JP6446807B2 - Method for producing organic compound using microorganism - Google Patents

Description

  The present invention relates to an efficient method for producing an organic compound using a microorganism.

  Fermentative production processes of organic compounds using saccharides as raw materials are widely used, and products obtained through the above processes are used as various industrial raw materials.

In patent document 1, in succinic acid fermentation production using microorganisms, the concentration of succinic acid alkali metal salt in an aqueous medium is set to a specific range, and then ammonia and / or ammonium salt is added to increase the concentration of succinic acid. A method for improving the production rate under conditions is disclosed.
Patent Document 2 discloses a method for improving the succinic acid production rate by adding hydroxyacetone in anaerobic fermentation using glycerol as a raw material. Glycerol is less consumed by E. coli because it is a highly reduced organic raw material compared to common sugars such as glucose. Therefore, in patent document 2, the consumption of glycerol of E. coli is promoted by converting a hydroxyacetone into 1,2-propanediol and activating a metabolic pathway that consumes reducing power, thereby improving productivity.
Non-Patent Document 1 discloses a method for supplying reducing power and improving succinic acid productivity by adding a compound serving as an electron carrier such as neutral red in succinic acid fermentation production using microorganisms. ing.

International Publication WO2013 / 069786 Pamphlet Special table 2009-532037 gazette

Journal of Bacteriology, 1999, 181 (8), p2403-2410.

  When an organic compound is produced using a microorganism, it is required to accumulate the organic compound at a high concentration. However, when the organic compound accumulates at a high concentration, stress due to osmotic pressure increases, metabolic activity due to product inhibition. There is a problem that the production rate decreases as the organic compound accumulates at a high concentration due to the decrease and the deterioration of the intracellular redox balance accompanying the environmental change.

In Patent Document 1, there is no description or suggestion about adding hydroxyacetone, and the method described in Patent Document 1 does not supply reducing power from other than the raw material, so that productivity is improved. There seems to be room for improvement.
Patent Document 2 is an invention directed to the case where glycerol is used as a raw material, and the case where a general saccharide is used as a raw material has not been studied. Moreover, in patent document 2, consumption of glycerol of E. coli is promoted by adding hydroxyacetone. That is, the added hydroxyacetone is converted to 1,2-propanediol, which causes consumption of reducing power. However, it is considered that adjustment of the redox balance in the cells of microorganisms, in particular, the supply of reducing power has not been achieved.

In Non-Patent Document 1, a compound such as neutral red is added to supply reducing power, but since these compounds are not subjected to a metabolic reaction that consumes reducing power, supply and consumption of reducing power. The balance cannot be adjusted. Further, in Non-Patent Document 1, since the reducing power is supplied from the initial stage of production where the reducing power is relatively satisfactory, the reducing power is excessively supplied at the time of low concentration accumulation, while the reducing power is insufficient. There is also a possibility that the supply of reducing power may be insufficient during high concentration accumulation.
An object of the present invention is to provide a method for efficiently producing an organic compound by improving the production rate under the condition that the organic compound is accumulated at a high concentration, particularly when producing an organic compound using a microorganism. is there.

As a result of intensive studies to solve the above problems, the present inventors have found that reduction power can be adjusted by producing an organic compound using a microorganism in the presence of hydroxyacetone. The present invention has been completed.
Since hydroxyacetone is converted into 1,2-propanediol or methylglyoxal by the metabolic reaction of microorganisms, it can be supplied as well as consuming reducing power. The present invention is based on the finding that the effect of hydroxyacetone can be obtained even under conditions where the organic compound is accumulated at a high concentration.

That is, the gist of the present invention resides in the following [1] to [11].
[1] A step of producing an organic compound by causing a microorganism having organic compound-producing ability or a processed product thereof to act on a saccharide-containing raw material in an aqueous medium in the presence of hydroxyacetone (hereinafter referred to as “fermentation step”). The manufacturing method of the organic compound characterized by having.
[2] The method for producing an organic compound according to [1], wherein the saccharide-containing raw material is a raw material containing hydroxyacetone.
[3] The method for producing an organic compound according to [1] or [2], wherein the saccharide-containing raw material is a lignocellulose decomposition raw material.
[4] The method for producing an organic compound according to [1] or [2], wherein the saccharide-containing raw material is a sucrose-containing raw material.
[5] The method for producing an organic compound according to any one of [1] to [4], wherein the fermentation step is performed in an anaerobic atmosphere.
[6] The organic according to any one of [1] to [5], wherein the aqueous medium contains at least one selected from the group consisting of carbonate ions, bicarbonate ions, and carbon dioxide gas. A method for producing a compound.
[7] The organic material according to any one of [1] to [6], wherein the organic compound is at least one selected from the group consisting of alcohols, amines, carboxylic acids, and phenols. A method for producing a compound.
[8] The method for producing an organic compound according to any one of [1] to [7], wherein the organic compound is an organic compound obtained through a biosynthetic pathway having pyruvic acid as an intermediate. .
[9] The microorganism according to any one of [1] to [8], wherein the microorganism is a microorganism having an activity of converting hydroxyacetone to 1,2-propanediol and an activity of converting hydroxyacetone to methylglyoxal. The manufacturing method of the organic compound in any one.
[10] The microorganism is a coryneform bacterium, Escherichia coli, Anaerobiospirillum genus bacteria, Actinobacillus genus bacteria, Mannheimia genus bacteria, Basfia genus Zymomonas (Zymomonas) zymonas The method for producing an organic compound according to any one of [1] to [9], wherein the organic compound is at least one selected from the group consisting of bacteria, genus Zymobacter, filamentous fungi, and yeast .
[11] The method for producing an organic compound according to any one of [1] to [10], further comprising a step of purifying the organic compound obtained in the fermentation step.

  According to the present invention, the production rate of the organic compound in the microorganism can be improved even under conditions where the target organic compound is accumulated at a high concentration.

The metabolic pathway of hydroxyacetone is shown.

  Hereinafter, the present invention will be specifically described. However, the present invention is not limited to the following embodiments, and various modifications can be made without departing from the scope of the present invention.

  The present invention is a process for producing an organic compound by allowing a microorganism having organic compound-producing ability or a treated product thereof to act on a saccharide-containing raw material in an aqueous medium in the presence of hydroxyacetone (hereinafter referred to as “fermentation process”). ) (Hereinafter also referred to as “the production method of the present invention”). Hereinafter, the production method of the present invention will be described in detail.

[Sugar-containing raw material]
The “sugar” of the “saccharide-containing raw material” in the present invention means a general saccharide, that is, an aldose having one or more aldehyde groups or a ketose having one or more ketone groups.
Hereinafter, the saccharides contained in the saccharide-containing raw material, the origin of the saccharide-containing raw material, and the production method will be described.

<Sugars in sugar-containing raw materials>
The saccharides in the present invention are not particularly limited, and so-called general saccharides can be used, but saccharides that can be utilized by microorganisms as a carbon source are preferable.
Specifically, monosaccharides having 3 carbon atoms (triose) such as glyceraldehyde; monosaccharides having 4 carbon atoms (tetrose) such as erythrose, threose, erythrulose; ribose, lyxose, xylose, arabinose, deoxyribose, xylulose 5 carbon monosaccharides (pentose) such as ribulose; 6 carbon atoms such as allose, talose, growth, glucose, altrose, mannose, galactose, idose, fucose, fucose, rhamnose, psicose, fructose, sorbose, tagatose Monosaccharide (hexose); monosaccharides having 7 carbon atoms such as cedoheptulose (heptose); disaccharides such as sucrose, lactose, maltose, trehanose, tulanose, cellobiose; trisaccharides such as raffinose, melezitose, maltotriose; Rakutoorigo sugars, galactooligosaccharides, oligosaccharides such Man'naorigo sugar; starch, dextrin, cellulose, hemicellulose, glucans and polysaccharides pentosan like.

  Among the saccharides described above, a saccharide containing a monosaccharide having 3 to 7 carbon atoms as a constituent component is preferable. Among these, at least one selected from the group consisting of hexose, pentose, and disaccharides containing these as constituents is more preferable. This is because they are abundant because they are constituents of nature and plants, and it is easy to obtain raw materials.

As hexose, glucose, fructose, mannose and galactose are preferable, and glucose is more preferable. As pentose, xylose and arabinose are preferable, and xylose is more preferable. Sucrose is preferred as the disaccharide containing hexose and pentose as constituent components. This is because glucose, xylose, and sucrose are the main constituents of nature and plants, and therefore it is easy to obtain raw materials.
In addition, the saccharide-containing raw material used in the present invention may contain one kind of saccharide alone, or may contain two or more kinds of saccharides.

<Sugar-containing raw material>
The saccharide-containing raw material used in the present invention is not particularly limited as long as it contains the saccharide, but may contain water or the like, if necessary, preferably water.
The saccharide-containing raw material used in the present invention is not particularly limited. For example, one or more saccharides dissolved in water to form an aqueous solution, or a plant containing one or more saccharides as a constituent, What decomposed | disassembled partially to saccharides, the plant body which contains 1 or more types of said saccharides as a structural component, or what extracted saccharides from the part can be used. Specifically, a lignocellulose decomposition raw material, a sucrose-containing raw material, a starch decomposition raw material, and the like, which will be described later, are mentioned.

The saccharide-containing raw material used in the present invention may be used by diluting with water or the like to lower the sugar concentration as necessary, or may be used by concentrating to increase the sugar concentration. Containing raw materials can also be added.
The concentration of the saccharide contained in the saccharide-containing raw material in the present invention is not particularly limited because it varies greatly depending on the origin of the saccharide-containing raw material, the type of sugar contained, and the like, but productivity of the fermentation production process and the chemical conversion process Is usually 0.1% by mass or more, preferably 2% by mass or more, and usually 80% by mass or less, preferably 70% by mass or less. However, when 2 or more types of saccharides are included, the total concentration is shown.

A preferred saccharide-containing raw material is a lignocellulose-decomposing raw material. That is, as the saccharide-containing raw material, those obtained by decomposing lignocellulose are preferable.
Lignocellulose is an organic substance composed of structural polysaccharide cellulose, hemicellulose, and polymer lignin of an aromatic compound. Lignocellulose is usually not edible and is usually discarded or incinerated, and is preferable in that it can be supplied stably and resources can be used effectively.
Lignocellulose decomposition materials include herbaceous biomass such as bagasse, corn stover, wheat straw, rice straw, switchgrass, napiergrass, Eliansus, Sasa and Susuki, and woody biomass such as waste wood, sawdust, bark and waste paper Etc. can be used suitably. Of these, bagasse, corn stover, and straw are preferable.

  The method for obtaining the saccharide-containing raw material from the above lignocellulose-decomposing raw material is not particularly limited. For example, after pretreatment of lignocellulose as necessary, enzyme, acid, subcritical water, supercritical water, etc. And a method of performing hydrolysis or thermal decomposition.

Moreover, a sucrose containing raw material is mentioned as a preferable saccharide containing raw material. That is, the saccharide-containing raw material preferably contains sucrose.
Further, sucrose is included in plants that can accumulate sucrose in cells. Hereinafter, such plants are referred to as “plants containing sucrose”. Examples of plants containing sucrose include those used as raw materials for sugar such as sugar cane, sugar beet, sugar maple, sugar beet, and sorghum, among which sugar cane and sugar beet are preferred.

  A method for obtaining a saccharide-containing raw material from a plant containing sucrose is not particularly limited, and examples thereof include a method of pressing or leaching after pulverizing the plant. In the present invention, plant juice containing sucrose thus obtained (for example, cane juice in the case of sugarcane), crude sugar, molasses, etc. can also be used as the saccharide-containing raw material.

Moreover, a starch decomposition raw material is mentioned as a preferable saccharide containing raw material. That is, the saccharide-containing raw material preferably contains starch.
Moreover, starch is contained in plants that can accumulate starch in cells, and hereinafter, such plants are referred to as “plants containing starch”. Examples of the plant containing starch include cassava, corn, potato, wheat, sweet potato, sago palm, rice, scrap, peanut, mung bean, bracken, green lily, etc. Among them, cassava, corn, potato and wheat are preferable.

  A method for obtaining a saccharide-containing raw material from a plant containing starch is not particularly limited, and examples thereof include a method of hydrolyzing starch extracted from the plant.

When saccharide is obtained from a saccharide-containing raw material, hydroxyacetone may be contained in a carbonyl compound that may be generated as a by-product or an impurity. It is preferable to use a saccharide-containing raw material containing such hydroxyacetone because it is not necessary to prepare hydroxyacetone separately or the amount of use can be reduced.
Examples of sugar-containing raw materials that can contain hydroxyacetone include lignocellulose-decomposing raw materials (for example, sugar liquid obtained by hydrolyzing or pyrolyzing lignocellulose), sucrose-containing raw materials (for example, sugarcane, sugar beet, sorghum, etc.) Cane juice, crude sugar, molasses) obtained by pressing or leaching

  Moreover, the saccharide-containing raw material used in the present invention may contain other components as long as the effects of the present invention are obtained. Although it does not specifically limit as another component, For example, by-products other than saccharides, impurities, etc. which arise when saccharides are obtained from a saccharide containing raw material are mentioned. Specifically, carbonyl compounds other than sugars, alcohol compounds such as aliphatic conjugated alcohols, sugar alcohols such as xylitol, ribitol, sorbitol, inositol, glycerol, phenol compounds derived from lignin, alkali metal compounds, alkaline earth metal compounds, Examples include inorganic compounds such as nitrogen compounds, sulfur compounds, halogen compounds, and sulfate ions. However, the solid matter derived from lignin or the like is preferably removed by filtration, adsorption or the like in consideration of handleability.

[Microorganisms capable of producing organic compounds]
The microorganism having the ability to produce an organic compound used in the present invention (hereinafter sometimes referred to as “the microorganism of the present invention”) is not particularly limited as long as it has the ability to produce the target organic compound. The “microorganism having the ability to produce organic compounds” in the present invention refers to a microorganism capable of producing and accumulating organic compounds in the medium when the microorganism is cultured in the medium.

<Organic compounds>
The organic compound produced by the microorganism of the present invention is not particularly limited as long as the microorganism is an organic compound that can be produced and accumulated in the medium. Specifically, ethanol, propanol, butanol, 1,3-propanediol Alcohols such as 1,3-butanediol, 2,3-butanediol, 1,4-butanediol, 1,5-pentanediol, 1,6-hexanediol, glycerol, erythritol, xylitol, sorbitol; Amines such as 5-pentamethylenediamine and 1,6-hexamethylenediamine; acetic acid, butyric acid, glycolic acid, lactic acid, 3-hydroxypropionic acid, pyruvic acid, succinic acid, fumaric acid, malic acid, oxaloacetic acid, cis- Aconitic acid, citric acid, isocitric acid, 2-oxoglutaric acid, 2-oxoi Carboxylic acids such as valeric acid, glutaric acid, itaconic acid, adipic acid, levulinic acid, quinic acid, shikimic acid, acrylic acid, methacrylic; alanine, valine, leucine, isoleucine, lysine, arginine, methionine, histidine, cysteine, serine, Amino acids such as threonine, glutamic acid, aspartic acid, glutamine, asparagine, phenylalanine, tyrosine, proline, tryptophan; phenols such as phenol, catechol, hydroquinone; benzoic acid, 4-hydroxybenzoic acid, protocatechuic acid, phthalic acid, isophthalic acid Aromatic carboxylic acids such as terephthalic acid; nucleosides such as inosine and guanosine; nucleotides such as inosinic acid and guanylic acid; unsaturated hydrocarbon compounds such as isobutylene, isoprene and butadiene And the like.

  Among these, alcohols, amines, carboxylic acids, and phenols are preferable because known methods can be employed for fermentation production and can be used as resin raw materials. Fatty acids having 2 to 10 carbon atoms More preferred are aliphatic alcohols and aliphatic carboxylic acids having 2 to 10 carbon atoms.

Moreover, since the hydroxyacetone used by this invention can be converted into pyruvic acid depending on microorganisms, the organic compound obtained through the biosynthetic pathway which uses pyruvic acid as an intermediate body is preferable. This is because not only saccharide-containing raw materials but also hydroxyacetone can be used as a carbon source for organic compound production.
Examples of organic compounds obtained through a biosynthetic pathway using pyruvic acid as an intermediate include alcohols such as ethanol, butanol, 1,3-butanediol, 2,3-butanediol, and 1,4-butanediol, Amines such as 5-pentamethylenediamine, acetic acid, lactic acid, succinic acid, fumaric acid, malic acid, oxaloacetic acid, cis-aconitic acid, citric acid, isocitric acid, 2-oxoglutaric acid, 2-oxoisovaleric acid, itaconic acid Carboxylic acids such as alanine, valine, lysine, glutamic acid, aspartic acid and the like. Among these, ethanol, 1,3-butanediol, 2,3-butanediol, 1,4-butanediol, and succinic acid are more preferable from the viewpoint of fermentation productivity.

<Microorganism>
The microorganism used in the present invention is not particularly limited as long as it is a microorganism having an ability to produce a target organic compound, but the activity of converting hydroxyacetone to 1,2-propanediol, and converting hydroxyacetone to methylglyoxal. Microorganisms having activity are preferable, and in addition to these, microorganisms having an activity of converting methylglyoxal into pyruvic acid are more preferable.

  The activity of converting hydroxyacetone to 1,2-propanediol refers to the activity of catalyzing the reaction of reducing hydroxyacetone to produce 1,2-propanediol. This activity is measured by a known method such as Niimi et al. (Niimi S, Suzuki N, Inui M, Yukawa H, Appl. Microbiol. Biotechnol., 2011, Vol. 90 (5), p1721-9). Can be confirmed. This enzyme reaction is usually a reversible reaction, and the reverse reaction activity can also be measured and confirmed.

  The activity of converting hydroxyacetone to methylglyoxal refers to the activity of catalyzing the reaction of oxidizing hydroxyacetone to produce methylglyoxal. This activity is measured by a known method such as Niimi et al. (Niimi S, Suzuki N, Inui M, Yukawa H, Appl. Microbiol. Biotechnol., 2011, Vol. 90 (5), p1721-9). Can be confirmed. This enzyme reaction is usually a reversible reaction, and the reverse reaction activity can also be measured and confirmed.

  The activity of converting methylglyoxal to pyruvic acid refers to the activity of catalyzing the reaction of oxidizing methylglyoxal to produce pyruvic acid. This activity can be confirmed by measurement by a known method, for example, the method of Ray et al. (Ray S, Ray M, J. Biol. Chem., 1982, Vol. 257 (18), p10566-70). . This enzyme reaction is usually a reversible reaction, and the reverse reaction activity can also be measured and confirmed.

In addition, if the above-mentioned activity of converting hydroxyacetone to 1,2-propanediol and the activity of converting hydroxyacetone to methylglyoxal are not inherently possessed, these activities can be imparted by breeding. it can.
Examples of means for imparting organic compound-producing ability through breeding include mutation treatment and gene recombination treatment, such as enhanced expression of enzyme genes in organic compound biosynthesis pathways and reduced expression of enzyme genes in byproduct biosynthesis pathways, etc. A known method can be employed.

  Although it does not specifically limit as a kind of microorganisms used by this invention, Coryneform bacteria, Escherichia coli, Anaerobiospirillum (Anaerobiospirillum (Anaerobiospirillum) genus bacteria, Actinobacillus (Actinobacillus) genus bacteria, Mannheimia (Bacteria) genus, Basfia (Basfia) It is preferably a microorganism selected from the group consisting of a genus bacterium, a genus Zymomonas, a bacterium belonging to the genus Zymobacter, a filamentous fungus, and a yeast. Among these, coryneform bacteria, Escherichia coli, Anaerobiospirillum genus bacteria, Actinobacillus genus bacteria, Mannheimia genus bacteria, Basfia genus bacteria, Zymobacter genus bacteria, filamentous form At least one selected from the group consisting of bacteria and yeasts is preferred, more preferably coryneform bacteria, Escherichia coli, and yeast, and particularly preferably coryneform bacteria.

  The coryneform bacterium is not particularly limited as long as it is classified as such, and examples include bacteria belonging to the genus Corynebacterium, bacteria belonging to the genus Brevibacterium, bacteria belonging to the genus Arthrobacter, and the like. Preferred examples include those belonging to the genus Corynebacterium and Brevibacterium, and more preferred are Corynebacterium glutamicum, Brevibacterium flavum, Brevibacterium ammoniagenes ( Bacteria classified as Brevibacterium ammoniagenes) or Brevibacterium lactofermentum.

  Particularly preferred specific examples of coryneform bacteria that can be used in the present invention include Brevibacterium flavum MJ-233 (FERM BP-1497), MJ-233 AB-41 (FERM BP-1498), Brevibacterium Ammonia Genes ATCC 6872, Corynebacterium glutamicum ATCC 31831, Brevibacterium lactofermentum ATCC 13869 and the like. Brevibacterium flavum is currently sometimes classified as Corynebacterium glutamicum (Lielbl W, Ehrmann M, Ludwig W, Schleifer KH, Int J Syst Bacteriol., 1991, Vol. 41, p255-260), in the present invention, Brevibacterium flavum MJ-233 strain and its mutant MJ-233 AB-41 strain are Corynebacterium glutamicum MJ-233 strain and MJ-233 AB-41, respectively. The same stock as the stock.

  The above-mentioned Brevibacterium flavum MJ-233 was founded on April 28, 1975 at the Institute of Microbial Technology of the Ministry of International Trade and Industry (currently National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center) (〒305-8856 Japan) No. 1 in Higashi 1-chome, 1-chome, Tsukuba City, Ibaraki Prefecture, Japan, with deposit number FERM P-3068, transferred to an international deposit based on the Budapest Treaty on May 1, 1981, under the deposit number of FERM BP-1497 It has been deposited.

  Examples of E. coli that can be used in the present invention include Escherichia coli. Examples of the genus Anaerobiospirillum that can be used in the present invention include Anaerobiospirillum succiniciproducens.

  Examples of the genus Actinobacillus that can be used in the present invention include Actinobacillus succinogenes. Examples of the genus Mannheimia that can be used in the present invention include Mannheimia succiniciproducens.

  Examples of the bacterium belonging to the genus Basfia that can be used in the present invention include Basfia succiniciproducens. Examples of the genus Zymomonas that can be used in the present invention include Zymomonas mobilis. Further, examples of the genus Zymobacter genus that can be used in the present invention include Zymobacter palmae.

  Examples of filamentous fungi that can be used in the present invention include Aspergillus, Penicillium, and Rhizopus. Among these, Aspergillus genus includes Aspergillus niger, Aspergillus oryzae, and Penicillium includes Penicillium chrysogenum and Penicillium simplicum simplicum. . In the genus Rhizopus, examples include Rhizopus oryzae.

  Examples of the yeast that can be used in the present invention include Saccharomyces, Shizosaccharomyces, Candida, Pichia, Kluyveromyces, and Yarrowia. Examples include the genus (Yarrowia) and the genus Zygosaccharomyces.

  Examples of the genus Saccharomyces include Saccharomyces cerevisiae, Saccharomyces uvarum, Saccharomyces bayanus and the like. Examples of the genus Shizosaccharomyces include Schizosaccharomyces pombe. Examples of the genus Candida include Candida albicans, Candida sonorensis, Candida glabrata, and the like. Examples of the Pichia genus include Pichia pastoris and Pichia stipitis.

  The genus Kluyveromyces includes Kluyveromyces lactis, Kluyveromyces marxianus, and Kluyveromyces thermotolerans. Etc. Moreover, as said Yarrowia genus (Yarrowia), Yarrowia lipolytica (Yarrowia lipolytica) etc. are mentioned. Examples of the genus Zygosaccharomyces include Zygosaccharomyces bailii and Zygosaccharomyces rouxii.

The microorganism is not only a wild strain, but also a mutant strain obtained by a normal mutation treatment such as UV irradiation or NTG treatment, a recombinant strain induced by a genetic technique such as cell fusion or gene recombination. Or a stock of
The microorganism used in the present invention may be a microorganism that inherently has an organic compound-producing ability, or may have been imparted with an organic compound-producing ability by breeding.
For example, if it is desired to give the above-mentioned microorganisms the ability to produce carboxylic acids such as succinic acid, fumaric acid, malic acid, etc., modifications such as those described below that reduce lactate dehydrogenase activity and modifications that enhance pyruvate carboxylase activity are required. Do it accordingly. In addition, when it is desired to impart alcohol-producing ability such as ethanol, butanol, and butanediol to the above-mentioned microorganism, modification as described below for reducing lactate dehydrogenase activity and modification for enhancing alcohol dehydrogenase activity are performed as necessary.
Hereinafter, the microorganisms used in the present invention will be specifically described, classified according to the type of organic compound to be produced.

  When the target organic compound is a carboxylic acid, it is preferable to use a microorganism modified so that lactate dehydrogenase (hereinafter also referred to as LDH) activity is reduced. Here, “LDH activity” refers to an activity (EC: 1.1.1.17) that catalyzes a reaction of reducing pyruvic acid to produce lactic acid. “LDH activity is reduced” means that LDH activity is reduced as compared to an unmodified strain. The LDH activity is preferably reduced to 30% or less per unit cell weight, more preferably 10% or less, compared to the unmodified strain. LDH activity may be completely lost. Decrease in LDH activity can be confirmed by measuring LDH activity by a known method such as the method of Kanarek et al. (Kanarek L, Hill RL, J Biol Chem., 1964, Vol. 239, p4202-4206). Can do.

The strain with reduced LDH activity is treated with the above-mentioned microorganism as a parent strain and treated with a mutagen usually used for mutagenesis such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG) or nitrous acid, Each can be obtained by selecting strains with reduced LDH activity. Moreover, you may modify | change using the gene which codes LDH. Specifically, this can be achieved by disrupting the ldh gene on the chromosome or altering an expression regulatory sequence such as a promoter or Shine-Dalgarno (SD) sequence.
Specific methods for producing strains with reduced LDH activity include methods using homologous recombination to chromosomes (see JP-A-11-206385, etc.) and methods using the sacB gene (Schafer A, Tauch A, Jager W). , Kalinowski J, Thierbach G, Puhler A, Gene 1994 Vol.145 (1), p69-73).

  When the target organic compound is a carboxylic acid, it may be a microorganism modified so that pyruvate carboxylase (hereinafter also referred to as PC) activity is enhanced. Here, “PC activity” refers to an activity (EC: 6.4.1.1) that catalyzes a reaction in which pyruvic acid is carboxylated to produce oxaloacetic acid. “PC activity is enhanced” means that PC activity is increased as compared to an unmodified strain. The PC activity is preferably increased 1.5 times or more per unit cell weight, more preferably 3 times or more, compared to the unmodified strain. The enhanced PC activity is measured by a known method, for example, the method of Fisher et al. (Fisher SH, Magasanik B, J Bacteriol., 1984, Vol. 158 (1), p55-62). Can be confirmed.

  The strain with enhanced PC activity is treated with the above-mentioned microorganism as a parent strain, and is treated with a mutagen that is usually used for mutagenesis such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG) or nitrous acid, Each can be obtained by selecting a strain having enhanced PC activity. Moreover, you may modify | change using the gene which codes PC. Specifically, it can be achieved by increasing the copy number of the pc gene. Increasing the copy number can be achieved by using a plasmid or making multiple copies on the chromosome by a known homologous recombination method. . The enhancement of PC activity can also be achieved by high expression by introducing mutations into the promoter of the pc gene on the chromosome or on the plasmid, replacement with a stronger promoter, and the like.

The pc gene used for enhancing PC activity is not particularly limited as long as it encodes a protein having PC activity, and examples thereof include a gene derived from Corynebacterium glutamicum.
In addition, pc genes derived from bacteria other than coryneform bacteria, other microorganisms, and animals and plants can also be used. A pc gene derived from a microorganism or animal or plant was isolated from a chromosome of a microorganism, animal or plant, etc., and the nucleotide sequence was determined based on a gene having a base sequence already determined, a gene encoding a protein having PC activity based on homology, etc. Things can be used. In addition, after the base sequence is determined, a gene synthesized according to the sequence can also 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.

  A PC expression vector is provided by inserting the gene encoding PC isolated as described above into a known expression vector so as to allow expression. By transforming with this expression vector, a strain with enhanced PC activity can be obtained. Alternatively, a PC activity-enhanced strain can also be obtained by integrating a DNA encoding PC into a chromosomal DNA of a host microorganism so as to allow expression, such as by homologous recombination. Note that transformation and homologous recombination can be performed according to ordinary methods known to those skilled in the art.

  When a PC gene is introduced on a chromosome or a plasmid, an appropriate promoter is incorporated upstream of the gene on the 5'-side, more preferably a terminator is incorporated downstream of the 3'-side. The promoter and terminator are not particularly limited as long as the promoter and terminator are known to function in microorganisms used as hosts, and may be the promoter and terminator of the pc gene itself, or other promoters. And a terminator. Vectors, promoters and terminators that can be used in these various microorganisms are described in detail in, for example, “Basic Course of Microbiology 8 Genetic Engineering / Kyoritsu Shuppan”.

  When the target organic compound is an alcohol, it is preferable to use a microorganism modified so that LDH activity is reduced, as in the carboxylic acid production method. Strains with reduced LDH activity can be prepared in the same manner as described above.

  Further, when the target organic compound is alcohol, it may be a microorganism modified so that alcohol dehydrogenase (hereinafter also referred to as ADH) activity is enhanced. Here, “ADH activity” refers to an activity (EC: 1.1.1.1, 1.1.1.2) that catalyzes a reaction in which an aldehyde is reduced to produce an alcohol. “ADH activity is enhanced” means that ADH activity is increased as compared to an unmodified strain. The ADH activity is preferably increased 1.5 times or more per unit cell weight, more preferably 3 times or more, compared with the unmodified strain. The enhanced ADH activity is known in the art, for example, Kotrbova-Kozak et al. (Kotrbova-Kozak A, Kotrba P, Inui M, Sajdok J, Yukawa H, Appl Microbiol Biotechnol., 2007, Vol. 76 (6 ), p1347-56), and can be confirmed by measuring ADH activity.

A strain with enhanced ADH activity can be prepared in the same manner as the method for enhancing PC activity described above.
The adh gene used for enhancing ADH activity is not particularly limited as long as it encodes a protein having ADH activity. For example, adhB gene derived from Zymomonas mobilis, adhE2 gene derived from Clostridium acetobutylicum Can be mentioned.
Furthermore, adh genes derived from bacteria other than the above, other microorganisms, and animals and plants can also be used. The adh gene derived from microorganisms or animals and plants was isolated from the chromosomes of microorganisms, animals and plants, etc., and the nucleotide sequences were determined based on the genes whose nucleotide sequences had already been determined, genes encoding ADH activity based on homology, etc. Things can be used. In addition, after the base sequence is determined, a gene synthesized according to the sequence can also 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.

  The microorganism used in the present invention may be a microorganism obtained by combining two or more kinds of modifications for imparting organic compound-producing ability. When making a plurality of modifications, the order does not matter.

  In the production method of the present invention, the processed product of the microorganism of the present invention can also be used. Examples of the processed microbial product include, for example, immobilized microbial cells obtained by immobilizing the microbial cells of the present invention described above with acrylamide, carrageenan, etc., crushed crushed cells, centrifugal supernatants thereof, or supernatants thereof. The fraction etc. which refine | purified partially by ammonium sulfate processing etc. are mentioned.

[Method for producing organic compound]
The production method of the present invention is a step of producing an organic compound by causing the microorganism of the present invention or a processed product thereof to act on a saccharide-containing raw material in an aqueous medium in the presence of hydroxyacetone (hereinafter referred to as “fermentation step”). However, it is preferable to have a step of recovering the produced organic compound (hereinafter referred to as “recovery step”) after the fermentation step.

When the microorganism of the present invention is used in the production method of the present invention, a slant cultured medium such as an agar medium may be directly used. However, prior to the fermentation step, the microorganism may be preliminarily used in a liquid medium. What was cultured may be used. That is, the fermentation process can be performed after the microorganisms of the present invention are proliferated in advance by performing seed culture and main culture described later.
In addition, the seed culture and main culture which will be described later and the fermentation process which will be described later can be performed simultaneously without distinction. Moreover, an organic compound can also be produced by reacting a saccharide-containing raw material in the presence of hydroxyacetone while growing a seed-cultured or main-cultured microorganism in a reaction solution.

<Seed culture>
The seed culture is performed in order to prepare the cells of the microorganism to be used for the main culture. As a medium used for seed culture, a normal medium used for culturing microorganisms can be used, but a medium containing a nitrogen source, an inorganic salt, or the like is preferable. Here, the nitrogen source is not particularly limited as long as it is a nitrogen source that can be assimilated and propagated by the present microorganism. Specifically, ammonium salt, nitrate, urea, soybean hydrolyzate, casein degradation product, peptone, yeast Various organic and inorganic nitrogen compounds such as 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, vitamins such as biotin, thiamine, pantothenic acid, inositol, and nicotinic acid, factors that promote growth such as nucleotides and amino acids are added as necessary.

  In seed culture, a carbon source may be added to the medium as necessary. The carbon source used for seed culture is not particularly limited as long as the microorganism can be assimilated and proliferated. Usually, carbohydrates such as galactose, lactose, glucose, fructose, xylose, arabinose, sucrose, starch, and cellulose; Fermentable carbohydrates such as polyalcohols such as glycerol, mannitol, xylitol and ribitol are used, among which glucose, sucrose, or fructose is preferable, and glucose or sucrose is particularly preferable. These carbon sources may be added alone or in combination.

  The seed culture is preferably performed at a general optimum growth temperature. The general optimum growth temperature means a temperature at which the growth rate is the fastest under the conditions used for the production of the organic compound. The specific culture temperature is usually 25 ° C to 40 ° C, preferably 30 ° C to 37 ° C. In the case of coryneform bacteria, the temperature is usually 25 ° C to 35 ° C, more preferably 28 ° C to 33 ° C, and particularly preferably about 30 ° C.

  The seed culture is preferably performed at a general optimum pH for growth. The general optimum growth pH refers to a pH having the fastest growth rate under the conditions used for the production of organic compounds. The specific culture pH is usually pH 4 to 10, preferably pH 6 to 8. In the case of coryneform bacteria, the pH is usually 6-9, with pH 6.5-8.5 being preferred.

  The culture time for seed culture is not particularly limited as long as a certain amount of cells can be obtained, but is usually 6 hours or more and 96 hours or less. In the seed culture, oxygen is preferably supplied by aeration or stirring.

  The bacterial cells after the seed culture can be used for the main culture described later. However, the seed culture may be omitted, and the cells cultured on the slope with a solid medium such as an agar medium may be directly used for the main culture. Moreover, you may repeat seed culture several times as needed.

<Main culture>
The main culture is performed in order to prepare the microbial cells to be subjected to the organic compound production reaction described later, and is mainly intended to increase the amount of the microbial cells. When performing the seed culture described above, the main culture is performed using the cells obtained by seed culture.

  The medium used for the main culture can be a normal medium used for culturing microorganisms, but is preferably a medium containing a nitrogen source, an inorganic salt, or the like. Here, the nitrogen source is not particularly limited as long as it is a nitrogen source that can be assimilated and propagated by the present microorganism. Specifically, ammonium salt, nitrate, urea, soybean hydrolyzate, casein degradation product, peptone, yeast Various organic and inorganic nitrogen compounds such as extract, meat extract and corn steep liquor can be mentioned. As the inorganic salt, various phosphates, sulfates, magnesium, potassium, manganese, iron, zinc, and other metal salts are used. In addition, vitamins such as biotin, thiamine, pantothenic acid, inositol, and nicotinic acid, factors that promote growth such as nucleotides and amino acids are added as necessary. In order to suppress foaming during culture, it is preferable to add an appropriate amount of a commercially available antifoaming agent to the medium.

  In the main culture, it is preferable to add a carbon source to the medium. The carbon source used in the main culture is not particularly limited as long as the microorganism can be assimilated and proliferated. Usually, carbohydrates such as galactose, lactose, glucose, fructose, xylose, arabinose, sucrose, starch, and cellulose; Fermentable carbohydrates such as polyalcohols such as glycerol, mannitol, xylitol and ribitol are used, among which glucose, sucrose, or fructose is preferable, and glucose or sucrose is particularly preferable.

Moreover, the starch saccharified liquid containing the said fermentable saccharide | sugar, molasses etc. are also used, and what is the saccharide liquid which the said fermentable saccharide extracted from plants, such as sugarcane, sugar beet, and a sugar maple, is preferable.
These carbon sources may be added alone or in combination.

  The concentration of the carbon source to be used is not particularly limited, but it is advantageous to add it in a range that does not inhibit growth, and is usually 0.1 to 10% (W / V), preferably 0.5 to the culture solution. It can be used within a range of ˜5% (W / V). Further, a carbon source may be additionally added in accordance with the decrease of the carbon source accompanying the growth.

  The main culture is preferably performed at a general optimum growth temperature. The specific culture temperature is usually 25 ° C to 40 ° C, preferably 30 ° C to 37 ° C. In the case of coryneform bacteria, the temperature is usually 25 ° C to 35 ° C, more preferably 28 ° C to 33 ° C, and particularly preferably about 30 ° C.

  Further, the main culture is preferably performed at a general optimum growth pH. The specific culture pH is usually pH 4 to 10, preferably pH 6 to 8. In the case of coryneform bacteria, the pH is usually 6-9, with pH 6.5-8.5 being preferred.

  The culture time for the main culture is not particularly limited as long as a certain amount of cells can be obtained, but is usually 6 hours or more and 96 hours or less. In the main culture, it is preferable to supply oxygen by aeration or stirring.

  In addition, in the main culture, as a method for preparing bacterial cells more suitable for the production of organic compounds, the culture is performed so that the carbon source depletion and sufficiency described in JP-A-2008-259451 are alternately repeated in a short time. Methods can also be used.

  The cells after the main culture can be used for the organic compound production reaction described later, but the culture solution may be used directly, or may be used after the cells are collected by centrifugation, membrane separation or the like.

<Fermentation process>
In the fermentation process, an organic compound is produced by allowing the above-described microorganism having an organic compound-producing ability or a processed product thereof to act on a saccharide-containing raw material in the presence of hydroxyacetone in an aqueous medium. The reaction that occurs in this fermentation process is hereinafter referred to as “organic compound production reaction”.
(Hydroxyacetone)
Hydroxyacetone is converted to 1,2-propanediol or methylglyoxal by the metabolic reaction of microorganisms. When converted to 1,2-propanediol, electron carriers such as NADH and NADPH are consumed, whereas when converted to methylglyoxal, electron carriers such as NADH and NADPH are generated. That is, the reducing power can be consumed or supplied by the reaction in which hydroxyacetone is metabolized in the cell.

  In addition, some microorganisms can be converted from hydroxyacetone to methylglyoxal and then further converted from methylglyoxal to pyruvic acid. Even in this metabolic reaction, electron carriers such as NADH and NADPH are generated, so that reducing power can be further supplied. Therefore, when hydroxyacetone is converted to pyruvic acid, the reducing power can be supplied more efficiently.

Therefore, in the fermentation process, the presence of hydroxyacetone can adjust the intracellular redox balance as described below.
In the fermentation process, when hydroxyacetone is present, when the reducing power supply from the saccharide-containing raw material is excessive, the reaction in which hydroxyacetone is converted to 1,2-propanediol becomes dominant and consumes the reducing power. Can do. Conversely, when the reducing power supply from the saccharide-containing raw material is insufficient, the reaction in which hydroxyacetone is converted to methylglyoxal becomes dominant, and the reducing power can be supplied. Furthermore, when the reducing power supply from the saccharide-containing raw material is in an appropriate state, the reaction to convert hydroxyacetone to 1,2-propanediol and the reaction to convert to methylglyoxal proceed so as to antagonize and reduce An appropriate state can be maintained without breaking the balance between supply and consumption of power.

  In the fermentation process, the reducing power is relatively satisfactory at the initial stage of production of organic compounds (at the time of low concentration accumulation), but the reducing power tends to be insufficient at the latter stage of production (at the time of high concentration accumulation). Therefore, in order to obtain the effect of improving the production rate under high concentration accumulation conditions, it is important to supply the reducing power at the timing when the reducing power in the latter stage of production starts to run short. If the fermentation process is performed in the presence of, the redox balance can be automatically adjusted as described above.

  In the fermentation of microorganisms, it is usually difficult to add an appropriate amount of a compound such as neutral red of Non-Patent Document 1 after determining the timing at which the supply of reducing power begins to be insufficient. It was very difficult to obtain the effect of improving the production rate under the condition that the target organic compound was accumulated at a high concentration.

  The concentration of hydroxyacetone in the aqueous medium at the start of the organic compound production reaction is not particularly limited as long as the effects of the present invention can be obtained, but is usually 0.01 g / L or more, preferably 3 in the aqueous medium. 0.0 g / L or more, more preferably 10 g / L or more, and usually 100 g / L or less, preferably 50 g / L or less, more preferably 30 g / L or less. The concentration of hydroxyacetone in the aqueous medium during the organic compound production reaction is not particularly limited as long as the effect of the present invention is obtained, but is 0.001 g / L or more, preferably 0.3 g / L in the aqueous medium. L or more, more preferably 1 g / L or more, and usually 10 g / L or less, preferably 5 g / L or less, more preferably 3 g / L or less.

Hydroxyacetone may be added directly into the aqueous medium, but in addition to or in place of this, a saccharide-containing raw material containing hydroxyacetone can also be used.
In addition, the timing of adding hydroxyacetone to the aqueous medium is not particularly limited as long as it is within a range in which the function of regulating the intracellular redox balance of the microorganism can be exerted. For example, the organic compound production reaction in the fermentation process It may be added in advance to the aqueous medium before the start of the step, or after the organic compound production reaction is started.

(Aqueous medium)
Moreover, an aqueous medium is an aqueous solution which performs the organic compound production reaction in a fermentation process, and it is preferable that it is an aqueous solution containing a nitrogen source, an inorganic salt, etc. so that it may mention later. In the aqueous medium, an organic compound production reaction can be performed by reacting the microorganism or a processed product thereof with a saccharide-containing raw material. In this specification, the aqueous medium means all liquids contained in the reaction vessel.

  The aqueous medium may be, for example, a medium for culturing microorganisms or a buffer solution such as a phosphate buffer, but the reaction solution is an aqueous solution containing a nitrogen source, an inorganic salt, or the like. It is preferable. Here, the nitrogen source is not particularly limited as long as it is a nitrogen source that can be assimilated by the microorganism to produce an organic compound. 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, vitamins such as biotin, thiamine, pantothenic acid, inositol, and nicotinic acid, factors that promote growth such as nucleotides and amino acids are added as necessary. In order to suppress foaming during the reaction, it is preferable to add an appropriate amount of a commercially available antifoaming agent to the reaction solution.

  The aqueous medium preferably contains carbonate ions, bicarbonate ions, or carbon dioxide gas (carbon dioxide gas) in addition to the saccharide-containing raw material, nitrogen source, inorganic salt, and the like described above. 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, potassium bicarbonate and the like.

  The concentration of carbonate ion or bicarbonate ion in the aqueous medium is usually 1 mM or more, preferably 2 mM or more, more preferably 3 mM or more, and usually 500 mM or less, preferably 300 mM or less, more preferably 200 mM or less. When carbon dioxide gas is contained, it is usually preferred to contain 50 mg or more, preferably 100 mg or more, more preferably 150 mg or more of carbon dioxide gas per liter of aqueous medium, while usually 25 g or less, preferably It is preferable to contain 15 g or less, more preferably 10 g or less of carbon dioxide gas.

  The pH of the aqueous medium during the organic compound production reaction is preferably adjusted to a range where the activity is most effectively exhibited according to the type of the microorganism used. Specifically, when coryneform bacteria are used, the pH of the reaction solution is usually 5.5 or higher, preferably 6 or higher, more preferably 6.6 or higher, and even more preferably 7.1 or higher. Usually, it is preferably 10 or less, preferably 9.5 or less, more preferably 9.0 or less.

  The pH of the aqueous medium is determined when the organic compound produced is an acidic substance, such as sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, ammonium carbonate, ammonium bicarbonate, sodium hydroxide, hydroxide It can be adjusted by adding calcium, magnesium hydroxide, ammonia (ammonium hydroxide), or a mixture thereof. When the organic compound to be produced is a basic substance, by adding an inorganic acid such as hydrochloric acid, sulfuric acid or phosphoric acid, an organic acid such as acetic acid, a mixture thereof, or by supplying carbon dioxide gas Can be adjusted.

(Sugar-containing raw material)
Examples of the saccharide-containing raw materials used in the present invention and preferred examples of the saccharide-containing raw materials are as described above.

  The use concentration of the saccharide-containing raw material is not particularly limited, but it is advantageous and preferable in terms of productivity if it is as high as possible within a range not inhibiting the formation of the organic compound. The concentration of the saccharide-containing raw material contained in the aqueous medium is usually 5% (W / V) or more, preferably 10% (W / V) or more, based on the concentration of the saccharide contained therein. On the other hand, it is usually 30% (W / V) or less, preferably 20% (W / V) or less. Moreover, you may add in addition of the saccharide containing raw material according to the reduction | decrease of the said saccharide containing raw material accompanying progress of the production reaction of an organic compound.

(Other conditions)
The amount of microbial cells used in the organic compound production reaction is not particularly limited, but the weight of wet cells is usually 1 g / L or more, preferably 10 g / L or more, more preferably 20 g / L or more. 700 g / L or less, preferably 500 g / L or less, more preferably 400 g / L or less.

  The time for the organic compound production reaction is not particularly limited, but is usually 1 hour or longer, preferably 3 hours or longer, and is usually 168 hours or shorter, preferably 72 hours or shorter.

  The organic compound production reaction may be performed at the same temperature as the optimum growth temperature of the microorganism to be used, but it is advantageous to carry out the reaction at a temperature higher than the optimum growth temperature, usually 2 ° C to 20 ° C, preferably It is carried out at a temperature 7 to 15 ° C higher. Specifically, in the case of coryneform bacteria, it is usually 35 ° C. or higher, preferably 37 ° C. or higher, more preferably 39 ° C. or higher, and usually 45 ° C. or lower, preferably 43 ° C. or lower, more preferably 41 It is below ℃. During the organic compound production reaction, it is not always necessary to set the temperature within the range of 35 ° C to 45 ° C.

  The organic compound generation reaction may be performed with aeration and stirring, but is preferably performed in an anaerobic atmosphere without aeration and without supplying oxygen. Under the anaerobic atmosphere here, for example, the container is sealed and reacted without aeration, the reaction is performed by supplying an inert gas such as nitrogen gas, or the inert gas containing carbon dioxide gas is vented. Can be obtained.

  The method for producing an organic compound of the present invention is not particularly limited, but can be applied to any of batch reaction, semi-batch reaction, and continuous reaction.

<Recovery process>
In the present invention, an organic compound is generated by the organic compound generation reaction and can be accumulated in the reaction solution. The accumulated organic compound may further include a step of recovering from the aqueous medium according to a conventional method. Specifically, for example, when the accumulated organic compound is a carboxylic acid such as succinic acid, fumaric acid or malic acid, solid substances such as cells are removed by centrifugation, filtration, etc., and then ion exchange is performed. The carboxylic acid can be recovered by desalting with a resin or the like, and purification from the solution by crystallization (crystallization) or column chromatography. If the accumulated organic compound is an alcohol such as ethanol, butanol, or butanediol, solids such as cells are removed by centrifugation, filtration, etc., and then concentrated by distillation or the like, and the solution is dehydrated into a membrane. For example, alcohol can be recovered.

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

The LC analysis method is shown below.
(Liquid phase chromatograph (LC) analysis-1)
Pump: Hitachi High-Technologies Corporation L-2130
Column oven: Hitachi High-Technologies L-2350
UV detector: Hitachi High-Technologies Corporation L-2400
RI detector: L-2490 manufactured by Hitachi High-Technologies Corporation
Column: ULTRON PS-80H 8.0ID × 300mmL manufactured by Shinwa Kako Co., Ltd.
Temperature: 60 ° C
Eluent: 0.11% by mass perchloric acid solution 1.0 mL / min Detection method: UV (210 nm), RI
Injection volume: 10 μL
(Liquid phase chromatograph (LC) analysis-2)
Pump: Shimadzu Corporation LC-10Ai
Column oven: CTO-10A manufactured by Shimadzu Corporation
UV detector: SPD-10A manufactured by Shimadzu Corporation
RI detector: Shimadzu RID-10A
Column: CK08EH 8.0ID × 300mmL manufactured by Mitsubishi Chemical Corporation
Temperature: 60 ° C
Eluent: 0.055 mass% perchloric acid solution 0.8 mL / min Detection method: UV (280 nm), RI
Injection volume: 10 μL
Succinic acid and pyruvic acid were quantified by LC analysis-1, and hydroxyacetone and 1,2-propanediol were quantified by LC analysis-2.
In LC analysis-2, the peaks of hydroxyacetone and 1,2-propanediol overlap with each other in UV (280 nm) and RI detection. However, since only hydroxyacetone has UV absorption (280 nm), hydroxyacetone was quantified by UV (280 nm), and 1,2-propanediol was quantified from RI after subtracting the RI conversion value.

Example 1
<Evaluation of succinic acid production in the presence of hydroxyacetone>
(A) Seed culture A 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 hydrate: 20 mg, manganese sulfate pentahydrate: 20 mg, D-biotin: 200 μg, thiamine hydrochloride: 200 μg, yeast extract: 1 g, casamino acid: 1 g, dissolved in 1,000 mL of distilled water] 1,000 mL After sterilization by heating at 121 ° C. for 20 minutes and cooling to room temperature, 15 mL was put into a 200 mL Erlenmeyer flask, and 600 μl of a 50% glucose aqueous solution previously sterilized was added. Brevibacterium flavum MJ233 / XylAB / PC-4 / ΔLDH strain (XylAB introduction, PC enhancement, LDH disruption strain: Japanese Patent Application No. 2013-161477) was inoculated and cultured with shaking at 30 ° C. for 5.1 hours.

(B) Main culture After adding 100 mL of A medium to a 500 mL Erlenmeyer flask and adding 4 mL of a 50% aqueous glucose solution sterilized in advance, the culture solution obtained in the seed culture of (A) above was treated with O.D. D. (660 nm) was inoculated to be 0.02, and cultured with shaking at 30 ° C. for 21.5 hours.

(C) Fermentation process (succinic acid production reaction)
The culture solution obtained in the main culture of (B) above was collected by centrifugation at 5,000 × g for 7 minutes, and the cell suspension [magnesium sulfate heptahydrate: 320 mg, ferrous sulfate 7 hydrate: 13 mg, manganese sulfate pentahydrate: 13 mg, phosphoric acid (85%): 410 mg, potassium hydroxide (48%): 540 mg, dissolved in 1000 mL of distilled water] D. A cell solution was prepared by suspending (660 nm) to be 60. Subsequently, glucose aqueous solution (500 g / L): 18 g, distilled water: 44 g, cell suspension: 1 mL, D-biotin aqueous solution (100 mg / L): 66 mg, thiamine hydrochloride (100 mg / L): 66 mg were mixed. A substrate solution was prepared. To the substrate solution, hydroxyacetone: 1.1 g, ammonium hydrogen carbonate: 960 mg, and a bacterial cell solution were added and reacted at 40 ° C. in an anaerobic atmosphere (the concentration of hydroxyacetone at the start of the reaction was 13.6 g / L). there were). The pH was maintained at 7.3 by adding a neutralizing agent [aqueous ammonia (28%): 97 g, ammonium bicarbonate: 32 g, dissolved in 250 mL of distilled water]. As a result, the accumulated amount of succinic acid after 16.8 hours was 3.74 g (31.7 mmol), the amount of pyruvic acid was 0.48 g (5.5 mmol), and the amount of hydroxyacetone was 0.30 g (4.1 mmol (3. 3g / L)), the concentration of 1,2-propanediol was 0.59 g (7.8 mmol), the accumulated amount of succinic acid after 24.1 hours was 4.69 g (39.7 mmol), and the amount of pyruvic acid was 0. .48 g (5.5 mmol), the amount of hydroxyacetone was 0.10 g (1.3 mmol (1.0 g / L)), and the amount of 1,2-propanediol was 0.71 g (9.3 mmol).

(Comparative Example 1)
The succinic acid production reaction was performed under the same conditions as in Example 1 except that hydroxyacetone was not added to the substrate solution. As a result, the accumulated amount of succinic acid after 16.8 hours was 3.74 g (31.7 mmol), the amount of pyruvic acid was 0.33 g (3.8 mmol), the amount of hydroxyacetone was 0 g (0 mmol), and 1,2-propane. The amount of diol is 0 g (0 mmol), the accumulated amount of succinic acid after 24.1 hours is 4.02 g (34.1 mmol), the amount of pyruvic acid is 0.25 g (2.9 mmol), the amount of hydroxyacetone is 0 g (0 mmol) ), The amount of 1,2-propanediol was 0 g (0 mmol).

  As a result of Example 1 and Comparative Example 1, the amounts of succinic acid, pyruvic acid, hydroxyacetone and 1,2-propanediol are shown in Table 1.

  The succinic acid production rate, hydroxyacetone consumption rate, and 1,2-propanediol production rate of Example 1 and Comparative Example 1 were calculated from the values in Table 1 and shown in Table 2.

From Table 2, in Example 1 to which hydroxyacetone was added, the succinic acid production rate in the initial stage of production (reaction 0 to 16.8 hours) was almost the same as that of Comparative Example 1, but the latter half of production (reaction 16. The production rate of succinic acid in 8 hours to 24.1 hours) was increased by 3.4 times.
In Example 1, the hydroxyacetone consumption rate in the latter stage of production is about 68% of the rate in the early stage of production, whereas the 1,2-propanediol production rate in the latter stage of production is only 43% of the rate in the early stage of production. The degree dropped significantly. This indicates that the reaction rate at which hydroxyacetone is converted to 1,2-propanediol is greatly reduced in the latter stage of production, and most of it is converted to methylglyoxal, and the reaction that supplies reducing power is dominant. It was suggested that
From the above results, it was clarified that by performing the succinic acid production reaction in the presence of hydroxyacetone, the shortage of reducing power at the time of high concentration accumulation was eliminated, and the production rate was improved.

  According to the method for producing an organic compound of the present invention, the production rate of an organic compound under a high concentration accumulation condition can be improved by adjusting the intracellular redox balance of a microorganism, and a desired organic compound can be produced with high production efficiency. can do.

Claims (9)

It has a step (hereinafter referred to as “fermentation step”) of producing an organic compound by allowing a microorganism having organic compound-producing ability or a treated product thereof to act on a saccharide-containing raw material in an aqueous medium in the presence of hydroxyacetone. ,
The organic compound is an organic compound obtained through a biosynthetic pathway having pyruvic acid as an intermediate;
The microorganism is a microorganism having an activity of converting hydroxyacetone to 1,2-propanediol, an activity of converting hydroxyacetone to methylglyoxal, and an activity of converting methylglyoxal to pyruvic acid, and starting the fermentation process Characterized in that the concentration of hydroxyacetone in the aqueous medium is at least 13.6 g / L ,
The said saccharide is a manufacturing method of the organic compound containing 1 or more types selected from glucose, fructose, and sucrose .   The method for producing an organic compound according to claim 1, wherein the saccharide-containing raw material is a raw material containing hydroxyacetone.   The method for producing an organic compound according to claim 1 or 2, wherein the saccharide-containing raw material is a lignocellulose decomposition raw material.   The method for producing an organic compound according to claim 1, wherein the saccharide-containing raw material is a sucrose-containing raw material.   The said fermentation process is performed in anaerobic atmosphere, The manufacturing method of the organic compound as described in any one of Claims 1-4 characterized by the above-mentioned.   The said aqueous medium contains at least 1 type chosen from the group which consists of carbonate ion, bicarbonate ion, and a carbon dioxide gas, The manufacture of the organic compound as described in any one of Claims 1-5 characterized by the above-mentioned. Method. The said organic compound is at least 1 type chosen from the group which consists of alcohol, amines, carboxylic acids, and phenols, The manufacture of the organic compound as described in any one of Claims 1-6 characterized by the above-mentioned. Method. Examples of the microorganism include coryneform bacteria, Escherichia coli, Anaerobiospirillum genus bacteria, Actinobacillus genus bacteria, Mannheimia genus bacteria, Basfia genus bacteria, Zymomonas (Zymomonas) (Zymobacter) bacteria, and wherein the filamentous fungus, and at least one is selected from the group consisting of yeast, a manufacturing method of an organic compound according to any one of claims 1-7. The method for producing an organic compound according to any one of claims 1 to 8 , further comprising a step of purifying the organic compound obtained in the fermentation step.

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