JP5602982B2 - 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 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 to produce succinic acid as a stationary cell without aeration of oxygen. Methods are also known (Patent Documents 3 and 4). In this case, it is economical because the amount of organic nitrogen added is small and sufficient growth can be achieved with a simple medium. However, the amount of succinic acid produced, the concentration produced, and the production per cell There was room for improvement, such as speed improvement and simplification of the manufacturing process. In addition, fermentative production of succinic acid using a bacterium having enhanced activity of phosphoenolpyruvate carboxylase has been reported (see, for example, Patent Document 5), but isocitrate lyase, which is an enzyme of the glyoxylate cycle, and The production of succinic acid using bacteria in which malate synthase has been activated has not been reported so far.
US Pat. No. 5,143,834 US Pat. No. 5,504,004 JP-A-11-113588 JP 11-196888 A Japanese Patent Laid-Open No. 11-196887 International Journal of Systematic Bacteriology (1999), 49,207-216

  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 inventors have found that bacteria having enhanced activities of isocitrate lyase and malate synthase, which are key enzymes of the glyoxylate cycle, or treated products thereof, carbonate ions, In order to complete the present invention, it is found that by acting on an organic raw material in a reaction solution containing bicarbonate ion or carbon dioxide gas, the consumption rate of the organic raw material, the generation rate of succinic acid, or the yield is increased. It came.

  That is, according to the present invention, the following inventions are provided.

  (1) A succinic acid obtained by allowing a bacterium having enhanced activities of isocitrate lyase and malate synthase or a processed product of the bacterium to act on an organic raw material in a reaction solution containing carbonate ion, bicarbonate ion or carbon dioxide gas. And producing the succinic acid, and recovering the succinic acid.

(2) The method according to (1), wherein the bacterium is modified so that the activities of isocitrate lyase and malate synthase are enhanced.

  (3) The bacterium is a bacterium modified by placing an isocitrate triase-encoding gene and a malate synthase-encoding gene under the control of a promoter that functions in a medium containing saccharide as a main carbon source. There is a method of (2).

  (4) The method according to any one of (1) to (3), wherein the bacterium is further modified so that lactate dehydrogenase activity is reduced to 10% or less as compared to an unmodified strain.

  (5) The method according to any one of (1) to (4), wherein the bacterium is any bacterium selected from the group consisting of a coryneform bacterium, a Bacillus bacterium, or a Rhizobium bacterium.

  (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 or sucrose.

(8) A method for producing a succinic acid-containing polymer, comprising a step of producing succinic acid by any one of methods (1) to (7) and a step of polymerizing the obtained succinic acid.
The present invention also provides the following inventions.
The gene encoding isocitrate triase and the gene encoding malate synthase are under the control of the fructose-1,6-diphosphate aldolase gene promoter, which is a promoter that functions in a medium containing saccharides as the main carbon source. By placing
Coryneform bacteria modified so that the activities of socitrate lyase and malate synthase are enhanced or a processed product of the coryneform bacteria is treated with carbonate ion, bicarbonate ion or carbon dioxide gas.
A method for producing succinic acid, comprising reacting glucose or sucrose in an anaerobic atmosphere in a reaction solution containing succinic acid to produce succinic acid, and recovering the 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 with enhanced activities of isocitrate lyase and malate synthase. Hereinafter, isocitrate triase and malate synthase may be referred to as ICL and MS, respectively.
"ICL and MS activities are enhanced" means that the activity of these enzymes is enhanced compared to non-modified strains such as wild strains, or normal culture conditions, for example, culture using saccharides as the main carbon source. This includes the case where the activities of these enzymes are enhanced as compared to when cultured under conditions. The activity of ICL and MS is preferably enhanced 1.5 times or more per unit cell weight, more preferably enhanced 2 times or more, compared with the unmodified strain or normal culture conditions.
ICL is an enzyme that catalyzes the reaction of converting isocitrate into succinic acid and glyoxylic acid, and ICL activity can be measured by the method of Reinscheid et al. (J. Bacteriology 176 3474, 1994) as described later.
MS is an enzyme that catalyzes a reaction for producing malic acid from glyoxylic acid and acetyl CoA, and MS activity can be measured by the method of Dixon et al. (Biochemical J. 72 3P, 1959).

The activity of ICL and MS can be enhanced by gene recombination, for example, by increasing the copy number of the gene encoding ICL and the gene encoding MS, or by replacing the promoter of these genes.
On the other hand, ICL and MS are induced by adding acetic acid or fatty acid to the medium, and it is reported that their expression is suppressed at the transcription level when sugars such as glucose are used as the main carbon source. (Gerstmeir R. et al. Journal of Biotechnology 104 (2003) 99-1
twenty two). Therefore, activation of these enzymes can also be performed by adding acetic acid or a fatty acid to the medium and culturing the cells. In this case, it is preferable to add acetic acid or fatty acid to the medium at a concentration of 1 mM to 1 M.

The parent strain of the bacterium that can be used in the present invention is not particularly limited as long as it can produce succinic acid, but is preferably a coryneform bacterium, a Bacillus bacterium, or a Rhizobium bacterium, and more preferably a coryneform bacterium. .
Examples of Bacillus bacteria include Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus, Bacillus stearothermophilus, etc., and Rhizobium genus Examples of bacteria include Rhizobium etli.
Examples of coryneform bacteria include microorganisms belonging to the genus Corynebacterium, microorganisms belonging to the genus Brevibacterium, or microorganisms belonging to the genus Arthrobacter, and among these, those belonging to the genus Corynebacterium or Brevibacterium More preferably, Corynebacterium glutamicum, Brevibacterium flavum, Brevibacterium ammoniagenes or Brevibacterium lactofermentum And microorganisms belonging to

Particularly preferred specific examples of the parent strains of the above bacteria include Brevibacterium flavum MJ-233 (FERM BP-1497), MJ-233 AB-41 (FERM BP-1498), Brevibacterium ammoniagenes ATCC6872, Coryne And bacteria glutamicum ATCC31831 and Brevibacterium lactofermentum ATCC13869. Since 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, the Brevibacterium flavum MJ-233 strain and the mutant MJ-233 AB-41 strain are the Corynebacterium glutamicum MJ-233 strain, respectively. And the same strain as the MJ-233 AB-41 strain.
Brevibacterium flavum MJ-233 was established on April 28, 1975 by the Ministry of International Trade and Industry, Ministry of International Trade and Industry, Microbial Industrial Technology Research Institute (currently the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center) (305-8566 Japan) Deposited as FERM P-3068 at Tsukuba City, Ibaraki Prefecture, 1-chome, 1-chome, 1st 6th), transferred to an international deposit based on the Budapest Treaty on May 1, 1981, and deposited with a deposit number of FERM BP-1497 Has been.

  The bacterium used as a parent strain in the method of the present invention is not only a wild strain, but also a mutant obtained by normal mutation treatment such as UV irradiation or NTG treatment, genetic techniques such as cell fusion or gene recombination. Any strain such as a recombinant strain to be induced may be used. The host of the 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 ICL gene and the MS gene are used for modification so that the ICL activity and the MS activity are enhanced, the gene that can be used is not particularly limited as long as it encodes a protein having the ICL activity or the MS activity. The ICL gene is the nucleotide sequence of nucleotide numbers 471 to 1766 of SEQ ID NO: 9, and the MS gene is a gene derived from Brevibacterium flavum MJ-233 having the nucleotide sequence of nucleotide numbers 468 to 2684 of SEQ ID NO: 13. Can do. In addition, as long as these genes encode proteins having ICL activity or MS activity, 90% or more, preferably DNA that hybridizes with DNA having the above base sequence under stringent conditions, or the above base sequence. May be a homologue such as DNA having a homology of 95% 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.
Further, the ICL gene may be a DNA encoding a protein having 90% or more, preferably 95% or more, more preferably 99% or more homology with the amino acid sequence of SEQ ID NO: 10 and having ICL activity. The MS gene may be DNA encoding a protein having MS activity with 90% or more, preferably 95% or more, more preferably 99% or more homology with the amino acid sequence of SEQ ID NO: 14.

  In addition, ICL genes and MS genes derived from bacteria other than Brevibacterium flavum MJ-233, or from other microorganisms or animals and plants can also be used. ICL genes and MS genes derived from microorganisms or animals and plants are based on genes whose homology has already been determined, homology, etc., and genes encoding proteins having ICL activity and MS activity from the chromosomes of microorganisms, animals and plants, etc. Those isolated and determined in nucleotide sequence can be used. In addition, after the base sequence is determined, a gene synthesized according to the sequence can be used. These can be obtained by amplifying a region containing the promoter and the ORF portion by a hybridization method or a PCR method.

In order to enhance the activity of ICL and MS, for example, the above DNA may be incorporated into a plasmid capable of functioning in the host microorganism so as to be expressed and introduced into the host microorganism. The ICL gene and the MS gene may be introduced using different plasmids, or both genes may be loaded on the same plasmid.
The plasmid vector into which the ICL gene and the MS gene can be incorporated is not particularly limited as long as it contains at least a gene that controls the replication growth function in the host bacterium. Specific examples of plasmids that can be used to introduce genes into coryneform bacteria include plasmid pCRY30 described in JP-A-3-210184; JP-A-2-72876 and US Pat. No. 5,185,262. Plasmids pCRY21, pCRY2KE, pCRY2KX, pCRY31, pCRY3KE and pCRY3KX described in the publication; plasmids pCRY2 and pCRY3 described in JP-A-1-191686; PHM1519 described in JP-A-77895; pAJ655, pAJ611 and pAJ1844 described in JP-A-58-192900; pCG1 described in JP-A-57-134500; pCG2 described in JP-A-58-35197 It can be mentioned pCG4 and pCG11 like described in JP 57-183799 JP.
Among them, plasmid vectors used in the host-vector system of coryneform bacteria have a gene responsible for the replication and replication function of the plasmid in coryneform bacteria and a gene responsible for the plasmid stabilization function in coryneform bacteria. For example, plasmids pCRY30, pCRY21, pCRY2KE, pCRY2KX, pCRY31, pCRY3KE and pCRY3KX are preferably used.

  In addition, DNA cleavage, ligation, and other methods such as chromosomal DNA preparation, PCR, plasmid DNA preparation, transformation, setting of oligonucleotides used as primers, etc. adopt ordinary methods well known to those skilled in the art. can do. These methods are described in Sambrook, J., Fritsch, EF, and Maniatis, T., “Molecular Cloning A Laboratory Manual, Second Edition”, Cold Spring Harbor Laboratory Press, (1989), etc.

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

In the introduction by the above recombinant plasmid or homologous recombination on the chromosome, the promoter for expressing the ICL gene and the MS gene or the promoter used for replacing the promoter of the ICL gene and the MS gene on the chromosome is a host bacterium. The promoter is not particularly limited as long as it can function, but a promoter whose transcriptional activity is not suppressed under anaerobic conditions is preferable. Examples thereof include a tac promoter used in Escherichia coli and a trc promoter. In the case of culturing bacterial cells using saccharides as a main carbon source, a promoter that functions under the above conditions can be used. For example, a fructose-1,6-bisphosphate aldolase (FBA) promoter (for example, a sequence) Base number 1-460 of number 9 can be used.
Thus, the expression levels of the ICL gene and the MS gene can be regulated by appropriately selecting a promoter.
As mentioned above, although the example using coryneform bacteria was described, also when using other bacteria, the activity enhancement of ICL and MS can be achieved by the same method.

  In this reaction, it is more effective to use a bacterial strain modified to reduce lactate dehydrogenase (also referred to as LDH) activity in addition to the enhancement of ICL and MS activities. 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 a method using a sacB gene (Schafer, A. et al. Gene 145 (1994) 69-73). Coryneform bacterium with reduced LDH activity and enhanced expression of ICL gene and MS gene creates a bacterium with disrupted LDH gene and transforms the bacterium with a recombinant vector containing ICL gene and MS gene Can be obtained. However, either the modification operation for reducing LDH activity or the modification operation for enhancing activity of ICL and MS may be performed first.

The bacterium used in the present invention may be a bacterium modified so that the activity of pyruvate carboxylase (hereinafter also referred to as PC) is enhanced in addition to the enhancement of ICL and MS activities. “The PC activity is enhanced” means that the PC activity is increased by 1.5 times or more, more preferably by 3 times or more, with respect to an unmodified strain such as a wild strain or a parent strain. Such a bacterium can be obtained, for example, by introducing the PC gene into a coryneform bacterium having enhanced expression of the ICL and MS genes. In addition, any modification may be performed first for the introduction of the PC gene and the modification for enhancing the ICL and MS activities.

The introduction of the PC gene can be performed, for example, by highly expressing the PC gene in coryneform bacteria in the same manner as described in JP-A-11-196888. As a specific PC gene gene, for example, a PC gene derived from Corynebacterium glutamicum (Peters-Wendisch, PG et al. Microbiology, vol. 144 (1998) p915-927) can be used.
Also, bacteria other than Corynebacterium glutamicum, or PC genes derived from other bacteria or animals and plants can be used. In particular, the following bacterial or animal or plant-derived PC genes have known sequences (shown below), and by amplifying the ORF portion by hybridization or PCR as described above, Can be acquired.
Human [Biochem.Biophys.Res.Comm., 202, 1009-1014, (1994)]
Mouse [Proc.Natl.Acad.Sci.USA., 90, 1766-1779, (1993)]
Rat [GENE, 165, 331-332, (1995)]
Yeast; Saccharomyces cerevisiae
[Mol.Gen.Genet., 229, 307-315, (1991)]
Schizosaccharomyces pombe
[DDBJ Accession No .; D78170]
Bacillus stearothermophilus
[GENE, 191, 47-50, (1997)]
Rhizobium etli
[J. Bacteriol., 178, 5960-5970, (1996)]

  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 the cells after culturing them under normal aerobic conditions. As a medium used for culture, a medium usually used for culture of microorganisms can be used. For example, a general medium in which a natural nutrient source such as meat extract, yeast extract or peptone is added to a composition composed of inorganic salts such as ammonium sulfate, potassium phosphate and magnesium sulfate can be used. The cultured cells are collected by centrifugation, membrane separation, etc. and used for the reaction.

  In the present invention, a treated product of bacterial cells can also be used. Examples of treated cells include, for example, immobilized cells obtained by immobilizing cells with acrylamide, carrageenan, etc., crushed materials obtained by crushing cells, centrifugation supernatants thereof, or partial supernatants thereof by ammonium sulfate treatment, etc. Examples include purified fractions.

  The organic raw material used in the production method of the present invention is not particularly limited as long as it is a carbon source that can be assimilated by the microorganism to produce succinic acid. , Starch, cellulose and other carbohydrates; fermentable carbohydrates such as glycerin, mannitol, xylitol, ribitol and other polyalcohols are used, among which glucose, fructose, glycerol and sucrose are preferred, and glucose and sucrose are 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 within the above range depending on the alkaline substance, carbonate, urea, etc. as necessary even during the reaction. Adjust to.

  As the reaction solution used in the present invention, water, a buffer solution, a medium, or the like is used, and a medium is most preferable. The medium can contain, for example, the above organic raw materials and carbonate ions, bicarbonate ions, or carbon dioxide gas, and can be reacted under anaerobic conditions. Carbonate ions or bicarbonate ions are supplied from magnesium carbonate, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, which can also be used as a neutralizing agent. It is also possible to supply from the salt or carbon dioxide gas. Specific examples of the carbonate or bicarbonate salt include magnesium carbonate, ammonium carbonate, sodium carbonate, potassium carbonate, ammonium bicarbonate, sodium bicarbonate, and potassium bicarbonate. Carbonate ions and bicarbonate ions are added at a concentration of 1 to 500 mM, preferably 2 to 300 mM, more preferably 3 to 200 mM. When carbon dioxide gas is contained, 50 mg to 25 g, preferably 100 mg to 15 g, more preferably 150 mg to 10 g of carbon dioxide gas is contained per liter of the solution.

  The optimal growth temperature for bacteria used in this reaction is usually 25 ° C to 35 ° C. The temperature during the reaction is usually 25 ° C to 40 ° C, preferably 30 ° C to 37 ° C. Although the quantity of the microbial cell used for reaction is not prescribed | regulated, 1-700 g / L, Preferably it is 10-500 g / L, More preferably, 20-400 g / L is used. The reaction time is preferably 1 hour to 168 hours, more preferably 3 hours to 72 hours.

  When culturing bacteria, it is necessary to supply oxygen by aeration and agitation. On the other hand, the 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 gas-containing inert gas is aerated, or the like is used. it can.

  Succinic acid accumulated in the reaction solution (culture solution) can be recovered (separated and purified) from the reaction solution according to a conventional method. Specifically, after removing solids such as bacterial cells by centrifugation, filtration, etc., desalting with an ion exchange resin or the like, 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 Coryneform Bacterial Expression Vector Compatible with pTZ4>
(A) Introduction of streptomycin / spectinomycin resistance gene Coryneform bacteria vector that can coexist with pTZ4 (coryneform bacteria vector: JP-A-2005-95169) is a plasmid vector pC2 having a replication region compatible with pTZ4. (Plasmid 36, 62 (1996)) was constructed by replacing the kanamycin resistance gene with a streptomycin / 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 by SEQ ID NO: 1 and SEQ ID NO: 2), 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, 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 separating by gel electrophoresis with 0.8% agarose (SeaKem GTG agarose: manufactured by FMCBioProducts) and then visualized by staining with ethidium bromide to detect a 937 bp fragment. The target DNA fragment is recovered from the gel using QIAQuick Gel Extraction Kit (manufactured by QIAGEN). After recovery, the DNA fragment is phosphorylated at the 5 ′ end with T4 polynucleotide kinase (T4 Polynucleotide Kinase: manufactured by Takara Bio Inc.). did.
After cleaving pC2 with restriction enzymes HindIII and NruI, the ends of the DNA fragments blunted at both ends with Klenow Fragment (Takara Bio) were mixed with the streptomycin / spectinomycin resistance gene prepared above, Ligation kit ver. 2 (manufactured by Takara Bio Inc.). 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.
As a result of the above PCR analysis using the synthetic DNA of SEQ ID NO: 2 as a primer, it was confirmed that a streptomycin / spectinomycin resistance gene was inserted, and this 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 (manufactured by Takara Bio Inc .: CAGATCTG), and ligated kit ver. After ligation using 2 (manufactured by Takara Bio Inc.), Escherichia coli (DH5α strain) was transformed with the obtained plasmid DNA and smeared on an LB agar medium containing 50 μg / mL spectinomycin. From the obtained colonies, after liquid culture, plasmid DNA was prepared by a conventional method, a plasmid that was cleaved with the restriction enzyme BglII was selected, and this was designated as pC3.1.

(B) Introduction of Multicloning Site An α-peptide gene containing a LacZ multicloning site was prepared by PCR using E. coli plasmid pT7Blue (Novagen) as a template and the synthetic DNAs shown in SEQ ID NO: 3 and SEQ ID NO: 4 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.
The amplification product was confirmed by separation by 1.0% agarose (SeaKem GTG agarose: manufactured by FMCBioProducts) gel electrophoresis and then visualization by ethidium bromide staining to detect a 5777 bp fragment. The target DNA fragment is recovered from the gel using QIAQuick Gel Extraction Kit (manufactured by QIAGEN). After recovery, the DNA fragment is phosphorylated at the 5 ′ end with T4 polynucleotide kinase (T4 Polynucleotide Kinase: manufactured by Takara Bio Inc.). did.
After cleaving pC3.1 with restriction enzymes PstI and HpaI, the DNA fragment whose end was blunted with Klenow Fragment (manufactured by Takara Bio Inc.) was mixed with the α-peptide gene fragment prepared above, and the ligation kit ver. 2 (manufactured by Takara Bio Inc.). Escherichia coli (DH5α strain) was transformed with the obtained plasmid DNA and smeared on an LB agar medium containing 50 μg / 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. 1).

<Construction of Isocitrate Triase Activation Plasmid>
(A) Extraction of genomic DNA of Brevibacterium flavum MJ233 strain 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 0.5 g O, 6 mg FeSO ₄ .7H ₂ O, 6 mg MnSO ₄ · 4-5H ₂ O, 200 μg biotin, 100 μg thiamine, 1 g yeast extract, 1 g casamino acid, 20 g glucose, 1 L distilled water] in 10 mL, Brevi Bacterium flavum MJ-233 strain is cultured until the late logarithmic growth phase, and the obtained cells are 10 mM NaCl / 20 mM Tris buffer (pH 8.0) / 1 mM EDTA.2Na solution containing lysozyme at a concentration of 10 mg / mL. Suspended in 0.15 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 volume of phenol / chloroform solution, shake gently for 10 minutes at room temperature, and then centrifuge the whole volume (
5,000 × g, 20 minutes, 10 to 12 ° C.), the supernatant fraction was collected, sodium acetate was added to a concentration of 0.3 M, and then twice the amount of 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) Preparation of Fructose-1,6-Diphosphate Aldolase Gene Promoter Fragment The fructose-1,6-bisphosphate aldolase gene promoter fragment was obtained using the DNA prepared in Example 2 (A) as a template. PCR using synthetic DNA (SEQ ID NO: 5 and SEQ ID NO: 6) designed based on the sequence of the gene of Corynebacterium glutamicum ATCC13032 (GenBank Database Accession No. AP005276) for which the entire genome sequence has been reported went.
Composition of reaction solution: Template DNA 1 μL, PfxDNA polymerase (manufactured by Invitrogen) 0.5 μL, 1-fold concentration buffer, 0.4 μM each primer, 1 mM MgSO ₄ , 0.2 μM dNTPs were mixed to make a total volume of 50 μL. Reaction temperature conditions: DNA thermal cycler PTC-200 (manufactured by MJ Research) was used, and a cycle consisting of 94 ° C. for 15 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 the heat retention at 72 ° C. in the final cycle was 3 minutes.
The amplification product was confirmed by separation by 1.0% agarose (SeaKem GTG agarose: manufactured by FMC BioProducts) gel electrophoresis and visualization by ethidium bromide staining, and a fragment of about 0.45 kb was detected. Recovery of the target DNA fragment from the gel was performed using QIAQuick Gel Extraction Kit (manufactured by QIAGEN).

(C) Preparation of Isocitrate Triase Gene Fragment The acquisition of isocitrate triase gene fragment was performed using the DNA prepared in (A) of Example 2 as a template, and Corynebacterium glutamicum ATCC13032 strain for which the entire genome sequence has been reported. PCR was performed using synthetic DNA (SEQ ID NO: 7 and SEQ ID NO: 8) designed based on the sequence of the gene (GenBank Database Accession No. AP005276).
Composition of reaction solution: Template DNA 1 μL, PfxDNA polymerase (manufactured by Invitrogen) 0.5 μL, 1-fold concentration buffer, 0.4 μM each primer, 1 mM MgSO ₄ , 0.2 μM dNTPs were mixed to make a total volume of 50 μL. Reaction temperature condition: DNA thermal cycler PTC-200 (manufactured by MJ Research) was used, and a cycle consisting of 94 ° C. for 15 seconds, 60 ° C. for 20 seconds and 72 ° C. for 1 minute 30 seconds was repeated 35 times. However, the heat retention at 94 ° C. in the first cycle was 1 minute, and the heat retention at 72 ° C. in the final cycle was 3 minutes.
Confirmation of the amplification product was carried out by separating by gel electrophoresis of 0.75% agarose (SeaKem GTG agarose: manufactured by FMCBioProducts) and then visualized by ethidium bromide staining to detect a fragment of about 1.3 kb. Recovery of the target DNA fragment from the gel was performed using QIAQuick Gel Extraction Kit (manufactured by QIAGEN).

(D) Binding of the fructose-1,6-bisphosphate aldolase gene promoter fragment and isocitrate triase gene fragment by crossover PCR and insertion into the vector The DNA fragments prepared in (B) and (C) above, respectively. Using the mixed sample as a template, both fragments were bound by crossover PCR using DNA primers described in SEQ ID NO: 5 and SEQ ID NO: 8.
Composition of reaction solution: Template DNA 1 μL, PfxDNA polymerase (manufactured by Invitrogen) 0.5 μL, 1-fold concentration buffer, 0.4 μM each primer, 1 mM MgSO ₄ , 0.2 μM dNTPs were mixed to make a total volume of 50 μL. Reaction temperature conditions: DNA thermal cycler PTC-200 (manufactured by MJ Research) was used, and a cycle consisting of 94 ° C. for 15 seconds, 60 ° C. for 20 seconds and 72 ° C. for 2 minutes was repeated 25 times. However, the heat retention at 94 ° C. in the first cycle was 1 minute, and the heat retention at 72 ° C. in the final cycle was 3 minutes.
The amplification product was confirmed by separation by 0.75% agarose (SeaKem GTG agarose: manufactured by FMCBioProducts) gel electrophoresis and visualization by ethidium bromide staining, and a fragment of about 1.8 kb was detected. Recovery of the target DNA fragment from the gel was performed using QIAQuick Gel Extraction Kit (manufactured by QIAGEN).
The DNA fragment thus prepared was phosphorylated at the 5 ′ end with T4 polynucleotide kinase (T4 Polynucleotide Kinase: manufactured by Takara Bio Inc.), and then BamHI linker (manufactured by Takara Bio Inc.) was used as a ligation kit ver. 2 (manufactured by Takara Bio Inc.). Subsequently, it was treated with the restriction enzyme BamHI to prepare a chimera gene fragment consisting of the fructose-1,6-bisphosphate aldolase gene promoter and isocitrate triase gene having a BamHI cleavage end.
After cleaving pMJPC1 (Japanese Patent Laid-Open No. 2005-95169) with restriction enzyme KpnI, the ends were blunted with Klenow Fragment (Takara Bio), and pBamHI linker (Takara Bio: CGGATCCG) was ligated with the ligation kit ver. 2 (made by Takara Bio Inc.). Next, this was treated with BamHI, the end was dephosphorylated with Calf Intestine Alkaline Phosphatase (Takara Bio), and then separated by 0.75% agarose (SeaKem GTG agarose: FMCBioProducts) gel electrophoresis, followed by odor The 6.4 kb fragment was recovered from the gel using a QIAQuick Gel Extraction Kit (manufactured by QIAGEN).
A vector fragment thus prepared and a chimeric gene comprising the promoter of the fructose-1,6-bisphosphate aldolase gene having the BamHI cleavage end and the isocitrate triase gene prepared above were ligated with a ligation kit ver. 2 (manufactured by Takara Bio Inc.).
Escherichia coli (DH5α strain) was transformed with the obtained plasmid DNA and smeared on an LB agar medium containing 50 μg / mL kanamycin. The obtained colonies were subjected to liquid culture, and plasmid DNA was prepared by a conventional method. Next, as a result of treating the plasmid DNA with the restriction enzyme BamHI, a clone producing a DNA fragment of about 1.8 kb was selected and named pICL2.0 (construction procedure is shown in FIG. 2).
The base sequence of the insert fragment of pICL2.0 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: 9 and 10, respectively.

<Construction of Malate Synthase Activation Plasmid>
(A) Preparation of Malate Synthase Gene Fragment The malate synthase gene fragment was obtained from the Corynebacterium glutamicum ATCC13032 strain in which the entire genome sequence was reported using the DNA prepared in (A) of Example 2 as a template. PCR was performed using synthetic DNA (SEQ ID NO: 11 and SEQ ID NO: 12) designed based on the gene sequence (GenBank Database Accession No. AP005276).
Composition of reaction solution: Template DNA 1 μL, PfxDNA polymerase (manufactured by Invitrogen) 0.5 μL, 1-fold concentration buffer, 0.4 μM each primer, 1 mM MgSO ₄ , 0.2 μM dNTPs were mixed to make a total volume of 50 μL. Reaction temperature condition: DNA thermal cycler PTC-200 (manufactured by MJ Research) was used, and a cycle consisting of 94 ° C. for 15 seconds, 60 ° C. for 20 seconds and 72 ° C. for 2 minutes 30 seconds was repeated 35 times. However, the heat retention at 94 ° C. in the first cycle was 1 minute, and the heat retention at 72 ° C. in the final cycle was 5 minutes.
The amplification product was confirmed by separation by 0.75% agarose (SeaKem GTG agarose: manufactured by FMCBioProducts) gel electrophoresis and then visualization by ethidium bromide staining, and a fragment of about 2.3 kb was detected. Recovery of the target DNA fragment from the gel was performed using QIAQuick Gel Extraction Kit (manufactured by QIAGEN).

(B) Binding of fructose-1,6-bisphosphate aldolase gene promoter fragment and malate synthase gene fragment by crossover PCR and insertion into vector Prepared in Example 2 (B) and (A) above, respectively. Using the sample mixed with DNA fragments as a template, both fragments were bound by crossover PCR using DNA primers described in SEQ ID NO: 5 and SEQ ID NO: 12.
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, 60 ° C. for 20 seconds and 72 ° C. for 3 minutes was repeated 25 times. However, the heat retention at 94 ° C. in the first cycle was 1 minute, and the heat retention at 72 ° C. in the final cycle was 5 minutes.
The amplification product was confirmed by separation by 0.75% agarose (SeaKem GTG agarose: manufactured by FMC BioProducts) gel electrophoresis and visualization by ethidium bromide staining, and a fragment of about 2.7 kb was detected. The target DNA fragment was recovered from the gel using QIAQuick Gel Extraction Kit (manufactured by QIAGEN).
The chimeric gene fragment consisting of the fructose-1,6-bisphosphate aldolase gene promoter and malate synthase gene thus prepared was phosphorylated at the 5 ′ end with T4 polynucleotide kinase (Treasure Bio Inc.). The DNA fragment prepared by treating pC3.14 constructed in Example 1 with the restriction enzyme EcoRV and the ligation kit ver. 2 (manufactured by Takara Bio Inc.).
Escherichia coli (DH5α strain) was transformed with the obtained plasmid DNA, and 50 μg / mLX-Gal
And LB agar medium containing 50 μg / mL spectinomycin. From the obtained colonies, those exhibiting a blue color were selected and named pMS2.0 (construction procedure is shown in FIG. 3).
The base sequence of the insert fragment of pMS2.0 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 deduced amino acid sequence are shown in SEQ ID NOs: 13 and 14, respectively.

<Preparation of glyoxylic acid cycle activated strain>
(A) Introduction of pICL2.0 and pMS2.0 Plasmid DNAs used for transformation of Brevibacterium flavum MJ233 / ΔLDH strain (strain in which LDH gene is disrupted: JP-A-2005-95169) are pICL2.0 and pMS2. It was prepared from Escherichia coli MJ110 strain transformed with 0.
Transformation of the Brevibacterium flavum MJ233 / ΔLDH strain was carried out by the electric pulse method (Res. Microbiol. Vol. 144, p. 181-185, 1993), and the obtained transformant was treated with 25 μg / mL kanamycin and 10 μg / It was smeared on an LBG agar medium containing 10 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]. The strain grown on this medium was named Brevibacterium flavum MJ233 / ICL / MS / ΔLDH.

(B) Measurement of ICL 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), centrifuged at 10,000 g for 15 minutes, and then the supernatant was separated. The resulting supernatant is further 40,000 rpm,
Ultracentrifugation was performed for 60 minutes, and isocitrate triase activity was measured using the supernatant after ultracentrifugation.
Enzyme activity was measured using a reaction solution containing 50 mM MOPS-NaOH (pH 7.3), 5 mM DTT, 15 mM MgCl ₂ , 1 mM EDTA, 5 mM isocitrate, 0.2 mM NADH, 18 U lactate dehydrogenase (pig heart isoenzyme I) and the above enzyme extract. The reaction was carried out at 25 ° C. 1 U was defined as the amount of enzyme that catalyzes the decrease of 1 μmol of NADH per minute.
The ICL specific activity of Brevibacterium flavum MJ233 / ICL / MS / ΔLDH according to the above measurement method was 0.709 U / mg-protein. In addition, in the cells obtained by culturing the parent strain Brevibacterium flavum MJ233 / ΔLDH strain in the same manner using A medium containing 2% glucose, the ICL activity is 0.0445 U / mg-protein, and the enhanced strain is about 16 It was confirmed that the ICL activity increased twice.

<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 4 (A) was added. / ICL / MS / ΔLDH strain was inoculated and seed-cultured at 30 ° C. for 24 hours. However, streptomycin and kanamycin were not added when the target strain Brevibacterium flavum MJ233 / ΔLDH strain was cultured.
The obtained whole culture solution was collected by centrifugation at 10,000 g for 5 minutes, and the cell suspension medium (magnesium sulfate 7 hydrate: 1 g, ferrous sulfate 7 hydrate: 40 mg, manganese sulfate. Hydrate: 40 mg, D-biotin: 400 μg, thiamine hydrochloride: 400 μg, monoammonium phosphate: 0.8 g, diammonium phosphate: 0.8 g, potassium chloride: 0.3 g, and distilled water: 1000 mL) at OD660 The suspension was suspended so that its absorbance was 80. Add 0.5 ml of the above-mentioned cell suspension and 0.5 ml of substrate solution (glucose: 200 g, magnesium carbonate: 194 g, and distilled water: 1000 ml) to a 4 ml reactor, 20% carbon dioxide gas, 80% nitrogen atmosphere Then, after reacting at 35 ° C. for 5 hours or 24 hours, the mixture was centrifuged under the above conditions, and the succinic acid concentration of the supernatant was analyzed.
In the reaction for 5 hours, glucose remained in the medium, and the concentration of succinic acid in the reaction solution of Brevibacterium flavum MJ233 / ΔLDH was 22.5 g / L, whereas Brevibacterium flavum MJ233. In the / ICL / MS / ΔLDH strain, 25.9 g / L of succinic acid was accumulated. The succinic acid productivity of the strain in which ICL and MS were activated was improved by about 15%.
In addition, at 24 hours of reaction, the residual glucose concentration is less than 1 g / L, and the yield to sugar of the Brevibacterium flavum MJ233 / ICL / MS / ΔLDH strain calculated from the analyzed succinic acid concentration is the parent strain. There was a 7.2% increase over the Brevibacterium flavum MJ233 / ΔLDH strain.
From these results, it became clear that the activation of ICL and MS has a remarkable effect of improving the productivity and yield of succinic acid.

The figure which shows the construction procedure of plasmid pC3.14. The figure which shows the construction procedure of pICL2.0. The figure which shows the construction procedure of pMS2.0.

Claims (3)

The gene encoding isocitrate triase and the gene encoding malate synthase are under the control of the fructose-1,6-diphosphate aldolase gene promoter, which is a promoter that functions in a medium containing saccharides as the main carbon source. by placing, the isocitrate lyase and coryneform bacterium or treated product of the coryneform bacterium in which the activity of malate synthase has been modified to enhance the reaction solution containing carbonate ion, bicarbonate ion or carbon dioxide gas A method for producing succinic acid, comprising reacting glucose or sucrose in an anaerobic atmosphere to produce succinic acid and recovering the succinic acid. The method according to claim 1 , wherein the bacterium is further modified so that lactate dehydrogenase activity is reduced to 10% or less compared to an unmodified strain. The manufacturing method of a succinic-acid containing polymer including the process of manufacturing a succinic acid by the method of Claim 1 or 2 , and the process of polymerizing the obtained succinic acid.

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