JP2006320208A - Method for producing succinic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To efficiently produce succinic acid by a fermentation method. <P>SOLUTION: An organic raw material is treated with a bacterium modified so as to enhance maleate dehydrogenase activity or the treated material of the bacterium in a reaction liquid containing carbonate ion, bicarbonate ion or carbon dioxide gas to produce succinic acid. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to the production of succinic acid using bacteria such as coryneform bacteria.

  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, 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, the fermentation production of non-amino organic acid using a bacterium with enhanced activity of phosphoenolpyruvate carboxylase has been reported (for example, see Patent Document 5), but the bacterium with enhanced activity of malate dehydrogenase. The production of non-amino organic acids using has not been previously reported.
US Pat. No. 5,142,834 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

  An object of the present invention is to provide a method for producing succinic acid with higher production efficiency.

  As a result of intensive studies to solve the above problems, the inventor of the present invention contains a bacterium modified so as to enhance malate dehydrogenase activity or a processed product thereof containing carbonate ion, bicarbonate ion or carbon dioxide gas. It has been found that the consumption rate of the organic raw material, the generation rate of succinic acid, or the yield is increased by acting on the organic raw material in the reaction solution, and the present invention has been completed.

That is, according to the present invention, the following inventions are provided.
(1) Bacteria modified so as to enhance malate dehydrogenase activity or a processed product of the bacteria is allowed to act on an organic raw material in a reaction solution containing carbonate ion, bicarbonate ion, or carbon dioxide gas to produce succinic acid. A method for producing succinic acid, comprising producing the succinic acid and recovering the succinic acid.
(2) The method according to (1), wherein the bacterium is any one selected from the group consisting of coryneform bacteria, Bacillus bacteria, and Rhizobium bacteria.
(3) The method according to (1) or (2), wherein the bacterium is a bacterium further modified so that lactate dehydrogenase activity is reduced to 10% or less compared to an unmodified strain.
(4) The method according to any one of (1) to (3), wherein the bacterium is further modified so that pyruvate carboxylase activity is enhanced.
(5) The method according to any one of (1) to (4), wherein the bacterium is a bacterium further modified so that fumarate reductase activity is enhanced.
(6) The method according to any one of (1) to (5), wherein the organic raw material is allowed to act in an anaerobic atmosphere.
(7) The method according to any one of (1) to (6), wherein the organic raw material is glucose.
(8) A method for producing a succinic acid-containing polymer, comprising a step of producing succinic acid by the method according to any one of (1) to (7) and a step of polymerizing the obtained succinic acid.

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

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

  Bacteria that can be used in the production method of the present invention are bacteria that have been modified so that malate dehydrogenase activity is enhanced. Here, “malate dehydrogenase activity” refers to the activity of catalyzing the reaction of reducing oxaloacetate and converting it to malate, and “maleate dehydrogenase activity is enhanced” refers to an unmodified strain of malate dehydrogenase. In comparison, it means that the malate dehydrogenase activity is enhanced. The malate dehydrogenase activity is preferably enhanced by 1.5 times or more, more preferably enhanced by 2 times or more per unit cell weight, compared to a malate dehydrogenase non-modified strain. Malate dehydrogenase activity can be measured by a method for measuring a decrease in NADH as described below.

  The enhancement of the malate dehydrogenase activity can be performed, for example, by modifying the parent bacterial strain by a genetic recombination method using a malate dehydrogenase gene.

Bacteria that can be used in the production method of the present invention are not particularly limited as long as they have the ability to produce succinic acid, but coryneform bacteria, Bacillus bacteria, or Rhizobium bacteria are preferable, and coryneform bacteria are more preferable. .
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 And more preferably, Corynebacterium glutamicum, Brevibacterium flavum, Brevibacterium amniagenes or Brementac fermentum bacterium. Bacteria belonging to

Particularly preferred specific examples of the bacterial parent strain used for obtaining the bacterium used in the production method of 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, and the like. 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 respectively Corynebacterium glutamicum MJ-233 strain. And MJ-233 AB-41 strain.
Brevibacterium flavum MJ-233 was released on April 28, 1975 at the Institute of Microbial Technology of the Ministry of International Trade and Industry (currently National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center) (305-8566) Contract number FERM at Tsukuba City, Ibaraki Prefecture, Japan
Deposited as P-3068, transferred to an international deposit under the Budapest Treaty on May 1, 1981, and deposited with a deposit number of FERM BP-1497.

Examples of Bacillus bacteria include Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus, Bacillus stearothermophilus, and the like.
Examples of Rhizobium bacteria include Rhizobium etli.

  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.

  When the malate dehydrogenase (MDH) gene is used to modify so that malate dehydrogenase activity is enhanced, the gene that can be used is not particularly limited as long as it encodes a protein having malate dehydrogenase activity. For example, SEQ ID NO: 32 And a gene derived from Brevibacterium flavum MJ-233 having the base sequence shown in FIG. This gene may be a DNA that hybridizes with a DNA having the above base sequence under stringent conditions, or a DNA having 90% or more, preferably 95% or more, more preferably 99% or more homology with the above base sequence. It may be a homologue. Here, the stringent conditions are 60 ° C., 1 × SSC, 0.1% SDS, preferably 60 ° C., 0.1 × SSC, 0.1%, which are the usual washing conditions for Southern hybridization. A condition for hybridization at a salt concentration corresponding to SDS is mentioned.

  In addition, MDH genes derived from bacteria other than Brevibacterium flavum MJ-233, or from other microorganisms or animals and plants can also be used. For the MDH gene derived from microorganisms or animals and plants, a gene encoding a protein having MDH activity based on a gene whose homology has already been determined, homology, etc., was isolated from the chromosomes of microorganisms, animals and plants, and the nucleotide sequence was determined. Things 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.

  By inserting the DNA encoding malate dehydrogenase isolated as described above into a known expression vector, a malate dehydrogenase expression vector is provided. By culturing a transformant transformed with this expression vector, a strain with enhanced malate dehydrogenase activity can be obtained. Alternatively, a malate dehydrogenase-enhanced strain can also be obtained by incorporating DNA encoding malate dehydrogenase into a chromosomal DNA of a host bacterium so that it can be expressed, such as by homologous recombination. Note that transformation and homologous recombination can be performed according to ordinary methods known to those skilled in the art.

  When the malate dehydrogenase gene is introduced, an appropriate promoter is incorporated upstream of the gene 5′-side, more preferably a terminator is incorporated downstream of the 3′-side. The promoter and terminator are not particularly limited as long as they are known to function in bacteria used as hosts, and may be the promoter and terminator of the malate dehydrogenase gene itself. These promoters and terminators may be substituted. Vectors, promoters and terminators that can be used in these various bacteria are described in detail in, for example, “Microbiology Basic Course 8 Genetic Engineering / Kyoritsu Shuppan”.

When a coryneform bacterium is used, the expression of MDH in the coryneform bacterium can be enhanced by introducing a DNA fragment containing the MDH gene into a plasmid vector containing at least a gene responsible for the replication and replication function of the plasmid in the coryneform bacterium. Possible recombinant plasmids can be obtained. The plasmid vector capable of introducing the MDH gene into the coryneform bacterium is not particularly limited as long as it contains at least a gene that controls the replication and proliferation function in the coryneform bacterium. Specific examples thereof include, for example, a plasmid pCRY30 described in JP-A-3-210184; plasmids pCRY21, pCRY2KE, pCRY2KX described in JP-A-2-72876 and US Pat. No. 5,185,262. pCRY31, pCRY3KE and pCRY3KX; plasmids pCRY2 and pCRY3 described in JP-A-1-191686; pAM330 described in JP-A-58-67679; pHM1519 described in JP-A-58-77895; PAJ655, pAJ611 and pAJ1844 described in JP-A-58-192900; pCG1 described in JP-A-57-134500; pCG2 described in JP-A-58-35197; described in JP-A-57-183799 PCG And pCG11; Plasmid vol.36 (1)
Examples thereof include pC2 described in p62-66 (1996) or a modified plasmid thereof. The enhancement of MDH activity is enhanced by introducing, replacing, or amplifying the MDH gene on the chromosome by a known homologous recombination method, introducing a mutation into the promoter of the MDH gene on the chromosome, or replacing it with a stronger promoter. It can also be performed by expression.

  In the integration into the recombinant plasmid or chromosome, the promoter for expressing the MDH gene may be any promoter as long as it functions in coryneform bacteria, and may be the promoter of the MDH gene itself to be used. The MDH gene expression level can also be controlled by appropriately selecting a promoter. As mentioned above, although the example using coryneform bacteria was described, also when using other bacteria, the enhancement of MDH activity can be achieved by the same method.

  In the case of using a Bacillus genus bacterium, examples of the vector include a pUB110 series plasmid and a pC194 series plasmid, and can also be integrated into a chromosome. As the promoter and terminator, promoters and terminators of enzyme genes such as alkaline protease, neutral protease, α-amylase and the like can be used.

In the production method of the present invention, it is more effective to use a bacterial strain modified so that lactate dehydrogenase (LDH) activity is reduced in addition to enhancement of MDH activity. Here, “the lactate dehydrogenase activity is reduced” means that the lactate dehydrogenase activity is reduced as compared with the unmodified strain of lactate dehydrogenase. It is preferable that the lactate dehydrogenase activity is reduced to 10% or less per microbial cell as compared with the lactate dehydrogenase non-modified strain. In addition, lactate dehydrogenase activity may be completely deficient. Reduction of lactate dehydrogenase activity is confirmed by measuring lactate dehydrogenase activity by a known method (L. Kanarek and RL Hill, J. Biol. Chem. 239, 4202 (1964)). Can do. As a specific method for producing a mutant having reduced lactate dehydrogenase activity of coryneform bacteria, a method by homologous recombination to a chromosome described in JP-A-11-206385, or Examples of the present specification And the method using the SacB gene described in (Schafer, A. et al. Gene 145 (1994) 69-73) and the like. The lactate dehydrogenase gene used for reducing lactate dehydrogenase activity is not particularly limited as long as it is a gene encoding a protein having lactate dehydrogenase activity. For example, a gene described in JP-A-11-206385 may be used. it can.
A coryneform bacterium with reduced lactate dehydrogenase activity and enhanced expression of the MDH gene is prepared, for example, by producing a bacterium with a disrupted LDH gene as in Example 2 described later, and comprising the bacterium containing the MDH gene. It can be obtained by transformation with a replacement vector. However, either the modification operation for reducing LDH activity or the modification operation for enhancing MDH activity may be performed first.

  In addition, in the production method of the present invention, bacteria modified so as to enhance the activity of pyruvate carboxylase may be used in addition to the enhancement of MDH activity. “The activity of pyruvate carboxylase is enhanced” means that the activity of pyruvate carboxylase is increased relative to an unmodified strain such as a wild strain or a parent strain. The activity of pyruvate carboxylase can be measured, for example, by a method for measuring a decrease in NADH as described later.

  The PC gene used for modification so that the activity of pyruvate carboxylase (PC) is enhanced is a gene whose base sequence has already been determined, or a protein having PC activity by the method shown below. The DNA fragment to be encoded can be isolated from the chromosomes of microorganisms, animals and plants, and the base sequence determined. In addition, after the base sequence is determined, a gene synthesized according to the sequence can be used.

A DNA fragment containing a PC gene is present on a chromosome derived from a microorganism, animal or plant. The basic operation for preparing the PC gene from these source microorganisms and animals and plants is described as follows, taking as an example one derived from a coryneform bacterium having a known sequence. The PC gene is present on the chromosome of the coryneform bacterium Corynebacterium glutamicum ATCC 13032 (Peters-Wendisch, PG et al. Microbiology, vol. 144 (1998) p915-927), and their sequences are known. (GenBank Database Accession)
No. AP005276) (SEQ ID NO: 15), which can be isolated and obtained by PCR.

  For example, when PCR is performed using oligonucleotides having the nucleotide sequences shown in SEQ ID NOS: 13 and 14 as primers for PCR and a chromosome derived from Corynebacterium glutamicum as a template, a PC gene consisting of about 3.7 kb is amplified. Can be made. At this time, by adding an appropriate restriction enzyme site to the 5 ′ end of the primer used for PCR, it can be ligated to an appropriate site of the vector described later, and the resulting recombinant vector is used for coryneform type. Can be introduced into bacteria.

  Even if the gene sequence is unknown, the protein can be purified using PC activity as an indicator, a probe can be synthesized from its N-terminal amino acid sequence or partially degraded sequence, and the gene fragment can be isolated by commonly used hybridization techniques. . It is also possible to synthesize a probe or primer based on the amino acid sequence of a region conserved among PC proteins, and obtain a fragment by hybridization or PCR. The DNA fragment sequence of the obtained fragment can be determined by a usual method.

  In the present invention, the DNA fragment containing the PC gene used for enhancing PC activity is not only isolated from Corynebacterium glutamicum chromosomal DNA, but also a commonly used DNA synthesizer such as Applied Biosystems. It may be synthesized using a 394 DNA / RNA synthesizer manufactured by (Applied Biosystems). In addition, as described above, the PC gene obtained from coryneform bacterial chromosomal DNA has the nucleotide sequence of SEQ ID NO: 15 as long as it does not substantially impair the function of the encoded PC, that is, the property involved in carbon dioxide fixation. , Some bases may be replaced with other bases, may be deleted, or bases may be newly inserted, and a part of the base sequence may be rearranged. Any of these derivatives may be used in the present invention. DNA that hybridizes under stringent conditions with DNA having the nucleotide sequence of SEQ ID NO: 15 or DNA having homology of 90% or more, preferably 95% or more, more preferably 99% or more with the nucleotide sequence of SEQ ID NO: 15. In addition, DNA encoding a protein having PC activity can also be suitably used. Here, as stringent conditions, it corresponds to 60 ° C., 1 × SSC, 0.1% SDS, preferably 0.1 × SSC, 0.1% SDS, which is a normal washing condition for Southern hybridization. The conditions for hybridizing at a salt concentration are included.

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

Human [Biochem.Biophys.Res.Comm., 202, 1009-1014, (1994)]
Mouse [Proc.Natl.Acad.Sci.USA., 90, 1766-1779, (1993)]
Rat [GENE, 165, 331-332, (1995)]
Yeast; Saccharomyces cerevisiae
[Mol.Gen.Genet., 229, 307-315, (1991)]
Schizosaccharomyces pombe
[DDBJ Accession No .; D78170]
Bacillus stearothermophilus
[GENE, 191, 47-50, (1997)]
Rhizobium etli
[J. Bacteriol., 178, 5960-5970, (1996)]
The DNA fragment containing the PC gene can be expressed by inserting it into an appropriate expression plasmid such as pUC118 (Takara Shuzo) and introducing it into an appropriate host microorganism such as Escherichia coli JM109 (Takara Shuzo). Confirmation of the activity of the expressed PC gene product, pyruvate carboxylase (SEQ ID NO: 16), was carried out by using a crude enzyme solution from the transformant and the method of Magasanik [J. Bacteriol., 158, 55-62, (1984)]. This can be confirmed by directly measuring the PC activity and comparing with the PC activity of the crude enzyme solution extracted from the non-transformed strain. Recombination capable of high expression of PC in coryneform bacteria by introducing a DNA fragment containing the PC gene into an appropriate plasmid, for example, a plasmid vector containing at least a gene responsible for the replication and replication function of the plasmid in coryneform bacteria. A plasmid can be obtained. Here, in the above recombinant plasmid, the promoter for expressing the PC gene can be a promoter possessed by coryneform bacteria, but is not limited thereto, and has a base sequence for initiating transcription of the PC gene. Any promoter can be used. For example, a TZ4 promoter as described in Example 3 described later can be mentioned.

As the plasmid vector into which the PC gene can be introduced, the same plasmid vectors as those described above that can be used for the introduction of the MDH gene can be used.

  A recombinant vector obtained by inserting the PC gene into a suitable plasmid vector replicable in an aerobic coryneform bacterium as described above, such as a coryneform bacterium such as Brevibacterium flavum MJ- By transforming 233 strain (FERM BP-1497), 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. By transforming the obtained bacterium with a recombinant vector containing the MDH gene, a coryneform bacterium with enhanced expression of the PC gene and the MDH gene can be obtained. Either the MDH gene or the PC gene may be introduced first. Transformation can be performed by, for example, the electric pulse method (Res. Microbiol., Vol. 144, p.181-185, 1993).

In addition, in the production method of the present invention, in addition to the enhancement of MDH activity, a bacterium modified so as to enhance the fumarate reductase activity may be used.
Here, “fumarate reductase activity” refers to the activity of catalyzing the reaction of reducing fumaric acid and converting it to succinic acid, and “fumarate reductase activity is enhanced” refers to a fumarate reductase non-modified strain. In comparison, the fumarate reductase activity is enhanced. The fumarate reductase activity can be measured by a method for measuring a decrease in K ₃ Fe (CN) ₆ as described later.

  Enhancement of fumarate reductase activity can be performed by modifying a parent bacterial strain by, for example, a gene recombination method using a fumarate reductase gene.

The modification by the genetic recombination method using the fumarate reductase gene can be performed by the same method as the MDH gene enhancement described above.
The type of fumarate reductase gene that can be used to enhance the fumarate reductase activity is not particularly limited as long as it encodes a protein having fumarate reductase activity. For example, the nucleotide sequence of SEQ ID NO: 19 A gene that hybridizes with a polynucleotide having the base sequence under stringent conditions can be used. The nucleotide sequence of SEQ ID NO: 19 is the nucleotide sequence of the fumarate reductase gene of Escherichia coli, and encodes four types of subunits (SEQ ID NOs: 20, 21, 22, 23) that constitute FRD.

  In addition, E. coli fumarate reductase is an enzyme responsible for the reverse reaction of succinate dehydrogenase acting in the forward direction of the TCA cycle, but is involved in fumarate respiration under anaerobic conditions. It is known that the gene expression is suppressed at the transcriptional level (Jones, HM, Gunsalus, RP, J. Bacteriol., 1985, Vol. 164, p1100-1109). Therefore, since it is considered that fumarate reductase has an excessively enhanced activity, the growth of bacterial cells may be deteriorated. Therefore, in the present invention, the fumarate reductase activity is enhanced to such an extent that the growth of bacterial cells is not significantly inhibited. It is desirable.

  In the production method of the present invention, when a bacterium modified so that the activities of malate dehydrogenase, pyruvate carboxylase, and fumarate reductase are enhanced and the lactate dehydrogenase activity is reduced is used for producing succinic acid. It is valid. Such a bacterium can be obtained, for example, by preparing a coryneform bacterium having a disrupted LDH gene and then transforming with a recombinant vector containing a PC gene, FRD gene, and MDH gene. Any modification operation using these genes may be performed first.

When using the above-mentioned bacterium for the production reaction of succinic acid, a slant-cultured solid medium such as an agar medium may be used for the direct reaction, but the bacterium previously cultured in a liquid medium (seed culture) is used. Is preferred. The succinic acid can be produced by reacting the seed-cultured bacteria with the organic raw material while growing them in 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. In this case, the medium normally used for culture of bacteria can be used as the culture medium. 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 the bacterium can assimilate and produce succinic acid. Usually, galactose, lactose, glucose, fructose, glycerol, sucrose, sucrose are used. , Starch, cellulose and other carbohydrates; fermentable carbohydrates such as glycerin, mannitol, xylitol, ribitol and other polyalcohols are used, among which glucose, fructose and glycerol are preferred, and glucose is particularly preferred.

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

  The reaction solution containing the organic raw material is not particularly limited, and may be, for example, a medium for culturing bacteria or a buffer solution such as a phosphate buffer solution. The reaction solution is preferably an aqueous solution containing a nitrogen source, an inorganic salt, and the like. Here, the nitrogen source is not particularly limited as long as 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.

  As succinic acid is produced, the pH of the reaction solution decreases. Therefore, the pH of the reaction solution is preferably 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.

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 contains, for example, the above-described organic raw materials and carbonate ions, bicarbonate ions, or carbon dioxide gas, and can be reacted preferably 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 the salt from carbon dioxide or carbon dioxide (CO ₂ ). Specific examples of the carbonate or bicarbonate salt include magnesium carbonate, ammonium carbonate, sodium carbonate, potassium carbonate, ammonium bicarbonate, sodium bicarbonate, and potassium bicarbonate. Carbonate ions and bicarbonate ions are added at a concentration of 0.001 to 5M, preferably 0.1 to 3M, and more preferably 1 to 2M. When carbon dioxide gas is contained, 50 mg to 25 g, preferably 100 mg to 15 g, more preferably 150 mg to 10 g of carbon dioxide gas is contained per liter of the solution.

  The 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.

  At the time of cultivation for growing bacteria, it is necessary to supply oxygen by aeration and agitation. On the other hand, during the succinic acid production reaction, aeration and stirring may be performed, but an anaerobic atmosphere is preferable. An anaerobic atmosphere can be achieved by suppressing the amount of oxygen supplied to the reaction solution, by reducing the oxygen concentration of the gas to be aerated, or by reducing the oxygen concentration to 0, or by not supplying oxygen. Specifically, for example, a method of sealing the container to make it non-ventilated, venting an inert gas such as nitrogen gas, or venting an inert gas containing carbon dioxide can be used. Although the dissolved oxygen concentration in the reaction solution at this time is kept low, specifically, the dissolved oxygen concentration is usually 0 to 2 ppm, preferably 0 to 1 ppm, more preferably 0 to 0.5 ppm. desirable.

  Succinic acid accumulated in the reaction solution (culture solution) can be recovered 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, succinic acid can be separated and purified from the solution by crystallization or column chromatography. it can.

Furthermore, in this invention, after manufacturing a succinic acid with the method of this invention mentioned above, a succinic-acid containing polymer can be manufactured by performing a polymerization reaction using the obtained succinic acid as a raw material. In recent years, with the increase in the number of environmentally friendly industrial products, attention has been focused on polymers using plant-derived raw materials, and the succinic acid produced in the present invention is used after being processed into polymers such as polyester and polyamide. I can do it. 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 succinic acid obtained by the production method of the present invention or a composition containing the succinic acid can be used for food additives, pharmaceuticals, cosmetics and the like.

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

<Construction of vector for gene disruption>
(A) Extraction of Bacillus subtilis genomic DNA In 10 mL of LB medium [composition: tryptone 10 g, yeast extract 5 g, NaCl 5 g dissolved in 1 L of distilled water], Bacillus subtilis ISW1214 is cultured until the late logarithmic growth phase, The cells were collected. 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) Amplification and cloning of SacB gene by PCR Acquisition of SacB gene of Bacillus subtilis using the DNA prepared in (A) as a template, and using the previously reported base sequence of the gene (GenBank Database Accession No. X02730) This was performed by PCR using the synthetic DNA (SEQ ID NO: 1 and SEQ ID NO: 2) designed based on the group.
Composition of reaction solution: 1 μL of template DNA, 0.2 μL of Pfx DNA polymerase (manufactured by Invitrogen), 1 × concentration attached buffer, 0.3 μM each primer, 1 mM MgSO ₄ and 0.25 μM dNTPs were mixed to make a total volume of 20 μL.
Reaction temperature conditions: DNA thermal cycler PTC-200 (manufactured by MJ Research) was used, and a cycle consisting of 94 ° C. for 20 seconds and 68 ° C. for 2 minutes was repeated 35 times. However, the heat retention at 94 ° C. in the first cycle was 1 minute 20 seconds, and the heat retention at 68 ° C. in the final cycle was 5 minutes.
Confirmation of the amplified product was carried out by separating by 0.75% agarose (SeaKem GTG agarose: manufactured by FMC BioProducts) gel electrophoresis and then visualized by ethidium bromide staining, and a fragment of about 2 kb was detected. The DNA fragment of interest was recovered from the gel using a QIAQuick Gel Extraction Kit (manufactured by QIAGEN).
The recovered DNA fragment was phosphorylated at the 5 ′ end with T4 polynucleotide kinase (T4 Polynucleotide Kinase: manufactured by Takara Shuzo), and then ligated kit ver. 2 (manufactured by Takara Shuzo Co., Ltd.) was used to bind to the EcoRV site of the E. coli vector (pBluescript II: manufactured by STRATEGENE), and E. coli (DH5α strain) was transformed with the resulting plasmid DNA. The recombinant Escherichia coli thus obtained was smeared on an LB agar medium [10 g tryptone, 5 g yeast extract, 5 g NaCl, and 15 g agar dissolved in 1 L distilled water] containing 50 μg / mL ampicillin and 50 μg / mL X-Gal. .
Clones that formed white colonies on this medium were then transferred to LB agar medium containing 50 μg / mL ampicillin and 10% sucrose and cultured at 37 ° C. for 24 hours. Among these clones, those that could not grow on a medium containing sucrose were subjected to liquid culture by a conventional method, and then plasmid DNA was purified. Strains in which the SacB gene is functionally expressed in E. coli should be unable to grow on sucrose-containing media. The obtained plasmid DNA was cleaved with restriction enzymes SalI and PstI, whereby an inserted fragment of about 2 kb was observed, and the plasmid was named pBS / SacB.

(C) Construction of chloramphenicol resistant SacB vector 500 ng of E. coli plasmid vector pHSG396 (Takara Shuzo: chloramphenicol resistant marker) was reacted with restriction enzyme PshBI10units at 37 ° C. for 1 hour, followed by phenol / chloroform extraction and ethanol precipitation. It was collected. After blunting both ends with Klenow fragment (manufactured by Takara Shuzo), ligation kit ver. MluI linker (Takara Shuzo) was ligated and circularized using 2 (Takara Shuzo), and Escherichia coli (DH5α strain) was transformed. The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 34 μg / mL chloramphenicol. Plasmid DNA was prepared from the obtained clones by a conventional method, and a clone having a restriction enzyme MluI cleavage site was selected and named pHSG396Mlu.
On the other hand, pBS / SacB constructed in (B) above was cleaved with restriction enzymes SalI and PstI, and the ends were blunted with Klenow fragment. Ligation kit ver. After linking the MluI linker using 2 (Takara Shuzo), a DNA fragment of about 2.0 kb containing the SacB gene was separated and recovered by 0.75% agarose gel electrophoresis. This SacB gene fragment was cleaved with the restriction enzyme MluI, and then the pHSG396Mlu fragment dehydrated with alkaline phosphatase (Alkaline Phosphatase Calf intestine) and ligation kit ver. 2 (manufactured by Takara Shuzo) was used to transform E. coli (DH5α strain). The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 34 μg / mL chloramphenicol. The colonies thus obtained were then transferred to an LB agar medium containing 34 μg / mL chloramphenicol and 10% sucrose and cultured at 37 ° C. for 24 hours. Among these clones, plasmid DNA was purified by a conventional method for those that could not grow on a medium containing sucrose. As a result of analyzing the thus obtained plasmid DNA by MluI digestion, it was confirmed that it had an inserted fragment of about 2.0 kb, and this was named pCMB1.

(D) Acquisition of kanamycin resistance gene The kanamycin resistance gene is obtained by PCR using the DNA of E. coli plasmid vector pHSG299 (Takara Shuzo: Kanamycin resistance marker) as a template and the synthetic DNAs shown in SEQ ID NOs: 3 and 4 as primers. Went by. Composition of reaction solution: Template DNA 1 ng, Pyrobest DNA polymerase (Takara Shuzo) 0.1 μL, 1 × concentration buffer, 0.5 μM each primer, 0.25 μM dNTPs were mixed to make a total volume of 20 μL.
Reaction temperature condition: DNA thermal cycler PTC-200 (manufactured by MJ Research) was used, and a cycle consisting of 94 ° C. for 20 seconds, 62 ° C. for 15 seconds, and 72 ° C. for 1 minute 20 seconds was repeated 20 times. However, the heat retention at 94 ° C. in the first cycle was 1 minute 20 seconds, and the heat retention at 72 ° C. in the final cycle was 5 minutes.
Confirmation of the amplification product was carried out by separating by 0.75% agarose (SeaKem GTG agarose: manufactured by FMCBioProducts) gel electrophoresis and then visualized by ethidium bromide staining to detect a fragment of about 1.1 kb. The DNA fragment of interest was recovered from the gel using a QIAQuick Gel Extraction Kit (manufactured by QIAGEN). The recovered DNA fragment was phosphorylated at the 5 ′ end with T4 polynucleotide kinase (T4 Polynucleotide Kinase: manufactured by Takara Shuzo).

(E) Construction of kanamycin-resistant SacB vector The approximately 3.5 kb DNA fragment obtained by cleaving pCMB1 constructed in (C) above with restriction enzymes Van91I and ScaI was separated and recovered by 0.75% agarose gel electrophoresis. did. This was mixed with the kanamycin resistance gene prepared in (D) above, and ligation kit ver. 2 (manufactured by Takara Shuzo), and Escherichia coli (DH5α strain) was transformed with the obtained plasmid DNA. The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 50 μg / mL kanamycin.
It was confirmed that the strain grown on this kanamycin-containing medium was unable to grow on the sucrose-containing medium. Further, the plasmid DNA prepared from the same strain produced fragments of 354, 473, 1807, and 1997 bp by digestion with the restriction enzyme HindIII. Therefore, it was determined that the structure shown in FIG. 1 was correct, and the plasmid was designated as pKMB1. Named.

<Preparation of LDH gene disruption strain>
(A) Extraction of Brevibacterium flavum MJ233-ES strain genomic DNA A medium [urea 2 g, (NH ₄ ) ₂ SO ₄ 7 g, KH ₂ PO ₄ 0.5 g, K ₂ HPO ₄ 0.5 g, MgSO _{4. 7H 2 O 0.5g, FeSO 4 ·} 7H 2 O 6mg, MnSO 4 · 4-5H 2 O6mg, biotin 200 [mu] g, thiamine 100 [mu] g, yeast extract 1g, casamino acid 1g, glucose 20g, dissolved in distilled water 1L] in 10mL Brevibacterium flavum strain MJ-233 was cultured until the late logarithmic growth phase, and genomic DNA was prepared from the cells obtained by the method shown in (A) of Example 1 above.

(B) Cloning of lactate dehydrogenase gene The MJ233 strain lactate dehydrogenase gene was obtained by using the DNA prepared in (A) above as a template and a synthetic DNA designed on the basis of the base sequence of the gene described in JP-A-11-206385 ( It was performed by PCR using SEQ ID NO: 5 and SEQ ID NO: 6). Composition of reaction solution: Template DNA 1 μL, Taq DNA polymerase (Takara Shuzo) 0.2 μL, 1 × concentration attached buffer, 0.2 μM each primer, 0.25 μM dNTPs were mixed to make the total volume 20 μL.
Reaction temperature conditions: DNA thermal cycler PTC-200 (manufactured by MJ Research) was used, and a cycle consisting of 94 ° C. for 20 seconds, 55 ° C. for 20 seconds and 72 ° C. for 1 minute was repeated 30 times. However, the heat retention at 94 ° C. in the first cycle was 1 minute 20 seconds, and the heat retention at 72 ° C. in the final cycle was 5 minutes.
Confirmation of the amplification product was carried out by separating by 0.75% agarose (SeaKem GTG agarose: manufactured by FMCBioProducts) gel electrophoresis and then visualized by ethidium bromide staining to detect a fragment of about 0.95 kb. The DNA fragment of interest was recovered from the gel using a QIAQuick Gel Extraction Kit (manufactured by QIAGEN).
The recovered DNA fragment was mixed with a PCR product cloning vector pGEM-TEasy (Promega) and ligation kit ver. After ligation using 2 (Takara Shuzo), 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 SacI and SphI, an inserted fragment of about 1.0 kb was recognized, which was designated as pGEMT / CgLDH.

(C) Construction of plasmid for disrupting lactate dehydrogenase gene The coding region of lactate dehydrogenase consisting of about 0.25 kb was excised by cutting the pGEMT / CgLDH prepared in (B) above with restriction enzymes EcoRV and XbaI. The ends of the remaining approximately 3.7 kb DNA fragment were blunted with Klenow fragment, and ligated kit ver. 2 (manufactured by Takara Shuzo) was cyclized to transform E. coli (DH5α strain). The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 50 μg / mL ampicillin.
The strain grown on this medium was subjected to liquid culture by a conventional method, and then the plasmid DNA was purified. By cutting the obtained plasmid DNA with restriction enzymes SacI and SphI, a clone in which an insert fragment of about 0.75 kb was recognized was selected and named pGEMT / ΔLDH.
Next, a DNA fragment of about 0.75 kb generated by cleaving the above pGEMT / ΔLDH with restriction enzymes SacI and SphI is separated and recovered by 0.75% agarose gel electrophoresis, and a lactate dehydrogenase gene fragment containing a defective region Was prepared. This DNA fragment was mixed with pKMB1 constructed in Example 1 cleaved with restriction enzymes SacI and SphI, and ligation kit ver. After ligation using 2 (Takara Shuzo), Escherichia coli (DH5α strain) was transformed with the obtained plasmid DNA. The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 50 μg / mL kanamycin and 50 μg / mL X-Gal.
Clones that formed white colonies on this medium were subjected to liquid culture by a conventional method, and then plasmid DNA was purified. The obtained plasmid DNA was cleaved with restriction enzymes SacI and SphI, so that an insert fragment of about 0.75 kb was selected and named pKMB1 / ΔLDH (FIG. 2).

(D) Preparation of lactate dehydrogenase gene disruption strain derived from Brevibacterium flavum MJ233-ES strain Plasmid DNA used for transformation of Brevibacterium flavum MJ-233 strain was obtained by the calcium chloride method (Journal of) using pKMB1 / ΔLDH. (Molecular Biology, 53, 159, 1970).
Brevibacterium flavum MJ-233 strain (FERM BP-1497) can be obtained by conventional methods (Wolf H et al., J. Bacteriol. 1983, 156 (3) 1165-1170, Kurusu Y et al., Agric Biol Chem. The endogenous plasmid was removed (curing) according to 1990, 54 (2) 443-7), and the obtained plasmid curing strain Brevibacterium flavum MJ233-ES strain was used for the subsequent transformation.
The Brevibacterium flavum MJ233-ES strain was transformed by the electric pulse method (Res. Microbiol., Vol. 144, p.181-185, 1993), and the obtained transformant was treated with 50 μg / mL kanamycin. LBG agar medium containing [tryptone 10 g, yeast extract 5 g, NaCl 5 g, glucose 20 g, and agar 15 g dissolved in 1 L of distilled water] was smeared.
Since the strain grown on this medium is a plasmid in which pKMB1 / ΔLDH cannot be replicated in Brevibacterium flavum MJ233-ES, the lactate dehydrogenase gene of the plasmid and Brevibacterium flavum MJ-233 strain As a result of homologous recombination with the same gene on the genome, the kanamycin resistance gene and SacB gene derived from the plasmid should be inserted on the same genome.
Next, the homologous recombination strain was subjected to liquid culture in an LBG medium containing kanamycin 50 μg / mL. A portion equivalent to about 1 million cells of this culture was smeared on a 10% sucrose-containing LBG medium. As a result, about 10 strains that were considered to be sucrose-insensitive due to elimination of the SacB gene by the second homologous recombination were obtained.
Among the strains thus obtained, those in which the lactate dehydrogenase gene is replaced with a mutant derived from pKMB1 / ΔLDH and those in which the wild type is restored are included. Confirmation of whether the lactate dehydrogenase gene is a mutant type or a wild type is easily confirmed by subjecting the cells obtained by liquid culture in LBG medium to direct PCR reaction and detecting the lactate dehydrogenase gene. it can. When the lactate dehydrogenase gene is analyzed using primers (SEQ ID NO: 7 and SEQ ID NO: 8) for PCR amplification, a wild-type DNA fragment of 720 bp and a mutant type having a deletion region should have a DNA fragment of 471 bp.
As a result of analyzing the strain that became insensitive to sucrose by the above method, a strain having only the mutant gene was selected, and the strain was named Brevibacterium flavum MJ233 / ΔLDH.

(E) Confirmation of lactate dehydrogenase activity The Brevibacterium flavum MJ233 / ΔLDH strain prepared in (D) above was inoculated in medium A and cultured with aerobic shaking at 30 ° C. for 15 hours. The obtained culture is centrifuged (3,000 × g, 4 ° C., 20 minutes) to recover the cells, and then sodium-phosphate buffer [composition: 50 mM sodium phosphate buffer (pH 7.3)] Washed with.
Next, 0.5 g (wet weight) of washed cells was suspended in 2 mL of the sodium-phosphate buffer, and the cells were crushed by applying an ultrasonic crusher (manufactured by Branson) under ice cooling. The crushed material was centrifuged (10,000 × g, 4 ° C., 30 minutes), and the supernatant was obtained as a crude enzyme solution. As a control, a crude enzyme solution of Brevibacterium flavum MJ233-ES strain was similarly prepared and subjected to the following activity measurement.
Confirmation of the enzyme activity of lactate dehydrogenase was carried out by measuring the oxidation of coenzyme NADH to NAD + with the production of lactic acid using pyruvate as a substrate for both crude enzyme solutions as a change in absorbance at 340 nm [L. Kanarek and R.L.Hill, J. Biol. Chem. 239, 4202 (1964)]. The reaction was performed at 37 ° C. in the presence of 50 mM potassium-phosphate buffer (pH 7.2), 10 mM pyruvic acid, 0.4 mM NADH. As a result, the lactate dehydrogenase activity in the crude enzyme solution prepared from Brevibacterium flavum MJ233 / ΔLDH was 10 minutes compared to the lactate dehydrogenase activity in the crude enzyme solution prepared from Brevibacterium flavum MJ233-ES strain. 1 or less.

<Construction of Coryneform Bacterial Expression Vector>
(A) Preparation of Promoter Fragment for Coryneform Bacteria Utilizing the DNA fragment (hereinafter referred to as TZ4 promoter) described in SEQ ID NO: 4 of JP-A-7-95891 reported to have strong promoter activity in coryneform bacteria It was decided. This promoter fragment was obtained by using a synthetic DNA designed based on the sequence described in SEQ ID NO: 4 of JP-A-7-95891 using Brevibacterium flavum MJ233 genomic DNA prepared in Example 2 (A) as a template. It was carried out by PCR using SEQ ID NO: 9 and SEQ ID NO: 10).
Composition of reaction solution: 1 μL of template DNA, 0.2 μL of Pfx DNA polymerase (manufactured by Invitrogen), 1 × concentration attached buffer, 0.3 μM each primer, 1 mM MgSO ₄ and 0.25 μM dNTPs were mixed to make a total volume of 20 μL.
Reaction temperature conditions: DNA thermal cycler PTC-200 (manufactured by MJ Research) was used, and a cycle consisting of 94 ° C. for 20 seconds, 60 ° C. for 20 seconds, and 72 ° C. for 30 seconds was repeated 35 times. However, the heat retention at 94 ° C. in the first cycle was 1 minute 20 seconds, and the heat retention at 72 ° C. in the final cycle was 2 minutes.
The amplification product was confirmed by separation by 2.0% agarose (SeaKem GTG agarose: manufactured by FMC BioProducts) gel electrophoresis and visualization by ethidium bromide staining, and a fragment of about 0.25 kb was detected. The DNA fragment of interest was recovered from the gel using a QIAQuick Gel Extraction Kit (manufactured by QIAGEN).
The recovered DNA fragment was phosphorylated at the 5 ′ end with T4 polynucleotide kinase (T4 Polynucleotide Kinase: manufactured by Takara Shuzo), and then ligated kit ver. 2 (Takara Shuzo) was used to bind to the SmaI site of E. coli vector pUC19 (Takara Shuzo), and Escherichia coli (DH5α strain) was transformed with the resulting plasmid DNA. The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 50 μg / mL ampicillin and 50 μg / mL X-Gal.
Six clones that formed white colonies on this medium were subjected to liquid culture by a conventional method, and then the plasmid DNA was purified and the nucleotide sequence was determined. Among them, a clone in which the TZ4 promoter was inserted so as to have a transcriptional activity in the reverse direction to the lac promoter of pUC19 was selected and named pUC / TZ4.
Next, DNA prepared by cleaving pUC / TZ4 with restriction enzymes BamHI and PstI
The fragment was mixed with a DNA linker consisting of synthetic DNA (SEQ ID NO: 11 and SEQ ID NO: 12) phosphorylated at the 5 ′ end and having sticky ends against BamHI and PstI at both ends, respectively, and ligation kit ver. After ligation using 2 (Takara Shuzo), Escherichia coli (DH5α strain) was transformed with the obtained plasmid DNA. This DNA linker contains a ribosome binding sequence (AGGAGG) and a cloning site (PacI, NotI, ApaI in this order from the upstream).
Clones that formed white colonies on this medium were subjected to liquid culture by a conventional method, and then plasmid DNA was purified. From the obtained plasmid DNA, one that was cleaved by the restriction enzyme NotI was selected and named pUC / TZ4-SD.
The thus constructed pUC / TZ4-SD was digested with the restriction enzyme PstI, blunted with Klenow fragment, and then cleaved with the restriction enzyme KpnI to obtain a promoter fragment of about 0.3 kb. Separated and collected by 0% agarose gel electrophoresis.

(B) Construction of Coryneform Bacterial Expression Vector pHSG298par-rep described in JP-A-12-93183 is used as a plasmid that can stably replicate independently in coryneform bacteria. This plasmid comprises a replication region and a region having a stabilizing function of the natural plasmid pBY503 possessed by Brevibacterium stachynis IFO12144, a kanamycin resistance gene derived from E. coli vector pHSG298 (Takara Shuzo), and a replication region of E. coli. The DNA prepared by cleaving pHSG298par-rep with the restriction enzyme SseI, blunting the ends with Klenow fragment, and then cleaving with the restriction enzyme KpnI is mixed with the TZ4 promoter fragment prepared in (A) above and ligated. Kit ver. After ligation using 2 (Takara Shuzo), Escherichia coli (DH5α strain) was transformed with the obtained plasmid DNA. The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 50 μg / mL kanamycin.
The strain grown on this medium was subjected to liquid culture by a conventional method, and then the plasmid DNA was purified. From the obtained plasmid DNA, one that was cleaved by the restriction enzyme NotI was selected, and the plasmid was named pTZ4 (construction procedure is shown in FIG. 3).

<Preparation of a strain with enhanced pyruvate carboxylase activity>
(A) Acquisition of pyruvate carboxylase gene The acquisition of pyruvate carboxylase gene derived from Brevibacterium flavum MJ233 strain has been reported for the whole genome sequence using the DNA prepared in (A) of <Example 2> as a template. Corynebacterium glutamicum 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 the ATCC 13032 strain (GenBank Database Accession No. AP005276). Composition of reaction solution: 1 μL of template DNA, 0.2 μL of Pfx DNA polymerase (manufactured by Invitrogen), 1 × concentration attached buffer, 0.3 μM each primer, 1 mM MgSO ₄ , 0.25 μM dNTPs were mixed to make a total volume of 20 μL. Reaction temperature conditions: DNA thermal cycler PTC-200 (manufactured by MJ Research) was used, and a cycle consisting of 94 ° C. for 20 seconds and 68 ° C. for 4 minutes was repeated 35 times. However, the heat retention at 94 ° C. in the first cycle was 1 minute and 20 seconds, and the heat retention at 68 ° C. in the final cycle was 10 minutes. After completion of the PCR reaction, 0.1 μL of Takara Ex Taq (Takara Shuzo) was added, and the mixture was further incubated at 72 ° C. for 30 minutes.
Confirmation of the amplification product was carried out by separating by 0.75% agarose (SeaKem GTG agarose: manufactured by FMC BioProducts) gel electrophoresis and then visualized by ethidium bromide staining, and a fragment of about 3.7 kb was detected. Recovery of the target DNA fragment from the gel is performed using the QIAQuick Gel Extraction Kit (manufactured by QIAGEN)
It was performed using.
The recovered DNA fragment was mixed with a PCR product cloning vector pGEM-TEasy (Promega) and ligation kit ver. After ligation using 2 (Takara Shuzo), 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 PacI and ApaI, an insertion fragment of about 3.7 kb was observed, which was designated as pGEM / MJPC.
The base sequence of the insert fragment of pGEM / MJPC was determined using a base sequencing device (Model 377XL) manufactured by Applied Biosystems and a Big Dye Terminator cycle sequence kit ver3. The resulting DNA base sequence and deduced amino acid sequence are set forth in SEQ ID NO: 15. Since this amino acid sequence shows extremely high homology (99.4%) with that derived from Corynebacterium glutamicum ATCC13032, the insert fragment of pGEM / MJPC is a pyruvate carboxylase gene derived from Brevibacterium flavum MJ233. I determined that there was.

(B) Construction of Pyruvate Carboxylase Activity Enhancement Plasmid A pyruvate carboxylase gene fragment consisting of about 3.7 kb generated by cleaving the pGEM / MJPC prepared in (A) above with restriction enzymes PacI and ApaI It was separated and collected by% agarose gel electrophoresis.
This DNA fragment was mixed with pTZ4 constructed in <Example 3> cleaved with restriction enzymes PacI and ApaI, and ligation kit ver. After ligation using 2 (Takara Shuzo), Escherichia coli (DH5α strain) was transformed with the obtained plasmid DNA. The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 50 μg / mL kanamycin.
The strain grown on this medium was subjected to liquid culture by a conventional method, and then the plasmid DNA was purified. The obtained plasmid DNA was cleaved with restriction enzymes PacI and ApaI, and a plasmid in which an insertion fragment of about 3.7 kb was recognized was selected and named pMJPC1 (FIG. 4).

(C) Transformation into Brevibacterium flavum MJ233 / ΔLDH strain Plasmid DNA for transformation with pMJPC1 capable of replicating in Brevibacterium flavum MJ233 strain is the Escherichia coli (DH5α strain transformed with (B) above. ).
Transformation into Brevibacterium flavum MJ233 / ΔLDH strain was performed by the electric pulse method (Res. Microbiol., Vol. 144, p.181-185, 1993), and the obtained transformant was kanamycin 50 μg / mL. In an LBG agar medium [tryptone 10 g, yeast extract 5 g, NaCl 5 g, glucose 20 g, and agar 15 g dissolved in 1 L of distilled water].
From the strain grown on this medium, liquid culture was performed by a conventional method, and then plasmid DNA was extracted and analyzed by restriction enzyme digestion. As a result, it was confirmed that the strain retained pMJPC1. The strain was named as bacterial flavum MJ233 / PC / ΔLDH strain.

(D) Pyruvate carboxylase enzyme activity The transformant Brevibacterium flavum MJ233 / PC / ΔLDH obtained in (C) above was cultured overnight in 100 ml of A medium containing 2% glucose and 25 mg / L kanamycin. . The obtained cells were collected, washed with 50 ml of 50 mM potassium phosphate buffer (pH 7.5), and resuspended in 20 ml of the same composition. The suspension was crushed with SONIFIER 350 (manufactured by BRANSON), and the centrifuged supernatant was used as a cell-free extract. The pyruvate carboxylase activity was measured using the obtained cell-free extract. Enzyme activity is measured at 100 mM Tris / H
Cl buffer (pH 7.5), 0.1 mg / 10 ml biotin, 5 mM magnesium chloride, 50 mM sodium bicarbonate, 5 mM sodium pyruvate, 5 mM adenosine triphosphate, 0.32 mM NADH, 20 units / 1.5 ml malate dehydrogenase (manufactured by WAKO) , Derived from yeast) and a reaction solution containing an enzyme at 25 ° C. 1 U was defined as the amount of enzyme that catalyzes the decrease of 1 μmol of NADH per minute. The specific activity in the cell-free extract in which pyruvate carboxylase was expressed was 0.2 U / mg protein. It should be noted that pyruvate carboxylase activity was not detected by this activity measurement method in cells obtained by similarly culturing the parent strain MJ233 / ΔLDH strain using A medium.

<Cloning of E. coli fumarate reductase gene>
(A) Escherichia coli DNA extraction Escherichia coli JM109 strain was cultured in 10 mL of LB medium until the late logarithmic growth phase, and genomic DNA was prepared by the method shown in Example 1 (A) above. did.

(B) Cloning of E. coli fumarate reductase gene The E. coli fumarate reductase gene was obtained using the DNA prepared in (A) above as a template and the sequence of the gene of E. coli K12-MG1655 strain for which the entire genome sequence was reported ( This was performed by PCR using synthetic DNA (SEQ ID NO: 17 and SEQ ID NO: 18) designed based on GenBank Database Accession No. U00096).
Composition of reaction solution: 1 μL of template DNA, 0.2 μL of Pfx DNA polymerase (manufactured by Invitrogen), 1 × concentration attached buffer, 0.3 μM each primer, 1 mM MgSO ₄ and 0.25 μM dNTPs were mixed to make a total volume of 20 μL.
Reaction temperature condition: DNA thermal cycler PTC-200 manufactured by MJ Research was used, and a cycle consisting of 94 ° C. for 20 seconds and 68 ° C. for 4 minutes was repeated 35 times. However, the heat retention at 94 ° C. in the first cycle was 1 minute and 20 seconds, and the heat retention at 68 ° C. in the final cycle was 10 minutes. After completion of the PCR reaction, 0.1 μL of Takara Ex Taq (Takara Shuzo) 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, and a fragment of about 3.8 kb was detected. The DNA fragment of interest was recovered from the gel using a QIAQuick Gel Extraction Kit (manufactured by QIAGEN).
The recovered DNA fragment was mixed with PCR product cloning vector pT7Blue T-Vector (manufactured by Novagen), and ligation kit ver. After ligation using 2 (Takara Shuzo), 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 HindIII and KpnI, an insert of about 3.9 kb was observed, which was named pFRD6.0.
The base sequence of the insert fragment of pFRD6.0 was determined using a base sequence decoding device (Model 377XL) manufactured by Applied Biosystems and Big Dye Terminator cycle sequence kit ver3. The resulting DNA base sequence and the deduced amino acid sequence are shown in SEQ ID NOs: 19, 20 to 23.

<Preparation of pyruvate carboxylase and fumarate reductase activity-enhanced strain>
(A) Modification of restriction enzyme site of pMJPC1 After completely cutting pMJPC1 constructed in Example 3 with restriction enzyme KpnI, alkaline phosphatase (Alkaline Phosphatase Calf intestine) is reacted to dephosphorylate the 5 ′ end. A DNA linker consisting of synthetic DNA (SEQ ID NO: 24 and SEQ ID NO: 25) phosphorylated at the 5 ′ end was mixed with the DNA fragment prepared by treatment, and ligation kit ver. After ligation using 2 (Takara Shuzo), Escherichia coli (DH5α strain) was transformed with the obtained plasmid DNA. The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 50 μg / mL kanamycin.
The strain grown on this medium was subjected to liquid culture by a conventional method, and then the plasmid DNA was purified. From the obtained plasmid DNA, one that was cleaved by the restriction enzyme NdeI was selected and named pMJPC1.1.

(B) Construction of pyruvate carboxylase and fumarate reductase activity-enhancing plasmid After cutting pFRD6.0 prepared in Example 5 with restriction enzyme Hind III, the ends were blunted with Klenow fragment, and then with restriction enzyme KpnI. A DNA fragment of about 3.9 kb generated by cleavage was separated and recovered by 0.75% agarose gel electrophoresis. The fragment containing the E. coli fumarate reductase gene prepared in this way was digested with pMJPC1.1 prepared in (A) above with the restriction enzyme NdeI, blunted with Klenow fragment, and then digested with the restriction enzyme KpnI. Mixed with the DNA prepared by ligation kit ver. After ligation using 2 (Takara Shuzo), Escherichia coli (DH5α strain) was transformed with the obtained plasmid DNA. The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 50 μg / mL kanamycin.
The strain grown on this medium was subjected to liquid culture by a conventional method, and then the plasmid DNA was purified. The obtained plasmid DNA was digested with restriction enzyme Hind III to generate fragments of 505, 2132, 2675, 3775, and 4193 bp. Therefore, it was determined that the structure shown in FIG. 5 was correct, and the plasmid was designated as pFRPC1.1. Named.

(C) Transformation of Brevibacterium flavum MJ233 / ΔLDH strain Transformation of Brevibacterium flavum MJ233 / ΔLDH strain using pFRPC1.1 was performed by the method described in (C) of Example 4, A strain confirmed to retain the plasmid pFRPC1.1 was obtained and named Brevibacterium flavum MJ233 / FRD / PC / ΔLDH strain.

(D) Measurement of FRD enzyme activity The transformant Brevibacterium flavum MJ233 / FRD / PC / ΔLDH obtained in (B) above was cultured overnight in 100 ml of A medium containing 2% glucose and 25 mg / L kanamycin. It was. 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. Fumarate reductase activity was measured using the obtained cell-free extract. The enzyme activity was measured by reacting at 25 ° C. in a reaction solution containing 33 mM Tris hydrochloric acid buffer (pH 7.5), 0.1 mM EDTA, 20 mM Na succinate, 2 mM K ₃ Fe (CN) ₆ and the enzyme. . 1 U was defined as the amount of enzyme that catalyzes the decrease of 2 μmol of K ₃ Fe (CN) ₆ per minute. The specific activity of fumarate reductase in the cell-free extract expressing plasmid pFRPC1.1 was 0.02 U / mg protein. Note that the specific activity of the parent strain MJ233 / ΔLDH strain cultured in the same manner using A medium was 0.01 U / mg protein.

<Construction of Coryneform Bacterial Expression Vector Compatible with pTZ4>
(A) Introduction of Streptomycin / Spectinomycin Resistance Gene A coryneform bacterial vector that can coexist with pTZ4 is a plasmid vector pC3 (Plasmid 36 62 (1996)) having a replication region compatible with pTZ4. It was constructed by replacing with the spectinomycin resistance gene.
The streptomycin / spectinomycin resistance gene (E. coli Tn7) was obtained by PCR using the plant transformation binary vector pLAN421 (Plant Cell Reports 10 286 (1991)) having the same gene as a template.
Composition of reaction solution: template DNA 10 ng, Pfx DNA polymerase (manufactured by Invitrogen)
0.2 μL, 1 × concentration attached buffer, 0.3 μM each primer (synthetic DNA shown in SEQ ID NO: 26 and SEQ ID NO: 27), 1 mM MgSO ₄ , and 0.25 μM dNTPs were mixed to make a total volume of 20 μL.
Reaction temperature conditions: DNA thermal cycler PTC-200 (manufactured by MJ Research) was used, and a cycle consisting of 94 ° C. for 20 seconds, 60 ° C. for 20 seconds, and 72 ° C. for 60 seconds was repeated 20 times. However, the heat retention at 94 ° C. in the first cycle was 1 minute and 20 seconds, and the heat retention at 72 ° C. in the final cycle was 2 minutes.
Confirmation of the amplification product was carried out by separation by 0.8% agarose (SeaKem GTG agarose: manufactured by FMCBioProducts) gel electrophoresis and visualization by ethidium bromide staining to detect a 937 bp fragment. Recovery of the target DNA fragment from the gel was performed using QIAQuick Gel Extraction Kit (manufactured by QIAGEN), and the recovered DNA fragment was phosphorylated at the 5 ′ end with T4 polynucleotide kinase (T4 Polynucleotide Kinase: manufactured by Takara Shuzo). .
After cleaving pC3 with restriction enzymes HindIII and NruI, the ends of the DNA fragments whose ends were blunted with Klenow Fragment (manufactured by Takara Shuzo) were mixed with the streptomycin / spectinomycin resistance gene prepared above and ligated. Kit ver. 2 (Takara Shuzo) was used. Escherichia coli (DH5α strain) was transformed with the obtained plasmid DNA, and 50 μg / mL spectinomycin was smeared on an LB agar medium. From the obtained colony, after liquid culture, plasmid DNA was prepared by a conventional method and analyzed by the above PCR using the synthetic DNAs of SEQ ID NO: 26 and SEQ ID NO: 27 as a result. As a result, the streptomycin / spectinomycin resistance gene was inserted. And was named pC3.
Next, the DNA fragment prepared by cleaving pC3 with restriction enzymes BamHI and PvuII was blunted with Klenow fragment, mixed with pBglII linker (Takara Shuzo: CAGATCTG), and ligated kit ver. After ligation using 2 (Takara Shuzo), Escherichia coli (DH5α strain) was transformed with the obtained plasmid DNA and smeared on an LB agar medium containing 50 μg / mL spectinomycin. From the obtained colonies, after liquid culture, plasmid DNA was prepared by a conventional method, and a plasmid cleaved with the restriction enzyme BglII was selected and named pC3.1.

(B) Introduction of Multicloning Site An α-peptide gene containing a LacZ multicloning site was prepared by PCR using Escherichia coli plasmid pT7Blue (Novagen) as a template and synthetic DNAs represented by SEQ ID NO: 28 and SEQ ID NO: 29 as primers.
Composition of reaction solution: template DNA 10 ng, Pfx DNA polymerase (manufactured by Invitrogen)
0.2 μL, 1 × concentration buffer, 0.3 μM each primer, 1 mM MgSO ₄ and 0.25 μM dNTPs were mixed to make a total volume of 20 μL.
Reaction temperature conditions: DNA thermal cycler PTC-200 (manufactured by MJ Research) was used, and a cycle consisting of 94 ° C. for 20 seconds, 60 ° C. for 20 seconds and 72 ° C. for 30 seconds was repeated 20 times. However, the heat retention at 94 ° C. in the first cycle was 1 minute and 20 seconds, and the heat retention at 72 ° C. in the final cycle was 2 minutes.
Confirmation of the amplified product is 1.0% agarose (SeaKem GTG agarose: FM).
The product was separated by gel electrophoresis and visualized by ethidium bromide staining, and a 5777 bp fragment was detected. Recovery of the target DNA fragment from the gel was performed using QIAQuick Gel Extraction Kit (manufactured by QIAGEN), and the recovered DNA fragment was phosphorylated at the 5 ′ end with T4 polynucleotide kinase (T4 Polynucleotide Kinase: manufactured by Takara Shuzo). .
After cleaving pC3.1 with restriction enzymes PstI and HpaI, the DNA fragment whose end was blunted with Klenow Fragment (manufactured by Takara Shuzo) was mixed with the gene fragment of α-peptide prepared above, and ligation kit ver . 2 (Takara Shuzo) was used. Escherichia coli (DH5α strain) was transformed with the obtained plasmid DNA and smeared on an LB agar medium containing 50 μg / mLX-Gal and 50 μg / mL spectinomycin. From the obtained colonies, those exhibiting blue color were selected, and after liquid culture, plasmid DNA was prepared by a conventional method. This plasmid DNA was confirmed to have an EcoRV cleavage site derived from the inserted LacZ multicloning site, and named pC3.14 (construction procedure is shown in FIG. 6).

(A) Cloning of MDH gene and construction of MDH-enhancing plasmid Malate dehydrogenase gene derived from Brevibacterium flavum MJ233 strain containing the promoter region was obtained using the DNA prepared in (A) of Example 2 as a template, and the entire genome It was carried out by PCR using synthetic DNA (SEQ ID NO: 30 and SEQ ID NO: 31) designed on the basis of the gene sequence (GenBank Database Accession No. AP005276) of Corynebacterium glutamicum ATCC13032 strain whose sequence was reported.
These synthetic DNAs each contain a recognition sequence for restriction enzymes EcoRV or SacI. 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 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 3 minutes.
The amplified product was purified using Rapid PCR Purification System (manufactured by Marigen) and then cleaved with restriction enzymes EcoRV and SacI. The resulting DNA fragment of about 1.2 kb was separated by gel electrophoresis with 0.8% agarose (SeaKem GTG agarose: manufactured by FMCBioProducts) and then visualized by staining with ethidium bromide, and Gel Extraction System (manufactured by Marigen). ) Was used to recover from the gel. This DNA fragment was mixed with DNA prepared by cleaving the plasmid pC3.14 described in Example 7 with restriction enzymes EcoRV and SacI, and ligation kit ver. After ligation using 2 (Takara Shuzo), 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 spectinomycin.
The strain grown on this medium was subjected to liquid culture by a conventional method, and then the plasmid DNA was purified. By cleaving the obtained plasmid DNA with the restriction enzyme EcoRI, a DNA fragment with an approximately 1 kb DNA fragment was selected and named pC3-MDH (FIG. 7).
The base sequence of the insert fragment of pC3-MDH was determined using a base sequence decoding device (Model 377XL) manufactured by Applied Biosystems and Big Dye Terminator Cycle Sequence Kit ver3. The resulting DNA base sequence and deduced amino acid sequence are shown in SEQ ID NO: 7.

(B) Production of MDH-enhanced strain Plasmid DNA used for transformation of Brevibacterium flavum MJ233 / FRD / PC / ΔLDH strain was prepared from E. coli strain MJ110 transformed with pC3-MDH.
Transformation of Brevibacterium flavum MJ233 / FRD / PC / ΔLDH strain was performed by the electric pulse method (Res. Microbiol. Vol. 144, p. 181-185, 1993), and the obtained transformant was 25 μg / mL. LBG agar medium containing kanamycin and 10 μg / mL streptomycin [10 g tryptone, 5 g yeast extract, 5 g NaCl, 20 g glucose, and 15 g agar dissolved in 1 L distilled water] was smeared. The strain grown on this medium was named Brevibacterium flavum MJ233 / MDH / FRD / PC / ΔLDH.

(C) Measurement of MDH activity The transformant obtained in (B) above was cultured overnight in 100 ml of A medium containing 2% glucose, 25 mg / L kanamycin and 10 mg / L streptomycin. After harvesting the resulting culture broth 10 ml, was washed twice with 50 mM Hepes / NaOH buffer solution (pH 7.5) 10 ml containing MgCl ₂ of 5mM, 50 mM Hepes / NaOH containing MgCl ₂ of glycerol and 5mM of 4.5M It was resuspended in 2 ml of buffer solution (pH 7.5). The suspension was crushed with a bioraptor (manufactured by Cosmo Bio) and centrifuged at 10,000 g × 15 minutes to separate the supernatant. The obtained supernatant was further subjected to ultracentrifugation at 40,000 rpm × 60 min, and malate dehydrogenase activity was measured using the supernatant after ultracentrifugation.
The enzyme activity was measured by reacting at 25 ° C. in a reaction solution containing 100 mM Tris / HCl buffer (pH 7.5), 5 mM MgCl ₂ , 5 mM oxalacetic acid, 0.32 mM NADH, and enzyme. 1 U was defined as the amount of enzyme that catalyzes the decrease of 1 μmol of NADH per minute.
The MDH specific activity of Brevibacterium flavum MJ233 / MDH / FRD / PC / ΔLDH according to the above measurement method was 20.39 U / mg-protein. In addition, MDH activity is 1.22U / mg-protein in the same cell culture of Brevibacterium flavum MJ233 / FRD / PC / ΔLDH, which is the parent strain, using A medium containing 2% glucose and 25 mg / L kanamycin. Thus, it was confirmed that MDH activity increased about 17 times in the enhanced strain.

<Bacterial cell reaction (magnesium carbonate neutralization reaction)>
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 8 (B) was added. / MDH / FRD / PC / ΔLDH strain was inoculated and seed-cultured at 30 ° C. for 24 hours. However, when the target strain Brevibacterium flavum MJ233 / FRD / PC / ΔLDH prepared in Example 6 (B) was cultured, the addition of streptomycin was excluded.
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, and distilled water: 1000 mL) at OD660 The suspension was suspended so that its absorbance was 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 20% carbon dioxide gas, 80% nitrogen atmosphere The reaction was allowed to proceed at 35 ° C. for 18 hours. In this reaction condition, glucose in the medium remained.
After the reaction, the mixture was centrifuged under the conditions described above, and the succinic acid concentration of the supernatant was analyzed. As a result, in the case of Brevibacterium flavum MJ233 / FRD / PC / ΔLDH, 83.4 g / L, Brevibacterium flavum MJ233 / The MDH / FRD / PC / ΔLDH strain was 97.6 g / L, and it was shown that succinic acid productivity was improved by about 20% by enhancing MDH.

<Bacteria reaction (neutralization of ammonium hydrogen carbonate)>
Bacterial cells cultured and collected according to the method shown in Example 9 were prepared by adding 0.5 ml of a cell suspension prepared with an OD660 absorbance of 40 to a substrate solution (glucose: 40 g, ammonium bicarbonate: 63 g). 0.5 mL of distilled water: 1000 mL) was added, and the mixture was reacted at 35 ° C. for 3 hours under 20% carbon dioxide gas and 80% nitrogen atmosphere. In this reaction condition, glucose in the medium remained.
After the reaction, the mixture was centrifuged under the conditions described above, and the succinic acid concentration in the supernatant was analyzed. As a result, in the Brevibacterium flavum MJ233 / FRD / PC / ΔLDH strain, 13.1 g / L, Brevibacterium flavum MJ233 / In the MDH / FRD / PC / ΔLDH strain, it was 14.9 g / L, and it was shown that enhancement of MDH is effective for improving succinic acid productivity in neutralization with ammonium bicarbonate as well as neutralization with magnesium carbonate. It was.

The figure which shows construction of plasmid pKMB1. The figure which shows the construction of plasmid pKMB1 / ΔLDH. The figure which shows construction of plasmid pTZ4. The figure which shows construction of plasmid pMJPC1. The figure which shows construction of plasmid pFRPC1.1. The figure which shows construction of plasmid pC3.14. The figure which shows construction of plasmid pC3-MDH.

Claims (8)

A bacterium modified to enhance malate dehydrogenase activity 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 to produce succinic acid, A method for producing succinic acid, comprising recovering the succinic acid. The method according to claim 1, wherein the bacterium is any bacterium selected from the group consisting of coryneform bacteria, Bacillus bacteria, and Rhizobium bacteria. The method according to claim 1 or 2, wherein the bacterium is a bacterium further modified so that lactate dehydrogenase activity is reduced to 10% or less compared to an unmodified strain. The method according to any one of claims 1 to 3, wherein the bacterium is a bacterium further modified so that pyruvate carboxylase activity is enhanced. The method according to any one of claims 1 to 4, wherein the bacterium is a bacterium further modified so that fumarate reductase activity is enhanced. The method according to claim 1, wherein the organic raw material is allowed to act in an anaerobic atmosphere. The method according to any one of claims 1 to 6, wherein the organic raw material is glucose. The manufacturing method of a succinic-acid containing polymer including the process of manufacturing a succinic acid by the method as described in any one of Claims 1-7, and the process of polymerizing the obtained succinic acid.

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