JP2008259451A - Method for preparing organic acid-producing microorganism strain and method for producing organic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for obtaining bacterial strain capable of efficiently producing an organic acid. <P>SOLUTION: Disclosed is a method for preparing a bacterial strain having an organic acid-producing ability, wherein a microorganism is cultured by repeating every 2 to 25 min an operation in which pH or dissolved oxygen concentration is decreased under the predetermined value by adding carbon resources when the pH or dissolved oxygen concentration of the culture medium exceeds the predetermined value. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for preparing a microbial cell so that organic acid production efficiency is improved, and a method for producing an organic acid using the microbial cell prepared by the method.

  In the case of producing an organic acid such as succinic acid by fermentation, anaerobic bacteria such as Anaerobiospirillum genus and Actinobacillus genus are usually used (for example, Patent Document 1 or 2, non-patent document 1). 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 organic acids as a stationary cell without aeration of oxygen. A method is also known (see, for example, Patent Document 3 or 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. The production speed per hit was not enough, and there was room for improvement.
US Pat. No. 5,143,834 US Pat. No. 5,504,004 JP-A-11-113588 JP 11-196888 A International Journal of Systematic Bacteriology, vol. 49, p207-216, 1999

  An object of the present invention is to provide a method for preparing a microbial cell so that the organic acid production efficiency is improved, and a method for producing an organic acid using the microbial cell prepared by the method.

  As a result of intensive studies to solve the above-mentioned problems, the present inventor cultivated microbial cells having improved organic acid production efficiency by culturing so that carbon source depletion and satisfaction are alternately repeated in a short time. It was found that can be obtained. More specifically, the operation of adding a carbon source and lowering the pH or dissolved oxygen concentration below the set value when the pH or dissolved oxygen concentration of the culture solution exceeds the set value is repeated every 2 to 25 minutes. The present inventors have found that the cells obtained by culturing microorganisms can be used very efficiently in the reaction for producing an organic acid, and have completed the present invention.

That is, according to the present invention, the following inventions are provided.
(1) A method for preparing a cell of a microorganism having an organic acid-producing ability, wherein an operation of adding a carbon source and lowering the pH below a set value when the pH of the culture solution exceeds the set value is 2 to 2. A method comprising culturing a microorganism repeatedly every 25 minutes.
(2) A method for preparing cells of microorganisms capable of producing an organic acid, wherein the carbon source is added and / or the oxygen aeration rate when the dissolved oxygen concentration of the culture solution exceeds 0.05 ppm A method of culturing a microorganism by repeating the operation of lowering the dissolved oxygen concentration to 0.05 ppm or less every 2 to 25 minutes by adjusting.
(3) The method of (1) or (2), wherein the carbon source is glucose or sucrose.
(4) The method according to any one of (1) to (3), wherein the carbon source is added within a period of ½ or less of the repetition time.
(5) The method according to any one of (1) to (4), wherein the microorganism is a coryneform bacterium.
(6) An organic acid is produced by allowing the microbial cell prepared by any one of the methods (1) to (5) to act on an organic raw material in a reaction solution containing the organic raw material, A method for producing an organic acid, comprising collecting the organic acid.
(7) The method according to (6), wherein the reaction solution contains carbonate ion, bicarbonate ion, or carbon dioxide gas.
(8) The method according to (6) or (7), wherein the organic raw material is allowed to act in an anaerobic atmosphere.
(9) The method according to any one of (6) to (8), wherein the organic acid is succinic acid.
(10) A method for producing an organic acid-containing polymer, comprising a step of producing an organic acid by any one of the methods (6) to (9), and a step of carrying out a polymerization reaction using the organic acid obtained in the step as a raw material. .

  By using the microbial cells prepared by the method of the present invention, an organic acid such as succinic acid can be efficiently produced.

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

1. Microorganism used in the present invention In the method of the present invention, a microorganism having the ability to produce an organic acid is used. Here, “having the ability to produce an organic acid” means that an organic acid can be produced and accumulated in the medium when the microorganism is cultured in the medium.
Examples of organic acids include dicarboxylic acids such as lactic acid, succinic acid, malic acid, fumaric acid, oxaloacetic acid, 2-oxoglutaric acid and cis-aconitic acid, tricarboxylic acids such as citric acid and isocitric acid, pyruvic acid, acetic acid and the like. Among them, succinic acid, malic acid, fumaric acid, citric acid, isocitric acid, 2-oxoglutaric acid, cis-aconitic acid and pyruvic acid are preferable, and succinic acid is particularly preferable. preferable.

As the microorganism having an organic acid-producing ability, an aerobic microorganism, a facultative anaerobic microorganism or a microaerobic microorganism can be used.
The aerobic microorganisms include Coryneform Bacterium, Bacillus genus, Rhizobium genus, Arthrobacter genus, Mycobacterium genus, Rhodococcus Examples include genus bacteria, Nocardia genus bacteria, and Streptomyces genus bacteria, with coryneform bacteria being more preferred.
The coryneform bacterium is not particularly limited as long as it is classified as such, and examples thereof include bacteria belonging to the genus Corynebacterium, bacteria belonging to the genus Brevibacterium, and bacteria belonging to the genus Arthrobacter. Are those belonging to the genus Corynebacterium or Brevibacterium, more preferably, Corynebacterium glutamicum, Brevibacterium flavum, Brevibacterium ammoniagenes (Brevibacterium) ammoniagenes) or bacteria classified as Brevibacterium lactofermentum.

Particularly preferred specific examples of the parent strain of the microorganism used in the present invention include Brevibacterium flavum MJ-233 (FERM BP-1497), MJ-233 AB-41 (FERM BP-1498), Brevibacterium ammoniagenes. ATCC6872, Corynebacterium glutamicum ATCC31831, Brevibacterium lactofermentum ATCC13869, etc. are mentioned. 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.
Brevibacterium flavum MJ-233 was established on April 28, 1975, by the Ministry of International Trade and Industry, Institute of Industrial Science, 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.

Examples of facultative anaerobic microorganisms include bacteria belonging to the family Enterobacteriaceae belonging to genera such as Escherichia, Salmonella, Enterobacter and the like.
Microaerobic microorganisms include Aquaspirillum, Spirillum, Asospirillum, Oceanospirillum, Campylobacter, Bdellovibrio, and Vampirovibrio (Vampirovibrio) And microorganisms belonging to

The microorganism used in the production method of the present invention is not only a wild strain, but also a mutant strain obtained by normal mutation treatment such as UV irradiation or NTG treatment, or a genetic technique such as cell fusion or gene recombination. Any strain such as a recombinant strain may be used.
Examples of means for imparting organic acid-producing ability by breeding include mutation treatment and gene recombination. For each organic acid, a known method such as enhancement of biosynthetic enzyme gene expression or degradation of degradation enzyme gene expression is employed. For example, in the case of imparting the ability to produce succinic acid, modifications such as those described below that reduce lactate dehydrogenase activity and means for enhancing pyruvate carboxylase activity may be mentioned.

Bacteria modified to reduce lactate dehydrogenase (also referred to as LDH) activity can be obtained by, for example, a method using homologous recombination on a chromosome described in JP-A-11-206385 or a method using a sacB gene. (Schafer, A. et al. Gene 145 (1994) 69-73). In addition, “LDH activity is reduced” means that LDH activity is reduced as compared to an unmodified strain. LDH activity may be completely lost. The decrease in LDH activity was confirmed by a known method (L. Kanarek and RL Hill, J. Biol. Chem. 239,
4202 (1964)) and can be confirmed by measuring LDH activity.

Bacteria modified so that the activity of pyruvate carboxylase (hereinafter also referred to as PC) is enhanced can be obtained, for example, in the same manner as described in Japanese Patent Application Laid-Open No. 11-196888 by using the plasmid for the pc gene in a host bacterium. It can be constructed by high expression. Moreover, it may be integrated on the chromosome by homologous recombination, or the expression of the pc gene can be enhanced by promoter replacement. Transformation can be performed by, for example, the electric pulse method (Res. Microbiol., Vol. 144, p.181-185, 1993).
“The PC activity is enhanced” means that the PC activity is preferably increased by 1.5 times or more, more preferably by 3 times or more per unit cell weight relative to an unmodified strain such as a wild strain or a parent strain. Say. PC
The enhanced activity can be confirmed by measuring the PC activity by a known method (Magasanik's method [J. Bacteriol., 158, 55-62, (1984)]).

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

By introducing a DNA fragment containing the pc gene as described above into an appropriate plasmid, for example, a plasmid vector containing at least a gene that controls replication and replication of the plasmid in the host bacterium, high expression of the pc gene in the host bacterium can be achieved. Possible recombinant plasmids can be obtained. Here, in the above recombinant plasmid, the promoter for expressing the pc gene may be any promoter as long as it is a base sequence for initiating transcription of the pc gene. For example, tac promoter, trc promoter, TZ4 Examples include promoters.
Specific examples of plasmids that can be used for introducing genes into coryneform bacteria include, for example, 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 specification gazette; plasmids pCRY2 and pCRY3 described in JP-A-1-191686; pAM330 described in JP-A-58-66779 PHM1519 described in JP-A-58-77895; pAJ655, pAJ611 and pAJ1844 described in JP-A-58-192900; pCG1 described in JP-A-57-134500; described in JP-A-58-35197 of CG2; may be mentioned pCG4 and pCG11 like described in JP-57-183799 publication. 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.
Other organic acid-producing bacteria can also be constructed by enhancing the expression of the biosynthetic enzyme gene or decreasing the expression of the degradation enzyme gene in the same manner as described above.
Regarding the organic acids exemplified above, many organic acid producing bacteria such as wild strains, mutant strains, and genetically modified strains are known, and any organic acid producing bacteria can be used in the method of the present invention.

2. In the present invention, the organic acid-producing microorganism as described above is cultured in the medium so that the cycle of increasing or decreasing the pH or dissolved oxygen concentration of the medium is repeated every 2 to 25 minutes. By doing so, the microbial cell suitable for manufacture of an organic acid is prepared.
That is, when organic acid-producing microorganisms are cultured, if the sugar (glucose, fructose, sucrose, etc.) added to the medium as a carbon source is consumed, the pH of the medium is decreased, the dissolved oxygen concentration is decreased or substantially zero. Although a certain state is maintained, if the consumption of the carbon source further progresses to depletion, the pH of the medium and the dissolved oxygen concentration increase with the depletion of the carbon source. Using this increase in pH or dissolved oxygen concentration as an indicator, carbon source addition or oxygen supply is performed in a short time, and the cycle of increasing or decreasing pH or dissolved oxygen concentration is adjusted every 2 to 25 minutes. . In addition, the time per cycle of the ascending and descending cycle may be 2 to 25 minutes, and does not need to be repeated at the same interval each time.
Usually, the cells are seeded in the initial medium, and the above cycle is repeated when the carbon source is depleted. The time for culturing by repeating the cycle may be a time for obtaining microbial cells suitable for the production of an organic acid, but is preferably 6 to 40 hours, for example.

2-1. In the case of controlling by pH In the first embodiment of the present invention, a reference value for pH is set in advance, and when the pH of the medium exceeds the set value, the pH is set by adding a carbon source. The operation of lowering the value below is repeated every 2 to 25 minutes.
Here, the set value of pH may be a value that allows growth of microorganisms and a pH increase exceeding the set value when the carbon source is depleted, but is usually between pH 6 and 8, at the start of culture. The pH value of the medium can also be set. Also, when adding an alkali such as ammonia to the medium as a nitrogen source or the like, do not consider the increase in pH when the alkali is added, or add the carbon source only when the pH increases due to the depletion of the carbon source, Or when adding alkali continuously and controlling so that pH does not fall below a fixed value, the alkali control value is made into a set value, and when it exceeds the set value, a carbon source is added.
The carbon source may be added while the pH exceeds the set value, and may not be immediately after the set value is exceeded. The timing of adding the carbon source is also adjusted by the concentration of the carbon source to be added, the cycle interval, and the like, but it is preferable to add it when the pH is higher than the set value by 0.05 or more.
For example, a mode in which the pH control value (set value) by adding alkali is set to pH 7.50, and when the pH exceeds 7.50 or more (when the pH becomes 7.55 or more), the carbon source is added is exemplified. The

The addition amount of the carbon source is an amount such that the interval from the addition of the carbon source until the pH that has fallen below the set value rises again and exceeds the set value is 2 to 25 minutes. The amount varies depending on the interval, the oxygen supply amount, the amount of alkali in the medium, etc., but it is preferable to add such that the carbon source concentration in the medium at the time of addition is 0.2 to 4 g / L.
The type of carbon source to be added is not particularly limited as long as it can be assimilated by microorganisms, but glucose or sucrose is preferable.
The carbon source is added when the pH exceeds the set value, but is preferably added intermittently, more preferably added within a time of ½ or less of the interval, and 1 of the interval. More preferably, it is added within a time of / 3 or less. For example, when the carbon source addition interval is 3 minutes, the carbon source is preferably added within 1 minute.
When controlling by pH, the amount of aeration during culture is not particularly limited.

2-2. In the case of controlling by the dissolved oxygen concentration In the second embodiment of the present invention, when the dissolved oxygen concentration of the medium exceeds 0.05 ppm, the carbon source is added and / or the oxygen supply amount is controlled. , Dissolved oxygen concentration 0.05
The operation of lowering to ppm or less is repeated every 2 to 25 minutes.

The amount of carbon source added is such that the interval from when the carbon source is added until the dissolved oxygen concentration that has fallen below 0.05 ppm again exceeds 0.05 ppm is 2 to 25 minutes. The specific amount varies depending on the interval, the oxygen supply amount, the amount of alkali in the medium, etc., but is preferably added so that the carbon source concentration in the medium immediately after addition is 0.2 to 4 g / L. .
The type of carbon source to be added is not particularly limited as long as it can be assimilated by microorganisms, but glucose or sucrose is preferable.
The carbon source is added when the dissolved oxygen concentration exceeds 0.05 ppm, but it is preferably added intermittently, preferably within a period of 1/2 or less of the interval. More preferably, it is added within 1/3 or less of the above. For example, when the carbon source addition interval is 3 minutes, it is preferable to add the carbon source within the first minute.
In this case, it is preferable that the aeration amount during the culture be an oxygen supply amount at which the dissolved oxygen concentration can be 0.05 ppm or less when the carbon source is not depleted.

On the other hand, when controlling the dissolved oxygen concentration of the medium with the oxygen supply amount, when the dissolved oxygen concentration exceeds 0.05 ppm, the oxygen supply amount is reduced by limiting the aeration amount and / or the stirring speed, etc. There is a method in which the dissolved oxygen concentration is lowered to 0.05 ppm or less, and after 2 to 25 minutes, the dissolved oxygen concentration is increased by raising the original oxygen supply amount.
In this case, the carbon source may be added at a rate such that carbon source depletion occurs.

  In any case where the increase in pH or dissolved oxygen concentration is used as an indicator, the culture may be performed so that the cycle of increase or decrease in pH or dissolved oxygen concentration is repeated as a result. There is no need to add a carbon source while monitoring the dissolved oxygen concentration.

In addition, the culture medium used for said microbial cell preparation culture | cultivation may contain components other than a carbon source, and the normal culture medium component used for culture | cultivation 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 culture temperature is usually 25 ° C to 40 ° C, preferably 30 ° C to 37 ° C.
The microbial cell prepared as described above is used in the following organic acid production reaction.
The cells after the cell preparation and culture may be used in the following organic acid production reaction as they are in the culture solution, or may be used for the organic acid production reaction after being collected by centrifugation, membrane separation, or the like.

3. Method for Producing Organic Acid The method for producing an organic acid according to the present invention is a method for producing an organic acid by allowing the microbial cell prepared by the above method to act on an organic raw material in a reaction solution containing the organic raw material. Is a method for producing an organic acid. Examples of organic acids that can be produced and examples of preferred organic acids are as described above.

  In the present invention, a processed product of microbial cells can also be used. Examples of the treated product of cells include immobilized cells obtained by fixing cells with acrylamide, carrageenan, and the like.

  The organic raw material used for the production of the organic acid is not particularly limited as long as it is a carbon source that can be assimilated by the above microorganisms to produce an organic acid. Carbohydrates such as starch and cellulose; fermentable carbohydrates such as polyalcohols such as glycerin, mannitol, xylitol, and ribitol are used, and among these, glucose or sucrose is preferable, and glucose is particularly preferable.

  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 concentration of the organic raw material used is not particularly limited, but it is advantageous to make it as high as possible within the range that does not impede the production of organic acid, 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 microorganisms or a buffer solution such as a phosphate buffer solution. The reaction solution is preferably an aqueous solution containing a nitrogen source, an inorganic salt, and the like. Here, the nitrogen source is not particularly limited as long as it is a nitrogen source that can be assimilated by microorganisms to produce succinic acid. Various organic and inorganic nitrogen compounds such as peptone, yeast extract, meat extract, corn steep liquor and the like can be mentioned. As the inorganic salt, various phosphates, sulfates, magnesium, potassium, manganese, iron, zinc, and other metal salts are used. In addition, factors that promote growth such as vitamins such as biotin, pantothenic acid, inositol, and nicotinic acid, nucleotides, and amino acids can be 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 reaction solution preferably contains carbonate ions, bicarbonate ions, or carbon dioxide gas (carbon dioxide gas) in addition to the organic raw material, nitrogen source, inorganic salt, and the like. Carbonate ions or bicarbonate ions are supplied from magnesium carbonate, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, etc., which can also be used as a neutralizing agent. It can also be supplied from these salts 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 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. The pH in the organic acid production reaction is usually in the range of pH 5 to 10, preferably pH 6 to 9.5, and the pH of the reaction solution is within the above range depending on the alkaline substance, carbonate, urea, etc. as necessary during the reaction. Adjust to.

  The temperature during the organic acid production 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 in particular, it is 1-700 g / L, Preferably it is 10-500 g / L, More preferably, it is 20-400 g / L. The reaction time is preferably 1 hour to 168 hours, more preferably 3 hours to 72 hours.

  The organic acid generation reaction may be performed with aeration and stirring, but may be performed in an anaerobic atmosphere without aeration and without supplying oxygen. For example, an anaerobic atmosphere may be obtained by a method such as sealing a container and reacting without aeration, supplying an inert gas such as nitrogen gas to react, or venting an inert gas containing carbon dioxide gas. it can.

By the reaction as described above, an organic acid such as succinic acid, fumaric acid, malic acid or pyruvic acid is generated and accumulated in the reaction solution. The organic acid accumulated in the reaction solution can be collected from the reaction solution according to a conventional method. Specifically, for example, after removing solids such as bacterial cells by centrifugation, filtration, etc., desalting with an ion exchange resin or the like, and crystallization from the solution or purification by column chromatography, etc. Acid can be collected.

Furthermore, in this invention, after manufacturing organic acids, such as a succinic acid, by the above-mentioned method, an organic acid containing polymer can be manufactured by performing a polymerization reaction using the obtained organic 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. In particular, succinic acid produced in the present invention is processed into polymers such as polyester and polyamide. Can be used. Specific examples of succinic acid-containing polymers include succinic acid polyesters obtained by polymerizing diols such as butanediol and ethylene glycol and succinic acid, succinic acid polyamides obtained by polymerizing diamines such as hexamethylenediamine and succinic acid, and the like. Is mentioned.
The organic acid obtained by the production method of the present invention or the composition containing the organic acid can be used for food additives, pharmaceuticals, cosmetics and the like.

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

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

(B) Construction of a plasmid for PC promoter substitution The DNA fragment of the N-terminal region of the pyruvate carboxylase gene derived from Brevibacterium flavum MJ233 strain was obtained using the DNA prepared in (A) above as a template and the entire genome sequence was reported. This was performed by PCR using synthetic DNA (SEQ ID NO: 1 and SEQ ID NO: 2) designed based on the sequence of the gene of Corynebacterium glutamicum ATCC13032 strain (Cgl0689 of GenBank Database Accession No. BA00000036). The DNA of SEQ ID NO: 1 used was phosphorylated at the 5 ′ end. Composition of reaction solution: Template DNA 1 μL, Pfx DNA polymerase (manufactured by Invitrogen) 0.2 μL, 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, 60 ° C. for 20 seconds, and 72 ° C. for 1 minute was repeated 35 times. However, 1
The heat retention at 94 ° C. in the cycle was 1 minute 20 seconds, and the heat retention at 72 ° C. in the final cycle was 4 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 to detect a fragment of about 0.9 kb. Recovery of the target DNA fragment from the gel was carried out using QIAQuick Gel Extraction Kit (manufactured by QIAGEN), and this was used as the PC gene N-terminal fragment.

On the other hand, a TZ4 promoter fragment constitutively highly expressed from Brevibacterium flavum MJ233 strain is a PCR using the plasmid pMJPC1 (Japanese Patent Laid-Open No. 2005-95169) as a template and the synthetic DNAs described in SEQ ID NO: 3 and SEQ ID NO: 4. It was prepared by. The DNA of SEQ ID NO: 4 was phosphorylated at the 5 ′ end. 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 condition: 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 25 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 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 0.5 kb was detected. Recovery of the target DNA fragment from the gel was performed using QIAQuick Gel Extraction Kit (manufactured by QIAGEN), and this was used as the TZ4 promoter fragment.

  The PC gene N-terminal fragment prepared above and the TZ4 promoter fragment were mixed, and ligation kit ver. 2 (manufactured by Takara Shuzo), cleaved with restriction enzyme PstI, separated by 1.0% agarose (SeaKem GTG agarose: manufactured by FMC BioProducts) gel electrophoresis, and a DNA fragment of about 1.0 kb was isolated from QIAQuick Gel Extraction Kit (Manufactured by QIAGEN) was used as a TZ4 promoter :: PC gene N-terminal fragment. Further, this DNA fragment and E. coli plasmid pHSG299 (Takara Shuzo) were mixed with DNA prepared by cutting with PstI, and ligation kit ver. 2 (Takara Shuzo) were used for connection. 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 kana machine and 50 μg / mL X-Gal. Clones that formed white colonies on this medium were subjected to liquid culture by a conventional method, and then plasmid DNA was purified. The obtained plasmid DNA was cleaved with the restriction enzyme PstI, whereby an inserted fragment of about 1.0 kb was recognized, and this was named pMJPC17.1.

The DNA fragment in the 5 ′ upstream region of the pyruvate carboxylase gene derived from Brevibacterium flavum MJ233 strain was obtained using the DNA prepared in Example 1 (A) as a template and the entire genome sequence reported. It was carried out by PCR using synthetic DNA (SEQ ID NO: 5 and SEQ ID NO: 6) designed based on the sequence of the gene of Glutamicum ATCC13032 strain (GenBank Database Accession No. BA00000036). Composition of reaction solution: Template DNA 1 μL, PfxDNA polymerase (manufactured by Invitrogen) 0.2 μL, 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, 60 ° C. for 20 seconds, and 72 ° C. for 30 seconds was repeated 35 times. However, the heat retention at 94 ° C. in the first cycle was 1 minute 20 seconds, and the heat retention at 72 ° C. in the final cycle was 5 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 0.7 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 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 pUC119 (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. 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 subjected to a PCR reaction using the synthetic DNAs shown in SEQ ID NO: 7 and SEQ ID NO: 6 as primers. Composition of reaction solution: 1 ng of the above plasmid, Ex-Taq DNA polymerase (manufactured by Takara Shuzo Co., Ltd.) 0.2 μL, 1 × concentration attached 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, 60 ° C. for 20 seconds and 72 ° C. for 50 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. As a result of confirming the presence or absence of the inserted DNA fragment in this way, a plasmid that recognized an amplification product of about 0.7 kb was selected and named pMJPC5.1.

  Next, the above-mentioned pMJPC17.1 and pMJPC5.1 were cleaved with restriction enzyme XbaI and mixed, and ligation kit ver. 2 (Takara Shuzo) were used for connection. The DNA fragment cleaved with restriction enzyme SacI and restriction enzyme SphI was separated by 0.75% agarose (SeaKem GTG agarose: manufactured by FMCBioProducts) gel electrophoresis, and a DNA fragment of about 1.75 kb was prepared by QIAQuick Gel Extraction Kit (manufactured by QIAGEN Gel Extraction Kit). ). A DNA fragment in which the TZ4 promoter is inserted between the 5 ′ upstream region and the N-terminal region of the PC gene, Mixed and ligation kit ver. 2 (Takara Shuzo) were used for connection. 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 kana machine and 50 μg / mL X-Gal. Clones that formed white colonies on this medium were subjected to liquid culture by a conventional method, and then plasmid DNA was purified. By cutting the obtained plasmid DNA with restriction enzymes SacI and SphI, an insert fragment of about 1.75 kb was observed, which was named pMJPC17.2 (FIG. 1).

(C) Preparation of PC-enhanced strain Plasmid DNA used for transformation of Brevibacterium flavum MJ233 / ΔLDH (Japanese Patent Laid-Open No. 2005-95169) was obtained using the plasmid DNA of pMJPC17.3 using the calcium chloride method (Journal of Molecular Biology, 53 , 159, 1970) and re-prepared from E. coli strain JM110. The Brevibacterium flavum MJ233 / ΔLDH strain was transformed by the electric pulse method (Res. Microbiol., Vol. 144, p.181-185, 1993), and the resulting transformant contained 25 μg / mL of kanamycin. 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] was smeared. Since the strain grown on this medium is a plasmid in which pMJPC17.2 cannot replicate in the Brevibacterium flavum MJ233 strain, the PC gene of the plasmid and the same gene on the genome of Brevibacterium flavum MJ233 strain As a result of homologous recombination with the kanamycin, the kanamycin resistance gene and sacB gene derived from the plasmid should be inserted on the genome. Next, the homologous recombination strain was subjected to liquid culture in an LBG medium containing 25 μg / mL kanamycin. A portion equivalent to about 1 million cells of this culture was smeared on a 10% sucrose-containing LBG medium. As a result, sacB gene was removed by the second homologous recombination and sucrose-insensitive sucrose strains were obtained. Among the strains thus obtained, those in which the TZ4 promoter derived from pMJPC17.2 is inserted upstream of the PC gene and those that have returned to the wild type are included. Confirmation of whether the PC gene is promoter-substituted type or wild type can be easily confirmed by subjecting the cells obtained by liquid culture in LBG medium to direct PCR reaction and detecting the PC gene. . When analyzed using primers (SEQ ID NO: 8 and SEQ ID NO: 9) for PCR amplification of the TZ4 promoter and PC gene, a 678 bp DNA fragment should be observed in the promoter-substituted form. As a result of analyzing the sucrose-insensitive strain by the above method, a strain into which the TZ4 promoter was inserted was selected, and the strain was named Brevibacterium flavum MJ233 / PC-4 / ΔLDH.

(D) Measurement of pyruvate carboxylase enzyme activity The transformant Brevibacterium flavum MJ233 / PC-4 / ΔLDH strain obtained in (C) above was overnight in 100 mL of A medium containing 2% glucose and 25 mg / L kanamycin. Culture was performed. 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 of the cell-free extract with enhanced pyruvate carboxylase expression was 0.1 U / mg protein. In addition, in the cells cultured in the same manner as the parent strain MJ233 / ΔLDH strain, it was below the detection limit of this activity measurement method.
Hereinafter, the Brevibacterium flavum MJ233 / PC-4 / ΔLDH strain was used as an organic acid-producing bacterium for microbial cell preparation culture and organic acid production reaction.

[Example 1]
<Seed culture>
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: 5 g, casamino acid: 5 g, and distilled water: 100 mL of a medium of 1000 mL are placed in a 500 mL Erlenmeyer flask at 120 ° C. for 20 minutes. Heat sterilized. This was cooled to room temperature, 4 mL of a 50% aqueous glucose solution sterilized in advance was added, and Brevibacterium flavum MJ233 / PC-4 / ΔLDH constructed above was inoculated and seed-cultured at 30 ° C. for 24 hours.

<Main culture>
Ammonium sulfate: 1.0 g, monopotassium phosphate: 1.5 g, dipotassium phosphate 1.5 g, potassium chloride: 1.67 g, magnesium sulfate heptahydrate: 0.5 g, ferrous sulfate, heptahydrate Product: 40 mg, manganese sulfate / hydrate: 40 mg, D-biotin: 1.0 mg, thiamine hydrochloride: 1.0 mg, yeast extract 10 g, antifoaming agent (CE457: manufactured by NOF Corporation): 1.0 g and distilled water: 400 mL of 1000 mL of medium was placed in a 1 L fermentor and sterilized by heating at 120 ° C. for 20 minutes. After cooling to room temperature, 5 mL of 72% aqueous glucose solution sterilized in advance was added, and 20 mL of the seed culture solution was added thereto, and the mixture was kept at 30 ° C. The pH was maintained at 7.0% or less using 9.3% ammonia water, main culture was performed at 300 mL / min for aeration and 600 rpm for 20 hours with stirring. The dissolved oxygen concentration gradually decreased immediately after the start of the culture, and became almost 0 after 4.5 hours from the start of the culture. Thereafter, since the dissolved oxygen concentration increased 6.5 hours after the start of the culture, when 667 μL of a 72% glucose aqueous solution sterilized in advance was added, it rapidly decreased again and became almost zero. Since an increase in the dissolved oxygen concentration was observed after about 23 minutes, 667 μL of a 72% aqueous glucose solution sterilized in advance was added and lowered again. Thereafter, a similar increase was observed about every 23 minutes, but it was reduced by the same method each time. The OD660 after 20 hours of culture was 78.0. FIG. 2 shows changes over time in pH and dissolved oxygen concentration at this time (up to 20 hours after the start of culture).

<Organic acid production culture>
Monoammonium phosphate: 84.4 mg, diammonium phosphate: 75.8 mg, potassium chloride 149.1 mg, magnesium sulfate heptahydrate: 0.2 g, ferrous sulfate heptahydrate: 8 mg, manganese sulfate Hydrate: 8 mg, D-biotin: 80 μg, thiamine hydrochloride: 80 μg and distilled water: 200 mL of medium was placed in a 500 mL Erlenmeyer flask and sterilized by heating at 120 ° C. for 20 minutes. After cooling to room temperature, it was placed in a 1 L jar fermenter. To 200 mL of this suspension, 90 mL of the culture solution obtained by the above main culture, 72% glucose solution sterilized in advance: 75 mL, and 125 mL of sterilized water were added and mixed, and kept at 35 ° C. The pH was maintained at 7.6 using an aqueous solution of ammonium carbonate: 154 g, 28% aqueous ammonia: 239 ml, distilled water: 650 mL, and an organic acid production reaction was performed while stirring at 200 rpm. At 13 hours after the start of the reaction, the production succinic acid concentration was 32.5 g / L, the production malic acid concentration was 3.1 g / L, the production fumaric acid concentration was 0.2 g / L, and the production acetic acid concentration was 2.2 g / L. It was.

[Example 2]
When seed culture and main culture (bacterial cell preparation) were performed in the same manner as in Example 1, the dissolved oxygen concentration gradually decreased immediately after the start of the culture, and became almost zero 4.5 hours after the start of the culture. Thereafter, since it increased 6.5 hours after the start of culture, when 50 μL of a 72% aqueous glucose solution sterilized in advance was added, it rapidly decreased again and became almost zero. Since an increase in dissolved oxygen concentration was observed after about 2 minutes, 50 μL of a 72% aqueous glucose solution sterilized in advance was added and lowered again. Thereafter, a similar increase was observed about every 2 minutes, but it was reduced by the same method each time. The OD660 after 20 hours of culture was 82.9.
When the organic acid production reaction was carried out in the same manner as described above using the culture solution thus obtained, the succinic acid concentration at 13 hours after the start of the reaction was 27.3 g / L, the production malic acid concentration was 2.0 g / L, and the production The fumaric acid concentration was 0.2 g / L, and the production acetic acid concentration was 4.5 g / L.

[Comparative Example 1]
When seed culture and main culture (bacterial cell preparation) were performed in the same manner as in Example 1, the dissolved oxygen concentration gradually decreased immediately after the start of the culture, and became almost zero 4.5 hours after the start of the culture. Then, since it rose 6.5 hours after the start of culture, a 72% aqueous glucose solution sterilized in advance was continuously added at 0.21 μL / second. After this, the dissolved oxygen concentration was more than 2 ppm for most of the time. The OD660 after 20 hours of culture was 72.3.
When the organic acid production reaction was carried out in the same manner as described above using the culture solution thus obtained, the succinic acid concentration at 13.5 hours after the start of the reaction was 15.5 g / L, the production malic acid concentration was 1.2 g / L, and the production The fumaric acid concentration was 0.1 g / L, and the production acetic acid concentration was 1.9 g / L.

[Comparative Example 2]
In accordance with Example 1, seed culture and main culture (bacterial cell preparation) were performed. However, in the main culture, 72 mL of a 72% glucose aqueous solution previously sterilized was added, and 20 mL of the aforementioned seed culture solution was added thereto, and the mixture was kept at 30 ° C. The main culture was performed using 9.3% ammonia water so that the pH was not lower than 7.0, aeration was 300 mL / min, and stirring was 600 rpm. The dissolved oxygen concentration gradually decreased immediately after the start of culture, and became almost 0 after 4.5 hours of culture.
The culture was continued for 20 hours after the start of culture. The OD660 after 20 hours of culture was 51.3.
When the organic acid production reaction was performed in the same manner as described above using the culture solution thus obtained, the succinic acid concentration at 16.9 hours after the start of the reaction was 16.9 g / L, the production malic acid concentration was 0.4 g / L, and the production The fumaric acid concentration was 0 g / L and the production acetic acid concentration was 2.2 g / L.

The figure which shows the construction procedure of plasmid pMJPC17.2. The underlined number indicates a primer consisting of the sequence of SEQ ID NO. The graph which shows the time-dependent change of pH of the culture medium in Example 1, and dissolved oxygen concentration.

Claims (10)

A method for preparing cells of a microorganism having an organic acid-producing ability, wherein an operation of adding a carbon source and lowering the pH below a set value when the pH of the culture solution exceeds the set value is performed every 2 to 25 minutes And culturing the microorganism repeatedly. A method for preparing cells of microorganisms capable of producing an organic acid, wherein a carbon source is added and / or an oxygen aeration rate is adjusted when the dissolved oxygen concentration in a culture solution exceeds 0.05 ppm Thus, the microorganism is cultured by repeating the operation of lowering the dissolved oxygen concentration to 0.05 ppm or less every 2 to 25 minutes. The method according to claim 1 or 2, wherein the carbon source is glucose or sucrose. The method according to any one of claims 1 to 3, wherein the carbon source is added within a period of ½ or less of the repetition time. The method according to any one of claims 1 to 4, wherein the microorganism is a coryneform bacterium. An organic acid is produced by allowing the microorganism cells prepared by the method according to any one of claims 1 to 5 to act on the organic raw material in a reaction solution containing the organic raw material, and collecting the organic acid A method for producing an organic acid, comprising: The method according to claim 6, wherein the reaction liquid contains carbonate ion, bicarbonate ion, or carbon dioxide gas. The method according to claim 6 or 7, wherein the organic raw material is allowed to act in an anaerobic atmosphere. The method according to any one of claims 6 to 8, wherein the organic acid is succinic acid. The manufacturing method of an organic acid containing polymer including the process of manufacturing an organic acid by the method as described in any one of Claims 6-9, and the process of performing a polymerization reaction using the organic acid obtained at the said process as a raw material.

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