JP4647391B2 - Highly efficient production method of dicarboxylic acid by coryneform bacteria - Google Patents

Description

  The present invention relates to a method for producing dicarboxylic acids such as succinic acid and fumaric acid by an aerobic coryneform bacterium. More specifically, the present invention relates to a highly efficient production method of dicarboxylic acid using aerobic coryneform bacteria under specific production conditions and methods.

Dicarboxylic acids such as succinic acid and fumaric acid and derivatives thereof are used in a wide range of fields such as polymer synthesis raw materials, cosmetics, pharmaceutical raw materials, and food additives. For example, succinic acid is expected to increase in demand in the future as a biodegradable plastic raw material, and its ester derivative as a clean cleaning solvent that does not cause environmental pollution.
On the other hand, these dicarboxylic acids are currently produced by a chemical synthesis method using petrochemical resource raw materials, but biological production methods using sugars such as glucose, which are renewable resources, as raw materials are more environmentally friendly. From the viewpoint of technology, its practical application is strongly desired.

  Conventionally, many methods for producing organic acids such as dicarboxylic acids by biological production methods have been proposed. For example, in a method for producing carboxylic acid from a carbon source by anaerobic metabolic reaction in an anaerobic atmosphere after incubating E. coli in an aerobic atmosphere and rapidly growing the cells, an aerobic growth medium and In a method for improving the yield of a carboxylic acid product by defining a carbon source concentration in an anaerobic reaction medium (see Patent Document 1), an organic acid production method in an aerobic atmosphere by a coryneform bacterium that is an aerobic bacterium, A method of defining the carbon source (raw material carbohydrate) concentration and reaction temperature of the aerobic reaction medium (see Patent Documents 2 and 3) is also known.

In order to obtain a large amount of cells used for organic acid production, synthesis including nutrient sources necessary for cell growth such as carbon sources, nitrogen sources, inorganic salts, vitamins, etc. when growing in an aerobic atmosphere Alternatively, culturing in a natural medium is widely performed.
As the carbon source, generally, sugars such as glucose, sucrose and fructose, alcohols such as ethanol, and amino acids such as glutamic acid are generally used.
However, if the acetic acid and / or acetate is used as the carbon source used in the culture growth under the aerobic condition as in the present invention, it is less under the reducing condition than when the normal carbon source such as glucose is used. It has not been found so far that the productivity of the dicarboxylic acid obtained by the reaction is improved and it becomes a highly efficient production technique of dicarboxylic acid.

Such an effect is observed only in the aerobic coryneform bacterium used in the present invention, and in the aerobic coryneform bacterium that has not lost the lactate dehydrogenase activity, it is used at the time of culture growth under aerobic conditions. Glucose and acetic acid and / or acetate as a carbon source show almost no difference in dicarboxylic acid productivity (see below).
JP 2000-515389 Japanese Patent Laid-Open No. 2003-235592 JP 2003-235593 A

The present invention relates to a method for producing a dicarboxylic acid in a method for culturing and growing an aerobic coryneform bacterium having lost lactate dehydrogenase activity under aerobic conditions, and producing dicarboxylic acid in a reaction medium under reducing conditions by the grown cells. Provides a method for producing a dicarboxylic acid that can be carried out more efficiently than the prior art.
The term “high efficiency” as used herein means that the production rate from saccharides to dicarboxylic acid in the dicarboxylic acid production reaction step is high, and the concentration of dicarboxylic acid reached in the production reaction medium is high.
Improvement in the production rate will increase the production capacity of dicarboxylic acid per reactor. If the concentration of dicarboxylic acid reached can be increased, the downstream product separation and production process can be streamlined. In terms of consumption, the industrialization effect of the present invention is very large.

  As a result of repeating various studies to solve the above problems, the present inventors cultured and proliferated aerobic coryneform bacteria in which lactate dehydrogenase activity was lost under aerobic conditions, and under reducing conditions by the proliferated cells. In the method for producing dicarboxylic acid in the reaction medium of (2), it was found that dicarboxylic acid can be produced with high efficiency by using acetic acid and / or acetate as a carbon source in the medium for culture growth. The present invention has been completed.

That is, the present invention
(1) A method of culturing and growing an aerobic coryneform bacterium having lost lactate dehydrogenase activity under aerobic conditions, and producing dicarboxylic acid in a reaction medium under reducing conditions using the obtained bacterial cells or treated products thereof A method for producing a highly efficient dicarboxylic acid, characterized in that acetic acid and / or acetate is used as a carbon source in the medium for culture growth,
(2) The above-mentioned (1) comprising reacting the microbial cells of the aerobic coryneform bacterium separated or recovered or a processed product thereof with a saccharide in a reaction medium under reducing conditions after culture growth, and collecting the generated dicarboxylic acid. A process for producing the dicarboxylic acid according to claim 1,
(3) The method for producing a dicarboxylic acid according to (1) above, wherein the aerobic coryneform bacterium is Corynebacterium or Brevibacterium,
(4) The aerobic coryneform in which the lactate dehydrogenase activity has disappeared is Corynebacterium glutamicum R / Δ1dh (National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary, Accession No. FERM P-20532) A process for producing a dicarboxylic acid according to (1), characterized in that
(5) The method for producing a dicarboxylic acid according to the above (1), wherein the acetate is a compound selected from ammonium acetate, sodium acetate and potassium acetate,
(6) In the above (5), the compound selected from ammonium acetate, sodium acetate and potassium acetate is formed in a culture growth medium by a reaction between acetic acid and a corresponding basic compound. A process for producing the dicarboxylic acid according to claim 1,
(7) The method for producing a dicarboxylic acid according to (2) above, wherein the saccharide is selected from glucose, xylose, arabinose, cellobiose, sucrose, maltose and dextrin,
(8) The reaction medium under reducing conditions contains carbonate ion, bicarbonate ion or carbon dioxide gas, and the dicarboxylic acid produced is selected from succinic acid, fumaric acid and malic acid (1) The method for producing dicarboxylic acid according to (1), and (9) the method for producing dicarboxylic acid according to (1) above, wherein the oxidation-reduction potential of the reaction medium under reducing conditions is -200 mV to -500 mV. ,
It is.

  According to the present invention, dicarboxylic acids such as succinic acid, fumaric acid, malic acid and the like that are used in a wide range of fields such as polymer synthetic raw materials, cosmetic applications, pharmaceutical raw materials and food additive applications, etc. Highly efficient biological production becomes possible. For this reason, the present invention is very effective in industrialization both economically and in terms of production energy consumption.

The microorganism used in the present invention is an aerobic coryneform bacterium having lost lactate dehydrogenase activity.
First, as an aerobic coryneform bacterium, an aerobic, gram-positive, non-acidic acid, which is defined in Burgy's Manual Determinative Bacteriology [(Bargeys Manual of Determinative Bacteriology, 8, 599, 1974)]. And a gonococcal microorganism having no spore-forming ability, such as a microorganism belonging to the genus Corynebacterium and the genus Brevibacterium.
Specific examples thereof include Corynebacterium glutamicum R (Independent Administrative Institution, National Institute of Advanced Industrial Science and Technology, Patent Biodeposition Center Accession No. FERM P-18976), ATCC13032, ATCC13058, ATCC13059, and the like.
In addition, the aerobic coryneform bacterium may be a wild-type mutant that exists in nature (for example, accession number FERM P-18777, accession number FERM P-18978 described in JP-A-2004-89029).

One of the requirements for realizing the effect of the present invention is that the lactate dehydrogenase activity of these aerobic coryneform bacteria is eliminated.
Here, “loss of lactate dehydrogenase activity” means that the gene function of the lactate dehydrogenase gene is lost (gene disruption = all or part of the lactate dehydrogenase gene is disrupted or mutated, the promoter of the gene or It means that it has no lactate dehydrogenase expression activity due to modification or removal of the gene expression unit such as ribosome binding site), and the formation or function of lactate dehydrogenase enzyme protein that is its expression form has been lost Means.

In the present invention, as long as the lactate dehydrogenase activity is lost, known transformation techniques preferable for dicarboxylic acid production can be additionally used.
The disruption of the lactate dehydrogenase gene of the present invention can be carried out by a method selected from homologous recombination method, transposon insertion method and mutagen introduction method. Transposon insertion method and mutagen introduction method are random disruption of genes on the chromosome. A homologous recombination method is preferable in order to efficiently destroy the target lactate dehydrogenase gene.
One example of a method for producing a lactate dehydrogenase gene disruption strain of coryneform bacteria by homologous recombination is described in detail in the Examples, but can be usually performed by the following operation method and procedure.

A) DNA extraction from microorganisms;
Genomic DNA extraction from coryneform bacteria was performed by the method of Sambrook et al. (Sambrook, J., EF Fritsch, and, except that the cells were pretreated with lysozyme at a concentration of 4 mg / ml at 37 ° C. for 30 minutes. T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

B) Cloning of target lactate dehydrogenase gene and preparation of plasmid for destruction;
The lactate dehydrogenase gene can be cloned by PCR using primers designed from conserved amino acid sequences between known lactate dehydrogenase genes or by hybridization using known lactate dehydrogenase genes. In the case of the Corynebacterium glutamicum R strain, the entire genome sequence (Hiroshi Nonaka, Kaori Nakata, Naoko Okai, Mariko Wada, Yumiko Sato, Masayuki Inui, Hideaki Yukawa Analysis ”of Japan Society for Agricultural Chemistry, April 2003, Yokohama, Japan Agricultural Chemical Society 2003 Annual Meeting Abstracts, p. 20) has been decided and can be used] Were designed Luo primers, the genomic DNA of coryneform bacterium as a template, a gene containing a full length of the lactate dehydrogenase gene can be amplified and obtained by PCR. On the other hand, the lactate dehydrogenase gene-disrupted strain is created by cloning the lactate dehydrogenase gene amplified by PCR into an E. coli vector such as pHSG398 that cannot replicate in coryneform bacteria, and then a unique strain located almost at the center of the lactate dehydrogenase gene A gene disruption plasmid is prepared by inserting a drug resistance gene such as kanamycin (used as a marker gene for gene disruption) into an appropriate restriction enzyme site.

C) Homologous recombination method by plasmid introduction;
The lactate dehydrogenase gene disruption strain was prepared by introducing the gene disruption plasmid into a coryneform bacterium by a highly efficient gene transfer method [electric pulse method (Y. Kurusu, et al., Agric. Biol. Chem. 54: 443-447]. , 1990, and AA Vertes, et al., Res. Microbiol. 144: 181-185, 1993)], and the lactate dehydrogenase gene is disrupted by homologous recombination to the chromosome. This can be done.
Confirmation of the destruction of the lactate dehydrogenase gene is based on the fact that a marker gene fragment such as a kanamycin resistance gene is inserted into the chromosome in addition to the lactate dehydrogenase gene fragment at the gene level by PCR or Southern hybridization. Then, it can confirm by having lost the enzyme activity of lactate dehydrogenase.

  Corynebacterium glutamicum R / Δldh, for example, is obtained as the coryneform bacterial strain in which the lactate dehydrogenase activity thus obtained has been lost. The deposit number is FERM P-20532 (date of deposit: May 10, 2005).

を意味するものとする。 In addition, Δ1dh described in the present specification and claims is

Means.

In the method for producing a dicarboxylic acid according to the present invention, the aerobic coryneform bacterium having lost the lactate dehydrogenase activity is first cultured and grown under aerobic conditions.
This aerobic culture growth can be conveniently carried out by culturing in a synthetic or natural medium containing nutrient sources necessary for the growth of bacteria such as carbon sources, nitrogen sources, inorganic salts, and vitamins under aerated conditions.

  In the present invention, it is essential to use acetic acid and / or acetate as the carbon source of the aerobic culture growth medium. The acetate is preferably selected from ammonium acetate, sodium acetate and potassium acetate, but is formed in the medium by reaction with basic compounds used for neutralization of the medium using acetic acid as a carbon source. It may be a salt such as

  As the nitrogen source, for example, ammonia, ammonium sulfate, ammonium chloride, ammonium nitrate, urea or the like can be used alone or in combination. As inorganic salts, for example, potassium monohydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, manganese sulfate and the like can be generally used. In addition, nutrients such as peptone, meat extract, yeast extract, corn steep liquor, casamino acid or various vitamins such as biotin or thiamine can be appropriately added to the medium as necessary.

  Culturing can be performed at a temperature of about 20 ° C. to about 40 ° C., preferably about 25 ° C. to about 35 ° C., under aerobic conditions such as aeration agitation or shaking. The carbon source concentration of acetic acid and / or acetate is about 1-20% (W / V), preferably about 2-5% (W / V). The culture period is usually about 1 to 7 days.

In the main culture growth step, acidic substances are produced or secreted in the medium as the bacteria grow, so the pH of the medium is adjusted to around 5-9, preferably 6.5-8.5. As the basic compound used for neutralization, ammonia, ammonium hydroxide, sodium or potassium hydroxide, carbonate compound, bicarbonate compound or a mixture thereof is used. Of these, sodium or potassium hydroxide, carbonate compound and bicarbonate compound are preferred.
The solution concentration of these basic compounds can be appropriately determined and used as long as the pH of the medium is adjusted within the above range.

Next, the cultured cells of the aerobic coryneform bacterium of the present invention are collected. The method for recovering and separating cultured cells from the culture is not particularly limited, and for example, known means such as centrifugation and membrane separation can be used.
A treatment such as immobilizing the collected cultured microbial cells with acrylamide or carrageenan can be added, and the resulting microbial cell treated product can be used in the next step.
The bacterial cells obtained from the culture growth step as described above are subjected to the dicarboxylic acid production step in the reaction medium under the following reducing conditions.

  In the dicarboxylic acid production process by the biochemical reaction under the reducing conditions of the present invention, the growth division of the aerobic coryneform bacterium of the present invention is suppressed, and a substantial complete suppression of secreted by-products accompanying the growth is realized. I can do it. From this point of view, when the microbial cells recovered from the culture growth process or the treated microbial cells are used in the reaction medium, the environmental state during the aerobic culture growth inside and outside the aerobic coryneform bacteria is also present in the reaction medium. It is recommended to use methods and conditions that are not applied. That is, it is preferable that the target dicarboxylic acid production reaction medium is produced in the aerobic culture growth step and does not substantially contain substances existing outside and inside the cells. More specifically, a secreted by-product generated in the culture growth process and released outside the cells, and a substance generated by the aerobic metabolic function in the cells and remaining in the cells are the dicarboxylic acid production reaction medium. It is recommended that the condition be substantially absent. In such a state, for example, the culture solution after culture growth is removed by centrifugation, membrane separation or the like, and / or the cells separated from the culture solution after culture are reduced for about 2 to 10 hours under reducing conditions. Realized by leaving it alone.

In this step, a reaction medium under reducing conditions is used. The reaction medium may be in a solid, semi-solid, or liquid state as long as it is under reducing conditions.
The reducing conditions in this step are defined by the oxidation-reduction potential of the reaction system, and the oxidation-reduction potential of the reaction medium is preferably about -200 mV to -500 mV, more preferably about -250 mV to -500 mV. It is.

  In the production reaction of the dicarboxylic acid of the present invention, it is not clear why the regulation of the redox potential of the production reaction system is effective for the efficient production of the target dicarboxylic acid, but the reason for the estimation is described below. I write. However, the present invention is not limited to the reason for the estimation.

  The target product of the present invention, dicarboxylic acid, is a compound produced by a biochemical reaction based on the metabolic function of bacteria. Various redox reactions are involved in biochemical reactions in microbial cells, and electrons are transferred. The oxidation-reduction potential is one of the scales showing the difficulty of accepting and donating electrons in the reaction system, but this potential is a variety of reactions (oxidation-reduction reactions) that constitute metabolic pathways occurring in microbial cells. And the state of electronic transfer between the inside and outside of the cell. The redox potential directly measured by the potentiometer is the potential between the reaction solution and the electrode, but the potential of the reaction solution correlates with the reaction occurring in the cell with a certain potential gradient through the cell membrane. That is, the oxidation-reduction potential reflects the sum of oxidation-reduction reactions in the entire reaction system including inside and outside the cell (including the contents and frequency of various reactions).

  Factors affecting the oxidation-reduction potential of the reaction system include various types and concentrations of the reaction system atmosphere gas, reaction temperature, reaction solution pH, and various inorganic and organic substances used to produce the target organic compounds present in the reaction solution. The compound concentration and composition can be considered. The oxidation-reduction potential of the reaction medium in the present invention is shown by integrating the above various influencing factors. Therefore, various chemical reactions are involved in the metabolic pathway to the target dicarboxylic acid, and these chemical reactions are under the influence of the above factors, but the efficiency is determined by a scale that defines the reaction state of a single redox potential. In particular, the desired dicarboxylic acid can be produced.

  The reduction state of the reaction medium can be easily estimated to some extent with a resazurin indicator (in the reduced state, decolorization from blue to colorless), but accurately, a redox potentiometer (for example, ORA Electrodes made by BROADELY JAMES) is used. Use to measure. In the present invention, it is preferable to maintain the reducing conditions immediately after adding the microbial cells or processed product to the reaction medium until collecting the dicarboxylic acid, but more preferably about 50% or more of the reaction time. It is desirable that the reaction medium be kept under reducing conditions for about 70% or more, more preferably about 90% or more.

  A known method may be used as a method for preparing the reaction medium under reducing conditions. For example, an aqueous solution for reaction medium may be used as a liquid medium for the culture medium instead of distilled water, etc., and the preparation method of the aqueous solution for reaction medium is, for example, preparation of a culture solution for absolute anaerobic microorganisms such as sulfate-reducing microorganisms. Method (Pfenning, N et. Al. (1981): The dissimilarity sulfate-reducing bacteria, In The Prokaryotes, A Handbook on Habitats, Isolation and Identity. 926-940, Berlin, Springer Verlag., “Agricultural Chemistry Experiment Vol.3, Department of Agricultural Chemistry, Faculty of Agriculture, Kyoto University, 1990, 26th edition, industrial books” "Publishing Co., Ltd.") can be used as a reference to obtain an aqueous solution under the desired reducing conditions.

  More specifically, the method for preparing the aqueous solution for reaction medium includes a method of removing dissolved gas by subjecting the aqueous solution for reaction medium to heat treatment or reduced pressure treatment. More specifically, the reaction medium aqueous solution is treated for about 1 to 60 minutes, preferably about 5 to 40 minutes under reduced pressure of about 10 mmHg or less, preferably about 5 mmHg or less, more preferably about 3 mmHg or less. Thus, the dissolved gas, particularly dissolved oxygen can be removed to prepare an aqueous solution for reaction medium under reducing conditions.

  Moreover, an appropriate reducing agent (for example, thioglycolic acid, ascorbic acid, cysteine hydrochloride, mercaptoacetic acid, thiolacetic acid, glutathione, sodium sulfide, etc.) can be added to prepare an aqueous solution for reaction medium under reducing conditions. . In some cases, an appropriate combination of these methods also provides a method for preparing an aqueous reaction medium solution under effective reducing conditions.

  As a method for maintaining the reduction conditions during the reaction, it is desirable to prevent oxygen contamination from outside the reaction system as much as possible, and a method in which the reaction system is sealed with an inert gas such as nitrogen gas or carbon dioxide gas is usually used. It is done. As a method for more effectively preventing oxygen contamination, in order to efficiently function the metabolic function of the aerobic coryneform bacterium of the present invention during the reaction, addition of a pH maintenance adjustment solution of the reaction system or various nutrients In some cases, it may be necessary to add a solution appropriately. In such a case, it is effective to remove oxygen from the added solution in advance.

  Factors affecting the oxidation-reduction potential of the reaction system include the type and concentration of the reaction system atmosphere gas, the reaction temperature, the reaction solution pH, and the concentration and composition of various inorganic and organic compounds used to produce the desired organic acid. Etc. are considered. The oxidation-reduction potential of the reaction medium in the present invention is shown by integrating the above various influencing factors.

  What is important in this dicarboxylic acid production process is that the pH of the reaction medium is lowered by the dicarboxylic acid produced in this process, by-product acidic substances, etc., so the pH of the reaction medium is around 5-9, preferably 6. In this case, a basic compound is used for neutralization. These basic compounds may be the same as those used in the culture growth step under aerobic conditions.

Moreover, the solution concentration of these basic compounds can be determined and used as long as the pH of the dicarboxylic acid production reaction medium is adjusted within the above range.
The reaction medium is supplied with sugars, carbon dioxide gas, carbonate ions or bicarbonate ions, which are raw materials for dicarboxylic acid production.

  Examples of the saccharide include monosaccharides such as glucose, galactose, fructose, mannose, xylose and arabinose, disaccharides such as cellobiose, sucrose or lactose, maltose, and polysaccharides such as dextrin or soluble starch. Of these, glucose is preferable. In this case, glucose is used in a concentration range of 0.5 to 500 g / L (liter).

  Carbonate ion or bicarbonate ion is supplied from sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate or carbon dioxide gas. These carbonate ions or bicarbonate ions are used in a concentration range of 1 to 500 mM, preferably 2 to 300 mM. When carbon dioxide gas is supplied, it is supplied so as to be contained in the solution at a concentration of 50 mg / L to 25 g / L, preferably 100 mg / L to 15 g / L.

  The composition of the reaction medium used for the dicarboxylic acid production reaction includes components necessary for maintaining the metabolic function of the aerobic coryneform bacterium of the present invention or its processed product, that is, carbon sources such as various sugars and carbonic acid sources. In addition, it contains a nitrogen source necessary for protein synthesis, other inorganic salts such as phosphorus, potassium or sodium, and a trace metal salt such as iron, manganese or calcium. These addition amounts can be appropriately determined depending on the required reaction time, the type of dicarboxylic acid produced or the type of aerobic coryneform bacterium used. Depending on the aerobic coryneform bacterium used, the addition of specific vitamins may be preferred. The carbon source, nitrogen source, inorganic salts, vitamins, and trace metal salts may be known ones, for example, those exemplified for the culture growth step. The reaction between the aerobic coryneform bacterium or its cell-treated product and the saccharide is preferably carried out under temperature conditions where the aerobic coryneform bacterium of the present invention or its cell-treated product can act, and the aerobic coryneform bacterium Or it can select suitably by the kind etc. of the microbial cell processed material. Usually, it is about 25 degreeC-35 degreeC. The production reaction of the dicarboxylic acid can be either batchwise or continuous.

  The dicarboxylic acid and its salts produced in the reaction medium as described above are separated and purified to collect the dicarboxylic acid. As the method, a known method used in a bioprocess can be used. As such known methods, there are salting out method, recrystallization method, organic solvent extraction method, esterification distillation separation method, chromatographic separation method or electrodialysis method of dicarboxylic acid production liquid, and the produced dicarboxylic acid and reaction liquid. The separation / purification sampling method can be appropriately determined according to the characteristics.

  EXAMPLES Hereinafter, although this invention is demonstrated with an Example, this invention is not limited to these Examples.

[Example 1]
a) Aerobic culture growth Coryneform bacterial strain (produced using the method described in Example 1 of International Publication No. WO2005 / 010182) in which the lactate dehydrogenase activity stored in a freezer at −80 ° C. has disappeared, ie coryne Corynebacterium glutamicum R / Δldh (FERM P-20532) was mixed with A-acetate agar medium (composition: urea 2 g, yeast extract 2 g, casamino acid 7 g, ammonium sulfate 7 g, primary phosphate Potassium (KH ₂ PO ₄ ) 0.5 g, dibasic potassium phosphate (K ₂ HPO ₄ ) 5 g, magnesium sulfate heptahydrate 0.5 g, iron sulfate 7 heptahydrate 6 mg, manganese sulfate monohydrate Product 4.2 mg, biotin 0.2 mg, thiamine 0.2 mg, ammonium acetate 20 g, agar 1.5 (W / V) and was applied to the distilled water 1L), 33 ℃, and allowed to stand at 12 hour dark.

Coryneform bacterial strains grown on the above plate were added to 10 ml of A-acetate medium (composition: the same composition as the above-mentioned A-acetate agar medium except that it does not contain agar) as a test tube culture medium. The cells were inoculated, and cultured with shaking at 33 ° C., 12 hours, 200 rpm.
The coryneform bacterial strain grown in this manner is subjected to 500 ml of A-acetic acid-U medium (composition: the same component composition as the above-mentioned A-acetic acid medium except that urea is not included), which is an aerobic culture growth medium. The jar fermenter having a volume of 1 L was transferred, and aerobic culture growth was carried out for 13 hours by aeration at 33 ° C., 1000 rpm, and sterilized air at 1 vvm.
During this time, NH ₄ OH (5N aqueous ammonia solution) was used to constantly maintain the pH in the jar fermenter tank at 7.5.
The cells grown in this manner were collected by centrifugation (4 ° C., 10 minutes, 5000 × G) and subjected to the succinic acid production reaction under the following reducing conditions.

b) Succinic acid production reaction under reducing conditions 150 g of wet cells recovered from the aerobic culture growth step of a) (about 30 g in terms of Dry Cell) was used as a BT-U medium (composition: succinic acid production reaction). 7 g of ammonium sulfate, 0.5 g of primary potassium phosphate (KH ₂ PO ₄ ), 0.5 g of dibasic potassium phosphate (K ₂ HPO ₄ ), 0.5 g of magnesium sulfate heptahydrate, iron sulfate · 7 hydrate 1 L reaction tank containing 6 mg of product, 4.2 mg of manganese sulfate monohydrate, 0.2 mg of biotin, 0.2 mg of thiamine, 36 g of glucose, 12.6 g of sodium bicarbonate (NaHCO ₃ ), 1 L of distilled water) In addition, the mixture was gently stirred at 33 ° C. to carry out a succinic acid production reaction under reducing conditions for 4 hours. The oxidation-reduction potential of the medium during the reaction decreased rapidly immediately after the start of the reaction, and thereafter maintained at about −400 mV, and the succinic acid production reaction was continued.
During this time, the pH in the reaction vessel was constantly maintained at 7.5 using an aqueous NH ₄ OH (5N aqueous ammonia) solution having a concentration of 5N (normal).
The amount of succinic acid produced in the reaction vessel was successively sampled and analyzed by liquid chromatography, and the change in production amount over time was examined.

  The results are shown in Table 1. The production rate in Table 1 is the production rate in the range of 0 to 4 hours after the start of the reaction.

[Reference Example 1]
As a medium used in the a) aerobic culture growth step in Example 1, A agar medium (composition: the same component composition as the above-mentioned A acetate agar medium except that ammonium acetate is not included and glucose is included 40 g), A Medium (composition: the same component composition as A agar medium except that agar is not included) and AU medium (composition: the same component composition as A medium except that urea is not included) The aerobic culture growth and succinic acid production reaction were performed under the same conditions as in Example 1 except that.
Table 1 shows the production rate in the succinic acid production reaction.

[Reference Example 2]
A) A ethanol agar medium (composition: the same component composition as the above-mentioned A acetate agar medium except that it does not contain ammonium acetate and contains 20 ml of ethanol) as the medium used in the aerobic culture growth step in Example 1 A ethanol medium (composition: the same component composition as the above-mentioned A ethanol agar medium except that it does not contain agar), and A-U ethanol medium (composition: the above A ethanol except that it does not contain urea) The aerobic culture growth and succinic acid production reaction were carried out under the same conditions as in Example 1 except that the same composition as the medium was used.
Table 1 shows the production rate in the succinic acid production reaction.

[Example 2]
As a medium used in the aerobic culture growth step in Example 1, A agar medium (composition: the same component composition as the above-mentioned A acetate agar medium except that ammonium acetate is not included and 20 g of sodium acetate is included), A medium (composition: the same component composition as A agar medium except that agar is not included), and AU medium (composition: the same component composition as A medium except that urea is not included) Aerobic culture growth and succinic acid production reaction were carried out under the same conditions as in Example 1. Table 1 shows the production rate in the succinic acid production reaction.

[Reference Example 3]
Corynebacterium glutamicum R strain (stored at −80 ° C. in a freezer) is a wild strain used for the production of the lactate dehydrogenase gene disruption strain of Example 1 and is a patent of the National Institute of Advanced Industrial Science and Technology. Except for using the deposit number FERM P-18976 at the biological depository center), the aerobic culture growth and the succinic acid production reaction under the reducing conditions were carried out under the same conditions and methods as in Example 1. went.
Table 2 shows the production rate in the succinic acid production reaction.

[Reference Example 4]
As a medium used in the aerobic culture growth step in Reference Example 3, A agar medium (composition: the same component composition as the above-mentioned A acetate agar medium except that ammonium acetate is not included and 40 g glucose is included), A Medium (composition: the same component composition as A agar medium except that agar is not included) and AU medium (composition: the same component composition as A medium except that urea is not included) The aerobic culture growth and succinic acid production reaction were performed under the same conditions as in Example 1 except that.
Table 2 shows the production rate in the succinic acid production reaction.

As shown in Table 1, in coryneform bacteria in which lactate dehydrogenase activity has been lost, succinic acid production rate under reducing conditions when using ammonium acetate as the carbon source in the aerobic culture growth medium, rather than using glucose and ethanol as the carbon source It is clear that is significantly improved.
Further, as shown in Example 2, it is clear that even when sodium acetate is used, a succinic acid production rate can be obtained under reducing conditions substantially equivalent to ammonium acetate.
Further, from Reference Examples 3 and 4 in Table 2, it is also clear that such an effect is not observed in the wild strain.

  According to the present invention, dicarboxylic acids such as succinic acid and fumaric acid can be efficiently produced. Therefore, the present invention can be used in a wide range of fields such as polymer synthesis raw materials, cosmetics, pharmaceutical raw materials, and food additives.

The time-dependent change of succinic acid production | generation and consumption of glucose in Example 1 is shown. The time-dependent change of succinic acid production | generation and glucose consumption in the reference example 1 is shown. The change with time of succinic acid production and glucose consumption in Reference Example 2 is shown. The time-dependent change of succinic acid production | generation and consumption of glucose in Example 2 is shown. The time-dependent change of succinic acid production | generation and glucose consumption in the reference example 3 is shown. The time-dependent change of succinic acid production | generation and glucose consumption in the reference example 4 is shown.

Claims (8)

Corynebacterium glutamicum, which has lost lactate dehydrogenase activity, is cultured and grown under aerobic conditions, and succinic acid is produced using saccharides as a raw material in the reaction medium under reducing conditions using the obtained bacterial cells or treated products A method for producing succinic acid with high efficiency, characterized in that acetic acid and / or acetate is used as a carbon source in a medium for culture growth under aerobic conditions . The succinic acid according to claim 1, which comprises reacting the microbial cells of Corynebacterium glutamicum or the treated product thereof and saccharides in a reaction medium under a reducing condition after culture growth, and collecting the succinic acid produced. Manufacturing method. Corynebacterium glutamicum , in which lactate dehydrogenase activity has been lost, is Corynebacterium glutamicum R / Δ1dh (National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary Accession No. FERM P-20532) Item 2. A method for producing succinic acid according to Item 1. The method for producing succinic acid according to claim 1, wherein the acetate is a compound selected from ammonium acetate, sodium acetate and potassium acetate. The succinic acid according to claim 4 , wherein the compound selected from ammonium acetate, sodium acetate and potassium acetate is formed in a culture growth medium by a reaction between acetic acid and a corresponding basic compound. Production method. Sugars, glucose, xylose, arabinose, cellobiose, sucrose, method for producing succinic acid according to claim 1, wherein a is selected from maltose and dextrin. The reaction medium under reducing conditions is, a carbonate ion, a manufacturing method of succinic acid according to claim 1, characterized in that it comprises containing a bicarbonate ion or carbon dioxide gas. The method for producing succinic acid according to claim 1, wherein the oxidation-reduction potential of the reaction medium under reducing conditions is -200 mV to -500 mV.

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