of (1) to (4), wherein the organic raw material is allowed to act in an anaerobic atmosphere.
(6) The method for producing a non-amino organic acid according to any one of (1) to (5), wherein the organic raw material is glucose or sucrose.
(7) The method for producing a non-amino organic acid according to any one of (1) to (6), wherein the non-amino organic acid is succinic acid.
(8) A non-amino organic comprising a step of producing a non-amino organic acid by any one of the methods (1) to (7), 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 or a processed product thereof that has been modified to reduce glutamate dehydrogenase activity while retaining glutamate synthase activity and glutamine synthetase activity above a certain level, by-product amino acids are reduced, The amount of non-amino organic acid produced 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 are those that retain a certain level of activity of glutamate synthase (hereinafter also referred to as GOGAT) and glutamine synthetase (hereinafter also referred to as GS). A bacterium modified to reduce glutamate dehydrogenase (hereinafter also referred to as GDH) activity and having a non-amino organic acid-producing ability.
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. Non-amino organic acids mean organic acids other than amino acids, but succinic acid, malic acid, fumaric acid, citric acid, isocitric acid, 2-oxoglutaric acid, cis-aconitic acid and pyruvic acid are preferred, and succinic acid is more preferred. .
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 that has been modified to reduce GDH activity. It is also possible to have a non-amino organic acid-producing ability by performing a modification that reduces the above. Examples of means for imparting the ability to produce non-amino organic acid by breeding include mutation treatment and gene recombination treatment. More specifically, modification or pyruvate carboxylase that reduces lactate dehydrogenase activity as described later is used. Modifications that enhance the activity are included.

  “Retaining GOGAT and GS activity above a certain level” means that the activity of GOGAT and GS in the bacterium of the present invention does not decrease to 30% or less of the activity in non-modified strains of these enzyme genes. These enzyme activities may be modified so as to show an activity higher than 30% of the activity in the non-modified strain, but the gene encoding these enzymes is modified such as gene disruption or mutation introduction to reduce the activity. Bacteria that are not present, retain the wild-type gene, and have the same GOGAT and GS activity as the unmodified strain are preferred.

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 kind of the parent strain is not particularly limited, but Coryneform Bacterium, Bacillus, Rhizobium, Escherichia, Lactobacillus, or Sacinobacillus ( Succinobacillus) bacteria are preferred, among which coryneform bacteria are more preferred.
Examples of Escherichia bacteria include Escherichia coli, and examples of Lactobacillus bacteria include Lactobacillus helveticus (J Appl Microbiol, 2001, 91, p846-852, Bacillus bacteria include Bacillus subtilis, Examples include Bacillus amyloliquefaciens, Bacillus pumilus, Bacillus stearothermophilus, and examples of Rhizobium bacteria include Rhizobium etli.
The coryneform bacterium is not particularly limited as long as it is classified as such, and examples thereof include bacteria belonging to the genus Corynebacterium, bacteria belonging to the genus 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 GDH activity is reduced. As described above, for GOGAT and GS, these enzyme activities may be modified so as to show a value higher than 30% of the activity in the unmodified strain, but these enzyme activities are comparable to those in the unmodified strain. In order to keep it active, it is not necessary to perform a modification operation.

In the present invention, “GDH activity” refers to the activity of catalyzing the reaction of generating glutamic acid from α-ketoglutaric acid and ammonia, and “GOGAT activity” refers to the reaction of generating two molecules of glutamic acid from glutamine and α-ketoglutaric acid. “GS activity” refers to the activity of catalyzing the reaction of producing glutamine from glutamic acid and ammonia.
The activity of GDH can be measured by the method described in the examples below. Moreover, GOGAT activity and GS activity can be measured by the method described in Journal of Fermentation and Bioengineering vol 70, p182-184 (1990).

  “Reduced GDH activity” means that GDH activity is reduced as compared to an unmodified strain of the GDH gene. The GDH activity is preferably reduced to 30% or less of the unmodified strain, more preferably 10% or less. GDH activity may be reduced below the detection limit. 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.

  For the strain with reduced GDH activity, the parent strain is treated with a mutagen such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG) or nitrous acid, which is usually used for mutagenesis, and the activity of the enzyme Can be obtained by selecting strains with reduced In addition, the enzyme activity may be reduced by modifying the gene encoding GDH. Specifically, this can be achieved by destroying a gene encoding GDH on the chromosome or modifying an expression regulatory sequence such as a promoter or Shine-Dalgarno (SD) sequence.

Hereinafter, a method for disrupting the GDH gene in coryneform bacteria will be described. Examples of the GDH gene on the chromosome include DNA containing the base sequence shown in SEQ ID NO: 7. The GDH gene can be obtained by synthesizing a synthetic oligonucleotide based on the above sequence and performing a PCR reaction using a 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 GDH 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 homology to the extent that homologous recombination occurs with the GDH gene on the chromosomal DNA of the coryneform bacterium to be disrupted, the homologous gene having homology with SEQ ID NO: 7 Can also be used. Here, the homology that causes homologous recombination is preferably 70% or more, more preferably 80% or more, still 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. “Stringent conditions” refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. Although it is difficult to quantify this condition clearly, for example, it corresponds to 65 ° C., 1 × SSC, 0.1% SDS, preferably 0.1 × SSC, 0.1% SDS. A condition of washing once at a salt concentration, more preferably 2 to 3 times may be mentioned.

  Using a gene as described above, for example, deleting a partial sequence of the GDH gene, producing a modified GDH gene modified so as not to produce normally functioning GDH, and coryneform with the DNA containing the gene By transforming bacteria and causing recombination between the deletion gene and the gene on the chromosome, the GDH gene on the chromosome can be destroyed. 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 reduction of GDH activity can also be performed by known mutagenesis methods such as PCR and mutagenesis. 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.

  The bacterium used in the present invention may be a bacterium modified so as to reduce lactate dehydrogenase (hereinafter also referred to as LDH) activity in addition to the reduction of GDH activity. Such bacteria are particularly effective when the non-amino organic acid is succinic acid. Such a bacterium can be obtained, for example, by preparing a bacterium in which the LDH gene is disrupted and further disrupting the GDH gene of the bacterium by the method described above. However, the modification operation for reducing LDH activity and the modification operation for reducing GDH activity may be performed first.

“LDH activity is reduced” means that LDH activity is reduced as compared to an LDH gene-unmodified strain. The LDH activity is preferably reduced to 10% or less per cell as compared with the unmodified strain. Also, LDH activity may be completely lost. The reduction in LDH activity can be confirmed by measuring LDH activity by a known method (L. Kanarek and 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).

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 reduction of GDH activity. “PC activity is enhanced” means that the PC activity is preferably increased by 100% or more, more preferably by 300% or more with respect to a non-modified strain such as a wild strain or a parent strain. 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 having the GDH gene disrupted. It should be noted that either the PC gene introduction or the modification operation for reducing the GDH activity 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. As a specific PC gene gene, for example, a PC gene derived from Corynebacterium glutamicum (Peters-Wendisch, PG et al. Microbiology, vol. 144 (1998) p915-927 (SEQ ID NO: 9)) is used. Can do. As long as the PC gene does not substantially impair the PC activity, in the base sequence of SEQ ID NO: 9, some bases may be substituted with other bases, or may be deleted, or A base may be newly inserted, or a part of the base sequence may be rearranged, and any of these derivatives can be used in the present invention. Furthermore, the DNA hybridizes with the DNA having the base sequence of SEQ ID NO: 9 under stringent conditions, or the base sequence of SEQ ID NO: 9 has a homology of 90% or more, preferably 95% or more, more preferably 99% or more. A DNA encoding a protein having PC activity can also be suitably used.

Moreover, bacteria other than Corynebacterium glutamicum, or PC genes derived from other bacteria or animals and plants can also be used. In particular, the following bacterial or animal or plant-derived PC genes have known sequences (shown below), and amplify the ORF portion 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, tac promoter, trc promoter and the like 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 the host bacterium. 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 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 enhancement of PC activity can also be achieved by replacing the promoter region of the PC gene with a strong promoter on the chromosome.
In addition, DNA cleavage, ligation, and other methods such as chromosomal DNA preparation, PCR, plasmid DNA preparation, transformation, setting of oligonucleotides used as primers, etc. adopt ordinary methods well known to those skilled in the art. can do. These methods are described in Sambrook, J., Fritsch, EF, and Maniatis, T., “Molecular Cloning A Laboratory Manual, Second Edition”, Cold Spring Harbor Laboratory Press, (1989) and the like.

2. Method for Producing Non-Amino Organic Acid In the method for producing a non-amino organic acid of the present invention, the bacterium or 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, A non-amino organic acid production method characterized in that a non-amino organic acid is produced and collected. 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 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 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 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 reaction for producing a non-amino organic acid such as succinic acid may be carried out by aeration and stirring, but it 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. Obtainable.

  By the bacterial 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.

Further, in the present invention, after producing a non-amino organic acid such as succinic acid by the above-described method of the present invention, a non-amino organic acid-containing polymer is produced by performing a polymerization reaction using the obtained non-amino organic acid as a raw material. can do. 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 non-amino organic acid obtained by the production method of the present invention or the composition containing the non-amino organic acid can also 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 GDH-deficient strain>
(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 GDH disruption Obtaining a DNA fragment lacking the internal sequence of the GDH gene derived from Brevibacterium flavum strain MJ233 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, SEQ ID NO: 5) designed based on the sequence around the gene of the Corynebacterium glutamicum ATCC13032 strain (GenBank Database Accession No. BA00000036) And by crossover PCR using SEQ ID NO: 6). PCR was performed using the DNA fragments in the N-terminal region of the GDH gene as primers with SEQ ID NOs: 1 and 2, and the DNA fragments in the C-terminal region with the synthetic DNAs in SEQ ID NOs: 3 and 4, respectively. Composition of reaction solution: 1 μL of template DNA, 0.5 μL of Pfx DNA polymerase (manufactured by Invitrogen), 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 carried out using the obtained two amplification products as templates and the synthetic DNAs of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 as primers. 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 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 synthetic DNAs of SEQ ID NOs: 5 and 6 are complementary to each other and contain sequences complementary to both N-terminal and C-terminal fragments of the GDH gene. The obtained DNA fragment lacking the internal sequence of the GDH gene was purified using QIAquick PCR Purification Kit (manufactured by QIAGEN) and then cleaved with restriction enzymes SacI and XbaI. The resulting DNA fragment of about 1.3 kb was detected by 0.8% agarose (SeaKem GTG agarose: manufactured by FMCBioProducts) gel electrophoresis and then visualized by ethidium bromide staining, and QIAquick Gel Extraction Kit (QIAGEN From the gel. This DNA fragment was mixed with DNA prepared by cleaving pKMB1 (including sacB gene: JP-A-2005-95169) with restriction enzymes SacI and XbaI, and ligation kit ver. 2 (Takara Shuzo) were used for connection. 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. By cutting the obtained plasmid DNA with restriction enzymes SacI and XbaI, an insert fragment of about 1.3 kb was recognized, and this was named pGDH1 (FIG. 1).

(C) Preparation of GDH-disrupted strain Plasmid DNA used for transformation of Brevibacterium flavum MJ233 / ΔLDH strain (LDH gene-disrupted strain: Japanese Patent Laid-Open No. 2005-95169) is calcium chloride using the above-mentioned plasmid DNA of pGDH1. It was re-prepared from E. coli strain JM110 transformed by the method (Journal of Molecular Biology, 53, 159, 1970). The Brevibacterium flavum MJ233 / ΔLDH strain was transformed by the electric pulse method (Res. Microbiol., Vol. 144, p.181-185, 1993), and the obtained transformant was treated with 50 μg / mL of kanamycin. It smeared on the containing LBG (LB + glucose) agar medium. Since the strain grown on this medium is a plasmid in which pGDH1 cannot be replicated in Brevibacterium flavum MJ233 strain, the GDH gene of the plasmid 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 kanamycin 50 μg / mL. 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 strains obtained in this manner, those in which the GDH gene has a deletion of the internal sequence derived from pGDH1 and those in which the wild type has been restored are included. Confirmation of whether the GDH gene is a mutant type or a wild type can be easily confirmed by subjecting the cells obtained by liquid culture in an LBG medium to a direct PCR reaction and detecting the GDH gene. When analyzed using primers (SEQ ID NO: 1 and SEQ ID NO: 4) for PCR amplification of the GDH gene, a DNA fragment of 1341 bp in the mutant type and 2556 bp in the wild type should be observed. As a result of analyzing the strain that became insensitive to sucrose by the above method, a strain lacking the internal sequence of the GDH gene was selected, and the strain was named Brevibacterium flavum MJ233 / ΔGDH / ΔLDH.

(D) Measurement of GDH activity The GDH gene-deficient strain Brevibacterium flavum MJ233 / ΔGDH / ΔLDH 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. GDH 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 Brevibacterium flavum MJ233 / ΔGDH / ΔLDH was 0.006 U / mg protein. In addition, the specific activity of GDH in the cells cultured in the same manner as the parent strain Brevibacterium flavum MJ233 / PC / ΔLDH (Japanese Patent Laid-Open No. 2005-95169) that was not gene-disrupted was 0.082 U / mg protein.

(E) Production of PC-enhanced strain The PC-enhanced strain is produced by introducing pMJPC1 (plasma for PC enhancement: JP-A 2005-95169) into Brevibacterium flavum MJ233 / ΔGDH / ΔLDH produced in (C) above. Went by.
Transformation was performed by introducing pMJPC1 plasmid DNA prepared from Escherichia coli DH5α strain by the electric pulse method described in (C) above. The obtained transformant was smeared on an LBG agar medium containing 25 μg / mL of kanamycin, liquid culture was carried out from a strain grown on this medium by a conventional method, and plasmid DNA was extracted and analyzed by restriction enzyme digestion. As a result, it was confirmed that the strain had pMJPC1, and this was named Brevibacterium flavum MJ233 / PC / ΔGDH / ΔLDH.

<Evaluation of GDH-deficient 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 Brevibacterium flavum MJ233 / PC / ΔGDH / ΔLDH prepared in (E) of Example 1 was added. And cultured at 30 ° C. for 24 hours.
The obtained whole culture solution was collected by centrifugation at 10,000 g for 5 minutes, and the cell suspension medium (magnesium sulfate 7 hydrate: 1 g, ferrous sulfate 7 hydrate: 40 mg, manganese sulfate. 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 ) So that the absorbance of OD660 is 80. Add 0.5 ml of the above-mentioned cell suspension and 0.5 mL of the substrate solution (glucose: 200 g, magnesium carbonate: 194 g, and distilled water: 1000 mL) to a 4 ml reactor, under 20% carbon dioxide gas and 80% nitrogen atmosphere. , The mixture was centrifuged at 35 ° C. for 8 hours, centrifuged under the above conditions, and the organic acid concentration of the supernatant was analyzed. As a result, succinic acid 48.2 g / L, alanine 1.65 g / L, valine 0.85 g / L was accumulating.
On the other hand, as a control strain, Brevibacterium flavum MJ233 / PC / ΔLDH strain (Japanese Patent Laid-Open No. 2005-95169) was reacted in the same manner as above. As a result, succinic acid 43.5 g / L, alanine 3.40 g / L, valine 1.20 g / L was accumulated.
A clear improvement in succinic acid accumulation and an amino acid reduction effect were observed due to GDH deficiency.

The figure which shows the construction procedure of plasmid pGDH1. The underlined numbers indicate the primers of each SEQ ID NO.

Claims (8)

A bacterium modified so that glutamate dehydrogenase activity is reduced to 30% or less compared to an unmodified strain of the enzyme, wherein glutamate synthase activity and glutamine synthetase activity are 30% compared to an unmodified strain of the enzyme. A non-amino organic acid is produced by allowing a bacterium that has not been reduced below 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. A method for producing a non-amino organic acid, comprising collecting the non-amino organic acid. The method for producing a non-amino organic acid according to claim 1, wherein the bacterium is a bacterium further modified so that lactate dehydrogenase activity is reduced. The method for producing a non-amino organic acid according to claim 1 or 2, wherein the bacterium is a bacterium further modified so that pyruvate carboxylase activity is enhanced. The bacterium of any one of claims 1 to 3, wherein the bacterium is any one selected from the group consisting of coryneform bacteria, Bacillus bacteria, Rhizobium bacteria, Escherichia bacteria, Lactobacillus bacteria, and Succinobacillus bacteria. A method for producing a non-amino organic acid according to claim 1. The method for producing a non-amino organic acid according to any one of claims 1 to 4, wherein the organic raw material is allowed to act in an anaerobic atmosphere. The method for producing a non-amino organic acid according to any one of claims 1 to 5, wherein the organic raw material is glucose or sucrose. The manufacturing method of the non-amino organic acid as described in any one of Claims 1-6 whose non-amino organic acid is a succinic acid. A non-amino organic acid comprising a step of producing a non-amino organic acid by the method according to claim 1 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 a containing polymer.

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