JP4575086B2 - Method for producing succinic acid - Google Patents

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 (see, for example, Patent Document 5), but the bacterium with enhanced activity of fumarate reductase. 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-mentioned problems, the present inventor contains a bacterium modified so that fumarate reductase is enhanced or a processed product thereof containing carbonate ion, bicarbonate ion, or carbon dioxide gas. It has been found that by acting on the organic raw material in the reaction solution, the consumption rate of the organic raw material, the generation rate of succinic acid, or the yield is increased, and the present invention has been completed.

That is, according to the present invention, the following inventions are provided.
(1) Succinic acid by allowing a bacterium modified to enhance fumarate reductase activity or a processed product of the bacterium to react with an organic raw material in a reaction solution containing carbonate ion, bicarbonate ion or carbon dioxide gas And producing the succinic acid, and collecting 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 modified so that the fumarate reductase activity is enhanced by using a succinate dehydrogenase gene derived from a coryneform bacterium.
(4) The method according to (1) or (2), wherein the bacterium is a bacterium modified so that fumarate reductase activity is enhanced by using a fumarate reductase gene derived from E. coli.
(5) The method according to any one of (1) to (4), wherein the bacterium is a bacterium further modified so that lactate dehydrogenase activity is reduced to 10% or less compared to an unmodified strain.
(6) The method according to any one of (1) to (5), wherein the bacterium is further modified so that pyruvate carboxylase activity is enhanced.
(7) The method according to any one of (1) to (6), wherein the organic raw material is allowed to act in an anaerobic atmosphere.
(8) The method according to any one of (1) to (7), wherein the organic raw material is glucose.
(9) A method for producing a succinic acid-containing polymer, comprising a step of producing succinic acid by any one of methods (1) to (8) 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 fumarate reductase activity is enhanced. Here, “fumarate reductase activity” refers to the activity of catalyzing the reaction of reducing fumaric acid to convert it to succinic acid, and “fumarate reductase activity is enhanced” refers to wild strains or fumaric acid reductase activity. It means that the fumarate reductase activity is enhanced as compared with the reductase non-modified strain. The fumarate reductase activity can be measured by a method for measuring a decrease in K ₃ Fe (CN) ₆ as described later. E. coli fumarate reductase is the enzyme responsible for the reverse reaction of succinate dehydrogenase that acts in the forward direction of the TCA cycle, but is involved in fumarate respiration under anaerobic conditions, and the transcription level under aerobic conditions. (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 that

  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. In addition, a gene encoding succinate dehydrogenase may encode a protein having fumarate reductase activity simultaneously with succinate dehydrogenase activity. Therefore, the expression level of a gene encoding a protein having both fumarate reductase activity and succinate dehydrogenase activity may be increased. For example, succinate dehydrogenase of coryneform bacteria can catalyze a reaction that produces succinate from fumaric acid, which is the reverse reaction. Succinate dehydrogenase activity can be measured by the method of Arkrell, B.A.C et al. (Meth Enzymol. 53, 466-483).

The parent strain of the bacterium that can be used in the present invention is not particularly limited as long as it has the ability to produce succinic acid, but is preferably a coryneform bacterium, a Bacillus bacterium, or a Rhizobium bacterium, and more preferably a coryneform bacterium. Examples of coryneform bacteria include microorganisms belonging to the genus Corynebacterium, microorganisms belonging to the genus Brevibacterium, or microorganisms belonging to the genus Arthrobacter, and among these, those belonging to the genus Corynebacterium or Brevibacterium And more preferably, Corynebacterium glutamicum, Brevibacterium flavum, Brevibacterium amniagenes or Brementac fermentum bacterium. And microorganisms belonging to

  Particularly preferred specific examples of the parent strains of the bacteria include Brevibacterium flavum MJ-233 (FERM BP-1497), MJ-233 AB-41 (FERM BP-1498), Brevibacterium ammoniagenes ATCC6872, Coryne And bacteria glutamicum ATCC 31831 and Brevibacterium lactofermentum ATCC 13869. Brevibacterium flavum is currently 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.

  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 genetically modified strain may be the same genus species as the parent strain or may be different from the genus species as long as it is a transformable microorganism. Bacteria are preferred as hosts.

When the fumarate reductase (FRD) gene is used for modification so that the fumarate reductase activity is enhanced, the gene that can be used is not particularly limited as long as it encodes a protein having fumarate reductase activity. For example, SEQ ID NO: 19 And a gene derived from Escherichia coli having the base sequence shown in FIG. This gene encodes four subunits (frdA, frdB, frdC and frdD; SEQ ID NOs: 20 to 23) constituting fumarate reductase (base numbers 440 to 2245, 2241 to 2975, and 2986 to 3381 of SEQ ID NO: 19). , And 3392 to 3751), respectively. The full length of this gene may be introduced into bacteria or may be introduced for each subunit gene. As long as each subunit gene encodes a subunit protein capable of forming a complex having FRD activity, DNA that hybridizes with the DNA having the base sequence under stringent conditions, or 90% with the base sequence It may be a homologue such as DNA having a homology of 95% or more, more preferably 99% or more, more preferably 99% or more. 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. Among such FRD gene homologs, those encoding a protein in which the amino acid corresponding to the 17th amino acid of the FRD B subunit (frdB) (SEQ ID NO: 21) is lysine are preferred. The gene having the nucleotide sequence shown in SEQ ID NO: 19 or a homologue thereof can be obtained by PCR or hybridization.
Further, if necessary, a mutation such that the amino acid corresponding to the 17th amino acid of frdB becomes lysine can be introduced by a known method.

Alternatively, a gene encoding a protein having both succinate dehydrogenase and fumarate reductase activities may be used. For example, the sdh gene derived from coryneform bacteria having the base sequence shown in SEQ ID NO: 28 can be mentioned. This gene is an operon gene containing genes (base numbers 1153 to 3171, 3174 to 3920 and 363 to 1133 of SEQ ID NO: 28) encoding the three subunits (sdhA, sdhB and sdhC) constituting succinate dehydrogenase, respectively. is there. The sdh operon of Corynebacterium glutamicum is described in Genebank accession No. NCgl0359 (sdhC) NCgl0360 (sdhA) NCgl0361 (sdhB).
The full length of this gene may be introduced into bacteria or may be introduced for each subunit gene. As long as each subunit gene encodes a subunit protein capable of forming a complex having FRD activity, DNA that hybridizes with the DNA having the base sequence under stringent conditions, or 90% with the base sequence It may be a homologue such as DNA having a homology of 95% or more, more preferably 99% or more, more preferably 99% or more. 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, as long as it has fumarate reductase activity, in the amino acid sequence shown to sequence number 20-23 and sequence number 29-31, the protein which has an amino acid sequence including substitution, deletion, insertion, or addition of one or several amino acids It may be a gene encoding. Here, several means, for example, 2 to 20, preferably 2 to 10, more preferably 2 to 5.

  In addition, FRD genes derived from bacteria other than E. coli and coryneform bacteria, or from other microorganisms or animals and plants can also be used. The FRD gene derived from a microorganism or animal or plant was isolated from a chromosome of a microorganism, animal or plant, etc., and the nucleotide sequence was determined based on a gene having a base sequence already determined, a gene encoding a protein having FRD activity based on homology, etc. 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.

When a coryneform bacterium is used, a DNA fragment containing the FRD gene is introduced 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 the coryneform bacterium. A recombinant plasmid capable of enhancing FRD expression can be obtained. The plasmid vector capable of introducing the FRD gene into the coryneform bacterium is not particularly limited as long as it contains at least a gene that controls the replication growth 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; can be exemplified pVK7 like described in JP-A-10-215883. Note that, as described above, if the activity of fumarate reductase is excessively enhanced, it is considered that the growth of the bacterial cells is deteriorated. Therefore, by selecting a plasmid having an appropriate copy number, the growth of the bacterial cells is not inhibited. It is preferable to adjust the expression level of the FRD gene to an extent. The enhancement of FRD activity can also be carried out by high expression by introducing, replacing, or amplifying the FRD gene on the chromosome by a known homologous recombination method.
Further, when the FRD gene has an operon structure, it can be achieved by introducing a mutation into the promoter region controlling its expression as described in WO0018935 in addition to the above method.

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

  In this reaction, it is more effective to use a bacterial strain modified so as to reduce lactate dehydrogenase activity in addition to enhancement of fumarate reductase 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 (Schafer, A. et al. Gene 145 (1994) 69-73) and the like. The coryneform bacterium with reduced lactate dehydrogenase activity and enhanced expression of the FRD gene of the present invention, for example, as in Example 2 described later, produces a bacterium with a disrupted LDH gene and uses the bacterium as an FRD gene. Can be obtained by transformation with a recombinant vector containing However, either the modification operation for reducing LDH activity or the modification operation for enhancing FRD activity may be performed first.

  In this reaction, in addition to the enhancement of fumarate reductase activity, a bacterium modified so as to enhance the activity of pyruvate carboxylase may be used. “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. Coryneform bacteria with enhanced expression of fumarate reductase and pyruvate carboxylase were prepared by using the fumarate reductase (FRD) gene and pyruvate carboxylase (PC) gene in the same manner as described in JP-A-11-196888. It can be produced by high expression in coryneform bacteria.

  The PC gene used in the method of the present invention is a gene whose base sequence has already been determined, or a DNA fragment encoding a protein having PC activity by the method shown below from a chromosome of a microorganism, animal or plant, etc. Those isolated and sequenced can be used. 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 specification, the size of the cleaved DNA fragment and the size of the plasmid can be obtained by cleaving Escherichia coli lambda phage DNA with restriction enzyme HindIII when using agarose gel electrophoresis. Based on a standard line drawn by the migration distance of DNA fragments of known molecular weight on the same agarose gel, and when polyacrylamide gel electrophoresis is used, DNA of Escherichia coli Phi-X 174 phage (φX174 phage) The size of each DNA fragment of the cleaved DNA fragment or plasmid can be calculated based on the standard line drawn by the migration distance on the same polyacrylamide gel of a DNA fragment of known molecular weight obtained by cleaving with restriction enzyme HaeIII it can. In determining the size of each DNA fragment, the result obtained by 1% agarose gel electrophoresis is adopted for the size of a fragment of 1 kb or more, and the size of a fragment of about 0.1 kb to less than 1 kb is used. Results obtained by 4% polyacrylamide gel electrophoresis were employed.

  In the present invention, the DNA fragment containing the PC gene used for enhancing the PC activity is not only isolated from Corynebacterium glutamicum chromosomal DNA, but also a DNA synthesizer commonly used, 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, bacteria other than Corynebacterium glutamicum, or PC genes derived from other microorganisms or animals and plants can also be used. In particular, the following PC genes derived from microorganisms or animals and plants are known in sequence (documents are shown below), and the ORF portion is amplified by hybridization as described above or by PCR. Can be obtained.

Human [Biochem.Biophys.Res.Comm., 202, 1009-1014, (1994)]
Mouse [Proc.Natl.Acad.Sci.USA., 90, 1766-1779, (1993)]
Rat [GENE, 165, 331-332, (1995)]
Yeast; Saccharomyces cerevisiae
[Mol.Gen.Genet., 229, 307-315, (1991)]
Schizosaccharomyces pombe
[DDBJ Accession No .; D78170]
Bacillus stearothermophilus
[GENE, 191, 47-50, (1997)]
Rhizobium etli
[J. Bacteriol., 178, 5960-5970, (1996)]
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 expressed PC gene product, pyruvate carboxylase (SEQ ID NO: 16), was carried out directly from the transformant by the crude enzyme solution & Magasanik method [J. Bacteriol., 158, 55-62, (1984)]. This can be confirmed by measuring the PC activity and comparing it 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.

  The plasmid vector into which the PC gene can be introduced is not particularly limited as long as it contains at least a gene that controls the replication growth function in coryneform bacteria. Specific examples thereof include, for example, a plasmid pCRY30 described in JP-A-3-210184; plasmids pCRY21, pCRY2KE, pCRY2KX described in JP-A-2-72876 and US Pat. No. 5,185,262. pCRY31, pCRY3KE and pCRY3KX; plasmids pCRY2 and pCRY3 described in JP-A-1-191686; pAM330 described in JP-A-58-67679; pHM1519 described in JP-A-58-77895; PAJ655, pAJ611 and pAJ1844 described in JP-A-58-192900; pCG1 described in JP-A-57-134500; pCG2 described in JP-A-58-35197; described in JP-A-57-183799 PCG And pCG11, etc. can be mentioned.

  Among them, plasmid vectors used in the host-vector system of coryneform bacteria have a gene responsible for the replication and replication function of the plasmid in coryneform bacteria and a gene responsible for the plasmid stabilization function in coryneform bacteria. For example, plasmids pCRY30, pCRY21, pCRY2KE, pCRY2KX, pCRY31, pCRY3KE and pCRY3KX are preferably used.

A recombinant vector obtained by inserting the PC gene into 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 FRD gene, a coryneform bacterium with enhanced expression of the PC gene and the FRD gene can be obtained. Either the FRD 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).

  Furthermore, in the present invention, the use of a bacterium modified so that the activities of fumarate reductase and pyruvate carboxylase are enhanced and the lactate dehydrogenase activity is reduced is particularly effective for the production of succinic acid. Such a bacterium can be obtained, for example, by transforming a coryneform bacterium having a disrupted LDH gene with a recombinant vector containing a PC gene and an FRD 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 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 after culturing the cells 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 microorganism to produce succinic acid, but is usually galactose, lactose, glucose, fructose, glycerol, sucrose, sucrose. , 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 saccharide | sugar are also used. These fermentable carbohydrates can be used alone or in combination. The use concentration of the organic raw material is not particularly limited, but it is advantageous to make it as high as possible within a range not inhibiting the production of succinic acid, and is usually 5 to 30% (W / V), preferably 10 to 20%. The reaction is carried out within the range of (W / V). Further, additional addition of organic raw materials may be performed in accordance with the decrease of the organic raw materials as the reaction proceeds.

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

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

  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-described 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 can also be supplied 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 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.

  When culturing bacteria, it is necessary to supply oxygen by aeration and agitation. On the other hand, the reaction for producing succinic acid may be carried out with aeration and stirring, but may be carried out in an anaerobic atmosphere without aeration and without supplying oxygen. The anaerobic atmosphere referred to here means that the reaction is performed while the dissolved oxygen concentration in the solution is kept low. In this case, the dissolved oxygen concentration is desirably 0 to 2 ppm, preferably 0 to 1 ppm, more preferably 0 to 0.5 ppm. As a method therefor, for example, a method in which the container is sealed and reacted without aeration, an inert gas such as nitrogen gas is supplied and reacted, a carbon dioxide-containing inert gas is aerated, and the like can be used. .

  Succinic acid accumulated in the reaction solution (culture solution) can be 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, 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. 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). Reaction solution composition:
Template DNA 1 μL, Taq DNA polymerase (Takara Shuzo) 0.2 μL, 1 × concentration buffer, 0.2 μM each primer, 0.25 μM dNTPs were mixed to make a total volume of 20 μL.

  Reaction temperature conditions: DNA thermal cycler PTC-200 (manufactured by MJ Research) was used, and a cycle consisting of 94 ° C. for 20 seconds, 55 ° C. for 20 seconds and 72 ° C. for 1 minute was repeated 30 times. However, the heat retention at 94 ° C. in the first cycle was 1 minute 20 seconds, and the heat retention at 72 ° C. in the final cycle was 5 minutes.

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

The recovered DNA fragment was mixed with a PCR product cloning vector pGEM-TEasy (Promega) and ligation kit ver. After ligation using 2 (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.

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

<Construction of Coryneform Bacterial Expression Vector>
(A) Preparation of Promoter Fragment for Coryneform Bacteria Utilizing the DNA fragment (hereinafter referred to as TZ4 promoter) described in SEQ ID NO: 4 of JP-A-7-95891 reported to have a strong promoter activity in coryneform bacteria It was decided. The promoter fragment was obtained by using a synthetic DNA designed based on the sequence described in SEQ ID NO: 4 of JP-A-7-95891 using Brevibacterium flavum MJ233 genomic DNA prepared in Example 2 (A) as a template. It was carried out by PCR using SEQ ID NO: 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 and 20 seconds, and the heat retention at 72 ° C. in the final cycle was 2 minutes.

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

The recovered DNA fragment was phosphorylated at the 5 ′ end with T4 polynucleotide kinase (T4 Polynucleotide Kinase: manufactured by Takara 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, a DNA fragment prepared by cleaving pUC / TZ4 with restriction enzymes BamHI and PstI consists of synthetic DNA (SEQ ID NO: 11 and SEQ ID NO: 12) phosphorylated at the 5 ′ end, and BamHI and A DNA linker having a sticky end against PstI was mixed, and ligation kit ver. After ligation using 2 (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. 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 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 strain,
It was determined that the insert of pGEM / MJPC was a pyruvate carboxylase gene derived from Brevibacterium flavum MJ233 strain.

(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 was measured at 100 mM Tris / HCl buffer (pH 7.5), 0.1 mg / 10 ml biotin, 5 mM magnesium chloride, 50 mM sodium bicarbonate, 5 mM sodium pyruvate, 5 mM adenosine triphosphate, 0.32 mM NADH, 20 units / The reaction was carried out at 25 ° C. in a reaction solution containing 1.5 ml malate dehydrogenase (manufactured by WAKO, derived from yeast) and the enzyme. 1 U was defined as the amount of enzyme that catalyzes the decrease of 1 μmol of NADH per minute. The specific activity 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.

(B) Transformation of Brevibacterium flavum MJ233 / ΔLDH strain Transformation of Brevibacterium flavum MJ233 / ΔLDH strain using pFRPC1.1 was carried out 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.

(C) 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. In addition, the specific activity was 0.01 U / mg protein in the bacterial cells obtained by similarly culturing the parent strain MJ233 / ΔLDH strain using A medium.

<Cloning of succinate dehydrogenase gene from coryneform bacteria>
Acquisition of the succinate dehydrogenase gene of Brevibacterium flavum MJ233 strain (hereinafter referred to as sdhC, sdhA, and sdhB) was obtained from Corynebacterium glutamicum ATCC13032 (GenBank Database Accession No.NC_003450) Synthetic DNA designed with reference to the nucleotide sequence of the gene was used as a primer for PCR. Specifically, DNA containing each gene of sdhC-sdhA-sdhB that forms an operon using chromosomal DNA of Brevibacterium flavum MJ233 as a template and synthetic DNA having the sequences of SEQ ID NOS: 26 and 27 as primers A fragment (SEQ ID NO: 28) was obtained by PCR. The PCR reaction was performed using KOD-PLUS- (Toyobo Co., Ltd.), followed by one cycle of incubation at 94 ° C for 5 minutes, followed by denaturation at 94 ° C for 15 seconds, association at 56 ° C for 30 seconds, and extension 72
A cycle consisting of 4 minutes at 25 ° C. was repeated 25 times. The generated PCR product was purified by a conventional method and then digested with SmaI. This DNA fragment was mixed with pVK7 (JP-A-10-215883) digested with SalI and then blunted with DNA branding Kit (manufactured by Takara Bio Inc.), and ligation Kit ver. Connected. Escherichia coli JM109 competent cells (manufactured by Takara Bio Inc.) were transformed with this plasmid, applied to LB medium containing 25 μg / ml of kanamycin (hereinafter abbreviated as Km), and cultured overnight. Thereafter, the emerged colonies were picked up, and single colonies were separated to obtain transformants. A plasmid was extracted from the obtained transformant, and the plasmid into which the target PCR product was inserted was named pVKSDH. The construction process of pVKSDH is shown in FIG.

<Construction of pyruvate carboxylase and succinate dehydrogenase activity enhancing plasmid>
A DNA fragment containing the three genes sdhC, sdhA and sdhB obtained by digesting pVKSDH constructed in Example 7 with XbaI and Sse8387I and a fragment obtained by digesting pHSG399 (Takara Bio Inc.) with XbaI and Sse8387I The DNA ligation kit ver.2 (manufactured by Takara Bio Inc.) was used for ligation. Escherichia coli JM109 competent cell (Takara Bio Inc.) is transformed with this DNA, and LB medium containing chloramphenicol (hereinafter abbreviated as Cm) 25 μg / ml, X-Gal 50 μg / ml, IPTG 1 mM And incubated overnight. Thereafter, the appeared white colonies were picked up, and single colonies were separated to obtain transformants. A plasmid was extracted from the obtained transformant, and a plasmid into which a DNA fragment containing sdhC, sdhA, and sdhB had been inserted was named pHSGSDH (FIG. 7).
On the other hand, by digesting pFRPC1.1, which is a plasmid carrying the fumarate reductase gene derived from Escherichia coli and the pyc gene derived from Brevibacterium flavum described in Example 6, with KpnI and NdeI, an Escherichia coli derived fuma The remaining region excluding the rate reductase gene can be recovered as one fragment. Therefore, pFRPC1.1 was digested with KpnI and NdeI, then blunt-ended, and the remaining partial DNA fragment excluding the fumarate reductase gene was recovered. The recovered DNA fragment was ligated after blunting a DNA fragment containing sdhC, sdhA, and sdhB obtained by digesting pHSGSDH with XbaI and Sse8387I. Using this DNA, Escherichia coli JM109 competent cells (manufactured by Takara Bio Inc.) were transformed, applied to an LB medium containing 25 μg / ml of Km, and cultured overnight. Thereafter, the emerged colonies were picked up, and single colonies were separated to obtain transformants. A plasmid was extracted from the resulting transformant, and the one from which the frdA, frdB, frdC, frdD genes were removed and the DNA fragment containing sdhC, sdhA, sdhB was inserted was named pSDHPC (FIG. 8).

<Construction of pyruvate carboxylase and succinate dehydrogenase-enhanced strain>
PVKSDH and pSDHPC obtained in Example 7 and Example 8 can autonomously replicate in cells of coryneform bacteria. Therefore, a coryneform bacterium was transformed with this plasmid to obtain a transformant. The MJ233 / ΔLDH strain constructed in Example 2 was transformed with pVKSDH and pSDHPC using the electric pulse method, and CM-Dex medium (glucose 5 g / L, polypeptone 10 g / L, yeast extract containing kanamycin 25 μg / ml). _{10g / L, KH 2 PO 4} 1g / L, MgSO 4 · 7H 2 O 0.4g / L, FeSO 4 · 7H 2 O 0.01g / L, MnSO 4 · 7H 2 O 0.01g / L, urea 3 g / L, Soybean hydrolyzate (1.2 g / L, pH 7.5 (KOH), and agar 15 g / L) was applied and cultured at 31.5 ° C. for about 24 hours. A strain grown on this medium is a strain into which the plasmid has been introduced. The transformants thus obtained were designated as MJ233 / SDH / ΔLDH and MJ233 / SDH / PC / ΔLDH, respectively. In order to prepare a control strain, plasmid pVK7 and plasmid pMJPC1 constructed in Example 4 were also introduced into the MJ233 / ΔLDH strain using the above method. The transformants thus obtained were designated as MJ233 / ΔLDH / pVK7 and MJ233 / PC / ΔLDH, respectively.

<Bacteria reaction (neutralization of ammonium carbonate, microaerobic reaction)>
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 heptahydrate: 20 mg, sulfuric acid Manganese hydrate: 20 mg, D-biotin: 200 μg, thiamine hydrochloride: 200 μg, yeast extract: 1 g, casamino acid: 1 g, and distilled water: 1000 mL of a medium of 100 mL is placed in a 500 mL Erlenmeyer flask at 120 ° C. for 20 minutes. Heat sterilized. This was cooled to room temperature, 4 mL of 50% glucose aqueous solution sterilized in advance and 50 μL of 5% kanamycin aqueous solution filtered aseptically were added, and the Brevibacterium flavum MJ233 / FRD / PC / ΔLDH strain prepared in Example 6 (B) Was seeded and cultured at 30 ° C. for 24 hours. Urea: 12 g, ammonium sulfate: 42 g, monopotassium phosphate: 1.5 g, dipotassium phosphate 1.5 g, magnesium sulfate heptahydrate: 1.5 g, ferrous sulfate heptahydrate: 60 mg, sulfuric acid Manganese hydrate: 60 mg, D-biotin: 600 μg, thiamine hydrochloride: 600 μg, yeast extract 3 g, casamino acid 3 g, antifoaming agent (Adecanol LG294: manufactured by Asahi Denka): 1 mL and distilled water: 2500 mL of 5 mL of medium The mixture was placed in a fermenter and sterilized by heating at 120 ° C. for 20 minutes. After cooling this to room temperature, 500 mL of a 12% aqueous glucose solution sterilized in advance was added, and the whole amount of the above-mentioned seed culture solution was added thereto, and the mixture was kept at 30 ° C. Main culture was performed at aeration of 500 mL / min and stirring at 500 rpm. Glucose was almost consumed after 12 hours.

  Magnesium sulfate heptahydrate: 0.2 g, Ferrous sulfate heptahydrate: 8 mg, Manganese sulfate / hydrate: 8 mg, D-biotin: 80 μg, Thiamine hydrochloride: 80 μg, Antifoam (Adecanol LG294 : Manufactured by Asahi Denka): 1 ml and distilled water: 200 mL of a medium was placed in a 500 mL Erlenmeyer flask and sterilized by heating at 120 ° C. for 20 minutes. After cooling to room temperature, the culture solution obtained by the above main culture was added to the cells collected by centrifugation at 8000 rpm for 5 minutes. D. It was resuspended so that (660 nm) was 60. 200 mL of this suspension and 200 mL of a 20% glucose solution sterilized in advance were mixed in a 1 L jar fermenter and kept at 35 ° C. The pH was kept at 7.6 using 2M ammonium carbonate, and the reaction was carried out with aeration at 100 mL / min and stirring at 400 rpm.

  About 20 hours after the start of the reaction, glucose was almost consumed. The sugar consumption rate was 5.00 g / L / h, the succinic acid production rate was 2.66 g / L / h, and the yield was 70.1%. On the other hand, when the Brevibacterium flavum MJ233 / PC / ΔLDH strain prepared in Example 4 (C) was reacted in the same manner as described above, the sugar consumption rate was 4.74 g / L / h, and succinic acid production The rate was 2.13 g / L / h and the yield was 58.7%.

<Bacteria reaction (neutralization of ammonium carbonate, anaerobic reaction)>
A reaction suspension was prepared in the same manner as in Example 10 above, the pH was maintained at 7.6 using 2M ammonium carbonate, and the reaction was carried out with stirring at 200 revolutions per minute without aeration. In about 40 hours after the start of the reaction, glucose was almost consumed. The sugar consumption rate was 2.50 g / L / h, the succinic acid production rate was 1.35 g / L / h, and the yield was 78.4%. On the other hand, when the Brevibacterium flavum MJ233 / PC / ΔLDH strain prepared in Example 4 (C) was reacted in the same manner as described above, the sugar consumption rate was 2.38 g / L / h, and succinic acid production The rate was 1.21 g / L / h and the yield was 74.4%.

<Bacteria reaction (sodium carbonate neutralization, microaerobic reaction)>
A suspension for reaction was prepared in the same manner as in Example 10, and the reaction was carried out in the same manner while maintaining the pH at 7.6 using 2M sodium carbonate. About 28 hours after the start of the reaction, glucose was almost consumed. The sugar consumption rate was 3.60 g / L / h, the succinic acid production rate was 2.27 g / L / h, and the yield was 82.8%. On the other hand, when the Brevibacterium flavum MJ233 / PC / ΔLDH strain prepared in Example 4 (C) was reacted in the same manner as above, the sugar consumption rate was 2.97 g / L / h, and succinic acid production The rate was 1.97 g / L / h and the yield was 88.0%.

<Bacteria reaction (sodium carbonate neutralization, anaerobic reaction)>
A reaction suspension was prepared in the same manner as in Example 10 above, the pH was maintained at 7.6 using 2M ammonium carbonate, and the reaction was carried out with stirring at 200 revolutions per minute without aeration. About 32 hours after the start of the reaction, glucose was almost consumed. The sugar consumption rate was 3.13 g / L / h, the succinic acid production rate was 1.80 g / L / h, and the yield was 97.1%. On the other hand, when the Brevibacterium flavum MJ233 / PC / ΔLDH strain prepared in Example 4 (C) was reacted in the same manner as above, the sugar consumption rate was 2.70 g / L / h, and succinic acid production The rate was 1.57 g / L / h and the yield was 88.6%.

<Bacterial cell reaction (magnesium carbonate neutralization, anaerobic culture)>
The culture for producing succinic acid was performed as follows using the Brevibacterium flavum MJ233 / ΔLDH / pVK7 strain and MJ233 / SDH / Δ LDH strain. Cells of MJ233 / ΔLDH / pVK7 and MJ233 / SDH / ΔLDH obtained by culturing in CM-Dex plate medium (containing kanamycin 25 μg / ml) were seeded in 3 ml of seed medium (glucose 10 g / L, (NH ₄ ) ₂ SO ₄ 2.5 g / L, KH ₂ PO ₄ 0.5 g / L, MgSO ₄・ 7H ₂ O 0.25 g / L, Urea 2 g / L, FeSO ₄・ 7H ₂ O 0.01 g / L, MnSO ₄・ 7H ₂ O 0.01g / L, biotin 50μg / L, VB1 · HCl 100μg / L, protocatechuic acid 15mg / L, CuSO ₄ 0.02mg / L, CaCl ₂ 10mg / L, pH7.0 (KOH)) Incubated in a test tube at 31.5 ° C for about 15 hours. Then, 3 ml of the main medium (glucose 100 g / L, (NH ₄ ) ₂ SO ₄ 5 g / L, KH ₂ PO ₄ 2 g / L, urea 3 g / L, FeSO ₄ · 7H ₂ O 0.01 g / L, Add MnSO ₄・ 7H ₂ O 0.01g / L, biotin 200μg / L, VB1 ・ HCl 200μg / L, MgCO ₃ 71.4g / L, final concentration after addition above, pH6.8 (NaOH)) to prevent aeration Therefore, the succinic acid production culture was performed by sealing with a silicon stopper. The culture was shaken at 31.5 ° C. for about 48 hours and terminated before the sugar in the medium disappeared. After completion of the culture, the accumulated amounts of succinic acid, by-product malic acid and fumaric acid in the culture solution were analyzed by liquid chromatography after the culture solution was appropriately diluted. The column used was two Shim-pack SCR-102H (Simazu) connected in series, and the sample was eluted at 40 ° C. with 5 mM p-toluenesulfonic acid. The eluate was neutralized with 20 mM Bis-Tris aqueous solution containing 5 mM p-toluenesulfonic acid and 100 μM EDTA, and the electrical conductivity was measured with CDD-10AD (Simazu). Fumaric acid was measured. The results at this time are shown in Table 2. In the MJ233 / SDH / ΔLDH strain, the yield was improved by about 4% compared to the MJ233 / ΔLDH / pVK7 strain in which the vector plasmid was introduced into the same host. In addition, malic acid accumulation decreased by 3.6 g / L, and fumaric acid accumulation decreased by 0.5 g / L.

(B) Cultivation Evaluation of SDH and PC Co-Amplification Strains Culture for succinic acid production was performed as follows using Brevibacterium flavum MJ233 / PC / ΔLDH strain and MJ233 / SDH / PC / ΔLDH strain. MJ233 / PC / ΔLDH strain and MJ233 / SDH / PC / ΔLDH strain obtained by culturing in CM-Dex plate medium were cultured in A medium (glucose 20 g / L, (NH ₄ ) ₂ SO ₄ 14 g / L, KH ₂ PO ₄ 0.5 g / L, K ₂ HPO ₄ 0.5 g / L, MgSO ₄・ 7H ₂ O 0.5 g / L, Urea 4 g / L, FeSO ₄・ 7H ₂ O 0.02 g / L, MnSO ₄・ 7H ₂ O 0.02g / L, biotin 200μg / L, VB1 · HCl 200μg / L, casamino acid 1g / L, yeast extract 1g / L) inoculated into 3ml, and aerobic conditions at 31.5 ° C for about 15 hours in a test tube Shaking culture was performed.
Then, the main liquid (glucose 200g / L, sodium sulfite 30g / L
In order to prevent aeration, 3 ml of MgCO _{3 (} 142.8 g / L) was added, and succinic acid production culture was carried out by sealing with a silicon stopper. The culture was shaken at 31.5 ° C. for about 48 hours and terminated before the sugar in the medium disappeared.

After completion of the culture, the accumulated amounts of succinic acid, by-product malic acid and fumaric acid in the culture solution were analyzed by liquid chromatography after the culture solution was appropriately diluted. The column used was two Shim-pack SCR-102H (Simazu) connected in series, and the sample was eluted at 40 ° C. with 5 mM p-toluenesulfonic acid. The eluate was 20 mM containing 5 mM p-toluenesulfonic acid and 100 μM EDTA.
Succinic acid, by-product malic acid and fumaric acid were measured by neutralizing with Bis-Tris aqueous solution and measuring electrical conductivity with CDD-10AD (Simazu). The results at that time are shown in Table 3.

In the MJ233 / SDH / PC / ΔLDH strain, the yield of succinic acid is improved by about 3% compared to MJ233 / PC / ΔLDH, which is a PC-only enhancement strain. / L decreased.
From this result and the result of the above (A), it was shown that the amplification of SDH is effective in improving the yield of succinic acid and reducing by-products of malic acid and fumaric acid during succinic acid production.

The figure which shows the construction procedure and restriction enzyme map of plasmid pKMB1. The figure which shows the construction | assembly procedure of plasmid pKMB1 / (DELTA) LDH. The figure which shows the construction procedure of plasmid pTZ4. The figure which shows the construction procedure of plasmid pMJPC1. The figure which shows the construction procedure of plasmid pFRPC1.1. The figure which shows the construction procedure of plasmid pVKSDH. The figure which shows the construction procedure of plasmid pHSGSDH. The figure which shows the construction procedure of plasmid pSDHPC.

Claims (6)

Transformation with a vector containing a succinate dehydrogenase gene derived from a coryneform bacterium or a fumarate reductase gene derived from E. coli, high expression of the gene on the chromosome by homologous recombination, or mutation in the promoter region of the gene the treated product of the modified coryneform bacterium or the coryneform bacterium to enhance fumaric acid reductase activity by introducing, on an organic raw material in a reaction solution containing carbonate ions or bicarbonate ions or carbon dioxide gas A method for producing succinic acid, characterized in that succinic acid is produced by the action, and the succinic acid is collected. The coryneform bacterium is further modified by replacing the lactate dehydrogenase gene on the chromosome with a mutant gene having a deletion region so that the lactate dehydrogenase activity is reduced to 10% or less compared to the unmodified strain. The method according to claim 1 , wherein the method is a coryneform bacterium. The coryneform bacterium is further modified so that pyruvate carboxylase activity by high expression of the gene is enhanced on chromosome by or homologous recombination transformed with a vector comprising a pyruvate carboxylase gene coryneform characterized in that it is a type bacterium, the method according to claim 1 or 2. The method according to any one of claims 1 to 3 , wherein the organic raw material is allowed to act in an anaerobic atmosphere. The method according to any one of claims 1 to 4 , 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 1-5 , and the process of polymerizing the obtained succinic acid.

Images