JP2008067627A - Non-amino organic acid producing bacteria and method for producing non-amino organic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a bacterial strain producing non-amino organic acids in high efficiency and a method for efficiently producing a non-amino organic acid and its polymer by using the strain. <P>SOLUTION: A non-amino organic acid is produced by treating an organic raw material with a bacterial strain modified to increase the enzymatic activities of citrate synthase, lactate dehydrogenase and pyruvate carboxylase, especially a coryne-form bacteria or a bacterial strain belonging to the genus Bacillus or Rhizobium or a treated material of the bacteria in a reaction liquid containing carbonate ion, bicarbonate ion or carbon dioxide gas to afford the non-amino organic acid, and recovering the produced non-amino organic acid. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a novel non-amino organic acid-producing bacterium and production of a non-amino organic acid using the same.

  When producing non-amino organic acids such as succinic acid by fermentation, anaerobic bacteria such as Anaerobiospirillum genus and Actinobacillus genus are usually used (Patent Documents 1 and 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 (Patent Documents 3 and 4). In this case, it is economical because the amount of organic nitrogen added is small and sufficient growth can be achieved using a simple medium. However, the amount of non-amino organic acid produced, the concentration, and the amount per cell There was room for improvement, such as improvement in production speed and simplification of the manufacturing process. In addition, fermentation production of non-amino organic acid using a bacterium with enhanced activity of phosphoenolpyruvate carboxylase has been reported (see, for example, Patent Document 5), but a bacterium with enhanced activity of citrate synthase. The production of non-amino organic acids using has not been previously reported.
US Pat. No. 5,143,834 US Pat. No. 5,504,004 JP-A-11-113588 JP 11-196888 A Japanese Patent Laid-Open No. 11-196887 International Journal of Systematic Bacteriology (1999), 49,207-216

  The subject of this invention is providing the manufacturing method of non-amino organic acid with higher production efficiency.

  As a result of intensive studies to solve the above-mentioned problems, the present inventor used a bacterium or a treated product thereof modified so as to enhance the activity of citrate synthase as a carbonate ion, bicarbonate ion or carbon dioxide gas. It has been found that by acting on organic raw materials in the contained reaction liquid, the consumption rate of organic raw materials, the production rate or yield of non-amino organic acids such as succinic acid are increased, and by-products such as acetic acid are reduced. The present invention has been completed.

That is, according to the present invention, the following inventions are provided.
(1) A bacterium having a non-amino organic acid-producing ability and modified so that citrate synthase activity is enhanced as compared with a non-modified strain.
(2) The bacterium according to (1), which is further modified so that lactate dehydrogenase activity is reduced as compared with an unmodified strain.
(3) The bacterium according to (1) or (2), further modified so that pyruvate carboxylase activity is enhanced as compared with an unmodified strain.
(4) Furthermore, the activity of one or more enzymes selected from the group consisting of acetate kinase, phosphotransacetylase, pyruvate oxidase, and acetyl CoA hydrolase was modified so as to be reduced compared to the unmodified strain ( The bacterium according to any one of 1) to (3).
(5) The bacterium of any one of (1) to (4) selected from the group consisting of coryneform bacteria, Bacillus bacteria, and Rhizobium bacteria.
(6) The non-amino organic acid obtained by allowing the bacterium of any one of (1) to (5) or a processed product of the bacterium to act on an organic raw material in a reaction solution containing carbonate ion, bicarbonate ion, or carbon dioxide gas. And producing the non-amino organic acid, wherein the non-amino organic acid is recovered.
(7) The method according to (6), wherein the organic raw material is allowed to act in an anaerobic atmosphere.
(8) The method according to (6) or (7), wherein the organic raw material is glucose or sucrose.
(9) The method for producing an organic acid according to any one of (6) to (8), wherein the non-amino organic acid is succinic acid.
(10) A method for producing a non-amino organic acid-containing polymer, comprising a step of producing a non-amino organic acid by any one of the methods (6) to (9) and a step of polymerizing the obtained non-amino organic acid.

  According to the production method of the present invention, a non-amino organic acid such as succinic acid can be produced quickly and efficiently. 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.

Hereinafter, embodiments of the present invention will be described in detail.
The bacterium of the present invention is a bacterium having a non-amino organic acid-producing ability and modified so that the activity of citrate synthase is enhanced. Hereinafter, citrate synthase is sometimes referred to as CS.
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 particularly preferred. .
“CS activity is enhanced” means that CS activity is enhanced as compared to a non-modified strain such as a wild-type strain. The CS activity is preferably enhanced 1.5 times or more per unit cell weight, and more preferably 2 times or more, compared to the unmodified strain.
CS is an enzyme that catalyzes the reaction of converting oxaloacetic acid to citric acid, and CS activity can be measured by the method described in Methods Enzymol. 13, 3-11 (1969).

  The bacterium of the present invention 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 enhance CS activity. It is also possible to have a non-amino organic acid-producing ability by performing a modification that enhances. Examples of means for imparting non-amino organic acid-producing ability by breeding include mutation treatment and gene recombination treatment. For example, when imparting succinic acid-producing ability, lactate dehydrogenase activity as described below is reduced. And other modifications that enhance pyruvate carboxylase activity.

The bacterium used in the present invention can be obtained by using a bacterium as shown below as a parent strain and modifying the parent strain. The kind of the parent strain is not particularly limited, but is preferably a coryneform bacterium, a Bacillus bacterium, or a Rhizobium bacterium, and more preferably a coryneform bacterium.
Examples of Bacillus bacteria include Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus, Bacillus stearothermophilus, etc., and Rhizobium genus Examples of bacteria include Rhizobium etli.
Examples of coryneform bacteria include bacteria belonging to the genus Corynebacterium, bacteria belonging to the genus Brevibacterium, or bacteria 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 Bacteria belonging to

Particularly preferred specific examples of the parent strains of the above bacteria include Brevibacterium flavum MJ-233 (FERM BP-1497), MJ-233 AB-41 (FERM BP-1498), Brevibacterium ammoniagenes ATCC6872, Coryne And bacteria glutamicum ATCC31831 and Brevibacterium lactofermentum ATCC13869. 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, the Brevibacterium flavum MJ-233 strain and the mutant MJ-233 AB-41 strain are the Corynebacterium glutamicum MJ-233 strain, respectively. And the same strain as the MJ-233 AB-41 strain.
Brevibacterium flavum MJ-233 was established on April 28, 1975 by the Ministry of International Trade and Industry, Ministry of International Trade and Industry, 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.

  The bacterium used as a parent strain in the method of the present invention is not only a wild strain, but also a mutant obtained by normal mutation treatment such as UV irradiation or NTG treatment, genetic techniques such as cell fusion or gene recombination. Any strain such as a recombinant strain to be induced may be used. The host of the above-mentioned genetically modified strain may be the same genus species as that of the transgene or a different genus species as long as it is a transformable microorganism. It is preferable to use an aerial bacterium as a host.

Enhancement of CS activity can be performed by a genetic recombination method, for example, by increasing the copy number of a gene encoding CS, or by replacing the promoter of this gene.
When the cs gene is used to modify the CS activity so as to enhance, the gene that can be used is not particularly limited as long as it encodes a protein having CS activity. For example, Brevibacterium having the base sequence of SEQ ID NO: 15 -A gene derived from Flavam MJ-233 strain (Cgl0829: 8000038..879151 of BA000036) can be mentioned. In addition, as long as the cs gene encodes a protein having CS activity, DNA that hybridizes with a DNA having a complementary sequence of the base sequence under stringent conditions, or 80% or more with the base sequence, preferably It may be a homologue such as DNA having a homology of 90% or more, more preferably 95% or more, particularly preferably 99% or more. Here, the stringent conditions are 60 ° C., 1 × SSC, 0.1% S, which is a condition for washing of ordinary Southern hybridization.
Conditions for hybridizing at a salt concentration corresponding to DS, preferably 0.1 × SSC, 0.1% SDS can be mentioned.
The cs gene encodes a protein having 80% or more, preferably 90% or more, more preferably 95% or more, particularly preferably 99% or more homology with the amino acid sequence of SEQ ID NO: 16, and having CS activity. It may be DNA.
Furthermore, the cs gene may be DNA encoding a protein having a CS activity, having a sequence in which one or several amino acids are substituted, deleted, inserted or added in the amino acid sequence of SEQ ID NO: 16. . Here, one or several means preferably means 1 to 20, more preferably 1 to 10, particularly preferably 1 to 5.

  In addition, a coryneform bacterium other than Brevibacterium flavum MJ-233, or a cs gene derived from other microorganisms or animals and plants can also be used. For cs genes derived from microorganisms or animals and plants, the genes whose base sequences have already been determined, or genes encoding proteins with CS activity based on homology, etc., are isolated from the chromosomes of microorganisms, animals and plants, and the nucleotide sequences are determined. Can be used. In addition, after the base sequence is determined, a gene synthesized according to the sequence can be used. These can be obtained by amplifying a region containing the promoter and the ORF portion by a hybridization method or a PCR method.

In order to increase the activity of CS, for example, the above DNA may be incorporated into a plasmid capable of functioning in the host microorganism so that it can be expressed and introduced into the host microorganism.
The plasmid vector into which the cs gene can be incorporated 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 to introduce genes into coryneform bacteria include 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 publication; plasmids pCRY2 and pCRY3 described in JP-A-1-191686; PHM1519 described in JP-A-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 It can be mentioned pCG4 and pCG11 like described in JP 57-183799 JP.
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.

  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), etc.

A recombinant vector obtained by inserting the cs gene into an appropriate site of a plasmid vector capable of replicating in the coryneform bacterium as described above, such as a coryneform bacterium such as Brevibacterium flavum (MJ-233 strain). By transforming (FERM BP-1497), coryneform bacteria with increased cs gene expression can be obtained.
Transformation is performed by, for example, the electric pulse method (Res. Microbiol., Vol. 144, p.181-185, 1993).
Etc.
The CS activity can also be enhanced by making multiple copies of the cs gene on the chromosome by a known homologous recombination method.
The CS activity can also be enhanced by replacing or modifying the cs gene promoter on the host chromosome. Examples of the promoter replacement method include a method using the sacB gene (Schafer, A. et al. Gene 145 (1994) 69-73).

In the introduction by the above-described recombinant plasmid or homologous recombination on the chromosome, the promoter for expressing the cs gene or the promoter used for replacing the promoter of the cs gene on the chromosome is one that can function in the host bacteria. The promoter is not particularly limited, but a promoter whose transcriptional activity is not suppressed under anaerobic conditions is preferable, and examples thereof include a tac promoter used in E. coli and a trc promoter.
As described above, the expression level of the cs gene can be controlled by appropriately selecting a promoter.
As mentioned above, although the example using coryneform bacteria was described, CS activity enhancement can be achieved by the same method also when using other bacteria.

  The bacterium of the present invention may be a bacterium modified so that lactate dehydrogenase (also referred to as LDH) activity is reduced in addition to the enhancement of CS activity. Here, “LDH activity is reduced” means that LDH activity is reduced as compared to an unmodified strain. The LDH activity is preferably reduced to 10% or less per unit cell weight as compared to the unmodified strain. LDH activity may be completely absent. The decrease in LDH activity can be confirmed by measuring LDH activity by a known method (L. Kanarek and RL Hill, J. Biol. Chem. 239, 4202 (1964)). As a specific method for producing a strain having a reduced LDH activity of a coryneform bacterium, a method by homologous recombination to a chromosome described in JP-A-11-206385 or a method using a sacB gene (Schafer, A. et al. Gene 145 (1994) 69-73). Coryneform bacterium with reduced LDH activity and enhanced cs gene expression can be obtained, for example, by preparing a bacterium with a disrupted LDH gene and transforming the bacterium with a recombinant vector containing the cs gene. it can. However, either the modification operation for reducing LDH activity or the modification operation for enhancing CS activity may be performed first.

  Further, the bacterium used in the present invention may be a bacterium modified so that the activity of pyruvate carboxylase (hereinafter also referred to as PC) is enhanced in addition to the enhancement of CS activity. “The PC activity is enhanced” means that the PC activity is preferably increased by 1.5 times or more, more preferably by 3 times or more per unit cell weight relative to an unmodified strain such as a wild strain or a parent strain. Say. The enhanced PC activity can be confirmed by measuring the PC activity by a known method (Magasanik's method [J. Bacteriol., 158, 55-62, (1984)]). Such a bacterium can be obtained, for example, by introducing the pc gene into a coryneform bacterium with enhanced expression of the cs gene. Note that any of the modification operations for enhancing PC activity and CS activity may be performed first.

The introduction of the pc gene can be performed in the same manner as the introduction of the cs gene. Specifically, for example, it can be carried out by highly expressing the pc gene in coryneform bacteria in the same manner as described in JP-A-11-196888. As a specific pc gene, for example, a pc gene derived from Corynebacterium glutamicum (Peters-Wendisch, PG et al. Microbiology, vol. 144 (1998) p915-927) (SEQ ID NO: 17) can be used. it can. Further, the pc gene is DNA that hybridizes under stringent conditions with DNA having the nucleotide sequence of SEQ ID NO: 17, or 80% or more, preferably 90% or more, more preferably 95% or more with the nucleotide sequence of SEQ ID NO: 17. Particularly preferably, DNA having a homology of 99% or more and encoding a protein having PC activity can also be suitably used.
Furthermore, pc genes derived from coryneform bacteria other than Corynebacterium glutamicum, or from other microorganisms or animals and plants can also be used. In particular, the microorganism or animal or plant-derived pc gene shown below has a known sequence (shown below), and by amplifying the ORF portion by hybridization or PCR as described above, Can be acquired.
Human [Biochem.Biophys.Res.Comm., 202, 1009-1014, (1994)]
Mouse [Proc.Natl.Acad.Sci.USA., 90, 1766-1779, (1993)]
Rat [GENE, 165, 331-332, (1995)]
Yeast; Saccharomyces cerevisiae
[Mol.Gen.Genet., 229, 307-315, (1991)]
Schizosaccharomyces pombe
[DDBJ Accession No .; D78170]
Bacillus stearothermophilus
[GENE, 191, 47-50, (1997)]
Rhizobium etli
[J. Bacteriol., 178, 5960-5970, (1996)]

  Furthermore, in addition to the enhancement of CS activity, the bacterium of the present invention comprises acetate kinase (hereinafter also referred to as ACK), phosphotransacetylase (hereinafter also referred to as PTA), pyruvate oxidase (hereinafter also referred to as POXB) and It may be a bacterium modified so that the activity of one or more enzymes selected from the group consisting of acetyl CoA hydrolase (hereinafter also referred to as ACH) is reduced.

Either PTA or ACK may decrease the activity, but it is more preferable to decrease both activities in order to efficiently reduce the byproduct of acetic acid.
“PTA activity” refers to the activity of catalyzing the reaction of transferring phosphoric acid to acetyl-CoA to produce acetyl phosphate. “Modified to reduce PTA activity” means that the PTA activity is lower than that of an unmodified strain such as a wild strain. The PTA activity is preferably reduced to 30% or less per unit cell weight, more preferably 10% or less per unit cell weight, compared to the unmodified strain. Moreover, PTA activity may be completely lost. The decrease in PTA activity can be confirmed by measuring PTA activity by the method of Klotzsch et al. (Klotzsch, HR, Meth Enzymol. 12, 381-386 (1969)).

  “ACK activity” refers to the activity of catalyzing the reaction of producing acetic acid from acetyl phosphate and ADP. “Modified to reduce ACK activity” means that ACK activity is lower than that of an unmodified strain, for example, a wild strain. The ACK activity is preferably reduced to 30% or less per microbial cell, and more preferably 10% or less per microbial cell, compared to the unmodified strain. Further, the ACK activity may be completely lost. The decrease in ACK activity can be confirmed by measuring the ACK activity by the method of Ramponi et al. (Ramponi G., Meth. Enzymol. 42, 409-426 (1975)).

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

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

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

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

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

  The decrease in ACH activity can be achieved by disrupting the ach gene according to a known method, for example, a method using homologous recombination or a method using the sacB gene (Schafer, A. et al. Gene 145 (1994) 69-73). It can be carried out. Specifically, it can be carried out according to the methods disclosed in WO2005 / 113744 and examples described later. Examples of the ach gene include GenBank Accession No. A gene having the base sequence of Cgl2569 (the 2729376..2730917 complementary strand of GenBank Accession No. BA000036) is mentioned, but it has homology to the extent that homologous recombination occurs with the ach gene on the chromosomal DNA of the host bacterium. Therefore, a homologous gene of the sequence can also be used. Here, the homology that causes homologous recombination is preferably 80% or more, more preferably 90% or more, and particularly preferably 95% or more. In addition, homologous recombination can occur as long as it is a DNA that can hybridize with the above gene under stringent conditions.

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

  When using the bacterium in the production reaction of a non-amino organic acid such as succinic acid, a slant culture in a solid medium such as an agar medium may be used for the direct reaction. However, the bacterium is previously cultured in a liquid medium (seed It is preferable to use those cultured. The bacterium thus seed-cultured can also be produced by reacting with an organic raw material while growing on a medium containing the organic raw material. Moreover, it can manufacture also by making the microbial cell obtained by making it grow react with an organic raw material in the reaction liquid containing an organic raw material. In order to use an aerobic coryneform bacterium in the method of the present invention, it is preferable to first use the cells after culturing them under normal aerobic conditions. As a medium used for culture, a medium usually used for culture of 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 cultured cells are collected by centrifugation, membrane separation, etc. and used for the reaction.

  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 present microorganism to produce a non-amino organic acid, but is usually galactose, lactose, glucose, fructose, glycerol, sucrose. Carbohydrates such as saccharose, starch and cellulose; fermentable carbohydrates such as polyalcohols such as glycerin, mannitol, xylitol and ribitol are used, among which glucose, fructose, glycerol and sucrose are preferred, especially glucose and sucrose Is preferred.

  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 the range not inhibiting the production of non-amino organic acid, and is usually 5 to 30% (W / V), preferably 10 to 10%. The reaction is carried out within the range of 20% (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 microorganism to produce a non-amino organic acid. Specifically, ammonium salt, nitrate, urea, soybean hydrolysate, casein Various organic and inorganic nitrogen compounds such as degradation products, peptone, yeast extract, meat extract, corn steep liquor and the like can be mentioned. 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 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 within the above range depending on the alkaline substance, carbonate, urea, etc. as necessary even during the reaction. Adjust to.

As the reaction solution used in the present invention, water, a buffer solution, a medium, or the like is used, and a medium is most preferable. The medium can contain, for example, the above organic raw materials and carbonate ions, bicarbonate ions, or carbon dioxide gas, and can be reacted under anaerobic conditions. Carbonate ions or bicarbonate ions are supplied from magnesium carbonate, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, which can also be used as a neutralizing agent. It is also possible to supply from the salt 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 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.

  When culturing bacteria, it is necessary to supply oxygen by aeration and agitation. On the other hand, the production reaction of the non-amino organic acid may be performed by aeration and stirring, but may be performed 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.

  The non-amino organic acid accumulated in the reaction solution (culture solution) can be recovered (separated and purified) from the reaction solution according to a conventional method. Specifically, after removing solids such as bacterial cells by centrifugation, filtration, etc., desalting with an ion exchange resin or the like, and separating and purifying the non-amino organic acid from the solution by crystallization or column chromatography be able to.

  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 be used for food additives, pharmaceuticals, cosmetics and the like.

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

<Preparation of PoxB deficiency, ACH deficiency, PTA deficiency, ACK deficiency, PC enhanced 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 1
g, dissolved in 1 g of casamino acid, 20 g of glucose, and 1 L of distilled water] In 10 mL, Brevibacterium flavum MJ-233 strain was cultured until the late logarithmic growth phase, and the cells were collected by centrifugation (10000 g, 5 minutes). . The obtained cells were suspended in 0.15 mL of a 10 mM NaCl / 20 mM Tris buffer solution (pH 8.0) / 1 mM EDTA · 2Na solution containing lysozyme at a concentration of 10 mg / mL. Next, proteinase K was added to the suspension so that the final concentration was 100 μg / mL, and the mixture was incubated at 37 ° C. for 1 hour. Further, sodium dodecyl sulfate was added to a final concentration of 0.5%, and the mixture was incubated at 50 ° C. for 6 hours for lysis. To this lysate, add an equal amount of phenol / chloroform solution, shake gently at room temperature for 10 minutes, and then centrifuge (5,000 × g, 20 minutes, 10-12 ° C.) to obtain a supernatant. The fraction was collected and sodium acetate was added to a concentration of 0.3 M, and then twice as much ethanol was added and mixed. The precipitate collected by centrifugation (15,000 × g, 2 minutes) was washed with 70% ethanol and then air-dried. To the obtained DNA, 5 mL of a 10 mM Tris buffer (pH 7.5) -1 mM EDTA · 2Na solution was added and left overnight at 4 ° C., and used as template DNA for subsequent PCR.

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

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

(D) Construction of plasmid for destroying pta-ack The DNA fragment lacking the internal sequence of pta-ack gene derived from Brevibacterium flavum MJ233 strain was obtained using the DNA prepared in (A) above as a template and the entire genome. Synthetic DNA (SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12) designed based on the sequence around the gene of Corynebacterium glutamicum ATCC13032 strain (GenBank Database Accession No. BA00000036) whose sequence has been reported ) Was used for crossover PCR. PCR was carried out using the DNA fragments of the 5 ′ terminal region of the pta-ack gene as SEQ ID NO: 9 and SEQ ID NO: 10, and the DNA fragments of the 3 ′ terminal region of the synthetic DNAs of SEQ ID NO: 11 and SEQ ID NO: 12, respectively. Composition of reaction solution: 1 μL of template DNA, 0.5 μL of Pfx DNA polymerase (manufactured by Invitrogen), 1 × concentration buffer, 0.4 μM each primer, 1 mM MgSO ₄ , 0.
2 μM dNTPs were mixed to make a total volume of 50 μL. Reaction temperature condition: DNA thermal cycler PTC-200 (manufactured by MJ Research) was used, and a cycle consisting of 94 ° C. for 15 seconds, 55 ° C. for 30 seconds, and 68 ° C. for 45 seconds was repeated 30 times. However, the heat retention at 94 ° C. in the first cycle was 2 minutes, and the heat retention at 68 ° C. in the final cycle was 3 minutes. Next, PCR was performed using the obtained two amplification products as templates and the synthetic DNAs of SEQ ID NO: 9 and SEQ ID NO: 12 as primers. Composition of reaction solution: Template DNA 1 μL, PfxDNA polymerase (manufactured by Invitrogen) 0.5 μL, 1 × concentration buffer, 0.4 μM each primer, 1 mM MgSO ₄ , 0.2 μM dNTPs were mixed to make a total volume of 50 μL. Reaction temperature condition: DNA thermal cycler PTC-200 (manufactured by MJ Research) was used, and a cycle consisting of 94 ° C. for 15 seconds, 55 ° C. for 30 seconds, and 68 ° C. for 1 minute 20 seconds was repeated 30 times. However, the heat retention at 94 ° C. in the first cycle was 2 minutes, and the heat retention at 68 ° C. in the final cycle was 3 minutes. The obtained DNA fragment lacking the internal sequence of the pta-ack gene was purified using QIAquick PCR Purification Kit (manufactured by QIAGEN), and then cleaved with restriction enzymes SacI and SphI. The resulting DNA fragment of about 1.1 kb was detected by separation 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 (Japanese Patent Laid-Open No. 2005-95169) with restriction enzymes SacI and SphI, and ligation kit ver. 2 (made by Takara Bio Inc.). Escherichia coli (DH5α strain) was transformed with the obtained plasmid DNA and smeared on an LB agar medium containing 50 μg / mL kana machine and 50 μg / mL X-Gal. Clones that formed white colonies on this medium were subjected to liquid culture by a conventional method, and then plasmid DNA was purified. The obtained plasmid DNA was cleaved with restriction enzymes SacI and SphI, and an insertion fragment of about 1.1 kb was observed, which was named pTACK1 (FIG. 3).

<Preparation of PoxB / ACH / PTA / ACK deficient strain>
(A) Preparation of PoxB-deficient strain The test strain for preparation of the poxB gene-deficient strain was Brevibacterium flavum MJ233 / ΔLDH (LDH disrupted strain: JP-A-2005-95169), and the plasmid DNA for transformation was It was prepared from Escherichia coli JM110 strain transformed with the calcium chloride method (Journal of Molecular Biology, 53, 159, 1970) using pOXB11 constructed in Example 1 (B). Brevibacterium is transformed by the electric pulse method (Res. Microbiol., Vol. 144, p.181-185, 1993), and the obtained transformant is smeared on an LBG agar medium containing 50 μg / mL of kanamycin. did. Since the strain grown on this medium is a plasmid in which pOXB11 cannot replicate in the Brevibacterium flavum MJ233 strain, the plasmid poxB gene and the same gene on the genome of Brevibacterium flavum MJ233 strain As a result of homologous recombination, the kanamycin resistance gene and sacB gene derived from the plasmid should be inserted on the same genome. Next, the homologous recombination strain was subjected to liquid culture in an LBG medium containing 25 μg / mL kanamycin. As a result of smearing about 1 million cells of this culture on LBG medium containing 10% sucrose, sacB gene was eliminated by the second homologous recombination, and several tens of strains considered to be insensitive to sucrose were obtained. I got it. Among the homologous recombination strains obtained in the second round as described above, those in which the poxB gene is replaced with a mutant type derived from pOXB11 and those in which the wild type is restored. Whether the poxB gene is a mutant type or a wild type can be determined by analyzing the poxB gene using primers (SEQ ID NO: 1 and SEQ ID NO: 4) for PCR amplification. In the case of a mutant having a deletion region of 1518 bp, a DNA fragment of 981 bp is generated. As a result of analyzing the strain that became insensitive to sucrose by the above method, a strain having only the mutant gene was selected, and the strain was named Brevibacterium flavum MJ233 / ΔPoxB / ΔLDH.

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

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

(D) Production of PC-enhanced strain pMJPC1 (PC-enhanced plasmid: JP-A 2005-95169) was introduced into Brevibacterium flavum MJ233 / ΔPTA / ΔACK / ΔACH / ΔPoxB / ΔLDH produced in (C) above, and PC Reinforced.
Transformation was performed by introducing pMJPC1 plasmid DNA prepared from E. coli DH5α strain by the electric pulse method described in (A) 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 same strain had pMJPC1, and this was named Brevibacterium flavum MJ233 / PC / ΔPTA / ΔACK / ΔACH / ΔPoxB / ΔLDH.

<Creation of CS enhanced strain>
(A) Construction of plasmid for enhancing CS The acquisition of citrate synthase gene derived from Brevibacterium flavum strain MJ233 containing the promoter region was reported using the DNA prepared in (A) of Example 1 as a template, and the entire genome sequence was reported. It was performed by PCR using synthetic DNA (SEQ ID NO: 13 and SEQ ID NO: 14) designed based on the sequence of the gene of Corynebacterium glutamicum ATCC13032 (GenBank Database Accession No. AP005276). 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 conditions: DNA thermal cycler PTC-200 (manufactured by MJ Research) was used, and a cycle consisting of 94 ° C. for 10 seconds and 68 ° C. for 3 minutes was repeated 35 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 10 minutes. After completion of the PCR reaction, 0.1 μL of Takara Ex Taq (Takara Bio) 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 FMCBioProducts) gel electrophoresis and then visualized by ethidium bromide staining to detect a fragment of about 2.1 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 mixed with PCR product cloning vector pT7Blue T-Vector (manufactured by Novagen), 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 plasmid DNA was purified. By cutting the obtained plasmid DNA with restriction enzymes PstI and KpnI, an insert fragment of about 2.1 kb was recognized, and this was named pCS3.0.
Next, pCS3.0 was cleaved with restriction enzymes PstI and KpnI to give about 4.4.
A DNA fragment of kb was separated and recovered by 0.75% agarose gel electrophoresis, mixed with DNA prepared by cleaving plasmid pC3.14 (Japanese Patent Laid-Open No. 2005-95169) with restriction enzymes PstI and KpnI, and ligated kit ver. 2 (made by Takara Bio Inc.). Escherichia coli DH5α strain was transformed with the plasmid DNA thus obtained, and smeared on an LB agar medium containing 50 μg / mL spectinomycin and 50 μg / mLX-Gal. White colonies grown 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 PstI and KpnI, so that an insert fragment of about 2.1 kb was selected and named pCS3.1 (FIG. 4).

(B) Production of CS-enhanced strain pCS3.1 constructed in (A) above was added to Brevibacterium flavum MJ233 / PC / ΔPTA / ΔACK / ΔACH / ΔPoxB / ΔLDH produced in (D) of Example 2 above. Introduced and enhanced CS.
Transformation was performed by introducing pCS3.1 plasmid DNA by the electric pulse method described in Example 2 (A). The obtained transformant was smeared on an LBG agar medium containing 10 μg / mL of streptomycin and 25 μg / mL of kanamycin, liquid culture was performed from a strain grown on this medium by a conventional method, and plasmid DNA was extracted and digested with restriction enzymes. As a result of analysis based on the above, it was confirmed that the same strain retained pCS3.1, and this was named Brevibacterium flavum MJ233 / CS / PC / ΔPTA / ΔACK / ΔACH / ΔPoxB / ΔLDH.

<Evaluation of CS enhanced 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, 50 μL of 5% kanamycin aqueous solution filtered aseptically and 50 μL of 2% aqueous streptomycin solution were added, and Brevibacterium flavum MJ233 prepared in Example 3 (B) was added. / CS / PC / ΔPTA / ΔACK / ΔACH / ΔPoxB / ΔLDH strain was inoculated and seed-cultured at 30 ° C. for 24 hours. However, addition of streptomycin was excluded when the target strain Brevibacterium flavum MJ233 / PC / ΔPTA / ΔACK / ΔACH / ΔPoxB / ΔLDH strain prepared in Example 2 (D) was cultured.
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 substrate solution (glucose: 200 g, magnesium carbonate: 194 g, and distilled water: 1000 mL) to 0.5 mL of the above cell suspension in a 4 mL reactor, and in a 5 to 6% carbon dioxide atmosphere, The reaction was carried out at 35 ° C. for 22 hours.
After the reaction, the mixture was centrifuged under the conditions described above, and the organic acid concentration of the supernatant was analyzed. As a result, the Brevibacterium flavum MJ233 / CS / PC / ΔPTA / ΔACK / ΔACH / ΔPoxB / ΔLDH strain Compared with the bacterial flavum MJ233 / PC / ΔPTA / ΔACK / ΔACH / ΔPoxB / ΔLDH strain, the yield of dicarboxylic acid (total amount of succinic acid, malic acid, fumaric acid) increased by 9.3%, and the dicarboxylic acid The amount of acetic acid produced as a by-product was reduced by 58%. The improvement of the dicarboxylic acid yield and the reduction effect of by-product acetic acid were recognized by the enhancement of CS.

The figure which shows the construction procedure of plasmid pOXB11. The figure which shows the construction procedure of plasmid pACH22. The figure which shows the construction procedure of plasmid pTACK1. The figure which shows the construction procedure of plasmid pCS3.1.

Claims (10)

A bacterium having a non-amino organic acid-producing ability and modified so that citrate synthase activity is enhanced compared to an unmodified strain. Furthermore, the bacteria of Claim 1 modified | changed so that lactate dehydrogenase activity might be reduced compared with an unmodified strain. Furthermore, the bacterium according to claim 1 or 2, which has been modified so that pyruvate carboxylase activity is enhanced as compared with an unmodified strain. Furthermore, it has been modified so that the activity of one or more enzymes selected from the group consisting of acetate kinase, phosphotransacetylase, pyruvate oxidase and acetyl-CoA hydrolase is reduced as compared to the unmodified strain. 4. The bacterium according to any one of 3. The bacterium according to any one of claims 1 to 4, which is selected from the group consisting of coryneform bacteria, Bacillus bacteria, or Rhizobium bacteria. The bacterium according to any one of claims 1 to 5 or a processed product of the bacterium is allowed to act on an organic raw material in a reaction solution containing carbonate ion, bicarbonate ion, or carbon dioxide gas, thereby allowing a non-amino organic acid to be produced. A method for producing a non-amino organic acid, characterized in that the non-amino organic acid is recovered. The method according to claim 6, wherein the organic raw material is allowed to act in an anaerobic atmosphere. The method according to claim 6 or 7, wherein the organic raw material is glucose or sucrose. The method for producing an organic acid according to any one of claims 6 to 8, wherein the non-amino organic acid is succinic acid. The manufacturing method of a non-amino organic acid containing polymer including the process of manufacturing a non-amino organic acid by the method as described in any one of Claims 6-9, and the process of polymerizing the obtained non-amino organic acid.

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